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UBC Theses and Dissertations

Identification and partial characterization of a transposon insertion mutant of Burkohderia multivorans… Steen, Barbara R. 1999

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IDENTIFICATION AND PARTIAL CHARACTERIZATION OF A TRANSPOSON INSERTION MUTANT OF BURKHOLDERIA MULTIVORANS WITH REDUCED RESISTANCE TO POLYMYXIN B b y B A R B A R A R. S T E E N B . Sc., T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1 9 9 0 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y i n T H E F A C U L T Y O F G R A D U A T E S T U D I E S D e p a r t m e n t o f M i c r o b i o l o g y a n d I m m u n o l o g y W e a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A S e p t e m b e r 1 9 9 9 © B a r b a r a R. S t e e n , 1 9 9 9 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of H v c r °k\ o t o 9) H The University of British Columbia Vancouver, Canada Date rhex-,,3 3 i °\°)°) DE-6 (2/88) Abstract Abstract Burkholderia multivorans i s a n a n t i b i o t i c r e s i s t a n t o p p o r t u n i s t i c p a t h o g e n t h a t i s b e i n g i s o l a t e d w i t h a n i n c r e a s e d f r e q u e n c y f r o m i m m u n o c o m p r o m i s e d p a t i e n t s . B. multivorans c a n c a u s e s e r i o u s l u n g i n f e c t i o n s i n p a t i e n t s w i t h c y s t i c f i b r o s i s b u t t h e m e c h a n i s m o f p a t h o g e n e s i s i s n o t u n d e r s t o o d . A s w e l l , B. multivorans c a u s e s l u n g i n f e c t i o n s i n c h r o n i c g r a n u l o m a t o u s d i s e a s e p a t i e n t s . N e u t r o p h i l s f r o m t h e s e p a t i e n t s a r e u n a b l e t o e l i c i t a n o x i d a t i v e b u r s t a n d a r e t h e r e f o r e d e p e n d e n t o n n o n o x i d a t i v e k i l l i n g m e c h a n i s m s f o r d e s t r o y i n g p h a g o c y t o s e d m i c r o b e s . B. multivorans i s r e s i s t a n t t o in vitro k i l l i n g b y c h r o n i c g r a n u l o m a t o u s d i s e a s e n e u t r o p h i l s a n d i s r e s i s t a n t t o k i l l i n g b y c a t i o n i c p e p t i d e s w h i c h a r e a m a j o r c o m p o n e n t o f n e u t r o p h i l n o n o x i d a t i v e k i l l i n g . T h e p u r p o s e o f t h e s e s t u d i e s w a s t o u n d e r s t a n d h o w B. multivorans r e s i s t s k i l l i n g b y c a t i o n i c p e p t i d e s . A t r a n s p o s o n i n s e r t i o n m u t a n t o f B. multivorans ( 2 6 D 7 ) w a s c r e a t e d t h a t h a d i n c r e a s e d s u s c e p t i b i l i t y t o p o l y m y x i n B , c o l i s t i n a n d t h e c a t i o n i c p e p t i d e C P 2 6 ( a c e c r o p i n - m e l i t t i n h y b r i d ) . I n o r d e r t o d e t e r m i n e t h e g e n e t i c n a t u r e o f t h i s m u t a t i o n , a c o s m i d l i b r a r y o f B. multivorans s t r a i n A T C C 1 7 6 1 6 w a s c o n s t r u c t e d a n d s c r e e n e d b y S o u t h e r n a n a l y s i s u s i n g a p r o b e s p e c i f i c f o r t h e D N A a d j a c e n t t o t h e t r a n s p o s o n i n s e r t i o n s i t e . S e q u e n c e a n a l y s i s o f t h e D N A at t h e s i t e o f t r a n s p o s o n i n s e r t i o n i n m u t a n t 2 6 D 7 s h o w e d f o u r o p e n r e a d i n g f r a m e s ( O R J F s ) . O R F 2 s h o w e d p r o t e i n s e q u e n c e s i m i l a r i t y t o s e v e r a l g l u c o s y l t r a n s f e r a s e s . O R F 3 h a d p r o t e i n s e q u e n c e s i m i l a r i t y t o m e t h y l t r a n s f e r a s e s a n d w a s t h e s i t e o f t h e t r a n s p o s o n i n s e r t i o n i n m u t a n t 2 6 D 7 . P h e n o t y p i c a n a l y s i s o f m u t a n t 2 6 D 7 s h o w e d t h a t t h e o u t e r m e m b r a n e o f t h i s m u t a n t w a s m o r e p e r m e a b l e t o 1 - A ^ - p h e n y l n a p h t h y l a m i n e i n t h e p r e s e n c e o f p o l y m y x i n t h a n t h e p a r e n t s t r a i n ; h o w e v e r , l i k e t h e p a r e n t s t r a i n , t h e m u t a n t r e m a i n e d i n c a p a b l e o f b i n d i n g i i Abstract d a n s y l a t e d p o l y m y x i n . N o d i f f e r e n c e w a s s e e n i n t h e l i p o p o l y s a c c h a r i d e l a d d e r s . T L C a n a l y s i s r e v e a l e d o n e f e w e r g l y c o l i p i d a n d a p o s s i b l e s h i f t i n o n e o f t h e p h o s p h a t i d y l e t h a n o l a m i n e s p o t s i n t h e m u t a n t . T h e r e s u l t s p r e s e n t e d h e r e s u g g e s t e d t h a t a g r o u p o f g e n e s w e r e i d e n t i f i e d t h a t e n c o d e d f o r p r o t e i n s p o s s i b l y i n v o l v e d i n l i p i d m o d i f i c a t i o n , i m p o r t a n t f o r r e s i s t a n c e t o c a t i o n i c p e p t i d e s . F u r t h e r s t u d i e s m a y i d e n t i f y t h e r o l e o f l i p i d m o d i f i c a t i o n i n t h i s i m p o r t a n t p h e n o t y p e o f t h e Burkholderia cepacia c o m p l e x . i i i Table of Contents Table of Contents Abstract 1 1 Table of contents iv List of Tables viii List of Figures i x Acknowledgements xii Dedication xiii List of Abbreviations xiv Chapter 1 Introduction 1 1.1 Burkholderia multivorans and the Burkholderia cepacia complex 1 1.2 The unusual genome of B. cepacia 2 1.3 Significance of B. cepacia 3 1.3.1 Environmental 3 1.3.2 Clinical 5 1.4 Virulence factors of B. cepacia 8 1.5 The Gram-negative outer membrane 11 1.5.1 General structure 11 1.5.2 Lipopolysaccharide 11 1.5.3 Membrane proteins 14 1.6 B. cepacia LPS and Host Interaction 14 1.7 Antibiotic Resistance of B. cepacia 16 1.7.1 Antibiotic uptake across the outer membrane 16 1.7.2 Antibiotic efflux pumps 17 1.7.3 Antibiotic modifying enzymes 18 iv Table of Contents 1.8 C a t i o n i c P e p t i d e s . 19 1.8.1 C a t i o n i c p e p t i d e s o f n e u t r o p h i l s 2 0 1.8.2 a - h e l i c a l p e p t i d e s 2 2 1.8.3 M e c h a n i s m o f a c t i o n 2 3 1.8.4 B a c t e r i a l r e s i s t a n c e t o c a t i o n i c p e p t i d e s a n d p o l y m y x i n 2 4 1.9 T h e s i s O b j e c t i v e s 2 6 Chapter 2 Materials and Methods 2 8 2.1 S t r a i n s , P l a s m i d s a n d G r o w t h C o n d i t i o n s 2 8 2.2 C o n s t r u c t i o n o f a Tn5-751 I n s e r t i o n M u t a n t L i b r a r y o f B. multivorans 2 8 2.3 P o l y m e r a s e C h a i n R e a c t i o n ( P C R ) 3 0 2.4 S o u t h e r n A n a l y s i s 3 0 2.5 S c r e e n i n g f o r a M u t a n t S u s c e p t i b l e t o P o l y m y x i n 31 2.6 D e t e r m i n a t i o n o f M i n i m a l I n h i b i t o r y C o n c e n t r a t i o n s ( M I C s ) 31 2.7 I s o l a t i o n o f C o s m i d C l o n e s 3 2 2.7.1 C o n s t r u c t i o n o f a C o s m i d L i b r a r y 3 2 2.7.2 C o n s t r u c t i o n o f a D N A P r o b e 3 3 2.7.3 S c r e e n i n g o f t h e B. multivorans g e n o m i c l i b r a r y 3 4 2.8 C l o n i n g o f t h e BamKl, Sail a n d Pstl F r a g m e n t s 3 4 2.9 C o m p l e m e n t a t i o n o f M u t a n t 2 6 D 7 3 4 2.10 N u c l e o t i d e S e q u e n c i n g a n d A n a l y s i s 3 5 2.11 B i o c h e m i c a l A n a l y s i s 3 6 2.12 G r a m S t a i n s 3 6 2.13 E l e c t r o n M i c r o s c o p y 3 6 2.14 N e u t r o p h i l K i l l i n g A s s a y 3 7 Table of Contents 2.15 Permeabilization of Whole Cells of B. cepacia to 1 -JV-phenylnaphylamine 37 2.16 Dansyl-polymyxin Binding Assay 38 2.17 Isolation of Membrane Proteins 39 2.18 Whole Cell Lysates and Proteinase K Digestion 39 2.19 SDS-PAGE 40 2.20 Silver Staining of Lipopolysaccharide (LPS) Ladders 40 2.21 Extraction of Lipids 41 2.22 Thin Layer Chromatography (TLC) 42 Chapter 3 Isolation and Partial Characterization of a Tn- 751S Insertion Mutant of B. multivorans, with Reduced Resistance to Polymyxin B 43 3.1 Objective 43 3.2 Transposition of Tn5-751 inB. multivorans 43 3.3 Isolation of Mutant 26D7 45 3.4 Initial Characterization of Mutant 26D7 49 3.5 Discussion 54 Chapter 4 Identification of Putative Genes Involved in Resistance to Polymyxin 57 4.1 Objective 57 4.2 Cloning of the Locus at the Transposon Insertion Site 57 4.3 Sequence Analysis of the Complementing Clone pDBS9 61 4.4 Identification of Putative Genes on the Complementing Fragment 67 4.4.1 ORF1 67 4.4.2 ORF2 70 4.4.3 ORF3 77 4.4.4 ORF4 79 vi Table of Contents 4.4.5 0 R F 5 83 4.5 S u b c l o n i n g o f Bam H I , Sal I a n d Pst I f r a g m e n t s i n t o p D N 1 8 85 4.6 C o m p l e m e n t a t i o n o f m u t a n t 2 6 D 7 8 5 4.7 D i s c u s s i o n 9 0 Chapter 5 Phenotypic Characterization of the Mutant 26D7 9 7 5.1 B a s i c B i o c h e m i s t r y a n d M i c r o b i o l o g y 9 7 5.2 N e u t r o p h i l K i l l i n g A s s a y 9 9 5.3 O u t e r M e m b r a n e P e r m e a b i l i t y 9 9 5.4 D a n s y l - p o l y m y x i n B i n d i n g t o t h e O u t e r M e m b r a n e 1 0 2 5.5 M e m b r a n e P r o t e i n s 1 0 2 5.6 L i p o p o l y s a c c h a r i d e L a d d e r s 1 0 6 5.7 T h i n L a y e r C h r o m a t o g r a p h y 1 0 9 5.8 D i s c u s s i o n I l l Chapter 6 General Discussion 115 References 1 2 0 Appendices 1 3 7 v i i List of Tables List of Tables Table 1. Strains and P lasmids 29 Table 2. M I C s o f p o l y m y x i n B and gentamicin for selected mutants 51 Table 3. M I C s o f cat ionic ant imicrobia l agents for the parent B. multivorans A T C C 17616 and mutant 2 6 D 7 52 Table 4. M I C s o f different famil ies o f antibiotics for the parent B. multivorans A T C C 17616 and mutant 2 6 D 7 53 Table 5. Genes and expect values f rom the results o f a g a p p e d - B L A S T P search us ing B. multivorans O R F 1 predicted protein sequence as the query sequence 69 Table 6. Genes and expect values from the results of a g a p p e d - B L A S T P search using B. multivorans O R F 2 predicted protein sequence as the query sequence 72 Table 7. Genes and expect values f rom the results o f a g a p p e d - B L A S T P search us ing B. multivorans O R F 3 predicted protein sequence as the query sequence 78 Table 8. Genes and expect values from the results of a g a p p e d - B L A S T P search us ing B. multivorans O R F 4 predicted protein sequence as the query sequence 82 Table 9. M I C s o f cat ionic ant imicrobia l agents for the parent B. multivorans A T C C 17616, mutant 2 6 D 7 and 2 6 D 7 + p D B S 9 89 Table 10. C a r b o n ass imi la t ion and b iochemica l tests for B. multivorans 98 Table 11. P r i m e r sequences 138 v i i i List of Figures List of Figures F i g u r e 1. P h a g o c y t o s i s , d e g r a n u l a t i o n a n d o x i d a t i v e r a d i c a l g e n e r a t i o n 7 F i g u r e 2. L i p i d A a n d i n n e r c o r e r e g i o n o f L P S f r o m Salmonella 12 F i g u r e 3. T r a n s p o s o n - d o n o r p l a s m i d p T G L 1 6 6 4 4 F i g u r e 4. P C R o f p u t a t i v e m u t a n t s u s i n g p r i m e r s s p e c i f i c f o r t h e t r a n s p o s o n 4 6 F i g u r e 5. S o u t h e r n b l o t a n a l y s i s o f p u t a t i v e m u t a n t s u s i n g a 3 2 P - l a b e l e d p r o b e s p e c i f i c f o r t h e t r a n s p o s o n 4 7 F i g u r e 6. R A P D - P C R o f m u t a n t s c o m p a r e d w i t h t h e p a r e n t B. multivorans s t r a i n A T C C 1 7 6 1 6 4 8 F i g u r e 7. S o u t h e r n b l o t a n a l y s i s o f s e l e c t e d p u t a t i v e p o l y m y x i n B s e n s i t i v e m u t a n t s u s i n g a p r o b e s p e c i f i c f o r t h e t r a n s p o s o n 5 0 F i g u r e 8. R e s t r i c t i o n e n z y m e m a p o f t h e 3.75 k b Sal I f r a g m e n t f r o m m u t a n t 2 6 D 7 t h a t h y b r i d i z e d t h e p r o b e s p e c i f i c f o r t h e t r a n s p o s o n 5 8 F i g u r e 9. S o u t h e r n b l o t a n a l y s i s o f c o l o n y d o t b l o t s o f t h e B. multivorans s t r a i n A T C C 1 7 6 1 6 c o s m i d l i b r a r y 6 0 F i g u r e 10. I d e n t i f i c a t i o n o f r e s t r i c t i o n e n z y m e d i g e s t e d c o s m i d f r a g m e n t s t h a t h y b r i d i z e t o t h e D N A p r o b e s p e c i f i c f o r t h e D N A a d j a c e n t t o t h e t r a n s p o s o n i n s e r t i o n i n m u t a n t 2 6 D 7 6 2 F i g u r e 11. N u c l e o t i d e s e q u e n c e o f t h e 6,029 b p D N A r e g i o n f r o m B. multivorans A T C C 1 7 6 1 6 n e a r t h e t r a n s p o s o n i n s e r t i o n s i t e i n m u t a n t 2 6 D 7 6 3 F i g u r e 12. D N A a n d c o r r e s p o n d i n g a m i n o a c i d s e q u e n c e f o r t h e i d e n t i f i e d B. multivorans p u t a t i v e O R F 1 6 8 ix List of Figures F i g u r e 13. A m i n o a c i d s e q u e n c e a l i g n m e n t f o r t h e O R F 1 p r e d i c t e d p r o t e i n s e q u e n c e a n d s i m i l a r s e q u e n c e s i d e n t i f i e d i n t h e g a p p e d - B L A S T P s e a r c h u s i n g t h e O R F 1 p r e d i c t e d p r o t e i n s e q u e n c e as t h e q u e r y s e q u e n c e 71 F i g u r e 14. A m i n o a c i d s e q u e n c e a l i g n m e n t f o r t h e O R F 2 p r e d i c t e d p r o t e i n s e q u e n c e a n d s i m i l a r s e q u e n c e s i d e n t i f i e d i n t h e g a p p e d - B L A S T P s e a r c h u s i n g t h e O R F 2 p r e d i c t e d p r o t e i n s e q u e n c e as t h e q u e r y s e q u e n c e 7 4 F i g u r e 15. P h y l o g e n e t i c t r e e o f p r o t e i n s e q u e n c e s s i m i l a r t o t h e O R F 2 p r e d i c t e d p r o t e i n s e q u e n c e o f B. multivorans 7 5 F i g u r e 16. A m i n o a c i d s e q u e n c e a l i g n m e n t f o r t h e O R F 3 p r e d i c t e d p r o t e i n s e q u e n c e a n d s i m i l a r s e q u e n c e s i d e n t i f i e d i n t h e g a p p e d - B L A S T P s e a r c h u s i n g t h e O R F 3 p r e d i c t e d p r o t e i n s e q u e n c e a s t h e q u e r y s e q u e n c e 8 0 F i g u r e 17. A m i n o a c i d s e q u e n c e a l i g n m e n t s f o r t h e O R F 4 p r e d i c t e d p r o t e i n s e q u e n c e a n d s i m i l a r s e q u e n c e s i d e n t i f i e d i n t h e g a p p e d - B L A S T P s e a r c h u s i n g t h e O R F 4 p r e d i c t e d p r o t e i n s e q u e n c e a s t h e q u e r y s e q u e n c e 8 4 F i g u r e 18. R e s t r i c t i o n m a p o f t h e Bam H I a n d Sal I f r a g m e n t s a n d t h e i r r e s p e c t i v e o r i e n t a t i o n s w h e n c l o n e d i n t o p D N 1 8 8 6 F i g u r e 19. C G D n e u t r o p h i l k i l l i n g a s s a y f o r B. multivorans A T C C 1 7 6 1 6 a n d m u t a n t 2 6 D 7 1 0 0 F i g u r e 2 0 . T h e e f f e c t o f i n c r e a s i n g c o n c e n t r a t i o n s o f p o l y m y x i n B o n t h e o u t e r m e m b r a n e p e r m e a b i l i t y o f B. multivorans A T C C 1 7 6 1 6 a n d m u t a n t 2 6 D 7 101 F i g u r e 2 1 . B i n d i n g o f d a n s y l - p o l y m y x i n ( D P X ) t o i n t a c t p a r e n t a n d m u t a n t c e l l s 1 0 3 F i g u r e 22. O u t e r a n d i n n e r m e m b r a n e p r o t e i n p r o f i l e s o f t h e p a r e n t s t r a i n , m u t a n t 2 6 D 7 , 2 6 D 7 p D B S 9 a n d 2 6 D 7 p D N 1 8 1 0 4 F i g u r e 23. E l e c t r o n m i c r o s c o p y o f B. multivorans A T C C 1 7 6 1 6 a n d m u t a n t 2 6 D 7 1 0 7 x List of Figures F i g u r e 24. L i p o p o l y s a c c h a r i d e p r o f i l e s f o r p a r e n t s t r a i n B. multivorans A T C C 1 7 6 1 6 , m u t a n t 2 6 D 7 , 2 6 D 7 p D B S 9 a n d 2 6 D 7 p D N 1 8 1 0 8 F i g u r e 2 5 . T w o - d i m e n s i o n a l t h i n - l a y e r c h r o m a t o g r a m s o f t o t a l e x t r a c t a b l e l i p i d s f o r t h e p a r e n t s t r a i n a n d m u t a n t 2 6 D 7 1 1 0 F i g u r e 2 6. B. multivorans s t r a i n A T C C 1 7 6 1 6 D N A p a r t i a l l y d i g e s t e d w i t h Sau 3 A 1 3 9 F i g u r e 2 7 . R e s t r i c t i o n d i g e s t s o f r a n d o m c l o n e s f r o m t h e B. multivorans A T C C 1 7 6 1 6 c o s m i d b a n k 1 4 0 x i Acknowledgements Acknowledgements I t h a n k m y s u p e r v i s o r , D a v i d S p e e r t f o r h i s a d v i c e , s u p p o r t a n d g u i d a n c e t h r o u g h o u t t h e p r o j e c t . S p e c i a l t h a n k s t o m y t h e s i s c o m m i t t e e B o b H a n c o c k , N i a m h K e l l y a n d R o b M c M a s t e r f o r t h e i r c o n s t a n t g u i d a n c e , a d v i c e a n d s u p p o r t . I g r a t e f u l l y a c k n o w l e d g e B o b H a n c o c k f o r h i s c o l l a b o r a t i o n . I t h a n k t h e C a n a d i a n C y s t i c F i b r o s i s F o u n d a t i o n f o r t h e i r f i n a n c i a l s u p p o r t . I w o u l d a l s o l i k e t o a c k n o w l e d g e t h e h e l p f u l s u g g e s t i o n s a n d g u i d a n c e f r o m F i o n a B r i n k m a n , L u c y B r o o k s , S u s a n F a r m e r , D e b o r a h H e n r y , E s h w a r M a h e n f h i r a l i n g a m , D a v i d S i m p s o n a n d R i c h a r d S t o k e s . x i i Dedication Dedication I w o u l d l i k e t o d e d i c a t e t h i s t h e s i s t o m y d a u g h t e r K a t h e r i n e w h o h a s b e e n p a t i e n t l y p l a y i n g b e s i d e m e w h i l s t I s t u d i e d a n d t o m y p a r e n t s w h o h a v e h e l p e d t o m a k e i t p o s s i b l e f o r m e t o b e a m o t h e r a n d a P h D s t u d e n t . x i i i List of Abbreviations List of Abbreviations A r a 4 N 4 - a m i n o - 4 - d e o x y a r a b i n o s a m e A T P a d e n o s i n e t r i p h o s p h a t e B L A S T B a s i c L o c a l A l i g n m e n t S e a r c h T o o l B P I b a c t e r i a l p e r m e a b i l i t y i n c r e a s i n g C C C P c a r b o n y l c y a n i d e - m - c h l o r o p h e n y l h y d r a z o n e C E M E c e c r o p i n - m e l i t t i n h y b r i d C F c y s t i c f i b r o s i s C F T R c y s t i c f i b r o s i s t r a n s m e m b r a n e r e g u l a t o r C G D c h r o n i c g r a n u l o m a t o u s d i s e a s e C L c a r d i o l i p i n C P - 2 6 c e c r o p i n - m e l i t t i n h y b r i d w i t h m o d i f i c a t i o n s C S A s u r f a c e c a r b o h y d r a t e a n t i g e n C - t e r m i n a l c a r b o x y - t e r m i n a l C T P c y t o s i n e t r i p h o s p h a t e D a n s y l 1 - d i m e t h y l a m i n o n a p h t h a l e n e - 5 - s u l f o n y l c h l o r i d e D M S O d i m e t h y l s u l f o x i d e D N A d e o x y r i b o n u c l e i c a c i d D P X d a n s y l - p o l y m y x i n d N T P d e x o y n u c l e o t i d e t r i p h o s p h a t e ( N c a n b e A , T, C o r G ) E D T A e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d E L I S A e n z y m e l i n k e d i m m u n o s o r b a n t a s s a y E T C e x t r a c e l l u l a r t o x i c c o m p l e x x i v List of Abbreviations E M B L E u r o p e a n M o l e c u l a r B i o l o g y L a b o r a t o r y g H B S S g e l H a n k ' s b a l a n c e d s a l t s o l u t i o n G T P g u a n o s i n e t r i p h o s p h a t e H E P E S A f - h y d r o x y e t h y l p i p e r a z i n e - j V - 2 - e t h a n e s u l f o n i c a c i d I F N - y i n t e r f e r o n g a m m a i N O S i n d u c i b l e n i t r i c o x i d e s y n t h e t a s e I S i n s e r t i o n s e q u e n c e K b k i l o b a s e k D a k i l o d a l t o n K D O 3 - d e o x y o c t - 2 - u l o s o n i c a c i d L B L u r i a B r i t a n i L P S l i p o p o l y s a c c h a r i d e M H M e u l l e r H i n t o n M I C m i n i m a l i n h i b i t o r y c o n c e n t r a t i o n N O n i t r i c o x i d e N P N 1 - A ^ - p h e n y l n a p h t h y l a m i n e N - t e r m i n a l a m i n o t e r m i n a l O D o p t i c a l d e n s i t y O F o x i d a t i v e f e r m e n t a t i v e O R F o p e n r e a d i n g f r a m e P C R p o l y m e r a s e c h a i n r e a c t i o n P E p h o s p h a t i d y l e t h a n o l a m i n e P E G p o l y e t h y l e n e g l y c o l P G P h o s p h a t i d y l g l y c e r o l x v List of Abbreviations P N P G p - n i t r o p h e n y l - P - D - g a l a c t o p y r a n o s i d e R A P D - P C R r a n d o m a m p l i f i e d p o l y m o r p h i c D N A p o l y m e r a s e c h a i n r e a c t i o n r R N A r i b o s o m a l r i b o n u c l e i c a c i d S D S s o d i u m d o d e c y l s u l p h a t e S D S - P A G E s o d i u m d o d e c y l s u l p h a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s T L C t h i n l a y e r c h r o m a t o g r a p h y x v i Chapter 1 - Introduction Chapter 1 Introduction 1.1 Burkholderia multivorans and the Burkholderia cepacia complex Burkholderia cepacia complex organisms are Gram-negative, non-spore-forming, aerobic bacillae. They are motile with peritrichous pili, 0.5-1.0 urn in width and 1.5-5.0 um in length with rounded ends. They are weakly oxidase positive and typically catalase positive although some catalase negative strains exist. The optimal growth temperature is considered to range between 30-35°C although some strains are found to grow at temperatures as high as 47°C. B. cepacia was first isolated and described by Burkholder in 1950 as a phytopathogen that causes sour skin rot in onions (Burkholder 1950). Based on the description in this paper it was placed in the genus Pseudomonas as Pseudomonas cepacia (cepia is Latin for onion). In 1973 the genus Pseudomonas was divided into five distinct groups based on ribosomal ribonucleic acid (rRNA) homologies, and P. cepacia was placed into rRNA homology group II (Palleroni et al. 1973). In 1992, on the basis of 16S rRNA sequence homology, DNA-DNA homology values, cellular lipid and fatty acid composition and phenotypic characteristics, Yabuuchi et al, proposed that P. cepacia and other members of the RNA homology group II be transferred to a new genus Burkholderia. Burkholderia cepacia was proposed as the type strain for this new genus (Yabuuchi et al. 1992). Vandamme et al, in 1997, studied the taxonomic structure of Burkholderia cepacia and its relationship with other Burkholderia species. Their data showed that the B. cepacia strains studied belonged to at least five distinct genomic species. This group of five phenotypically similar but genetically distinct species was referred to as the B. cepacia complex (Vandamme et al. 1997). Each group within this complex is called a genomovar. Genomovars share a low level of DNA hybridization (50-70% reassociation) and represent distinct species however, until a differential diagnostic test is available a nomenspecies is not proposed (Ursing et al. 1995). The name Burkholderia multivorans has been given to the B. 1 Chapter I - Introduction cepacia g e n o m o v a r II s t r a i n s a n d t h e g e n o m o v a r V s t r a i n s c o r r e s p o n d t o t h e p r e v i o u s l y d e s c r i b e d n e w s p e c i e s Burkholderia vietnamiensis ( G i l l i s et a l . 1 9 9 5 a n d V a n d a m m e et a l . 1 9 9 7 ) . T h e r e m a i n i n g B. cepacia g r o u p s a r e r e f e r r e d t o as g e n o m o v a r s I, III, a n d I V . T h e r o l e a n d p a t h o g e n i c p o t e n t i a l o f e a c h o f t h e s e g e n o m o v a r s i s u n d e r i n v e s t i g a t i o n . G e n o m o v a r I st r a i n s a r e m a i n l y e n v i r o n m e n t a l i s o l a t e s . B. multivorans ( g e n o m o v a r II) s t r a i n s c a u s e c l i n i c a l i n f e c t i o n s i n b o t h c y s t i c f i b r o s i s ( C F ) a n d c h r o n i c g r a n u l o m a t o u s d i s e a s e d ( C G D ) p a t i e n t s . G e n o m o v a r III s t r a i n s s e e m t o b e t h e c a u s e o f m o s t o f t h e f a t a l a n d e p i d e m i c o u t b r e a k s i n C F c e n t e r s a n d c a u s e w h a t i s o f t e n r e f e r r e d t o as ' c e p a c i a s y n d r o m e ' ( V a n d a m m e et a l . 1997, M a h e n t h i r a l i n g a m et a l . 1 9 9 7 ) . B. cepacia i s n o l o n g e r c o n s i d e r e d a n a u t h e n t i c p s e u d o m o n a d . It b e l o n g s t o t h e p - s u b c l a s s o f Proteobacteria r a t h e r t h a n t h e y - s u b c l a s s , w h e r e t h e g e n u s Pseudomonas h a s b e e n p l a c e d ( P a l l e r o n i et a l . 1 9 81 a n d P a l l e r o n i , 1 9 9 3 ) . B a c t e r i a o f t h e P-s u b c l a s s g e n e r a l l y s h a r e m o r e t h a n 8 6 % s i m i l a r i t y i n 1 6 S r R N A n u c l e o t i d e s e q u e n c e s . T h e e x t e n t o f 1 6 S r R N A s i m i l a r i t y b e t w e e n m e m b e r s o f t h e P - s u b d i v i s i o n a n d m e m b e r s o f t h e y-s u b d i v i s i o n o f Proteobacteria i s i n t h e r a n g e o f 8 1 - 8 3 % . M o s t o f t h e p u b l i s h e d w o r k o n o r g a n i s m s f r o m t h e B. cepacia c o m p l e x o c c u r r e d p r i o r t o t h e i d e n t i f i c a t i o n o f t h e d i f f e r e n t g e n o m o v a r s . F o r s i m p l i c i t y I w i l l r e f e r t o B. cepacia w h e n r e v i e w i n g p u b l i s h e d w o r k . T h e s t r a i n u s e d f o r t h i s s t u d y w a s a g e n o m o v a r II s t r a i n B. multivorans A T C C 1 7 6 1 6 . 1.2 The unusual genome of B. cepacia T h e B. cepacia g e n o m e c o n s i s t s o f t w o t o f i v e c h r o m o s o m e s ( r e p l i c o n s ) a n d a n o v e r a l l g e n o m e s i z e r a n g i n g f r o m 5 t o 9 M b w i t h t h e m a j o r i t y o f s t r a i n s c l o s e r i n s i z e t o 9 M b . M a n y i n s e r t i o n s e q u e n c e ( I S ) e l e m e n t s a r e l o c a t e d t h r o u g h o u t t h e B. cepacia g e n o m e a n d h a v e b e e n i d e n t i f i e d b a s e d o n t h e i r a b i l i t y t o p r o m o t e g e n o m i c r e a r r a n g e m e n t s . I n s e r t i o n s e q u e n c e s a r e s m a l l e l e m e n t s o f D N A f l a n k e d b y i n v e r t e d r e p e a t s e q u e n c e s a n d c o n t a i n a n o p e n r e a d i n g f r a m e 2 Chapter I - Introduction (ORF) which spans its entire length and codes for a transposase. This transposase is essential for the transposition of the insertion sequence from one DNA site to another. In B. cepacia, IS elements play a role in mediating the fusion of plasmids (Barsomian et al. 1986), rearranging plasmids (Gaffney et al. 1987), increasing the expression of neighboring genes (Scordalis et al. 1987) and activating the lac genes on a broad host range plasmid (Wood et al. 1990). As well, these IS elements have been shown to play a role in the recruitment of genes by transposition of adjacent genes specifically related to novel catabolic functions such as degradation of the herbicide 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) (Haugland et al. 1990, Daubaras et al. 1995). IS elements play a central role in transcription of this gene and likely have stimulated a rapid evolution of this degradative pathway (Hubner et al. 1997). A detailed study of a population of Burkholderia cepacia (213 isolates) suggests genetic recombination events do occur frequently in the environment (Wise et al. 1995). In B. cepacia, the presence of multiple chromosomes and IS elements, a wide variation in genome size, and its extraordinary genetic plasticity is reflected by the organisms nutritional versatility and ability to colonize both plants and animals. 1.3 Significance of B. cepacia 1.3.1 Environmental Due to the nutritional versatility of B. cepacia, it has possibilities as an agent for both bioremediation and agricultural disease control. B. cepacia is capable of degrading highly chlorinated compounds, which are toxic and found in complex pesticides and herbicides, some being carcinogenic. One example is 2,4,5-T, which is a potent herbicide, extensively used in many countries for weed control. This compound is not easily degraded and persists for long periods in the environment. This herbicide can act as the sole carbon and energy source for B. 3 Chapter 1 - Introduction cepacia a n d s e v e r a l e x p e r i m e n t s h a v e d e m o n s t r a t e d t h a t B. cepacia s t r a i n A C l 1 0 0 i s c a p a b l e o f r e m o v i n g s i g n i f i c a n t a m o u n t s o f 2,4,5-T f r o m c o n t a m i n a t e d s o i l ( D a u b a r a s e t a l . 1 9 9 6 ) . B. cepacia h a s a l s o b e e n s h o w n t o g r o w o n f l u o r e n e a n d a w i d e v a r i e t y o f o t h e r p o l y c y c l i c a r o m a t i c c o m p o u n d s , w h i c h a r e p o l l u t a n t s o f c o n c e r n b e c a u s e t h e y a r e t o x i c a n d p o t e n t c a r c i n o g e n s ( G r i f o l l et a l . 1 9 9 5 ) . O t h e r c o m p o u n d s B. cepacia i s c a p a b l e o f d e g r a d i n g a r e c a t e c h o l s ( O h et a l . 1 9 9 7 ) , p h t h a l a t e s , w a s t e s d i s c h a r g e d i n t o t h e e n v i r o n m e n t b y p l a s t i c s , p a p e r a n d p a i n t i n d u s t r i e s ( S a i n t et a l . 1 9 9 6 ) a n d t h e h e r b i c i d e d i c l o f o p - m e t h y l ( S m i t h - G r e e i e r et a l . 1 9 9 6 ) . B. cepacia i s k n o w n t o a n t a g o n i z e a n d r e p r e s s m a n y s o i l b o r n e p l a n t p a t h o g e n s . It i s c a p a b l e o f p r e v e n t i n g b l i g h t c a u s e d b y t h e f u n g u s Altermaria b y i n h i b i t i n g s p o r e g e r m i n a t i o n ( H o l m e s et a l . 1 9 9 8 ) a n d c a n k i l l t h e f u n g u s Aphanomyces euteiches, w h i c h c a u s e s r o o t r o t i n p e a s ( B o w e r s e t at. 1 9 9 3 , K i n g et a l . 1 9 9 6 ) . S t e m r o t o f p o i n s e t t i a i s c a u s e d b y Rhizobium solani a n d c a n a l s o b e c o n t r o l l e d i f p o i n s e t t i a c u t t i n g s a r e r o o t e d w i t h B. cepacia ( C a r t w r i g h t et a l . 1 9 9 5 ) . B. cepacia a p p e a r s t o b e a n a t t r a c t i v e a l t e r n a t i v e t o t o x i c f u n g i c i d e s ( w h i c h c a n n o t b e b r o k e n d o w n i n t h e e n v i r o n m e n t ) f o r p r e v e n t i o n o f p l a n t d i s e a s e s . A t t h i s t i m e , m a n y p a t e n t s a r e b e i n g s o u g h t i n t h e h o p e s o f u s i n g B. cepacia f o r a g r i c u l t u r a l a p p l i c a t i o n s a n d d e c o n t a m i n a t i o n o f s o i l s . T h i s p o s e s a p o t e n t i a l p r o b l e m d u e t o t h e e m e r g e n c e o f B. cepacia a s a h u m a n p a t h o g e n . T h e p o s s i b i l i t y o f B. cepacia b e i n g r e l e a s e d i n l a r g e q u a n t i t i e s i n t h e e n v i r o n m e n t f o r b o t h b i o r e m e d i a t i o n a n d a g r i c u l t u r a l u s e s i s a l a r m i n g f o r t h o s e i n d i v i d u a l s s u s c e p t i b l e t o i n f e c t i o n s c a u s e d b y t h i s s a m e o r g a n i s m . 4 Chapter 1 - Introduction 1.3.2 Clinical P r i o r t o 1 9 8 0 , r e p o r t s o f B. cepacia i n f e c t i o n s i n h u m a n s w e r e f e w a n d r e s t r i c t e d t o p a t i e n t s e x p o s e d t o c o n t a m i n a t e d d i s i n f e c t a n t a n d a n a e s t h e t i c s o l u t i o n s i n w h i c h B. cepacia i s c a p a b l e o f s u r v i v i n g f o r l o n g p e r i o d s o f t i m e ( G o v a n et a l . 1 9 9 6 ) . T h e i n c i d e n c e o f B. cepacia i n f e c t i o n w a s o n t h e r i s e i n t h e e a r l y 1 9 8 0 s a n d a n a l y s i s o f t h e U S A d a t a b a s e s o f n o s o c o m i a l i n f e c t i o n s c o n f i r m e d a l a r g e i n c r e a s e i n c l i n i c a l l y s i g n i f i c a n t B. cepacia i s o l a t i o n s at t h a t t i m e ( J a r v i s et a l . 1 9 8 7 , M a r t o n e et a l . 1 9 8 7 ) . T h e i n v e s t i g a t i o n s s h o w e d t h a t t h e m a i n s o u r c e o f n o s o c o m i a l o u t b r e a k s o f B. cepacia w e r e c o n t a m i n a t e d l i q u i d r e s e r v o i r s a n d m o i s t e n v i r o n m e n t a l s u r f a c e s . A p a r t f r o m b e i n g a n o s o c o m i a l p a t h o g e n , B. cepacia h a s a l s o e m e r g e d a s a n i m p o r t a n t p a t h o g e n i n t w o c o n g e n i t a l d i s e a s e s : c y s t i c f i b r o s i s ( I s l e s et a l . 1984, T h o m a s s e n et a l . 1 9 8 5 ) a n d c h r o n i c g r a n u l o m a t o u s d i s e a s e ( O ' N e i l et a l . 1 9 8 6 ) . C y s t i c f i b r o s i s ( C F ) i s t h e m o s t c o m m o n i n h e r i t e d l e t h a l d i s o r d e r o f C a u c a s i a n p o p u l a t i o n s w i t h 5 0 ,000 i n d i v i d u a l s a f f e c t e d , a f r e q u e n c y o f 1 i n 2,500 l i v e b i r t h s a n d a c a r r i e r f r e q u e n c y o f 1 i n 2 5 ( B y e et a l . 1 9 9 4 ) . I n 1 9 8 9 , t h e g e n e r e s p o n s i b l e f o r C F w a s i s o l a t e d b y t h e g r o u p s o f L a p - C h e e T s u i i n T o r o n t o , C a n a d a a n d F r a n c i s C o l l i n s i n M i c h i g a n , U S A ( K e r e m et a l . 1989, R i o r d a n et a l . 1 9 8 9 , a n d R o m m e r s et a l . 1 9 8 9 ) . T h e C F g e n e e n c o d e s a c o m p l e x 1 4 8 0 a m i n o a c i d p r o t e i n c a l l e d t h e c y s t i c f i b r o s i s t r a n s m e m b r a n e c o n d u c t a n c e r e g u l a t o r ( C F T R ) . C F T R i s a b l e t o c o n d u c t c h l o r i d e at a h i g h r a t e ( B e a r et a l . 1 9 9 2 ) a n d h a s b e e n l o c a l i z e d t o t h e a p i c a l s u r f a c e o f e p i t h e l i a l c e l l s i n t h e i n t e s t i n a l a n d r e s p i r a t o r y t r a c t ( T r e z i s e et a l . 1 9 9 1 ) . A m u t a t i o n i n C F T R r e s u l t s i n t h e f a i l u r e o f c A M P r e g u l a t e d c h l o r i d e i o n t r a n s p o r t o n t h e s u r f a c e o f e p i t h e l i a l c e l l s ( K n o w l e s et a l . 1 9 8 3 , Q u i n t o n et a l . 1 9 8 6 ) . I n n o r m a l c e l l s , t h e o s m o t i c m o v e m e n t o f w a t e r l i n k e d t o c h l o r i d e s e c r e t i o n p r o b a b l y r e p r e s e n t s o n e w a y o f h y d r a t i n g m u c o s a l s u r f a c e s . A s a l t i m b a l a n c e i s c a u s e d b y d e f e c t i v e c h l o r i d e i o n t r a n s p o r t i n C F e f f e c t i n g t h e e p i t h e l i a l c e l l s l i n i n g i n t e r n a l o r g a n s . T h i s d i s t u r b a n c e i n c h l o r i d e s e c r e t i o n c o m b i n e d w i t h a n i n c r e a s e i n 5 Chapter 1 - Introduction s o d i u m a b s o r p t i o n a n d w a t e r i n f l u x i n t o c e l l s c a u s e s d e h y d r a t i o n o f t h e m u c o s a l s u r f a c e s . T h i s l e a d s t o a b u i l d u p o f s t i c k y d e h y d r a t e d m u c u s i n m a l e s e x d u c t s , d u c t s o f t h e p a n c r e a s a n d t h e a i r w a y s o f t h e l u n g . I n t h e l u n g s , t h i s i m p a i r s m u c o c i l i a r y c l e a r a n c e a n d p r o b a b l y c o n t r i b u t e s t o t h e p e r s i s t e n t m i c r o b i a l c o l o n i z a t i o n o f t h e C F l u n g o f t e n r e s u l t i n g i n e p i s o d e s o f d e b i l i t a t i n g a n d u l t i m a t e l y f a t a l i n f e c t i o n ( S t e r n et a l . 1 9 9 6 ) . B y f a r t h e m o s t c o m m o n p a t h o g e n i n C F i s m u c o i d Pseudomonas aeruginosa f o l l o w e d b y w h a t i s c o n s i d e r e d t h e c o n t r o v e r s i a l n e w o p p o r t u n i s t i c p a t h o g e n B. cepacia. D u e t o a g g r e s s i v e a n t i b i o t i c t h e r a p y , t o d a y t h e p r o g n o s i s f o r n e w l y d i a g n o s e d p a t i e n t s i s g o o d w i t h a m e d i a n l i f e e x p e c t a n c y o f 3 0 y e a r s . P. aeruginosa l u n g i n f e c t i o n s i n C F a r e c h a r a c t e r i z e d b y e a r l y c o l o n i z a t i o n a n d a s s o c i a t e d w i t h a n i n c r e a s e i n t h e h o s t i m m u n e r e s p o n s e , f o l l o w e d b y e p i s o d e s o f p e r s i s t e n t , i n c r e a s i n g l y s e v e r e l u n g d i s e a s e . B. cepacia i n f e c t i o n s r o s e s i g n i f i c a n t l y i n C F c l i n i c s d u r i n g t h e 1 9 8 0 s s t a b i l i z i n g i n t h e l a s t 10 y e a r s . T h e p r e v a l e n c e o f B. cepacia i n d i f f e r e n t C F c l i n i c s r a n g e s f r o m 0 - 4 0 % p a t i e n t c o l o n i z a t i o n ( S a j j a n et a l . 1 9 9 2 a ) . I n f e c t i o n o f C F p a t i e n t s w i t h B. cepacia i s a s s o c i a t e d w i t h c l i n i c a l o u t c o m e s r a n g i n g f r o m e s s e n t i a l l y a s y m p t o m a t i c t o f a t a l n e c r o t i z i n g p n e u m o n i a ( G o v a n et a l . 1 9 9 6 ) . T h e r e i s s t r o n g e v i d e n c e t h a t t h i s h e t e r o g e n e i t y i n c l i n i c a l o u t c o m e s m a y b e r e l a t e d t o t h e f a c t t h a t B. cepacia i s a c o m p l e x o f f i v e g e n o m o v a r s ( V a n d a m m e et a l . 1 9 9 7 ) . G e n o m o v a r III s t r a i n s s e e m t o b e a s s o c i a t e d w i t h c a s e s o f e p i d e m i c s p r e a d a n d h i g h m o r t a l i t y r a t e i n t h e V a n c o u v e r , B.C. C F c l i n i c . B. multivorans s t r a i n s a r e a l s o c o m m o n i n C F p a t i e n t s a l t h o u g h t h e r e i s l i t t l e e v i d e n c e f o r p a t i e n t t o p a t i e n t s p r e a d a n d i t i s f o u n d m a i n l y i n t h e p e d i a t r i c p o p u l a t i o n ( M a h e n t h i r a l i n g a m et a l . 1 9 9 7 ) . H o w e v e r , a n o u t b r e a k a m o n g p e d i a t r i c p a t i e n t s o f B. multivorans a s s o c i a t e d w i t h s i g n i f i c a n t m o r t a l i t y h a s b e e n r e p o r t e d ( W h i t e f o r d et a l . 1 9 9 5 ) . A n o t h e r s e r i o u s p r o b l e m w i t h B. cepacia i s i t s r e s i s t a n c e t o p o t e n t a n t i - p s e u d o m o n a l d r u g s . T h i s h a s l e d t o a l a c k o f e f f e c t i v e a n t i m i c r o b i a l t h e r a p y f o r p a t i e n t s w i t h B. cepacia i n f e c t i o n s 6 Chapter I - Introduction a n d t h e p r e v a l e n c e o f t h i s o r g a n i s m m a y b e i n p a r t a r e s u l t o f a g g r e s s i v e a n t i b i o t i c t h e r a p y a n d s e l e c t i o n o f t h i s o p p o r t u n i s t ( S i m p s o n et a l . 1 9 9 4 ) . E v e n w h e n a p a r t i c u l a r B. cepacia i s o l a t e s h o w s s u s c e p t i b i l i t y t o a n t i m i c r o b i a l a g e n t s in vitro, a g g r e s s i v e t h e r a p y r a r e l y r e s u l t s i n c l i n i c a l i m p r o v e m e n t . M a n y s t u d i e s a r e u n d e r w a y t o t r y a n d u n d e r s t a n d t h e p a t h o g e n i c a n d v i r u l e n c e p r o p e r t i e s o f B. cepacia i n r e l a t i o n t o C F b u t i t i s s t i l l n o t c l e a r h o w B. cepacia i s a b l e t o e s t a b l i s h i n f e c t i o n i n t h e l u n g s o f t h e s e p a t i e n t s . A c l u e t o u n d e r s t a n d i n g t h e i n t e r a c t i o n o f B. cepacia w i t h t h e i m m u n e s y s t e m o f t h e h u m a n h o s t c o m e s f r o m a s e c o n d g e n e t i c d i s o r d e r , c h r o n i c g r a n u l o m a t o u s d i s e a s e ( C G D ) . C G D i s a r a r e d i s e a s e w i t h a n e s t i m a t e d f r e q u e n c y o f 1 i n 5 0 0 , 0 0 0 b i r t h s . C G D r e s u l t s from a m u t a t i o n t h a t r e n d e r s t h e p h a g o c y t i c N A D P H o x i d a s e e n z y m e d y s f u n c t i o n a l . P h a g o c y t i c c e l l s ( n e u t r o p h i l s , e o s i n o p h i l s , m o n o c y t e s a n d |ouwa«| plasma membrane m a c r o p h a g e s ) kill i n g e s t e d m i c r o -o r g a n i s m s b y r e l e a s i n g m i c r o b i c i d a l p r o t e i n s f r o m c y t o p l a s m i c g r a n u l e s a n d b y g e n e r a t i n g s u p e r o x i d e (O2") a n d o t h e r r e a c t i v e o x y g e n s p e c i e s i n t h e i n t r a c e l l u l a r p h a g o s o m a l c o m p a r t m e n t t h a t c o n t a i n s Figure 1. Phagocytosis, degranulation and oxidative radical generation. U n o p s o n i z e d t h e i n g e s t e d m i c r 0 0 r g a n i s m a s d e p i c t e d a n d m i c r o b e s o p s o n i z e d w i t h I g G a n t i b o d i e s a n d c o m p l e m e n t f r a g m e n t s a t t a c h t o t h e m figure L T h e e n z y m e ^ c a t a l y z e s p h a g o c y t i c c e l l . T h i s c a u s e s i n d u c t i o n o f p h a g o c y t o s i s , f o l l o w e d b y f u s i o n o f g r a n u l e s m e f o r m a t i o n o f s u p e r o x i d e i s N A D P H w i t h t h e p h a g o s o m a l m e m b r a n e a n d a c t i v a t i o n o f t h e N A D P H o x i d a s e . ( F r o m R o o s , 1 9 9 4 ) Q x i d a s e T h i g i § a m u l t i . c o m p o n e n t e n z y m e , c o n s i s t i n g o f f i v e s u b u n i t s . T w o o f t h e s e s u b u n i t s a r e i n t e g r a l m e m b r a n e p r o t e i n s a n d t h e o t h e r t h r e e s u b u n i t s a r e l o c a l i z e d i n t h e c y t o s o l o f r e s t i n g p h a g o c y t e s ( B a b i o r , 1 9 9 1 ) . I n a c t i v a t e d p h a g o c y t e s , t h e t h r e e c y t o s o l i c p r o t e i n s a r e t r a n s l o c a t e d t o t h e t w o m e m b r a n e p r o t e i n s • <§) neutrophil (^ ) Chapter 1 - Introduction ( C l a r k et a l . 1 9 9 0 ) a n d t o g e t h e r t h e s e f i v e p r o t e i n s f o r m a f u n c t i o n a l o x i d a s e c a p a b l e o f g e n e r a t i n g s u p e r o x i d e ( R o t r o s e n et a l . 1 9 9 3 ) . W i t h o u t a f u n c t i o n i n g N A D P H o x i d a s e e n z y m e t h e p h a g o c y t e s f r o m C G D p a t i e n t s a r e d e p e n d e n t o n n o n o x i d a t i v e k i l l i n g m e c h a n i s m s f o r d e s t r o y i n g i n v a d i n g m i c r o b e s a n d as a r e s u l t a r e u n a b l e t o k i l l c e r t a i n b a c t e r i a a n d f u n g i . T h e s e p a t i e n t s s u f f e r f r o m s e v e r e r e c u r r e n t b a c t e r i a l a n d f u n g a l i n f e c t i o n s o f t h e s u b c u t a n e o u s t i s s u e s , t h e l u n g s a n d t h e l y m p h n o d e s ( F o r r e s t et a l . 1 9 8 8 ) . B. cepacia i s a n i m p o r t a n t p a t h o g e n i n C G D c h i l d r e n a n d h a s b e e n r e p o r t e d t o c a u s e s i g n i f i c a n t m o r b i d i t y ( B o t t o n e , et a l . 1 9 7 5 , O ' N e i l et a l . 1986, L a c y et a l . 1 9 9 3 ) . S t u d i e s b y S p e e r t et a l , 1 9 9 4 s h o w e d t h a t n e u t r o p h i l s from C G D pa t i e n t s a r e u n a b l e t o k i l l B. cepacia in vitro b u t a r e s t i l l c a p a b l e o f k i l l i n g P. aeruginosa w h e r e n o r m a l n e u t r o p h i l s w e r e a b l e t o k i l l b o t h o r g a n i s m s e q u a l l y e f f i c i e n t l y . F r o m t h e s e s t u d i e s i t w a s c o n c l u d e d t h a t B. cepacia i s r e s i s t a n t t o t h e n o n o x i d a t i v e k i l l i n g m e c h a n i s m s o f C G D n e u t r o p h i l s a n d t h i s r e s i s t a n c e m a y b e a m a j o r c o n t r i b u t o r t o t h e a b i l i t y o f B. cepacia t o t h w a r t t h e i m m u n e s y s t e m o f i m m u n o c o m p r o m i s e d h o s t s a n d c a u s e s e r i o u s l u n g i n f e c t i o n s i n C G D . V i r u l e n c e o f B. cepacia i n C G D d o e s n o t a p p e a r t o h a v e t h e v a r i a t i o n t h a t i s s e e n i n C F n o r i s i t d e p e n d e n t o n g e n o m o v a r d e s i g n a t i o n . A n e x a m p l e o f t w o s t r a i n s r e c o v e r e d f r o m C G D p a t i e n t s w i t h s e v e r e i n f e c t i o n s w e r e f r o m g e n o m o v a r II (B. multivorans) a n d g e n o m o v a r V (B. vietnamiensis) ( S p e e r t et al. 1 9 9 9 ) . 1.4 Virulence Factors of B. cepacia M o s t o f t h e s t u d i e s d o n e o n v i r u l e n c e o f B. cepacia a r e r e l a t e d t o i t s p a t h o g e n e s i s i n t h e C F l u n g . T h e a b i l i t y o f a p o t e n t i a l p a t h o g e n t o a d h e r e t o t h e h o s t m u c o s a l o r e p i t h e l i a l c e l l s i s e s s e n t i a l i n t h e e s t a b l i s h m e n t o f a n i n f e c t i o n . A i r w a y m u c i n s ( m u c u s g l y c o p r o t e i n s ) a r e s y n t h e s i z e d a n d s e c r e t e d b y m u c o u s c e l l s o f t h e s u b m u c o s a l g l a n d s i n t h e r e s p i r a t o r y m u c o s a . P i l i a t e d B. cepacia f r o m s p u t a o f C F p a t i e n t s a r e a b l e t o b i n d t o p u r i f i e d m u c i n , a 5 5 - k D a 8 Chapter 1 - Introduction r e c e p t o r a n d a m e m b r a n e g l y c o l i p i d o n e p i t h e l i a l c e l l s ( S a j j a n et a l . 1 9 9 3 ) . T h e B. cepacia a d h e s i n w a s i d e n t i f i e d a s a 2 2 - k D a p r o t e i n l o c a l i z e d t o t h e p i l i p r e s e n t o v e r t h e e n t i r e s u r f a c e o f t h e c e l l , n o t s e e n i n n o n - m u c i n b i n d i n g s t r a i n s ( S a j j a n et a l . 1 9 9 2 b ) . T h e m a j o r s u b u n i t o f t h e 2 2 - k D a p i l i n a s s o c i a t e d a d h e s i n w a s p u r i f i e d a n d i m m u n o l o c a l i z e d t o p e r i t r i c h o u s l y a r r a n g e d p i l i a n d , d u e t o t h e i r u n u s u a l a p p e a r a n c e as l a r g e i n t e r t w i n e d f i b e r s , t h e y w e r e r e f e r r e d t o as c a b l e p i l i ( S a j j a n et a l . 1 9 9 5 ) . A s t u d y b y B u t l e r et a l i n G r e a t B r i t a i n o f 3 8 s t r a i n s o f B. cepacia s h o w e d o n l y s i x w e r e c a p a b l e o f m u c i n b i n d i n g b u t i t w a s t h e e p i d e m i c i s o l a t e t h a t h a d t h e h i g h e s t a f f i n i t y f o r m u c i n ( B u t l e r et a l . 1 9 9 2 ) . I n v a s i o n o f e p i t h e l i a l c e l l s b y B. cepacia h a s b e e n d e m o n s t r a t e d a n d e l e c t r o n m i c r o s c o p y s h o w e d i n t r a c y t o p l a s m i c b a c t e r i a e n c l o s e d b y m e m b r a n e -b o u n d v a c u o l e s w i t h n o l y s o s o m a l f u s i o n ( B u r n s et a l . 1 9 9 6 a ) . P r o d u c t i o n o f s i d e r o p h o r e s e n a b l e s b a c t e r i a t o c o m p e t e w i t h h o s t i r o n - b i n d i n g p r o t e i n s , t r a n s f e r r i n a n d l a c t o f e r r i n , c h e l a t i n g e s s e n t i a l i r o n r e q u i r e d f o r g r o w t h a n d p r o m o t i n g e s t a b l i s h m e n t a n d m a i n t e n a n c e o f i n f e c t i o n . B. cepacia p r o d u c e s at l e a s t f o u r d i f f e r e n t i r o n -b i n d i n g s i d e r o p h o r e s : 1. P y o c h e l i n n o t r e l a t e d t o t h e Pseudomonas s i d e r o p h o r e ( S o k o l et a l . 1 9 8 6 ) , 2. S a l i c y l i c a c i d ( V i s c a et a l . 1993, S o k o l et a l . 1 9 9 2 ) , 3. C e p a b a c t i n ( M e y e r et a l . 1 9 8 9 ) a n d 4. O r n i b a c t i n s ( S t e p h a n et a l . 1 9 9 3 ) . I n a s t u d y o f 4 3 B. cepacia i s o l a t e s f r o m C F p a t i e n t s , 4 9 % p r o d u c e d p y o c h e l i n a n d t h o s e p y o c h e l i n p o s i t i v e s t r a i n s w e r e i s o l a t e d from p a t i e n t s w i t h s e v e r e p u l m o n a r y i n f e c t i o n s ( S o k o l et a l . 1 9 8 6 ) . U s i n g a r a t m o d e l o f c h r o n i c l u n g i n f e c t i o n i n v o l v i n g t r a n s t r a c h e a l i n o c u l a t i o n o f r a t s w i t h a g a r b e a d s , S o k o l et a l , 1 9 8 8 s h o w e d t h a t e x o g e n o u s p y o c h e l i n s u p p l i e d t o t h e r a t s , i n c r e a s e d t h e v i r u l e n c e o f B. cepacia. D a r l i n g et a l , i n 1 9 9 8 s t u d i e d 61 C F i s o l a t e s ; 6 2 % p r o d u c e d p y o c h e l i n , 9 3 % p r o d u c e d s a l i c y l i c a c i d , 1 1 % p r o d u c e d c e p a b a c t i n a n d 9 1 % i s o l a t e s t e s t e d p r o d u c e d o r n i b a c t i n ( D a r l i n g et a l . 1 9 9 8 ) . F r o m t h e s e d a t a , i t i s s u g g e s t e d t h a t t h e s i d e r o p h o r e s i n B. cepacia a r e i m p o r t a n t i n c h e l a t i n g i r o n f r o m h o s t i r o n b i n d i n g p r o t e i n s a n d t h a t p y o c h e l i n m a y i n c r e a s e v i r u l e n c e o f B. cepacia. 9 Chapter 1 - Introduction E x t r a c e l l u l a r b a c t e r i a l p r o d u c t s h a v e b e e n i d e n t i f i e d a s i m p o r t a n t v i r u l e n c e f a c t o r s i n o t h e r o r g a n i s m s i n c l u d i n g P. aeruginosa a n d m a y b e i m p o r t a n t i n B. cepacia. I n 1984 , M c K e v i t t a n d W o o d s i d e n t i f i e d t h e o n l y p r o t e a s e t h a t h a s b e e n c h a r a c t e r i z e d f o r B. cepacia. T h i s p r o t e a s e i s a 3 7 - k D a e x t r a c e l l u l a r m e t a l l o p r o t e a s e ( A b e e t a l . 1 9 9 6 ) a n d , u p o n i n s t i l l a t i o n i n t o r a t l u n g s , c a u s e s p n e u m o n i a e x h i b i t e d b y n e u t r o p h i l i n f i l t r a t i o n a n d p r o t e i n a c e o u s e x u d a t i o n i n t h e l a r g e a i r w a y s ( M c K e v i t t et a l . 1 9 8 9 ) . A h e m o l y s i n from a h i g h l y t r a n s m i s s i b l e s t r a i n o f B. cepacia w a s i d e n t i f i e d a n d p a r t i a l l y c h a r a c t e r i z e d a s a l i p o p e p t i d e t o x i n . A t l o w c o n c e n t r a t i o n s , t h i s h e m o l y s i n i n d u c e s n u c l e o s o m a l d e g r a d a t i o n a n d a p o p t o s i s i n h u m a n n e u t r o p h i l s a n d m o u s e m a c r o p h a g e s ( H u t c h i s o n et a l . 1 9 9 8 ) . A s w e l l , t h e l i p a s e a c t i v i t y o f B. cepacia i s w e l l d o c u m e n t e d i n t h e l i t e r a t u r e a n d w a s first d e s c r i b e d i n Pseudomonas sp. i n 1941 b y S t a r r a n d B u r k h o l d e r ( S t a r r et a l . 1 9 4 1 ) . P u r i f i e d l i p a s e f r o m B. cepacia i s n o t c y t o t o x i c b u t i s c a p a b l e o f r e d u c i n g t h e p h a g o c y t i c f u n c t i o n o f r a t a l v e o l a r m a c r o p h a g e s ( S t r a u s et a l . 1 9 9 2 ) . A n e x t r a c e l l u l a r t o x i c c o m p l e x ( E T C ) c o n s i s t i n g o f a s u r f a c e c a r b o h y d r a t e a n t i g e n ( C S A ) , l i p o p o l y s a c c h a r i d e ( L P S ) a n d p r o t e i n ( S t r a u s et a l . 1 9 8 9 ) , a p p e a r s t o b e r e s p o n s i b l e f o r t h e l e t h a l i t y a n d e x t e n s i v e p u l m o n a r y n e c r o s i s a s s o c i a t e d w i t h l u n g i n f e c t i o n s c a u s e d b y B. cepacia ( S t r a u s et a l . 1 9 8 8 ) . T o x i c i t y o f t h e E T C w a s c o n s i d e r e d t o b e a s s o c i a t e d w i t h t h e L P S p o r t i o n o f t h e c o m p l e x b e c a u s e t h e r e w a s n o e f f e c t o n a c t i v i t y a f t e r b o i l i n g a n d p r o t e o l y s i s a n d L P S i s r e s i s t a n t t o b o t h o f t h e s e t r e a t m e n t s ( S t r a u s et a l . 1 9 8 8 ) . T h e s e s t u d i e s s u g g e s t t h a t B. cepacia c a n p r o d u c e a p r o t e a s e , a h e m o l y s i n , a l i p a s e a n d a n E T C t h a t a c t a s v i r u l e n c e f a c t o r s i m p o r t a n t i n a s s i s t i n g t h e b a c t e r i a i n e v a s i o n o f h o s t d e f e n s e s . 10 Chapter 1 - Introduction 1.5 The Gram-negative outer membrane 1.5.1 General structure T h e e n v e l o p e o f G r a m - n e g a t i v e b a c t e r i a c o n s i s t s o f t w o m e m b r a n e s s e p a r a t e d b y t h e p e r i p l a s m i c s p a c e a n d a l a y e r o f p e p t i d o g l y c a n . T h e c y t o p l a s m i c m e m b r a n e i s c o n s i d e r e d a t y p i c a l p h o s p h o l i p i d b i l a y e r w i t h t h e n e g a t i v e l y c h a r g e d p h o s p h a t e h e a d g r o u p s o n t h e t w o o u t e r s u r f a c e s o f t h e m e m b r a n e a n d t h e h y d r o p h o b i c f a t t y a c i d c h a i n s e x t e n d i n g i n t o t h e c e n t e r o f t h e m e m b r a n e p e r p e n d i c u l a r t o i t s p l a n e . I n t e g r a l a n d p e r i p h e r a l m e m b r a n e p r o t e i n s a r e f o u n d t h r o u g h o u t t h e i n n e r m e m b r a n e a n d t h e i r m a i n f u n c t i o n i s i n c e l l u l a r e n e r g y ( e l e c t r o n t r a n s p o r t ) , e x p o r t o f t o x i c c e l l u l a r b y p r o d u c t s a n d t r a n s p o r t o f n u t r i e n t s ( C r o n a n et a l . 1 9 8 7 ) . T h e p e p t i d o g l y c a n l a y e r i s c l o s e l y a s s o c i a t e d w i t h t h e i n n e r l e a f l e t o f t h e o u t e r m e m b r a n e a n d p r o v i d e s s t r u c t u r a l i n t e g r i t y t o t h e b a c t e r i a l c e l l . T h e o u t e r m e m b r a n e , i s a n a s y m m e t r i c a l b i l a y e r i n w h i c h t h e i n n e r l e a f l e t i s c o m p o s e d o f p h o s p h o l i p i d s a n d t h e o u t e r l e a f l e t c o n t a i n s L P S . T h e m o s t a b u n d a n t p h o s p h o l i p i d c o m p o n e n t s o f B. cepacia are, i n o r d e r o f a b u n d a n c e , p h o s p h a t i d y l e t h a n o l a m i n e , d i p h o s p h a t i d y l g l y c e r o l ( c a r d i o l i p i n ) a n d p h o s p h a t i d y l g l y c e r o l s i m i l a r t o t h a t f o u n d f o r P. aeruginosa ( K a w a i et a l . 1 9 8 8 ) . 1.5.2 Lipopolysaccharide L P S f o u n d i n t h e o u t e r l e a f l e t o f t h e o u t e r m e m b r a n e o f G r a m - n e g a t i v e b a c t e r i a , p l a y s a n i m p o r t a n t r o l e i n t h e i n t e r a c t i o n o f t h e o r g a n i s m w i t h i t s e n v i r o n m e n t . T h e h o s t d e f e n s e s y s t e m r e c o g n i z e s b a c t e r i a l L P S a n d i n r e s p o n s e p r o d u c e s a n t i b o d i e s t h a t s p e c i f i c a l l y r e c o g n i z e L P S s t r u c t u r e . L P S i s r e l e a s e d i n t o t h e e n v i r o n m e n t a n d o n c e r e l e a s e d L P S ( a l s o c a l l e d e n d o t o x i n ) c a n c a u s e f e v e r , h y p o t e n s i o n a n d t o x i c s h o c k s y n d r o m e i n t h e h o s t o r g a n i s m . L P S h a s b e e n s h o w n t o a c t i v a t e B - l y m p h o c y t e s , n e u t r o p h i l s a n d m a c r o p h a g e s t o r e l e a s e i m m u n e a c t i v a t i n g c y t o k i n e s . 11 Chapter 1 - Introduction L P S c o n s i s t s o f a t h r e e d i s t i n c t r e g i o n s : 1) Lipid A i s t h e h y d r o p h o b i c m e m b r a n e a n c h o r i n g r e g i o n o f L P S a n d r a t h e r t h a n c a r r y i n g t w o f a t t y a c i d r e s i d u e s t y p i c a l o f p h o s p h o l i p i d s , i t c a r r i e s f i v e t o s e v e n a t t a c h e d t o a p h o s p h o r y l a t e d g l u c o s a m i n e d i m e r . A l l t h e f a t t y a c i d s i n l i p i d A a r e s a t u r a t e d o r h y d r o x y l a t e d ( u n l i k e t h e u n s a t u r a t e d f a t t y a c i d s f o u n d i n p h o s p h o l i p i d s ) . 2 ) The rough core o l i g o s a c c h a r i d e i s d i v i d e d i n t o t h e o u t e r c o r e a n d i n n e r c o r e r e g i o n s . T h e i n n e r c o r e i s a s h o r t c h a i n o f s u g a r s , i n c l u d i n g s e v e r a l 2-k e t o - 3 - d e o x y o c t o n o i c a c i d ( K D O ) a n d h e p t o s e r e s i d u e s ( s e e F i g u r e 2 a s a n e x a m p l e o f L P S f r o m Salmonella). T h e o u t e r c o r e c o n s i s t s o f c o m m o n h e x o s e s ( G l u c o s e , D-galactose, N-ac e t y l - D - g l u c o s a m i n e a n d N-acetyl g a l a c t o s a m i n e ) a n d h a s a l s o b e e n t e r m e d t h e h e x o s e r e g i o n . T h i s r e g i o n o f L P S c o n t a i n s p h o s p h a t e r e s i d u e s a n d 16 r e s i d u e s c a n b e f o u n d p e r L P S m o l e c u l e i n P. aeruginosa. 3 ) A t t a c h e d t o t h e r o u g h c o r e i s t h e h y d r o p h i l i c O s i d e c h a i n (O-Antigen), c o m p o s e d o f m a n y r e p e a t i n g t e t r a - o r p e n t a s a c c h a r i d e u n i t s ( R i e t s c h e l et a l . 1 9 9 3 f o r a r e v i e w ) . T h i s O - a n t i g e n p o l y s a c c h a r i d e , t h e i m m u n o d o m i n a n t p o r t i o n o f L P S , i s o n l y p r e s e n t o n a p p r o x i m a t e l y 1 0 % o f a l l L P S m o l e c u l e s i n o n e b a c t e r i u m ( H a n c o c k et a l . 1 9 9 4 ) . S t r a i n s l a c k i n g t h e O - s i d e c h a i n a r e r e f e r r e d t o a s h a v i n g r o u g h c o l o n y m o r p h o l o g y a n d a r e s e n s i t i v e t o 12 Figure 2. Lipid A and inner core region of LPS from Salmonella. T h e n u m b e r s i n c i r c l e s r e p r e s e n t t h e n u m b e r o f c a r b o n a t o m s p r e s e n t i n e a c h a c y l c h a i n . R e s i d u e s R l c o n s t i t u t e A r a 4 N a n d R 2 c o n s t i t u t e s 2 - a m i n o e t h y l p y r o p h o s p h a t e s u b s t i t u t i o n s . ( F r o m R i e t s c h e l et a l . 1 9 9 3 ) Chapter I - Introduction k i l l i n g b y h u m a n s e r u m a n d s t r a i n s c a r r y i n g a n O - s i d e c h a i n o n t h e i r L P S a r e r e f e r r e d t o as h a v i n g s m o o t h c o l o n y m o r p h o l o g y a n d a r e r e s i s t a n t t o k i l l i n g b y h u m a n s e r u m . T h e d i s t r i b u t i o n a n d s t r u c t u r e o f L P S g i v e s t h e o u t e r m e m b r a n e m a n y o f i t s b a r r i e r p r o p e r t i e s . T h e p r e s e n c e o f t h e p h o s p h a t e r e s i d u e s i n c o r e r e g i o n o f L P S r e s u l t s i n a s t r o n g n e g a t i v e c h a r g e o n t h e s u r f a c e o f P. aeruginosa. T h i s s t r o n g n e g a t i v e c h a r g e a c r o s s t h e s u r f a c e o f P. aeruginosa r e s u l t s i n a l a y e r o f w a t e r m o l e c u l e s t h a t r e s t r i c t t h e u p t a k e o f h y d r o p h o b i c c o m p o u n d s t h r o u g h t h e o u t e r m e m b r a n e . A s w e l l , t h e s e p h o s p h a t e r e s i d u e s s t a b i l i z e t h e o u t e r m e m b r a n e b y f o r m i n g n o n - c o v a l e n t c r o s s - b r i d g e s w i t h a d j a c e n t L P S m o l e c u l e s w i t h t h e h e l p o f d i v a l e n t c a t i o n s ( M g 2 + o r C a 2 + ) ( R o t t e m et a l . 1 9 7 7 ) . T h e i n t e r a c t i o n o f t h e h y d r o p h o b i c l i p i d A c o m p o n e n t o f L P S w i t h m e m b r a n e p r o t e i n s a l s o a i d s i n s t a b i l i z a t i o n o f t h e m e m b r a n e ( N i k a i d o e t a l . 1 9 8 5 ) . 5 4 % o f B. cepacia C F i s o l a t e s p o s s e s s r o u g h L P S ( M c K e v i t t e t a l . 1 9 8 4 ) a n d a h i g h l y t r a n s m i s s i b l e v i r u l e n t s t r a i n w a s s h o w n t o b e s e r u m s e n s i t i v e ( B u t l e r et a l . 1 9 9 4 ) s u g g e s t i n g t h a t t h e O - a n t i g e n i s n o t i m p o r t a n t i n v i r u l e n c e o f B. cepacia. W e s t e r n b l o t t i n g a n d a b s o r p t i o n s t u d i e s h a v e s h o w n t h a t a l a r g e p r o p o r t i o n o f s e r u m a n t i b o d i e s f r o m C F p a t i e n t s i n f e c t e d w i t h B. cepacia r e a c t e d w i t h t h e c o r e L P S o f B. cepacia. T h e s e a n t i b o d i e s d o n o t c r o s s r e a c t c o n s i s t e n t l y w i t h d i f f e r e n t s t r a i n s o f B. cepacia o r w i t h t h e c o r e r e g i o n o f P. aeruginosa L P S ( N e l s o n et a l . 1 9 9 3 ) . T h i s s t r o n g l y s u g g e s t s s t r u c t u r a l d i f f e r e n c e s e x i s t n o t o n l y b e t w e e n B. cepacia a n d P. aeruginosa b u t a l s o a m o n g s t s t r a i n s o f B. cepacia. T h e L P S f r o m B. cepacia c o n t a i n s r e l a t i v e l y l o w a m o u n t s o f p h o s p h o r u s a n d m o r e h e p t o s e , w i t h g l u c o s e a n d r h a m n o s e b e i n g t h e m a j o r s a c c h a r i d e c o m p o n e n t s ( M a n i e l l o et a l . 1 9 7 9 ) . O n l y a s i n g l e K D O a n d t w o o r t h o p h o s p h a t e r e s i d u e s w e r e f o u n d i n t h e c o r e r e g i o n o f B. cepacia L P S . A 4-amino- 4 -d e o x y a r a b i n o s e ( A r a 4 N ) w a s p r o d u c e d u p o n d e p h o s p h o r y l a t i o n o f w h a t w a s a p h o s p h a t e e s t e r ( C o x et a l . 1 9 9 1 ) . B y a n a l o g y t o o t h e r L P S m o l e c u l e s i t i s l i k e l y t h a t t h e A r a 4 N i s 13 Chapter 1 - Introduction p h o s p h o d i e s t e r l i n k e d t o t h e 4 - p o s i t i o n o f a g l u c o s a m i n e r e s i d u e o f l i p i d A ( s e e R l i n F i g u r e 2, M e y e r et a l . 1 9 8 9 ) . T h e l o w n u m b e r o f p h o s p h a t e r e s i d u e s f o u n d i n t h e L P S o f B. cepacia a n d t h e p r e s e n c e o f a r a b i n o s a m i n e s u b s t i t u t e d p h o s p h a t e s r e d u c e s t h e a m o u n t o f n e g a t i v e c h a r g e s f o u n d i n t h e o u t e r l e a f l e t o f t h e o u t e r m e m b r a n e o f t h i s o r g a n i s m . 1 .5 .3 Membrane proteins B. cepacia p r o d u c e s f i v e m a j o r o u t e r m e m b r a n e p r o t e i n s : 4 0 - k D a , 3 6 - k D a , 2 4 . 5 - k D a , 17-k D a a n d 1 4 . 5 - k D a ( A n w a r , H. et a l . 1 9 8 3 ) . T h e m a j o r o u t e r m e m b r a n e p r o t e i n i s 8 1 - k D a ( O p c P O ) a n d d i s s o c i a t e s i n t o a 3 6 - k D a ( O p c P l ) a n d a 2 7 - k D a ( O p c P 2 , s a m e as 24.5 i n A n w a r ' s s t u d y ) p r o t e i n , f o r m i n g a n o n c o v a l e n t a s s o c i a t i o n t o m a k e u p t h e 8 1 - k D a p r o t e i n ( G o t o h et a l . 1 9 9 4 ) . It h a s b e e n s u g g e s t e d t h a t t h i s i s a p o r e f o r m i n g p r o t e i n ( p o r i n ) a n d r e d u c e d e x p r e s s i o n o f t h e s e p r o t e i n s i s a s s o c i a t e d w i t h h i g h e r r e s i s t a n c e t o p M a c t a m s ( A r a n o f f et a l . 1 9 8 8 ) . P o r i n s s e r v e as d i f f u s i o n c h a n n e l s t h a t f a c i l i t a t e t h e p a s s a g e o f h y d r o p h i l i c m o l e c u l e s a c r o s s t h e o u t e r m e m b r a n e . I f t h i s t r u l y i s a p o r i n , i t i s u n i q u e i n t h a t m o s t G r a m - n e g a t i v e p o r i n s a r e t r i m e r s n o t d i m e r s ( B e n z et a l . 1 9 8 8 ) . P a r r et a l , 1 9 8 7 s h o w e d t h a t t h e o u t e r m e m b r a n e p e r m e a b i l i t y c o e f f i c i e n t f o r B. cepacia t o t h e (3-lactam n i t r o c e f i n w a s 1 0 - f o l d l e s s t h a t t h a t f o r E. coli. A n t i b i o t i c r e s i s t a n c e a n d s p e c i f i c a l l y p - l a c t a m r e s i s t a n c e i n B. cepacia i s d u e t o t h e p o o r p e r m e a b i l i t y o f i t s o u t e r m e m b r a n e . T h e s e s t u d i e s o f B. cepacia o u t e r m e m b r a n e p r o t e i n s s u g g e s t t h a t p o r i n s i n t h e o u t e r m e m b r a n e o f B. cepacia h a v e v e r y s m a l l p o r e s c o m p a r e d t o E. coli a n d P. aeruginosa m a k i n g d i f f u s i o n o f h y d r o p h i l i c c o m p o u n d s d i f f i c u l t . 1.6 B. cepacia LPS and host interaction L P S c a u s e s a c t i v a t i o n o f h o s t i m m u n e c e l l s r e s u l t i n g i n t h e s y n t h e s i s a n d s e c r e t i o n o f c y t o k i n e s t h a t a r e r e q u i r e d f o r t h e r e g u l a t i o n o f i m m u n e r e s p o n s e s ( R i e t s c h e l et a l . 1 9 9 3 ) . T h e 14 Chapter 1 - Introduction c o n s e q u e n c e o f o v e r p r o d u c t i o n o f c y t o k i n e s i s s e v e r e as o b s e r v e d i n e n d o t o x i c s h o c k , w h i c h i s a s s o c i a t e d w i t h e l e v a t e d l e v e l s o f T N F - a i n t h e h o s t ( M a t h i s o n et a l . 1 9 8 8 ) . It h a s b e e n s u g g e s t e d t h a t e l e v a t e d l e v e l s o f T N F - a m a y p l a y a r o l e i n ' c e p a c i a s y n d r o m e ' ( G o v a n et a l . 1 9 9 6 ) . B. cepacia L P S i s f o u r t o f i v e t i m e s m o r e t o x i c t h a n P. aeruginosa L P S ( S h a w et a l . 1 9 9 5 ) . B. cepacia L P S c a n s t i m u l a t e I L - 8 r e l e a s e b y m o n o c y t e s . I L - 8 i s a c y t o k i n e i m p o r t a n t f o r t h e r e c r u i t m e n t o f n e u t r o p h i l s d u r i n g a n i n f e c t i o n ( P a l f r e y m a n et a l . 1 9 9 7 ) . A s w e l l , L P S f r o m B. cepacia c a u s e s i n c r e a s e d e x p r e s s i o n o f c o m p l e m e n t r e c e p t o r 3 ( C R 3 ) o n n e u t r o p h i l s a n d p r i m e s t h e r e s p i r a t o r y b u r s t , i n c o n t r a s t w i t h P. aeruginosa, w h i c h s h o w s l i t t l e o r n o n e u t r o p h i l p r i m i n g a c t i v i t y . T h i s i n c r e a s e d s u r f a c e e x p r e s s i o n o f C R 3 f a c i l i t a t e s m i g r a t i o n o f n e u t r o p h i l s t o t h e l u n g c i r c u l a t i o n a n d i s i n v o l v e d i n a d h e s i o n , p h a g o c y t o s i s a n d a c t i v a t i o n o f n e u t r o p h i l s ( H u g h e s et a l . 1 9 9 7 ) . T h e L P S p u r i f i e d f r o m C F i s o l a t e s o f B. cepacia i n d u c e s a c o n c e n t r a t i o n o f T N F - a s i m i l a r t o t h a t o f E. coli L P S ( c o n s i d e r e d t h e m o s t b i o l o g i c a l l y a c t i v e L P S ) , f o u r t i m e s t h e a c t i v i t y o f P. aeruginosa a n d e i g h t t i m e s t h e a c t i v i t y o f S. maltophilia. B. cepacia a l s o i n d u c e s t h e p r o d u c t i o n o f m o r e s u p e r o x i d e a n i o n t h a n P. aeruginosa a n d S. maltophilia ( Z u g h a i e r et a l . 1 9 9 9 a ) . I n a d d i t i o n , c e r t a i n e p i d e m i c s t r a i n s o f B. cepacia p r o d u c e a m e l a n i n -l i k e p i g m e n t t h a t a c t s a s a s c a v e n g e r f o r s u p e r o x i d e a n i o n a n d m a y t h e r e f o r e p r o t e c t B. cepacia f r o m o x i d a t i v e k i l l i n g b y p h a g o c y t i c c e l l s ( Z u g h a i e r et a l . 1 9 9 9 b ) . R e c e n t l y , i t h a s b e e n r e p o r t e d t h a t i n d u c i b l e n i t r i c o x i d e s y t h e t a s e ( i N O S ) m a y n o t b e e x p r e s s e d i n t h e b r o n c h i a l e p i t h e l i u m o f C F p a t i e n t s . S i n c e n i t r i c o x i d e ( N O ) h a s b e e n s h o w n t o c o n t r i b u t e t o t h e a n t i m i c r o b i a l a c t i v i t y o f r e a c t i v e o x y g e n s p e c i e s i n t h e n o r m a l l u n g , e v i d e n c e s u g g e s t s t h a t t h i s l a c k o f i N O S m a y c o n t r i b u t e t o t h e s u r v i v a l o f B. cepacia i n t h e C F l u n g ( K e l l e y et a l . 1 9 9 8 ) . B. cepacia c a n n o t b e k i l l e d b y e i t h e r s u p e r o x i d e a n i o n o r N O a l o n e r a t h e r t h e i r c o m b i n e d a c t i v i t y i s r e q u i r e d ( S m i t h et a l . 1 9 9 9 ) . I n t e r e s t i n g l y , t h e t r e a t m e n t o f C G D p a t i e n t s h a s b e e n s u c c e s s f u l w i t h t h e u s e o f i n t e r f e r o n - y ( I F N - y ) t h e r a p y ( I n t e r n a t i o n a l C h r o n i c G r a n u l o m a t o u s C o o p e r a t i v e S t u d y G r o u p , 15 Chapter 1 - Introduction 1991). IFN-y is a cytokine, which is known to induce NO production in neutrophils (Evans et al. 1996). These properties of B. cepacia LPS provide some of the first insights into the virulence of this organism. 1.7 Antibiotic Resistance of B. cepacia Bacteria have evolved a variety of mechanisms to escape the action of antibiotics such as membrane permeability changes causing restricted entry into the periplasm and/or cytoplasm, enhanced export of a drug via specific transport proteins, production of enzymes that modify and inactivate the antibiotic and modification of the target itself. Antibiotic resistance probably had its earliest origins in the soil organisms that produce antibiotics in order to protect themselves against the antibiotics they produce themselves. In general, B. cepacia is resistant to a wide range of antibiotics. Those resistance mechanisms that have been identified in B. cepacia are reviewed here. 1.7.1 Antibiotic uptake across the outer membrane Three possible pathways exist for the uptake of antibiotics across the outer membrane of Gram-negative bacteria and the path of uptake is dependent on the physical properties of the particular antibiotic. Hydrophobic antibiotics such as some P-lactams, quinolones, rifampicin and macrolides are usually excluded by Gram-negatives, due to the high negative charge of LPS (Nikaido, 1983). Hydrophilic antibiotics such as most P-lactams, tetracycline and chloramphenicol pass across the Gram-negative outer membrane through the water filled channels formed by porins. For example, porin deficient mutants have significantly increased MICs for some P-lactams. (Hancock et al. 1988). As mentioned, the putative porin proteins identified for B. cepacia are considered to have extremely small channels inhibiting the entry of 16 Chapter 1 - Introduction t h e s e h y d r o p h i l i c a n t i b i o t i c s . T h e ' s e l f - p r o m o t e d - u p t a k e ' p a t h w a y h a s b e e n p r o p o s e d as t h e r o u t e o f u p t a k e f o r p o l y c a t i o n i c a n t i b i o t i c s l i k e p o l y m y x i n , a m i n o g l y c o s i d e s a n d c a t i o n i c p e p t i d e s ( H a n c o c k , 1 9 8 1 ) . T h i s ' s e l f - p r o m o t e d u p t a k e ' m o d e l i n v o l v e s t h e i n t e r a c t i o n o f a p o l y c a t i o n i c m o l e c u l e w i t h t h e n e g a t i v e l y c h a r g e d L P S m o l e c u l e . T h e L P S m o l e c u l e s a r e s t a b i l i z e d i n t h e m e m b r a n e b y M g 2 + o r C a 2 + c r o s s b r i d g i n g o f t h e n e g a t i v e l y c h a r g e d p h o s p h a t e r e s i d u e s f o u n d o n L P S . P o l y c a t i o n i c a n t i m i c r o b i a l s h a v e a h i g h e r a f f i n i t y f o r L P S , d i s p l a c i n g t h e M g 2 + o r C a 2 + . D u e t o t h e b u l k y s i z e o f t h e c a t i o n i c p e p t i d e s , t h e y d i s r u p t t h e o u t e r m e m b r a n e a n d c a u s e a p e r m e a b i l i t y c h a n g e , p r o m o t i n g t h e i r o w n u p t a k e a n d t h e u p t a k e o f o t h e r w i s e e x c l u d e d m o l e c u l e s i n t o t h e c e l l ( H a n c o c k et a l . 1 9 8 4 , H a n c o c k et a l . 1 9 9 1 ) . It h a s b e e n c l e a r l y d e m o n s t r a t e d t h a t B. cepacia d o e s n o t h a v e a ' s e l f - p r o m o t e d - u p t a k e ' p a t h w a y ( M o o r e et a l . 1 9 8 6 ) i n h i b i t i n g t h e a b i l i t y o f a m i n o g l y c o s i d e s o r c a t i o n i c a n t i m i c r o b i a l a g e n t s t o p e n e t r a t e t h e c e l l . T h i s i s t h o u g h t t o b e d u e t o t h e l o w n u m b e r o f n e g a t i v e l y c h a r g e d p h o s p h a t e r e s i d u e s o n B. cepacia L P S . O n e o f t h e s e p h o s p h a t e s i s a t t a c h e d t o t h e l i p i d A p o r t i o n o f L P S a n d i s l i n k e d t o a n A r a 4 N s u g a r ( C o x et a l . 1 9 9 1 ) . T h i s A r a 4 N m a y f o r m a b r i d g e w i t h t h e a d j a c e n t p h o s p h a t e o n t h e n e x t L P S m o l e c u l e s u c h t h a t d i v a l e n t c r o s s b r i d g i n g i s n o t r e q u i r e d f o r t h e o u t e r m e m b r a n e o f B. cepacia. U.2 Antibiotic efflux pumps M u l t i d r u g e f f l u x p u m p s t h a t t r a v e r s e b o t h t h e i n n e r a n d o u t e r m e m b r a n e s o f G r a m -n e g a t i v e b a c t e r i a m a k e a m a j o r c o n t r i b u t i o n t o i n t r i n s i c r e s i s t a n c e o f t h e s e b a c t e r i a . T h e s e p u m p s a r e c o m p o s e d o f at l e a s t t h r e e c o m p o n e n t s , a r e e n e r g i z e d b y t h e p r o t o n - m o t i v e f o r c e a n d c a n p u m p o u t a w i d e v a r i e t y o f a n t i b i o t i c s ( N i k a i d o , 1 9 9 8 ) . A n e f f l u x s y s t e m i n v o l v i n g t h r e e p r o t e i n s w a s i d e n t i f i e d i n Pseudomonas aeruginosa ( P o o l e et a l . 1 9 9 3 ) . T h e s e p r o t e i n s w e r e d e s i g n a t e d M e x A , M e x B a n d O p r M ( s a m e as O p r K ) a n d a r e c r i t i c a l f o r t h e r e s i s t a n c e o f P. 17 Chapter I - Introduction aeruginosa t o q u i n o l o n e s , P-lactams ( o t h e r t h a n i m i p e n e m ) , t e t r a c y c l i n e a n d c h l o r a m p h e n i c o l w h i c h w a s d e m o n s t r a t e d b y s t u d y i n g k n o c k o u t m u t a n t s o f a l l t h r e e g e n e s i n v o l v e d . A n e f f l u x p u m p w i t h s i m i l a r i t y t o t h i s s y s t e m h a s b e e n i d e n t i f i e d i n B. cepacia t h a t i n v o l v e s a n o u t e r m e m b r a n e p r o t e i n d e s i g n a t e d O p c K , s i m i l a r t o O p r M , s h o w n t o b e r e s p o n s i b l e f o r i n t r i n s i c r e s i s t a n c e t o t e t r a c y c l i n e , c h l o r a m p h e n i c o l a n d c i p r o f o x a c i n ( B u r n s et a l . 1 9 9 6 b ) . 1.7.3 Antibiotic modifying enzymes E n z y m e s t h a t m o d i f y a n d i n a c t i v a t e p-lactam a n t i b i o t i c s h a v e b e e n i d e n t i f i e d i n B. cepacia. I n 1 9 7 9 , B e c k m a n a n d L e s s i e d i s c o v e r e d t h a t B. cepacia i s a b l e t o u t i l i z e p e n i c i l l i n G as t h e s o l e c a r b o n s o u r c e a n d e x t r a c t s o f t h e b a c t e r i a s h o w h i g h l e v e l s o f P - l a c t a m a s e a c t i v i t y . R e s i s t a n c e t o P - l a c t a m a n t i b i o t i c s h a s b e e n a t t r i b u t e d t o i n d u c i b l e p - l a c t a m a s e s i n m a n y G r a m -n e g a t i v e b a c t e r i a . I n d u c i b l e p - l a c t a m a s e s a r e c h r o m o s o m a l l y e n c o d e d a n d a r e n o r m a l l y r e p r e s s e d ; o r g a n i s m s c a n b e i n d u c e d t o e x p r e s s h i g h l e v e l s o f t h e s e P - l a c t a m a s e s a f t e r e x p o s u r e t o c e r t a i n p - l a c t a m c o m p o u n d s . A n i n d u c i b l e c h r o m o s o m a l P - l a c t a m a s e s h a s b e e n i d e n t i f i e d i n B. cepacia e n c o d e d b y t h e penA g e n e ( P r i n c e et a l . 1 9 8 8 ) w h i c h i s r e g u l a t e d b y a n u p s t r e a m o p e n r e a d i n g f r a m e c a l l e d penR ( P r o e n c a et a l . 1 9 9 3 , T r e p a n i e r et a l . 1 9 9 7 ) a n d c a n b e i n d u c e d b y i m i p e n e m ( T r e p a n i e r et a l . 1 9 9 7 ) . C a r b a p e n e m s s u c h as i m i p e n e m a n d m e r o p e n e m a r e c u r r e n t l y t h e m o s t p o t e n t a n d b r o a d s p e c t r u m P-lactam a n t i b i o t i c s a v a i l a b l e a n d a r e r e s i s t a n t t o m o s t P - l a c t a m a s e s ( L i v e r m o r e et a l . 1 9 9 2 ) b u t c a n b e h y d r o l y z e d b y B. cepacia ( S i m p s o n et a l . 1 9 9 3 ) . A z i n c - d e p e n d e n t c a r b a p e n e m a s e ( P C M - 1 ) h a s b e e n i d e n t i f i e d a n d p a r t i a l l y p u r i f i e d . P C M - 1 i s c a p a b l e o f h y d r o l y z i n g a w i d e r a n g e o f P-lactams i n c l u d i n g i m i p e n e m , m e r o p e n e m , b i a p e n e m , c e p h a l o r i d i n e , c e f t a z i d i m e , p e n z y l p e n i c i l l i n , a m p i c i l l i n a n d c a r b e n i c i l l i n ( B a x t e r et a l . 18 Chapter 1 - Introduction 1 9 9 4 ) . T h e h i g h l e v e l o f r e s i s t a n c e t o p - l a c t a m a n t i b i o t i c s i n B. cepacia i s d u e t o a c o m b i n a t i o n o f p o o r e n t r y i n t o t h e c e l l a n d t h e p r e s e n c e o f t h e s e i n a c t i v a t i n g e n z y m e s . 1.8 Cationic peptides W i t h t h e r a p i d e m e r g e n c e o f a n t i b i o t i c r e s i s t a n c e a n d s o m e b a c t e r i a h a v i n g d e v e l o p e d r e s i s t a n c e t o a l l a n t i b i o t i c s a v a i l a b l e ( N e u , 1 9 9 2 ) , t h e d e v e l o p m e n t o f a n e w c l a s s o f a n t i b i o t i c s h a s b e c o m e m o r e i m p o r t a n t . A n t i m i c r o b i a l p e p t i d e s h a v e a t t r a c t e d i n c r e a s i n g i n t e r e s t d u e t o t h e i r u n i q u e p r o p e r t i e s . T h e y a r e i m p o r t a n t c o m p o n e n t s o f n o n - s p e c i f i c h o s t d e f e n s e m e c h a n i s m s , e n c o d e d b y h o s t g e n e s a n d f o u n d t h r o u g h o u t t h e a n i m a l k i n g d o m ( B o m a n , 19 9 5 ) . T h e s e a n t i m i c r o b i a l p e p t i d e s a r e 12-45 a m i n o a c i d s i n l e n g t h a n d a r e a m p h i p a t h i c w i t h b o t h h y d r o p h o b i c a n d c a t i o n i c s u r f a c e s . T h e y a r e c a t i o n i c b e c a u s e o f t h e p r e s e n c e o f e x c e s s l y s i n e a n d a r g i n i n e r e s i d u e s r e s u l t i n g i n a n e t c h a r g e o f at l e a s t +2 at n e u t r a l p H . It i s t h e c a t i o n i c n a t u r e o f t h e s e p e p t i d e s t h a t a c c o u n t s f o r t h e i r a b i l i t y t o t a r g e t t h e n e g a t i v e l y c h a r g e d m e m b r a n e s o f m i c r o b e s . I n G r a m - n e g a t i v e b a c t e r i a c a t i o n i c p e p t i d e s l i k e p o l y m y x i n a n d a m i n o g l y c o s i d e s u s e t h e ' s e l f - p r o m o t e d - u p t a k e ' p a t h w a y t o c r o s s t h e o u t e r m e m b r a n e a n d r e a c h t h e i r p r o p o s e d t a r g e t , t h e c y t o p l a s m i c m e m b r a n e . T h e s e p e p t i d e s , l i k e p o l y m y x i n , i n t e r a c t e l e c t r o s t a t i c a l l y w i t h t h e n e g a t i v e l y c h a r g e d h e a d g r o u p s o f b a c t e r i a l p h o s p h o l i p i d s f o u n d i n t h e i n n e r m e m b r a n e , i n s e r t i n t o t h e m e m b r a n e f o r m i n g c h a n n e l s t h a t c a u s e l e a k a g e o f c e l l c o n t e n t s l e a d i n g t o c e l l d e a t h ( C h r i s t e n s e n et a l . , 1 9 88, L e h r e r et a l . 1 9 8 9 , W i m l e y et a l . 1 9 9 4 ) . T h e a m p h i p a t h i c n a t u r e o f t h e s e p e p t i d e s a p p e a r s t o b e i m p o r t a n t i n t h e i r i n t e r a c t i o n w i t h m e m b r a n e s a n d t h e i r f o r m a t i o n o f c h a n n e l s w i t h i n t h e s e m e m b r a n e s ( S a b e r w a l , 1 9 9 4 ) . A d v a n t a g e s o f c a t i o n i c p e p t i d e s f o r u s e i n a n t i m i c r o b i a l t h e r a p y a r e 1. T h e y a r e a b l e t o k i l l r a p i d l y . 2. T h e y h a v e a b r o a d s p e c t r u m o f a c t i v i t y ( a g a i n s t G r a m - p o s i t i v e a n d G r a m -19 Chapter 1 - Introduction n e g a t i v e b a c t e r i a , f u n g i a n d v i r u s e s ) . 3. It i s d i f f i c u l t t o s e l e c t f o r s p o n t a n e o u s l y r e s i s t a n t m u t a n t s in vitro. 4. T h e s e p e p t i d e s s h o w a c t i v i t y a g a i n s t m o s t a n t i b i o t i c r e s i s t a n t p a t h o g e n s ( H a n c o c k et a l . 1 9 9 8 ) . H o w e v e r , m a n y in vitro s t u d i e s h a v e s h o w n t h a t B. cepacia i s r e s i s t a n t t o a l l c a t i o n i c p e p t i d e s . 1.8.1 Cationic peptides of neutrophils N e u t r o p h i l s c o n s t i t u t e 5 0 - 7 0 % o f t h e t o t a l w h i t e b l o o d c e l l s i n h u m a n s . T h e y p l a y a v i t a l r o l e i n t h e h o s t i m m u n e r e s p o n s e b y i n g e s t i n g a n d k i l l i n g i n v a d i n g m i c r o o r g a n i s m s . T h e s e i n v a d i n g m i c r o b e s a r e d e s t r o y e d b y t w o p o s s i b l e m e c h a n i s m s c a l l e d t h e " o x y g e n - d e p e n d e n t " a n d t h e " o x y g e n - i n d e p e n d e n t " . T h e m i c r o b i c i d a l - c y t o t o x i c p r o t e i n s o f n o n o x i d a t i v e k i l l i n g a r e f o u n d i n n e u t r o p h i l c y t o p l a s m i c g r a n u l e s ( p r i m a r y a n d s e c o n d a r y ) a n d a r e d i s c h a r g e d i n t o t h e p h a g o c y t i c v a c u o l e c o n t a i n i n g t h e m i c r o b e as s h o w n i n F i g u r e 1 o n p a g e 7. D e f e n s i n s a r e t h e m o s t a b u n d a n t c a t i o n i c p e p t i d e f o u n d i n n e u t r o p h i l s c o m p r i s i n g 10-1 8 % o f t h e i r t o t a l p r o t e i n ( L e h r e r e t a l . 1 9 9 3 ) . D e f e n s i n s a r e c l a s s i f i e d i n t o t h r e e g r o u p s , t h e c l a s s i c a l a - d e f e n s i n s , p - d e f e n s i n s a n d i n s e c t / p l a n t d e f e n s i n s ( t h i o n i n s ) . c t - D e f e n s i n s a r e f o u n d p r i m a r i l y i n n e u t r o p h i l s a n d p a n e t h c e l l s w h e r e as t h e P - d e f e n s i n s a r e m o r e w i d e l y d i s t r i b u t e d . P - D e f e n s i n s h a v e b e e n f o u n d i n t h e e p i t h e l i a l c e l l s o f m a n y h u m a n ( Z h a o et a l . 1 9 9 6 ) a n d m u r i n e ( H u t t n e r et a l . 1 9 9 7 ) o r g a n s , h u m a n s k i n ( H a r d e r et a l . 1 9 9 7 ) , b o v i n e n e u t r o p h i l s ( S e l s t e d et a l . 1 9 9 3 ) , t o n g u e ( S c h o n w e t t e r et a l . 1 9 9 5 ) a n d t r a c h e a ( D i a m o n d et a l . 1 9 9 1 ) . T h e p l a n t d e f e n s i n s ( n o w c a l l e d a a n d p t h i o n i n s ) a r e c o m p a c t , L - s h a p e d m o l e c u l e s c o n t a i n i n g t w o l o n g d i s u l p h i d e l i n k e d c c - h e l i c e s a n d t w o s h o r t a n t i p a r a l l e l P-sheets ( B o h l m a n n et a l . 1 9 9 1 ) . D i f f e r e n t f r o m t h e i n s e c t d e f e n s i n s , t h e s t r u c t u r e o f a a n d P - d e f e n s i n s a r e d o m i n a t e d b y a n t i p a r a l l e l P-s h e e t s ( n o a - h e l i c e s ) , s t a b i l i z e d b y d i s u l f i d e c r o s s b r i d g e s a n d c o n t a i n s i x c y s t e i n e r e s i d u e s . T h e y c o n s i s t o f t h r e e r e g i o n s o f P-sheet s t r u c t u r e a n d a p r o t r u d i n g P - h a i r p i n h y d r o p h o b i c l o o p . 2 0 Chapter I - Introduction D e f e n s i n s a r e a c t i v e a g a i n s t a w i d e v a r i e t y o f m i c r o b e s i n c l u d i n g G r a m - p o s i t i v e a n d n e g a t i v e b a c t e r i a , f u n g i , s p i r o c h e t e s , m y c o b a c t e r i a a n d e n v e l o p e d ( n o t n a k e d ) v i r u s e s ( L e h r e r et a l . 1 9 9 3 ) . D e f e n s i n s p e r m e a b i l i z e t h e o u t e r a n d i n n e r m e m b r a n e o f E. coli s e q u e n t i a l l y , w i t h c e l l d e a t h o c c u r r i n g u p o n i n n e r m e m b r a n e d i s r u p t i o n ( L e h r e r et a l . 1 9 8 9 ) . D e f e n s i n s a r e c a p a b l e o f d i s r u p t i n g n e g a t i v e l y c h a r g e d b u t n o t n e u t r a l m e m b r a n e s ( C o c i a c i c h et a l . 1 9 9 3 ) a n d f o r m m u l t i m e r s w i t h i n t h e m e m b r a n e r e s u l t i n g i n v o l t a g e - d e p e n d a n t i o n c h a n n e l s ( K a g a n et a l . 1990, P a r d i et a l . 1 9 9 2 , W i m l e y et a l . 1 9 9 4 ) . A l s o f o u n d i n n e u t r o p h i l s a r e t h e h i g h m o l e c u l a r w e i g h t c a t i o n i c p r o t e i n s t h a t s h o w s i m i l a r a c t i v i t y t o t h e c a t i o n i c p e p t i d e s , p r o b a b l y d u e t o t h e i r p o s i t i v e c h a r g e s . T h e s e a r e b a c t e r i a l p e r m e a b i l i t y i n c r e a s i n g p r o t e i n ( B P I ) a n d t h e s e r p r o c i d i n s : p r o t e i n a s e 3, a z u r o c i d i n a n d c a t h e p s i n G . I n a d d i t i o n , a c l e a v a g e p r o d u c t o f l a c t o f e r r i n ( a l s o f o u n d i n n e u t r o p h i l g r a n u l e s ) h a s b a c t e r i a l l y s i s a c t i v i t y . B P I i s a 5 0 - k D a c a t i o n i c p r o t e i n t h a t c o n t a i n s 4 5 6 a m i n o a c i d r e s i d u e s . B P I i s c y t o t o x i c t o a w i d e r a n g e o f G r a m - n e g a t i v e b a c t e r i a , h o w e v e r , f o r S. typhimurium a n d E. coli, b a c t e r i a l s u s c e p t i b i l i t y v a r i e s a n d i s d e t e r m i n e d b y t h e s t r u c t u r e o f L P S a n d t h e l e n g t h o f t h e O - a n t i g e n ( W e i s s et a l . 1 9 8 6 ) . T h i s i s n o t t r u e f o r P. aeruginosa w h e r e a l l s t r a i n s , r e g a r d l e s s o f O - a n t i g e n l e n g t h , a r e e q u a l l y s u s c e p t i b l e ( S i e f f e r m a n e t a l . 1 9 9 1 ) . B i n d i n g o f B P I t o l i v e b a c t e r i a i s s h o w n t o i n c r e a s e t h e p e r m e a b i l i t y o f t h e o u t e r m e m b r a n e ( W e i s s et a l . 1 9 9 2 ) . L o n g e r O - a n t i g e n c h a i n s i m p e d e t h e a c c e s s o f B P I t o t h e c o r e r e g i o n o f L P S , t h e m a i n s i t e o f t h e n e g a t i v e c h a r g e s t o w h i c h B P I b i n d s ( K a s t o w s k y et a l . 1 9 9 2 ) . L a c t o f e r r i n i s a n 8 0 - k D a i r o n - b i n d i n g g l y c o p r o t e i n t h a t i s c l o s e l y r e l a t e d i n s t r u c t u r e t o t r a n s f e r r i n . T h e r e i s s t r o n g e v i d e n c e i n d i c a t i n g a d i r e c t k i l l i n g e f f e c t o f l a c t o f e r r i n , u n r e l a t e d t o i r o n b i n d i n g , w h i c h i s m e d i a t e d b y b i n d i n g t o t h e s u r f a c e o f s u s c e p t i b l e o r g a n i s m s ( A r n o l d et a l . 1 9 8 2 ) . G r a m - n e g a t i v e b a c t e r i a e x p o s e d t o l a c t o f e r r i n r e l e a s e L P S ( E l l i s o n e t a l . 1 9 8 8 ) a n d t h i s a n t i m i c r o b i a l a c t i v i t y i s m e d i a t e d b y t h e a c i d - p e p s i n c l e a v a g e p r o d u c t l a c t o f e r r i c i n , t h e c a t i o n i c 21 Chapter I - Introduction N - t e r m i n a l r e g i o n o f l a c t o f e r r i n ( B e l l a m y et a l . 1 9 9 2 ) . L a c t o f e r r i c i n i s h i g h l y b a s i c , h a s a s i n g l e d i s u l f i d e b o n d a n d n o i r o n - b i n d i n g c a p a c i t y a n d i t i s a c t i v e a g a i n s t b a c t e r i a , y e a s t a n d f u n g i ( B e l l a m y et a l . 1 9 9 2 b , B e l l a m y et a l . 1 993, Y a m a u c h et a l . 1 9 9 3 ) . T h e s e p e p t i d e s a n d p r o t e i n s a r e t h e m a j o r c o m p o n e n t s o f n e u t r o p h i l n o n o x i d a t i v e k i l l i n g . B. cepacia i s r e s i s t a n t t o n o n o x i d a t i v e k i l l i n g c o m p o n e n t s o f n e u t r o p h i l s a s s e e n i n k i l l i n g a s s a y s u s i n g C G D a n d n o r m a l n e u t r o p h i l s ( S p e e r t et a l . 1 9 9 4 ) . 1.8.2 a-Helical cationic peptides T h e a - h e l i c a l c a t i o n i c p e p t i d e s e x i s t i n a r a n d o m - c o i l c o n f i g u r a t i o n i n a q u e o u s s o l u t i o n s ( S t e i n e r , 1 9 8 2 ) b u t f o r m a h e l i x - t u r n - h e l i x s t r u c t u r e w h e n i n t e r a c t i n g w i t h m e m b r a n e s ( D e m s e y et a l . 1 9 9 0 ) . a - H e l i c a l p e p t i d e s a r e c h a r a c t e r i z e d b y o n e a m p h i p a t h i c a - h e l i x a n d o n e h y d r o p h o b i c a - h e l i x . C e c r o p i n s w e r e f i r s t i s o l a t e d f r o m t h e p u p a e o f s i l k m o t h , Hyalophora cecropia ( S t e i n e r et a l . 1 9 8 1 ) a n d s i m i l a r m o l e c u l e s h a v e s i n c e b e e n i s o l a t e d f r o m m a n y i n s e c t o r d e r s ( M e r r i f i e l d et a l . 1 9 9 4 ) , p i g i n t e s t i n e ( L e e et a l . 1 9 8 9 ) a n d m a r i n e p r o t o c h o r d a t e s ( Z h o a et a l . 1 9 9 7 ) . C e c r o p i n s c a u s e i n s t a n t l y s i s o f b a c t e r i a l c e l l s b y d e s t r u c t i o n o f t h e c y t o p l a s m i c m e m b r a n e ( B o m a n et a l . 1 9 9 3 ) a n d o n l y l y s e n e g a t i v e o r n e u t r a l c h a r g e d m e m b r a n e s f o r m i n g i o n c h a n n e l s . It i s t h e c a t i o n i c N - t e r m i n u s t h a t a i d s t h e b i n d i n g o f c e c r o p i n s t o t h e b a c t e r i a l m e m b r a n e ( W a d e et a l . 1 9 9 0 , C h r i s t e n s e n et a l . 1 9 8 8 ) . M e l i t t i n , i s o l a t e d f r o m b e e v e n o m t o x i n , h a s a s i m i l a r h e l i x - t u r n - h e l i x s t r u c t u r e , h o w e v e r , o p p o s i t e t o c e c r o p i n s , i t h a s a h y d r o p h o b i c C -t e r m i n u s a n d a c a t i o n i c N - t e r m i n u s ( P i e r s et a l . 1 9 9 4 ) . M a g a i n i n s w e r e i s o l a t e d from t h e s k i n o f t h e A f r i c a n frog, Xenopus laevis ( Z a s l o f f et a l . 1 9 8 7 ) a n d f o r m p o r e s i n t h e c e l l m e m b r a n e s o f s u s c e p t i b l e m i c r o o r g a n i s m s b u t n o t e u k a r y o t i c m e m b r a n e s ( B e c h i n g e r et a l . 1 9 9 7 , L u d k e et a l . 1 9 9 6 ) . B a c t e n e c i n s a r e p r o d u c e d i n b o v i n e n e u t r o p h i l s a n d e x i s t i n a n e x t e n d e d l o o p f o r m . T h e s e p e p t i d e s b i n d t o t h e o u t e r m e m b r a n e o f G r a m - n e g a t i v e b a c t e r i a a n d i n c r e a s e o u t e r 2 2 Chapter 1 - Introduction m e m b r a n e p e r m e a b i l i t y f o l l o w e d b y a n i n c r e a s e i n p e r m e a b i l i t y o f t h e i n n e r m e m b r a n e a n d i n h i b i t i o n o f t h e e l e c t r o n t r a n s p o r t c h a i n ( S k e r l a v a j et a l . 1 9 9 0 ) . 1.8.3 Mechanism of action T h e o v e r a l l m e c h a n i s m o f k i l l i n g G r a m - n e g a t i v e b a c t e r i a f o r c a t i o n i c p e p t i d e s a n d p o l y m y x i n h a s b e e n p r o p o s e d t o b e d i s r u p t i o n o f t h e c y t o p l a s m i c m e m b r a n e o f b a c t e r i a ( H a n c o c k e t a l . 1 9 9 8 ) . T h e m e c h a n i s m o f a c t i o n h a s b e e n p r o p o s e d t o i n v o l v e t h r e e s t e p s . F i r s t b i n d i n g o f t h e p e p t i d e t o t h e c e l l s u r f a c e , p r o b a b l y t h r o u g h d i s p l a c e m e n t o f t h e M g 2 + o r C a 2 + c a t i o n s c r o s s - l i n k i n g t h e n e g a t i v e c h a r g e s o n L P S . S e c o n d p e r m e a b i l i z a t i o n o f t h e o u t e r m e m b r a n e o c c u r s v i a t h e ' s e l f - p r o m o t e d - u p t a k e ' p a t h w a y d e s c r i b e d e a r l i e r i n s e c t i o n 1.7.1. T h i s i s f o l l o w e d b y p e r m e a b i l i z a t i o n o f t h e c y t o p l a s m i c m e m b r a n e r e s u l t i n g i n l o s s o f v i a b i l i t y o f t h e c e l l b y l y s i s a n d p o s s i b l e D N A d a m a g e ( L i c h t e n s t e i n et a l . 1 9 8 8 ) . I n m o d e l m e m b r a n e s y s t e m s , c a t i o n i c p e p t i d e s a g g r e g a t e t o f o r m m u l t i m e r s t h a t r e s u l t i n c h a n n e l s a c r o s s t h e i n n e r m e m b r a n e w i t h t h e h y d r o p h o b i c f a c e o f t h e p e p t i d e s p o s i t i o n e d o u t w a r d i n t e r a c t i n g w i t h t h e m e m b r a n e a n d t h e h y d r o p h i l i c f a c e s o r i e n t e d t o w a r d s t h e i n s i d e o f t h e f o r m e d c h a n n e l s . T h i s r e s u l t s i n l e a k a g e o f p r o t o n s , l o s s o f t h e p r o t o n m o t i v e f o r c e , l o s s o f m e m b r a n e p o t e n t i a l a n d l e a k a g e o f o t h e r c o m p o u n d s c a u s i n g c e l l d e a t h ( L e h r e r et a l . 1 9 8 9 ) . S e l e c t i v i t y o f c a t i o n i c p e p t i d e s f o r b a c t e r i a l m e m b r a n e s o v e r e u k a r y o t i c c e l l m e m b r a n e s i s d u e t o t h e i r c o m p o s i t i o n a l a n d c h a r g e d i f f e r e n c e s . B a c t e r i a l m e m b r a n e s a r e c o m p o s e d o f m o s t l y n e g a t i v e l y c h a r g e d l i p i d s s u c h a s L P S , p h o s p h a t i d y l g l y c e r o l a n d c a r d i o l i p i n . E u k a r y o t i c c e l l m e m b r a n e s a r e c o m p o s e d o f z w i t t e r i o n i c l i p i d s s u c h a s p h o s p h a t i d y l c h o l i n e a n d s p h i n g o m y e l i n ( a g l y c o l i p i d f o r w h i c h c e r a m i d e i s t h e l i p i d p r e c u r s o r ) . A s w e l l , c h o l e s t e r o l , a b u n d a n t i n e u k a r y o t i c m e m b r a n e s , i s t h o u g h t t o i n h i b i t i n s e r t i o n o f c a t i o n i c p e p t i d e s i n t o t h e m e m b r a n e . B a c t e r i a l c e l l s h a v e a l a r g e n e g a t i v e t r a n s m e m b r a n e p o t e n t i a l o f a p p r o x i m a t e l y - 1 4 0 m V w h e r e a s t h e m e m b r a n e p o t e n t i a l o f 23 Chapter 1 - Introduction e u k a r y o t i c c e l l s i s a p p r o x i m a t e l y t o - 2 0 m V . T h i s l a r g e m e m b r a n e p o t e n t i a l i s t h o u g h t t o a i d i n t h e i n s e r t i o n o f t h e s e p e p t i d e s i n t o t h e m e m b r a n e o f b a c t e r i a l b u t n o t e u k a r y o t i c c e l l s ( H a n c o c k , 1 9 9 7 ) . S p o n t a n e o u s r e s i s t a n c e t o c a t i o n i c p e p t i d e s h a s n o t b e e n o b s e r v e d a n d t h i s i s p r o b a b l y d u e t o t h e f a c t t h a t a n a l t e r a t i o n i n m e m b r a n e s t r u c t u r e t o p r e v e n t i n s e r t i o n a n d c h a n n e l f o r m a t i o n i s d i f f i c u l t t o a c h i e v e . 1.8.4 Bacterial resistance to cationic peptides and polymyxin P o l y m y x i n B , p r o d u c e d b y Bacillus polymyxa i s c o n s i d e r e d a c a t i o n i c a n t i b i o t i c w i t h a p o s i t i v e l y c h a r g e d r i n g s t r u c t u r e a n d a h y d r o p h o b i c l i p i d t a i l . L i k e c a t i o n i c p e p t i d e s , p o l y m y x i n b i n d s t o t h e o u t e r m e m b r a n e ( H a n c o c k et a l . 1 9 8 1 ) , i n d u c e s s l o u g h i n g o f t h e o u t e r m e m b r a n e ( P e t e r s o n et a l . 1 9 8 5 ) a n d i n c r e a s e s t h e p e r m e a b i l i t y o f t h e o u t e r m e m b r a n e t o p r o t e i n s a n d h y d r o p h o b i c c o m p o u n d s ( V a a r a et a l . 1 9 8 1 , 1 9 8 3 ) . I n a d d i t i o n t o t h i s , p o l y m y x i n b i n d s t o a n d d i s r u p t s t h e p a c k i n g a r r a n g e m e n t o f i s o l a t e d L P S ( P e t e r s o n et a l . 1 9 8 5 ) . R e s i s t a n c e o f G r a m -n e g a t i v e b a c t e r i a t o p o l y m y x i n h a s b e e n a c h i e v e d t h r o u g h c h a n g e s i n t h e L P S s t r u c t u r e . R e s i s t a n t m u t a n t s o f S. typhimurium h a v e i n c r e a s e d l e v e l s o f a r a b i n o s a m i n e ( a n d e t h a n o l a m i n e ) e s t e r i f i e d t o t h e l i p i d A p h o s p h a t e r e s i d u e s ( s e e F i g u r e 2 o n p a g e 12). E s t e r i f i c a t i o n o f a p h o s p h a t e r e s i d u e r e d u c e s t h e n e g a t i v e c h a r g e o f t h a t p h o s p h a t e , r e d u c i n g t h e o v e r a l l n e g a t i v e c h a r g e o f t h e L P S m o l e c u l e . S u c h m u t a n t s a n d t h e i r i s o l a t e d L P S b i n d l e s s p o l y m y x i n B t h a n t h e w i l d - t y p e d o e s ( V a a r a et a l . 1 9 8 1 ) . S i m i l a r o b s e r v a t i o n s w e r e s e e n f o r s e v e r a l s t r a i n s o f E. coli a n d S. typhimurium ( P e t e r s o n et a l . 1 9 8 7 ) . O n e o f t h e m a j o r c o n t r o l p o i n t s o f Salmonella sp. p a t h o g e n e s i s i s t h e P h o P - P h o Q t w o - c o m p o n e n t r e g u l a t o r y s y s t e m ( M i l l e r et a l . 1 9 8 9 ) t h a t i n d u c e s t h e e x p r e s s i o n o f r e s i s t a n c e t o c a t i o n i c p e p t i d e s a n d p o l y m y x i n . T h e a b i l i t y o f S. typhimurium t o s u r v i v e w i t h i n m a c r o p h a g e s i s d u e i n p a r t t o a c t i v a t i o n o f P h o P - P h o Q r e s u l t i n g i n e x p r e s s i o n o f r e s i s t a n c e t o a n t i m i c r o b i a l p e p t i d e s a n d t h i s e x p r e s s i o n o f r e s i s t a n c e t o 2 4 Chapter I - Introduction a n t i m i c r o b i a l p e p t i d e s c o r r e l a t e s w i t h v i r u l e n c e i n m i c e ( M i l l e r et a l . 1 9 8 9 ) . T h i s s u p p o r t s t h e h y p o t h e s i s t h a t r e s i s t a n c e t o a n t i m i c r o b i a l c a t i o n i c p e p t i d e s i s p o t e n t i a l l y a v i r u l e n c e m e c h a n i s m a s w a s s u g g e s t e d b y S p e e r t et a l , 1 9 9 4 w h e r e B. cepacia w a s s h o w n t o b e r e s i s t a n t t o n o n o x i d a t i v e k i l l i n g o f C G D n e u t r o p h i l s . O t h e r S. typhimurium m u t a n t s t h a t a r e r e s i s t a n t t o p o l y m y x i n m a p t o a l o c u s t h a t h a s b e e n d e s i g n a t e d pmrA ( p o l y m y x i n r e s i s t a n c e ) ( M a k e l a et a l . 1 9 7 8 ) . D N A s e q u e n c e a n a l y s i s o f t h i s l o c u s r e v e a l e d a n o p e r o n i n c l u d i n g orfl, pmrA, a n d pmrB ( R o l a n d et a l . 1 9 9 3 ) a n d P m r A - P m r B i s a t w o c o m p o n e n t r e g u l a t o r y s y s t e m c o n t r o l l i n g r e s i s t a n c e t o a n t i m i c r o b i a l p e p t i d e s t h a t i s r e g u l a t e d b y t h e P h o P - P h o Q s y s t e m ( G u n n et a l . 1 9 9 6 ) . M u t a n t s o f pmrA a r e r e s i s t a n t t o p o l y m y x i n a n d s h o w a n A r a 4 N e s t e r l i n k e d t o a p h o s p h a t e o f l i p i d A ( H e l a n d e r et a l . 1 9 9 4 ) . I n a d d i t i o n t o t h e s e s t u d i e s , a n o p e r o n , pmr¥ h a s b e e n s h o w n t o b e u n d e r t h e c o n t r o l o f P m r A - P m r B a n d i s d i r e c t l y i n v o l v e d i n t h e s u b s t i t u t i o n o f t h e 4' p h o s p h a t e o n t h e g l u c o s a m i n e o f l i p i d A s h o w n i n F i g u r e 2 o n p a g e 12 ( H e l a n d e r et a l . 1994, G u n n et a l , 1 9 9 8 ) . T h e r e i s a l s o e v i d e n c e t o s u p p o r t t h a t i n c r e a s e d a c y l a t i o n o f l i p i d A i n S. typhimurium r e s u l t s i n r e s i s t a n c e t o c a t i o n i c a n t i m i c r o b i a l a g e n t s ( G u o e t a l . 1 9 9 8 ) . M o s t o f t h e o t h e r g e n e t i c s t u d i e s t o d a t e o n r e s i s t a n c e t o c a t i o n i c p e p t i d e s a n d p o l y m y x i n h a v e m a p p e d t o t h e L P S b i o s y n t h e s i s o p e r o n as s e e n f o r Ralstonia solanacearum ( T i t a r e n k o e et a l . 1 9 9 7 ) , Brucella bronchisetica ( B a r e m a n n s et a l . 1 9 9 8 ) a n d S. typhimurium ( M a c i a s et a l . 1 9 9 0 ) . O r g a n i s m s t h a t a r e i n h e r e n t l y r e s i s t a n t t o p o l y m y x i n h a v e b e e n s h o w n t o c a r r y L P S , w h i c h h a s t h e 4 ' - p h o s p h a t e o n l i p i d A c o m p l e t e l y s u b s t i t u t e d w i t h 4 A r a N s u c h as Proteus mirabilis ( S i d o r c z y k et a l . 1 9 8 3 ) a n d Chromobacterium violaceum ( H a s e e t a l . 1 9 7 7 ) . A t w o c o m p o n e n t r e g u l a t o r y s y s t e m a s s e e n i n Salmonella h a s n o t b e e n i d e n t i f i e d i n B. cepacia. R e s i s t a n c e o f B. cepacia i s m o s t l i k e l y d u e i n p a r t t o t h e i n a b i l i t y o f p o l y m y x i n t o b i n d t o t h e L P S a n d t h e a b s e n c e o f t h e ' s e l f - p r o m o t e d - u p t a k e ' p a t h w a y . T h e c o n s t i t u t i v e p r e s e n c e o f 4 A r a N e s t e r l i n k e d t o a p h o s p h a t e o f l i p i d A i n t h e L P S o f B. cepacia, b y a n a l o g y t o t h e pmrA 2 5 Chapter 1 - Introduction m u t a n t s o f S. typhimurium, p r o b a b l y p l a y s a r o l e i n t h e r e s i s t a n c e o f B. cepacia t o c a t i o n i c p e p t i d e s . T h i s 4 A r a N l i n k e d t o B. cepacia L P S w o u l d r e d u c e i t s a b i l i t y t o b i n d c a t i o n i c p e p t i d e s o r p o l y m y x i n . T h e a c t u a l t a r g e t f o r b a c t e r i a l k i l l i n g b y p o l y m y x i n a n d c a t i o n i c p e p t i d e s i s t h e c y t o p l a s m i c m e m b r a n e w h e r e i n t e r a c t i o n c a u s e s c e l l l y s i s . O n e s t u d y b y M a n n i e l l o et a l . 1 9 7 8 s h o w e d t h a t s p h e r o p l a s t s o f B. cepacia a r e r e s i s t a n t t o l y s i s b y p o l y m y x i n s u g g e s t i n g t h a t r e s i s t a n c e t o p o l y m y x i n B m a y b e r e l a t e d t o a r e s i s t a n t c y t o p l a s m i c m e m b r a n e . A l t h o u g h c a t i o n i c p e p t i d e s a n d a n t i b i o t i c s a p p e a r t o b e u n a b l e t o k i l l B. cepacia, s e v e r a l s t u d i e s h a v e s h o w n t h a t c a t i o n i c p e p t i d e s a n d p o l y m y x i n a r e c a p a b l e o f r e d u c i n g t h e M I C s f o r o t h e r a n t i b i o t i c s t o B. cepacia ( R a j y a g u r u et a l . 1 9 9 7 , 1 9 9 8 ) . 1.9 Thesis Objective Burkholderia cepacia i s a n i m p o r t a n t o p p o r t u n i s t i c p a t h o g e n t h a t i n f e c t s i m m u n o c o m p r o m i s e d i n d i v i d u a l s a n d c a u s e s f a t a l i n f e c t i o n s f o r b o t h C F a n d C G D p a t i e n t s . V i r u l e n c e f a c t o r s h a v e b e e n s t u d i e d i n s o m e d e t a i l i n r e l a t i o n t o i t s p a t h o g e n i c i t y i n C F ; h o w e v e r , a s t r o n g c o r r e l a t i o n b e t w e e n t h e s t u d i e d v i r u l e n c e f a c t o r s a n d B. cepacia p a t h o g e n i c i t y i n C F d o e s n o t e x i s t . B. cepacia i s e x t r e m e l y r e s i s t a n t t o a l a r g e n u m b e r o f a n t i b i o t i c s , p r o b a b l y d u e t o i t s i m p e r m e a b l e o u t e r m e m b r a n e . O f p a r t i c u l a r i n t e r e s t i s t h e r e s i s t a n c e o f B. cepacia t o k i l l i n g b y n o n o x i d a t i v e m e c h a n i s m s o f n e u t r o p h i l s . T h e m a i n c o m p o n e n t s o f n o n o x i d a t i v e k i l l i n g a r e t h e c a t i o n i c p e p t i d e s a n d p r o t e i n s t o w h i c h B. cepacia i s r e s i s t a n t . F o r Salmonella a s t r o n g c o r r e l a t i o n e x i s t s b e t w e e n r e s i s t a n c e t o d e f e n s i n s a n d v i r u l e n c e i n a m o u s e m o d e l ( F i e l d s et a l . 1 9 8 9 ) . B y u n d e r s t a n d i n g w h y B. cepacia i s s o r e s i s t a n t t o c a t i o n i c p e p t i d e s o n e m i g h t g a i n i n s i g h t i n t o a p o s s i b l e v i r u l e n c e m e c h a n i s m f o r t h i s o r g a n i s m . T h e a i m o f t h i s s t u d y w a s t o i d e n t i f y a t r a n s p o s o n i n s e r t i o n m u t a n t o f B. cepacia t h a t h a d r e d u c e d s u s c e p t i b i l i t y t o c a t i o n i c p e p t i d e s . T h e s t r a i n u s e d i n t h i s s t u d y i s a g e n o m o v a r II s t r a i n , B. multivorans A T C C 1 7 6 1 6 , a n d t h e 2 6 Chapter 1 - Introduction p h e n o t y p e o f c a t i o n i c p e p t i d e r e s i s t a n c e i s s h a r e d b y a l l m e m b e r s o f t h e B. cepacia c o m p l e x . C h a r a c t e r i z a t i o n o f t h e t r a n s p o s o n i n s e r t i o n m u t a n t w a s c a r r i e d o u t a s f o l l o w s : 1. D N A s e q u e n c e a n a l y s i s o f t h e r e g i o n o f D N A s u r r o u n d i n g t h e s i t e o f t r a n s p o s o n i n s e r t i o n t o i d e n t i f y g e n e s t h a t m a y b e i n v o l v e d i n B. multivorans c a t i o n i c p e p t i d e r e s i s t a n c e . 2. C o m p a r i s o n o f o u t e r m e m b r a n e p r o t e i n p r o f i l e s , L P S l a d d e r s , p h o s p h o l i p i d s b y T L C , m e m b r a n e p e r m e a b i l i t y a n d L P S b i n d i n g o f p o l y m y x i n w e r e c o m p a r e d f o r t h e p a r e n t a n d m u t a n t s t r a i n s . B o t h t h e g e n e t i c a n d p h e n o t y p i c c h a r a c t e r i z a t i o n o f t h i s m u t a n t i n t h i s s t u d y h a s l e a d t o t h e i d e n t i f i c a t i o n o f a n o v e l m e c h a n i s m o f r e s i s t a n c e t h a t B. cepacia c o m p l e x o r g a n i s m s m a y r e q u i r e i n o r d e r t o r e s i s t k i l l i n g b y c a t i o n i c p e p t i d e s . 2 7 Chapter 2 - Materials and Methods Chapter 2 Materials and Methods 2.1 Strains, Plasmids and Growth Conditions T h e b a c t e r i a l s t r a i n s a n d p l a s m i d s u s e d i n t h i s s t u d y a r e l i s t e d i n T a b l e 1. F o r r o u t i n e b a c t e r i a l g r o w t h , L u r i a B r i t a n i ( L B ) b r o t h w a s u s e d . M u e l l e r - H i n t o n ( M H ) b r o t h ( D i f c o L a b o r a t o r i e s , D e t r o i t , M i c h , s e e a p p e n d i x A f o r r e c i p e s ) w a s u s e d f o r m i n i m a l i n h i b i t o r y c o n c e n t r a t i o n ( M I C ) d e t e r m i n a t i o n s . A l l c u l t u r e s w e r e i n c u b a t e d at 3 7 ° C o v e r n i g h t w i t h a e r a t i o n u n l e s s o t h e r w i s e s t a t e d . P l a s m i d s w e r e m a i n t a i n e d b y g r o w t h i n t h e p r e s e n c e o f t h e a p p r o p r i a t e a n t i b i o t i c . C o n c e n t r a t i o n s o f a n t i b i o t i c s w e r e : t e t r a c y c l i n e a n d k a n a m y c i n s u l f a t e ( B o e r h i n g e r M a n n h e i m , G m b H ) at 2 0 u.g/ml, a m p i c i l l i n ( N o v o p h a r m ) at 1 0 0 u g / m l f o r E. coli, t e t r a c y c l i n e at 1 0 0 u.g/ml f o r B. multivorans, t r i m e t h o p r i m ( I C N B i o m e d i c a l Inc.) at 1 0 0 u.g/ml f o r b o t h E. coli a n d B. multivorans. A l l s t r a i n s w e r e s t o r e d at - 7 0 ° C i n M H b r o t h w i t h 8 . 0 % d i m e t h y l s u l f o x i d e ( D M S O ) ( v / v ) . B. multivorans c u l t u r e s w e r e i n o c u l a t e d d i r e c t l y from t h e f r e e z e r s t o c k w h e n u s e d . 2.2 Construction of a Tn5-751S Insertion Mutant Library of B. multivorans. p T G L 1 6 6 ( c a r r y i n g t r a n s p o s o n T n 5 - 7 5 7 5 ) w a s t r a n s f e r r e d f r o m E. coli D H 5 a t o B. multivorans A T C C 1 7 6 1 6 b y c o n j u g a t i o n . E q u a l a m o u n t s o f d o n o r a n d r e c i p i e n t b a c t e r i a w e r e p l a t e d o n L B a g a r f o r f o u r h o u r s at 3 0 ° C . R e s i s t a n t B. multivorans t r a n s c o n j u g a n t s w e r e s e l e c t e d at 3 0 ° C o n L B a g a r c o n t a i n i n g t e t r a c y c l i n e a n d t r i m e t h o p r i m a n d 31 u g / m l p o l y m y x i n B s u l f a t e ( I C N B i o c h e m i c a l s Inc., 7 6 4 0 U n i t s / m g ) . T h i s l o w c o n c e n t r a t i o n o f p o l y m y x i n B a c t e d t o c o u n t e r s e l e c t t h e E. coli d o n o r s t r a i n . T h e s e l e c t e d p T G L I 6 6 c o n t a i n i n g d e r i v a t i v e s o f s t r a i n 1 7 6 1 6 w e r e p r o p a g a t e d f o r 6 0 h o u r s at 4 7 ° C i n L B b r o t h s o t h a t p T G L 1 6 6 w a s l o s t . A p p r o p r i a t e d i l u t i o n s w e r e t h e n p l a t e d o n s u p p l e m e n t e d m i n i m a l s a l t s a g a r ( s e e a p p e n d i x A 28 Chapter 2 - Materials and Methods TABLE 1. Strains and plasmids. A l l the strains and plasmids pertinent to this study and the relevant references are listed: Strain or plasmid Pertinent characteristics Source Strains B. multivorans A T C C 17616 26D7 JTC P. aeruginosa Ml E. coli D H 5 a X L 1-Blue M R HB1005 Plasmids pBluescript II SK+ SuperCos 1 pDN18 pRK2013 pTGL166 pBSl 5G6, 5H11,2F6, 2F9, 1G3 pBS15 pBS16 pBS4 pBSPl pDBS6 pDBS9 pDBSl pDBS2 B. cepacia genomovar II type strain Derivative of A T C C 17616 with Tn5-751S inserted in its chromosome. Reduced resistance to polymyxin. C G D isolate isolated from mouse intestine F-, (|)80dlacZAM15 A(lacZYA-argF)U169 deoRrecAl endAl hsdR17(rK\mK +) phoA supE44 X' thi-1 gyrA96 relAl . Cosmid library host strain cationic peptide sensitive strain high copy cloning vector high copy cosmid cloning vector Broad-host-range low copy TetR cloning vector Kan R transfer plasmid 53 kb broad-host-range Inc PI plasmid; temperature sensitive with respect to its replication due to a mutation in trf A; contains Tn5-751S, which confers resistance to both kanamycin and trimethoprim, as well as the nontransposable penicillin and tetracycline resistance markers. The Spel site within Tn5-751S was replaced by a Swal site. pBluescript II KS(+) derivative carrying the Kan R gene from Tn5-751 and flanking D N A from mutant 26D7. E. coli XL- lBlue M R carrying SuperCos 1 derivatives with 35 kb Sau3Al partially digested A T C C 17616 chromosomal D N A corresponding to the region of D N A adjacent to the transposon insertion in mutant 26D7 pBluescript II KS (+) carrying the 3.8 kb Sail chromosomal fragment from A T C C 17616. pBluescript II KS (+) carrying the 3.8 kb Sail chromosomal fragment in opposite orientation to pBS15. pBluescript II KS (+) carrying the 4.0 kb BamHl chromosomal fragment from A T C C 17616. pBluescript II KS (+) carrying the 7.0 kb Pstl chromosomal fragment from A T C C 17616. pDN18 carrying the 4.0 kb BamHl fragment from pBS4. pDN18 carrying the 4.0 kb BamHl fragment from pBS4 in the opposite orientation to pDBS6. pDN18 carrying Kpnl-Pstl subclone from pBS15. pDN18 carrying Kpnl-Pstl subclone from pBS16. A T C C This study Speert etal. 1994 Stieritz et al, 1975 Gibco B R L Stratagene Hancock Stratagene Stratagene Nunnetal, 1990. Figurski, 1979 Cheng et al, 1994. This study This study This study This study This study This study This study This study This study A T C C - American Type Culture Collection (Rockville, Md.) 29 Chapter 2 - Materials and Methods f o r r e c i p e ) c o n t a i n i n g t r i m e t h o p r i m . T r i m e t h o p r i m r e s i s t a n t c l o n e s ( c a r r y i n g Tn5-751S) w e r e s c r e e n e d f o r s e n s i t i v i t y t o t e t r a c y c l i n e t o e n s u r e t h e y h a d l o s t p T G L I 6 6 . C h r o m o s o m a l D N A from r a n d o m l y s e l e c t e d m u t a n t s w a s d i g e s t e d c o m p l e t e l y w i t h e a c h o f Sail, BamHl o r EcoRl. A p o l y m e r a s e c h a i n r e a c t i o n ( P C R ) p r o d u c t w a s m a d e u s i n g t h e p r i m e r s N 7 2 5 - 5 a n d N 7 2 7 - 3 ( s e e t a b l e o f p r i m e r s , a p p e n d i x B ) . T h e s e p r i m e r s a r e s p e c i f i c f o r t h e k a n a m y c i n c a s s e t t e f o u n d i n t r a n s p o s o n Tn5-751S a n d t h e r e s u l t i n g P C R p r o d u c t w a s u s e d f o r S o u t h e r n h y b r i d i z a t i o n ( p r o b e 1) t o i d e n t i f y d i g e s t e d c h r o m o s o m a l f r a g m e n t s c a r r y i n g t h e t r a n s p o s o n . 2.3 Polymerase Chain Reaction (PCR) A s i n g l e P C R c y c l e c o n s i s t e d o f d e n a t u r a t i o n at 9 4 ° C f o r o n e m i n , a n n e a l i n g at 6 0 ° C f o r 1 m i n a n d e x t e n s i o n at 7 2 ° C f o r t w o m i n . T h i s w a s r e p e a t e d f o r 3 0 c y c l e s . P C R w a s p e r f o r m e d i n a P e r k i n E l m e r D N A T h e r m a l C y c l e r u s i n g o n e u n i t o f T a q D N A p o l y m e r a s e , 2 0 n g o f t e m p l a t e D N A , 2 5 0 u M d e o x y n u c l e o s i d e t r i p h o s p h a t e s , 15 p m o l e s p r i m e r s a n d 3 m M M g C b i n a t o t a l v o l u m e o f 2 0 u l p e r r e a c t i o n . F o r m a k i n g r a d i o l a b e l e d p r o b e s , t h e P C R p r o d u c t w a s g e l p u r i f i e d u s i n g P r e p - A - G e n e ® D N A p u r i f i c a t i o n K i t ( B i o - R a d ) . A f t e r a s e c o n d r o u n d o f P C R a n d g e l p u r i f i c a t i o n , t h e P C R p r o d u c t w a s r a d i o l a b e l e d as d e s c r i b e d i n s e c t i o n 2.4. R A P D - P C R w a s d o n e as p e r M a h e n t h i r a l i n g a m et a l . 1 9 96. 2.4 Southern Analysis D N A f r a g m e n t s w e r e r e s o l v e d o n a 1 . 0 % a g a r o s e g e l a n d t r a n s f e r r e d t o a p o s i t i v e l y c h a r g e d n y l o n m e m b r a n e ( N y t r a n ® P l u s , B o e h r i n g e r M a n n h e i m ) b y a l k a l i t r a n s f e r . T h e g e l w a s s o a k e d i n t h e a l k a l i n e b u f f e r (0.5 N N a O H , 1.5 M N a C l ) f o r 3 0 m i n u t e s a n d t h e n p l a c e d o n a s h e e t o f s a r a n w r a p . A p i e c e o f n y l o n m e m b r a n e e q u a l i n d i m e n s i o n t o t h e g e l w a s p l a c e d c a r e f u l l y o n t o p o f t h e g e l . A p i e c e o f W h a t m a n f i l t e r p a p e r , f o l l o w e d b y a s t a c k o f p a p e r t o w e l s 3 0 Chapter 2 - Materials and Methods ( a l l c u t t o t h e s i z e o f t h e g e l ) , a g l a s s p l a t e a n d f i n a l l y a w e i g h t w e r e p l a c e d o n t o p o f t h e g e l s o t h a t t h e D N A w a s p u l l e d b y c a p i l l a r y a c t i o n u p w a r d o n t o t h e n y l o n m e m b r a n e . T h e m e m b r a n e w a s t h e n h y b r i d i z e d w i t h r a d i o l a b e l e d D N A at 5 5 ° C , o v e r n i g h t i n h y b r i d i z a t i o n b u f f e r ( 0 . 6 X S S C (3 M N a C l , 0.3 M s o d i u m c i t r a t e ) , 5 X D e n h a r d t ' s s o l u t i o n , 0 . 5 % s o d i u m d o d e c y l s u l f a t e ( S D S ) , 0.02 m g / m l s a l m o n s p e r m D N A ) . P r o b e s w e r e l a b e l e d w i t h [ c t - 3 2 P ] - d G T P b y r a n d o m p r i m i n g f o r 3 0 m i n u t e s at 3 7 ° C u s i n g K l e n o w f r a g m e n t o f D N A p o l y m e r a s e I o f E. coli ( G i b c o B R L ) . T h e r e a c t i o n w a s set u p i n 2 0 u l t o t a l w i t h 2 0 u C i o f [ a - 3 2 P ] - d G T P , 1 u l o f 5 m M d A T P , d T T P , d C T P a n d 1 U n i t o f K l e n o w . U n i n c o r p o r a t e d n u c l e o t i d e s w e r e r e m o v e d b y u s i n g a n S-4 0 0 H R s p i n c o l u m n ( P h a r m a c i a , B i o t e c h . ) . A f t e r h y b r i d i z a t i o n , t h e m e m b r a n e s w e r e w a s h e d t w i c e u n d e r l o w s t r i n g e n c y ( 2 x S S C , 0. 1 % S D S ) o n c e at 5 5 ° C , s e c o n d l y at 2 3 ° C , f o r 15 m i n u t e s e a c h a n d t w i c e u n d e r h i g h s t r i n g e n c y 0.2x S S C , 0 . 1 % S D S f o r 5 m i n u t e s at 2 3 ° C . T h e b l o t s w e r e e x p o s e d t o X - r a y f i l m ( X - O M A T A R f i l m , E a s t m a n K o d a k Co., R o c h e s t e r , N.Y.) at - 7 0 ° C w i t h i n t e n s i f y i n g s c r e e n s . 2.5 Screening for a Mutant Susceptible to Polymyxin P u t a t i v e m u t a n t s f r o m t h e r a n d o m i n s e r t i o n l i b r a r y ( s e c t i o n 2.2) w e r e p i c k e d . 6,000 w e r e g r i d d e d i n t o 9 6 w e l l E L I S A p l a t e s c o n t a i n i n g 2 0 0 u l o f L B b r o t h a n d t r i m e t h o p r i m . T h e s e w e r e i n c u b a t e d at 3 7 ° C f o r 16 h o u r s f o l l o w e d b y r e p l i c a p l a t i n g o n e a c h o f : s u p p l e m e n t e d m i n i m a l s a l t s m e d i a w i t h o u t a n t i b i o t i c o r p l a t e s c o n t a i n i n g e i t h e r 6 0 0 o r 1 2 0 0 U n i t s o f p o l y m y x i n . C l o n e s t h a t d i d n o t g r o w o n t h e t w o c o n c e n t r a t i o n s o f p o l y m y x i n w e r e s e l e c t e d o f f t h e m a s t e r p l a t e as p o t e n t i a l p o l y m y x i n s e n s i t i v e m u t a n t s . 2.6 Determination of Minimal Inhibitory Concentrations (MICs) T h e M I C s f o r a l l a n t i b i o t i c s a n d a n t i m i c r o b i a l p e p t i d e s w e r e d o n e b y t h e s t a n d a r d m i c r o t i t r e b r o t h d i l u t i o n m e t h o d a s d e s c r i b e d i n T h e N a t i o n a l C o m m i t t e e f o r C l i n i c a l L a b o r a t o r y 31 Chapter 2 - Materials and Methods S t a n d a r d s M a n u a l . T h e s e w e r e c o n d u c t e d i n p o l y p r o p y l e n e E L I S A p l a t e s ( F a l c o n ) u s i n g M H b r o t h w i t h o u t c a t i o n s u p p l e m e n t a t i o n . T h e f i r s t w e l l c o n t a i n e d a f i n a l c o n c e n t r a t i o n o f a n t i b i o t i c at 5 2 8 u g / m l f o l l o w e d b y s e r i a l d i l u t i o n s ( b y Vi) i n e a c h a d j a c e n t w e l l d o w n t o 0.5 u g / m l . T h e l a s t w e l l c o n t a i n e d n o a n t i b i o t i c . B a c t e r i a w e r e a d d e d t o e a c h w e l l at a c o n c e n t r a t i o n o f 1 0 5 b a c t e r i a p e r m l . M i c r o t i t r e p l a t e s w e r e r e a d a f t e r 2 4 a n d 4 8 h o u r s o f i n c u b a t i o n at 3 7 ° C a n d t h e l o w e s t c o n c e n t r a t i o n o f a n t i b i o t i c w h e r e n o g r o w t h w a s o b s e r v e d ( b y e y e ) w a s d e s i g n a t e d t h e M I C f o r t h a t a n t i b i o t i c . 2.7 Isolation of Cosmid Clones 2.7.1 Construction of a Cosmid Library of B. multivorans Genomic DNA A 1 7 6 1 6 g e n o m i c D N A l i b r a r y w a s c o n s t r u c t e d i n S u p e r C o s I c o s m i d a c c o r d i n g t o m a n u f a c t u r e r i n s t r u c t i o n s ( S t r a t a g e n e , L a J o l l a , C A ) . T o o b t a i n c h r o m o s o m a l D N A f r o m B. multivorans A T C C 1 7 6 1 6 , 2.0 m l o f o v e r n i g h t c u l t u r e w a s h a r v e s t e d b y c e n t r i f u g a t i o n at 7 , 0 0 0 x g f o r 10 m i n u t e s . P e l l e t s w e r e r e s u s p e n d e d i n 2 0 0 u l G E T b u f f e r (5 m M g l u c o s e , 10 m M E D T A , 2 5 m M T r i s , p H 8.0) f o l l o w e d b y t h e a d d i t i o n o f 2.8 m l o f l y s i s b u f f e r [ 1 . 0 % S D S , 5 0 m M T r i s - C l p H 8.0, 5 0 m M E D T A p H 8.0, 6 0 u g / m l R N A s e ( P h a r m a c i a ) a n d 3 0 u g / m l P r o t e i n a s e K ( B o e h r i n g e r M a n n h e i m ) ] . T h i s w a s t u m b l e d i n a 15.0 m l s c r e w c a p p e d t u b e at 3 7 ° C f o r o n e h o u r . A f t e r t h e a d d i t i o n o f 1.0 m l o f s a t u r a t e d a m m o n i u m a c e t a t e , t h e l y s a t e w a s s h a k e n v i g o r o u s l y a n d l e f t at r o o m t e m p e r a t u r e f o r o n e h o u r t o p r e c i p i t a t e p r o t e i n s a n d p o l y s a c c h a r i d e s . T h e d e b r i s w a s r e m o v e d b y c e n t r i f u g a t i o n . T h e D N A w a s e t h a n o l p r e c i p i t a t e d a n d c h l o r o f o r m e x t r a c t e d . T h i s D N A w a s p a r t i a l l y d i g e s t e d w i t h t h e r e s t r i c t i o n e n z y m e Sau3Al t o g i v e f r a g m e n t s i n t h e s i z e r a n g e o f 3 0 t o 4 0 k b . F r a g m e n t s o f t h i s s i z e w e r e o b t a i n e d b y d i g e s t i n g 1 0 u g o f c h r o m o s o m a l D N A w i t h 0.015 U n i t s o f Sau3A f o r 5 m i n u t e s ( s e e a p p e n d i x C f o r d e t a i l s ) . T h e s e w e r e l i g a t e d t o BamHl r e s t r i c t e d S u p e r C o s 1 w i t h a r a t i o o f 3.28 i n s e r t t o 1 3 2 Chapter 2 - Materials and Methods v e c t o r at 1 6 ° C f o r 4 h o u r s . T h e l i g a t e d D N A w a s p a c k a g e d u s i n g t h e G i g a p a c k ® I I - X L p a c k a g i n g e x t r a c t a c c o r d i n g t o t h e m a n u f a c t u r e r i n s t r u c t i o n s a n d t r a n s f e c t e d i n t o t h e h o s t s t r a i n E. coli X L I - B l u e M R ( S t r a t a g e n e , L a J o l l a , C A ) . T h e h o s t s t r a i n w a s g r o w n i n L B b r o t h s u p p l e m e n t e d w i t h 0 . 2 % (w/v) m a l t o s e a n d l O m M MgCi2. R a n d o m a m p i c i l l i n r e s i s t a n t t r a n s f e c t a n t s w e r e s e l e c t e d a n d r e s t r i c t i o n d i g e s t e d w i t h Bam H I t o e n s u r e t h e c o s m i d l i b r a r y c o n t a i n e d c o s m i d c l o n e s w i t h r a n d o m i n s e r t s ( s e e A p p e n d i x D ) . C l o n e s w e r e t h e n p i c k e d a n d g r i d d e d i n t o 9 6 w e l l E L I S A p l a t e s . 2.7.2 Construction of a Probe Specific to Chromosomal DNA at the Transposon Insertion Site C h r o m o s o m a l D N A w a s i s o l a t e d f r o m m u t a n t 2 6 D 7 a n d d i g e s t e d w i t h t h e r e s t r i c t i o n e n z y m e Sail w i t h t h e i n t e n t i o n o f l e a v i n g t h e k a n a m y c i n r e s i s t a n c e c a s s e t t e i n t a c t i n t h e t r a n s p o s o n . T h e e n t i r e Sail 2 6 D 7 c h r o m o s o m a l d i g e s t w a s l i g a t e d t o Sail r e s t r i c t e d p B l u e s c r i p t II S K + . E . coli D H 5 - a w a s e l e c t r o p l a t e d u s i n g a g e n e p u l s e r ( B i o R A D ) at 1 9 0 0 V , 2 5 u F c a p a c i t a n c e a n d 2 0 0 o h m r e s i s t a n c e , r e v i v e d f o r o n e h o u r i n S O B ( a p p e n d i x A ) a n d p l a t e d o n t o L B a g a r c o n t a i n i n g k a n a m y c i n . K a n a m y c i n r e s i s t a n t c l o n e s w e r e i s o l a t e d . R e s t r i c t i o n m a p p i n g a n d S o u t h e r n a n a l y s i s ( u s i n g p r o b e 1, d e s c r i b e d i n s e c t i o n 2.2) c o n f i r m e d t h a t a c l o n e ( p B S l ) c o n t a i n e d 2.3 k b o f t r a n s p o s o n D N A as w e l l as 1.45 k b o f f l a n k i n g c h r o m o s o m a l D N A . D N A s e q u e n c e f r o m t h e f l a n k i n g c h r o m o s o m a l f r a g m e n t w a s o b t a i n e d u s i n g p r i m e r T 3 a n d t n 5 - l . T h i s c h r o m o s o m a l s e q u e n c e w a s u s e d t o d e s i g n t h e p r i m e r s S e q l R a n d S e q l L ( p r o b e 2 ) . F o r p r i m e r s e q u e n c e s s e e a p p e n d i x B . T h e s e p r i m e r s g a v e a 1.2 k b P C R p r o d u c t s p e c i f i c f o r t h e c h r o m o s o m a l D N A a d j a c e n t t o t h e t r a n s p o s o n i n s e r t i o n s i t e . 33 Chapter 2 - Materials and Methods 2.7.3 Screening of the B. multivorans 17616 Genomic Library B r i e f l y , c o l o n i e s w e r e g r o w n o v e r n i g h t at 3 7 ° C o n n y l o n m e m b r a n e ( N y t r a n ® P l u s ) p l a c e d o n t o p o f L B a g a r c o n t a i n i n g a m p i c i l l i n . T h e m e m b r a n e w a s w a s h e d b y p l a c i n g o n W h a t m a n p a p e r s o a k e d i n 0. 5 N N a O H , 1. 5 M N a C l , 0. 1 % S D S f o r 15 m i n u t e s f o l l o w e d b y n e u t r a l i z a t i o n o n 1.0 M T r i s - H C l , p H 7.5, 1.5 M N a C l f o r 5 m i n u t e s a n d 2 x S S C f o r 15 m i n u t e s . C e l l u l a r d e b r i s w a s r e m o v e d b y g e n t l y r u b b i n g t h e s u r f a c e o f t h e m e m b r a n e i m m e r s e d i n 2 x S S C b y h a n d u s i n g a c l e a n g l o v e . T h e b l o t s w e r e p r o c e s s e d as d e s c r i b e d i n s e c t i o n 2.4 u s i n g r a d i o l a b e l e d p r o b e 2. 2.8 Cloning of the BamHl, Sal I and Pst\ Fragments T h e f i v e c o s m i d s i s o l a t e d f r o m t h e 1 7 6 1 6 c h r o m o s o m a l l i b r a r y w e r e d i g e s t e d w i t h s e v e r a l d i f f e r e n t r e s t r i c t i o n e n z y m e s . S o u t h e r n a n a l y s i s ( u s i n g p r o b e 2 ) w a s d o n e t o i d e n t i f y e n z y m e f r a g m e n t s from t h e c o s m i d c l o n e s c a r r y i n g t h e D N A o f i n t e r e s t . A 4.0 k b Bam H I , a 3.8 k b Sal I a n d a 9.0 k b PstI f r a g m e n t w e r e i d e n t i f i e d a n d g e l p u r i f i e d u s i n g P r e p - A - G e n e s D N A p u r i f i c a t i o n K i t ( B i o - R a d ) . T h e s e f r a g m e n t s w e r e s u b c l o n e d i n p B l u e s c r i p t II K S + a n d s u b s e q u e n t l y p D N 1 8 ( s e e T a b l e 1). 2.9 Complementation of Mutant 26D7 p D B S l , p D B S 2 , p D B S 6 , p D B S 9 a n d p D N 1 8 w e r e t r a n s f e r r e d i n t o m u t a n t 2 6 D 7 b y t r i p a r e n t a l m a t i n g s u s i n g p R K 2 0 1 3 as t h e h e l p e r p l a s m i d . M I C s w e r e p e r f o r m e d ( d e s c r i b e d i n s e c t i o n 2.6) i n t h e a b s e n c e o f v e c t o r s e l e c t i o n . T o e n s u r e t h e v e c t o r w a s n o t l o s t d u r i n g t h e p r o c e d u r e a f t e r t h e M I C w a s d e t e r m i n e d , t h e b a c t e r i a w e r e p l a t e d i n t h e p r e s e n c e a n d a b s e n c e o f t e t r a c y c l i n e . 3 4 Chapter 2 - Materials and Methods 2.10 Nucleotide Sequencing and Analysis p B S 1 5 p l a s m i d D N A w a s p u r i f i e d b y p o l y e t h y l e n e g l y c o l ( P E G ) p r e c i p i t a t i o n a s p e r t h e P R I S M ™ R e a d y R e a c t i o n D y e D e o x y ™ T e r m i n a t o r C y c l e S e q u e n c i n g K i t P r o t o c o l . S e q u e n c i n g w a s p e r f o r m e d b y a u t o m a t e d P C R s e q u e n c i n g u s i n g a n A p p l i e d B i o s y s t e m s 3 7 7 A u t o m a t e d D N A S e q u e n c e r a n d A m p l i T a q D y e D e o x y ™ T e r m i n a t o r C y c l e S e q u e n c i n g . A l l r e a c t i o n s w e r e d o n e i n t h e p r e s e n c e o f 5 . 0 % D M S O . C o m p l e t e s e q u e n c e o f t h e c o m p l e m e n t i n g D N A w a s o b t a i n e d b y c h r o m o s o m e w a l k i n g u s i n g c u s t o m p r i m e r s ( s e e a p p e n d i x C ) . P r i m e r s w e r e s y n t h e s i z e d o n a P e r k i n - E l m e r - A B I 3 94-8 s y n t h e s i z e r . S e q u e n c e s w e r e a s s e m b l e d u s i n g L a s e r g e n e f o r W i n d o w s , D N A S T A R Inc., M a d i s o n , W I . D N A s e q u e n c i n g a n d p r i m e r s y n t h e s i s w a s p e r f o r m e d w i t h t h e a s s i s t a n c e o f t h e N u c l e i c A c i d - P r o t e i n S e r v i c e U n i t at t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a . O p e n r e a d i n g f r a m e s w e r e i d e n t i f i e d u s i n g G e n e M a r k (http://genemark.biology.gatech.edu/GeneMark). S e q u e n c e s i m i l a r i t y s e a r c h e s w e r e d o n e u s i n g t h e N a t i o n a l C e n t e r f o r B i o t e c h n o l o g y I n f o r m a t i o n B a s i c L o c a l A l i g n m e n t S e a r c h T o o l ( N C B I -B L A S T , A l t s c h u l et a l . 1 9 9 0, http://www.ncbi.nlm.nih.gov/blast). S e q u e n c e a l i g n m e n t s w e r e d o n e u s i n g M u l t i p l e S e q u e n c e A l i g n m e n t s a c c e s s e d t h r o u g h t h e B a y o r C o l l e g e o f M e d i c i n e S e a r c h L a u n c h e r w e b s i t e u s i n g C L U S T A L W (http://www.hgsc.bcm.tmc.edu/SearchLauncherA. T h e F A S T A f o r m a t f r o m t h e s e a l i g n m e n t s w a s e n t e r e d i n t o B O X S H A D E (http://www:ch.embnet.org/sofrware/BOX form.html). P r o t e i n p r e d i c t i o n s w e r e d e t e r m i n e d u s i n g P S O R T (http://www.psort.nibb.ac.jb:8800/) a n d E M B L " p r o t e i n p r e d i c t " (http://www.embl-h e i d e l b e r g . d e / s e r v i c e s / i n d e x . h t m l # 5 ) p r o g r a m s . P r o t e i n p i s a n d m o l e c u l a r w e i g h t s w e r e p r e d i c t e d u s i n g p I / M W t o o l s at h t t p : / / w w w . e x p a s y . c h / t o o l s / p i _ t o o l . h t m l . F o r t h e p h y l o g e n e t i c a n a l y s i s t h e F A S T A f o r m a t o f t h e s e q u e n c e a l i g n m e n t f r o m C L U S T A L W w e r e e n t e r e d i n t o t h e P H Y L I P p a c k a g e ( F e l s e n t e i n , 1 9 9 3 ) . 3 5 Chapter 2 - Materials and Methods 2.11 Biochemical Analysis T h e A P I R a p i d N F T s y s t e m ( P M L M i c r o b i o l o g i c a l , R i c h m o n d , B C , C A N A D A ) w a s u s e d f o l l o w e d b y g l u c o s e , m a l t o s e , l a c t o s e , m a n n i t o l , x y l o s e a n d s u c r o s e o x i d a t i o n - f e r m e n t a t i o n ( O F ) r e a c t i o n s , a n d M o e l l e r l y s i n e d e c a r b o x y l a s e t e s t ( D i f c o ) . A l l i n c u b a t i o n s f o r t h e s e t e s t s w e r e p e r f o r m e d at 3 2 ° C i n a m b i e n t a i r . O F s u g a r s w e r e i n c u b a t e d f o r 3 d a y s . K e y p o s i t i v e b i o c h e m i c a l r e a c t i o n s f o r B. multivorans c o m p l e x are : ( i ) o x i d a t i o n o f g l u c o s e a n d x y l o s e a n d at l e a s t o n e p o s i t i v e r e a c t i o n a m o n g o x i d a t i o n o f m a l t o s e , l a c t o s e , s u c r o s e a n d / o r l y s i n e d e c a r b o x y l a s e ; ( i i ) w e a k l y p o s i t i v e o x i d a s e r e a c t i o n , d e f i n e d a s a f a i n t p u r p l e c o l o r o c c u r r i n g b e t w e e n 2 0 a n d 6 0 s e c o n d s w i t h t h e P a t h o t e c c y t o c h r o m e o x i d a s e s t r i p ( R e m e l , L e n e x a , K a n s ) ( a s t r o n g o x i d a s e r e a c t i o n w a s c o n s i d e r e d a d e e p p u r p l e c o l o r d e v e l o p i n g w i t h i n 10 s e c o n d s ; ( i i i ) p-n i t r o p h e n y l - P - D - g a l a c t o p y r a n o s i d e ( P N P G ) p o s i t i v e l y a n d n o n i t r o g e n g a s p r o d u c t i o n from t h e r e d u c t i o n o f n i t r a t e ( b o t h r e a d f r o m t h e A P I R a p i d N F T s t r i p ) . 2.12 Gram Stains B a c t e r i a w e r e h e a t f i x e d t o g l a s s s l i d e s a n d s t a i n e d f o r o n e m i n u t e w i t h c r y s t a l v i o l e t , o n e m i n u t e w i t h G r a m ' s i o d i n e , 15-30 s e c o n d s w i t h a c e t o n e - e t h y l a l c o h o l (1:1), f o l l o w e d b y c o u n t e r s t a i n o f 10 s e c o n d s w i t h S a f r a n i n . S l i d e s w e r e r i n s e d w i t h t a p w a t e r b e t w e e n e a c h step. 2.13 Electron Microscopy N e g a t i v e s t a i n i n g f o r e l e c t r o n m i c r o s c o p y w a s d o n e b y D r . T e r r y B e v e r i d g e i n t h e D e p a r t m e n t o f M i c r o b i o l o g y at t h e U n i v e r s i t y o f G u e l p h , as d e s c r i b e d i n M a r t i n et a l . , 1986. 3 6 Chapter 2 - Materials and Methods 2.14 Neutrophil Killing Assay P r e v i o u s l y d e s c r i b e d b y S p e e r t et a l . 1994. R 5 s e r u m , c o m p l e m e n t c o m p o n e n t 5 d e f i c i e n t s e r u m t h a t h a s b e e n a d s o r b e d t o s h e e p r e d b l o o d c e l l s , w a s u s e d b e c a u s e B. multivorans i s s u s c e p t i b l e t o c o m p l e m e n t - m e d i a t e d l y s i s . A d s o r p t i o n t o s h e e p r e d b l o o d c e l l s w a s d o n e p r e v i o u s l y f o r a n u n r e l a t e d e x p e r i m e n t i n t h e l a b a n d h a d n o e f f e c t o n t h i s p r o c e d u r e . N e u t r o p h i l s w e r e i s o l a t e d f r o m h u m a n b l o o d b y f i r s t d i l u t i n g h e p a r i n i z e d b l o o d 1:1 w i t h 0 . 9 % s a l i n e f o l l o w e d b y s e d i m e n t a t i o n i n 6 . 0 % D e x t r a n 7 0 i n 0 . 9 % s a l i n e ( M a c r o d e x - S a l i n e P h a r m a c i a ) 3:1 D e x t r a n t o b l o o d b y v o l u m e . N e u t r o p h i l s w e r e s e d i m e n t e d o u t o f t h e r e s u l t i n g p l a s m a b y F i c o l l - H y p a q u e d e n s i t y g r a d i e n t s e d i m e n t a t i o n at 7 5 0 x g f o r 3 0 m i n u t e s . T h e p e l l e t w a s w a s h e d i n H a n k ' s B a l a n c e d S a l t S o l u t i o n w i t h 0 . 1 % g e l a t i n ( g H B S S ) at 1 6 0 x g f o r 10 m i n u t e s . T h e r e m a i n i n g r e d b l o o d c e l l s i n t h e p e l l e t w e r e l y s e d b y r e s u s p e n d i n g t h e w a s h e d p e l l e t i n 0 . 8 7 % a m m o n i u m c h l o r i d e a n d i n c u b a t e d at 3 7 ° C ( r o t a t i n g ) f o r 10 m i n u t e s . T h e c e l l s w e r e t h e n w a s h e d t w i c e i n g H B S S a n d c o u n t e d o n a h e m o c y t o m e t e r a n d r e s u s p e n d e d i n g H B S S at 5 x l 0 6 c e l l s / m l . B a c t e r i a l c u l t u r e s w e r e a d j u s t e d t o a n OD620 o f 0.600 ( o r a n o p t i c a l d e n s i t y c o r r e s p o n d i n g t o 1 0 9 b a c t e r i a / m l ) . T h e b a c t e r i a l c e l l s a r e d i l u t e d 1/27 i n g H B S S t o a c o n c e n t r a t i o n o f 2 . 5 x 1 0 b a c t e r i a / m l . N e u t r o p h i l s a n d b a c t e r i a w e r e a d d e d (1:1) t o a t o t a l v o l u m e o f 5 0 0 u l g H B S S w i t h 1 0 % R 5 s e r u m i n 1 2 x 7 5 m m p o l y p r o p y l e n e s n a p c a p t u b e s a n d t u m b l e d f o r 1 2 0 m i n u t e s at 3 7 ° C . S a m p l e s o f 5 0 u l w e r e r e m o v e d at 0, 3 0 a n d 1 2 0 m i n u t e s , n e u t r o p h i l s l y s e d f o r 10 m i n u t e s i n 0 . 1 % g e l a t i n H B S S a n d v i a b l e b a c t e r i a l c o u n t s w e r e m a d e . F o r c o n t r o l s b a c t e r i a i n t h e a b s e n c e o f n e u t r o p h i l s a n d a s t e r i l e c o n t r o l o f n e u t r o p h i l s o n l y w e r e p l a t e d o u t . 3 7 Chapter 2 - Materials and Methods 2.15 Permeabilization of whole cells of B. multivorans to 1-iV-Phenylnaphthylamine 1 - A ^ - P h e n y l n a p h t h y l a m i n e ( N P N ) a s s a y s w e r e p e r f o r m e d a s d e s c r i b e d ( M o o r e , 1986, L o h , 1984, H a n c o c k , 1 9 8 4 ) . N P N w a s d i s s o l v e d i n a c e t o n e at a c o n c e n t r a t i o n o f 5 0 0 u M . C e l l s w e r e g r o w n t o l o g p h a s e (OD600 o f 0.600) a n d w e r e r e s u s p e n d e d t o OD600 o f 0.600 i n H E P E S ( A , - 2 - h y d r o x y e t h y l p i p e r a z i n e - A ^ , - 2 - e t h a n e s u l f o n i c a c i d ) b u f f e r ( p H 7.2) c o n t a i n i n g 5 u M C C C P . C C C P s t o p s a n y e n e r g y d e p e n d e n t e f f l u x o f N P N o u t o f t h e c e l l s s o t h a t a n y i n c r e a s e i n f l u o r e s c e n c e o b s e r v e d ( o n l y s e e n i n t h e m u t a n t ) r e m a i n s at a m a x i m u m . 10 u M N P N w a s a d d e d t o 1.0 m l o f C C C P t r e a t e d B. multivorans c e l l s p l u s p o l y m y x i n i n a q u a r t z c u v e t t e . E n h a n c e m e n t i n p e r m e a b i l i t y o f c e l l s t o N P N i n t h e p r e s e n c e o f i n c r e a s i n g c o n c e n t r a t i o n s o f p o l y m y x i n w a s m e a s u r e d a s i n c r e a s e d f l u o r e s c e n c e w i t h a P e r k i n - E l m e r f l u o r e s c e n c e s p e c t r o p h o t o m e t e r a t t a c h e d t o a P e r k i n - E l m e r C o l e m a n 165 s t r i p - c h a r t r e c o r d e r . E x c i t a t i o n a n d e m i s s i o n w a v e l e n g t h s f o r N P N w e r e s e t at 3 5 0 a n d 4 2 0 nm, r e s p e c t i v e l y , w i t h s l i t w i d t h s o f 5 nm. 2.16 Dansyl-polymyxin binding assay A f l u o r e s c e n t d e r i v a t i v e o f p o l y m y x i n w a s p r e p a r e d b y c o u p l i n g 1-d i m e t h y l a m i n o n a p h t h a l e n e - 5 - s u l f o n y l c h l o r i d e ( d a n s y l c h l o r i d e ) t o p o l y m y x i n s u l f a t e t o g i v e d a n s y l - p o l y m y x i n a s d e s c r i b e d b y S c h i n d l e r a n d T e u b e r 1 9 7 5 . B i n d i n g o f d a n s y l - p o l y m y x i n t o w h o l e c e l l s o f Burkholderia multivorans w a s m o n i t o r e d b y m e a s u r i n g f l u o r e s c e n c e i n t e n s i t y w i t h a P e r k i n - E l m e r 650-1 OS f l u o r e s c e n t s p e c t r o p h o t o m e t e r s e t w i t h a n e x c i t a t i o n , w a v e - l e n g t h o f 3 4 0 n m a n d a n e m i s s i o n w a v e l e n g t h o f 4 8 5 n m as d e s c r i b e d ( M o o r e et a l , 1 9 8 4 ) . C e l l s w e r e g r o w n t o m i d l o g p h a s e (OD600 0.5), h a r v e s t e d b y c e n t r i f u g a t i o n a n d r e s u s p e n d e d i n 5 m M H E P E S p H 7.2 o f 5 u M C C C P . B a c k g r o u n d f l u o r e s c e n c e w a s m e a s u r e d b y a d d i n g 5 0 0 p m o l e s 3 8 Chapter 2 - Materials and Methods o f d a n s y - p o l y m y x i n t o 1.0 m l o f b u f f e r 5-10 t i m e s i n t h e s a m e c u v e t t e a n d t h e a s s o c i a t e d i n c r e a s e i n f l u o r e s c e n c e w a s m o n i t o r e d . T h i s w a s t h e n d o n e w i t h w h o l e B. multivorans c e l l s . 2.17 Isolation of Membrane Proteins 2.0 l i t e r s o f b a c t e r i a l c u l t u r e w e r e g r o w n i n t o l a t e l o g p h a s e t o OD600 o f 0.800. C e l l s w e r e h a r v e s t e d b y c e n t r i f u g a t i o n at 5 , 9 3 0 x g f o r 10 m i n u t e s at 4 ° C . P e l l e t s w e r e r e s u s p e n d e d i n 0.01 v o l u m e s o f 2 0 % (w/v) s u c r o s e i n 10 m M T r i s p H 8.0, 5 0 u g / m l D N a s e I ( B o e r h i n g e r M a n n h e i m ) . T h e c e l l s w e r e f r o z e n t o e n h a n c e b r e a k a g e a n d p a s s e d t w i c e at 1 5 , 0 0 0 p s i t h r o u g h a F r e n c h P r e s s u r e c e l l . D e b r i s w a s r e m o v e d b y c e n t r i f u g a t i o n at 2 . 0 0 0 x g f o r 10 m i n u t e s at 4 ° C . A t w o s t e p s u c r o s e g r a d i e n t w a s d o n e t o s e p a r a t e i n n e r a n d o u t e r m e m b r a n e s u s i n g 14 m l 7 0 % s u c r o s e , 14 m l 5 0 % s u c r o s e a n d 12 m l o f s a m p l e i n 2 0 % s u c r o s e . C e n t r i f u g a t i o n w a s d o n e at 4 ° C o v e r n i g h t at 2 3 , 0 0 0 r p m s ( 8 9 , 0 0 0 x g ) i n a S W 27/8 r o t o r . I n n e r ( b e t w e e n t h e 2 0 % a n d 5 0 % s u c r o s e s t e p s ) a n d o u t e r ( b e t w e e n t h e 5 0 % a n d 7 0 % s u c r o s e s t e p s ) m e m b r a n e b a n d s w e r e i s o l a t e d . A t l e a s t 2 v o l u m e s o f d i s t i l l e d w a t e r w a s a d d e d t o t h e c o l l e c t e d s a m p l e s t o d i l u t e o u t t h e s u c r o s e t o b e l o w 2 0 % ( s u c r o s e i n t e r f e r e s w i t h t h e p r o t e i n a s s a y ) . T h e s e s a m p l e s w e r e c e n t r i f u g e d a t 4 7 , 0 0 0 r p m ( 2 2 8 , 0 0 0 x g ) i n 6 0 T i t u b e s f o r o n e h o u r . U s i n g a s y r i n g e t h e p e l l e t s w e r e r e s u s p e n d e d i n 1.0 m l o f d i s t i l l e d w a t e r . T h e B i o R a d p r o t e i n ( B r a d f o r d ) a s s a y w a s p e r f o r m e d a c c o r d i n g t o m a n u f a c t u r e r ' s i n s t r u c t i o n s , t o d e t e r m i n e t h e p r o t e i n c o n c e n t r a t i o n i n e a c h s a m p l e . A p p r o x i m a t e l y 10 o f e a c h s a m p l e w a s a n a l y z e d b y S D S - P A G E ( s e c t i o n 2.18). G e l s w e r e s t a i n e d w i t h C o o m a s s i e b r i l l i a n t b l u e i n 2 5 % (w/v) i s o p r o p a n o l - 7 % ( v / v ) a c e t i c a c i d . 2.18 Whole Cell Lysates and Proteinase K Digestion T h i s m e t h o d w a s o r i g i n a l l y d e s c r i b e d i n H i t c h c o c k et a l . , 1 9 8 3 . B a c t e r i a w e r e g r o w n o v e r n i g h t o n s o l i d L B m e d i a c o n t a i n i n g t h e a p p r o p r i a t e a n t i b i o t i c . C e l l s w e r e h a r v e s t e d w i t h a 3 9 Chapter 2 - Materials and Methods s t e r i l e s w a b a n d s u s p e n d e d i n 10.0 m l o f c o l d H a n k ' s B a l a n c e d S a l t S o l u t i o n ( H B S S ) p H 7.2 t o a t u r b i d i t y o f O D 6 o o 0.200. 1.5 m l o f t h i s w a s m i c r o f i i g e d f o r 1.5 m i n u t e s . T h e p e l l e t w a s r e s u s p e n d e d i n 5 0 u l o f l y s i n e b u f f e r ( 2 % S D S , 4 % 2 - m e r c a p t o e t h a n o l , 1 0 % g l y c e r o l , 1 M T r i s ( p H 6.8) a n d b r o m o p h e n o l b l u e . T h e s e w e r e h e a t e d at 1 0 0 ° C f o r 10 m i n . T o d i g e s t p r o t e i n , 2 5 |iig o f p r o t e i n a s e K ( B o e h r i n g e r M a n n h e i m G m B H ) s o l u b i l i z e d i n 10 u l o f l y s i n g b u f f e r w a s a d d e d t o t h e b o i l e d l y s a t e s a n d i n c u b a t e d at 6 0 ° C f o r 6 0 m i n u t e s . 1 jLtl o f e a c h s a m p l e w a s a n a l y z e d b y S D S - P A G E ( s e c t i o n 2.19) a n d s i l v e r s t a i n e d ( s e c t i o n 2.20). 2.19 SDS-PAGE P r e p a r a t i o n s o f b o t h t h e w h o l e c e l l l y s a t e s a n d t h e m e m b r a n e p r o t e i n s w e r e a n a l y z e d b y S D S - P A G E ( L a e m m l i , 1 9 7 0 ) . A 4 . 5 % s t a c k i n g w i t h a 1 2 . 5 % s e p a r a t i n g g e l c o n t a i n i n g S D S w a s u s e d f o r b o t h p r o t e i n a n d L P S l a d d e r s e p a r a t i o n . F o r a 12.5%) g e l t h e p r o t e i n s t a n d a r d s i n c l u d e d : M y o s i n 2 0 0 - k D a , P - g a l a c t o s i d a s e 1 1 6 - k D a , B S A 9 7 . 4 - k D a , o v a l b u m i n 6 6 - k D a , c a r b o n i c a n h y d r a s e 4 5 - k D a , s o y b e a n t r y p s i n i n h i b i t o r 3 1 - k D a , l y s o z y m e 2 1 . 5 - k D a , a p r o t i n i n 6.5-kDa ( B i o - R A D ) . 2.20 Silver Staining of Lipopolysaccharide T h i s m e t h o d w a s o r i g i n a l l y d e s c r i b e d i n H i t c h c o c k et a l . , 1 9 8 3 t o s t a i n L P S l a d d e r s f r o m w h o l e c e l l l y s a t e s ( s e c t i o n 2.18). A l l g l a s s w a r e a n d e l e c t r o p h o r e s i s p l a t e s w e r e c l e a n e d w i t h d e t e r g e n t , w a r m w a t e r a n d r i n s e d t h o r o u g h l y w i t h t a p w a t e r f o l l o w e d b y m i l l i Q w a t e r ( 1 5 o h m s ) P o l y a c r y l a m i d e m i n i g e l s w e r e f i x e d o v e r n i g h t i n 2 0 0 m l 2 5 % i s o p r o p a n o l i n 7 % a c e t i c a c i d . O x i d i z a t i o n w a s p e r f o r m e d f o r 5 m i n u t e s i n 1 5 0 m l m i l l i Q w a t e r , 1.05 g p e r i o d i c a c i d , 4.0 m l i s o p r o p a n o l , a n d 7 % a c e t i c a c i d . T h e g e l s w e r e t h e n w a s h e d 8 t i m e s i n m i l l i Q w a t e r f o r 3 0 m i n u t e s e a c h t i m e . S i l v e r s t a i n i n g w a s d o n e f o r 10 m i n u t e s u s i n g 1.0 m l c o n c e n t r a t e d 4 0 Chapter 2 - Materials and Methods a m m o n i u m h y d r o x i d e , 2 8 m l 0.1 N N a O H , 115 m l m i l l i Q w a t e r a n d 5.0 m l 2 0 % s i l v e r n i t r a t e . W h e n m a k i n g t h e s i l v e r s t a i n t h e a m m o n i u m h y d r o x i d e w a s f r e s h a n d t h e s i l v e r n i t r a t e w a s a d d e d s l o w l y d r o p b y d r o p s o t h a t t h e s o l u t i o n c l e a r e d w i t h e a c h d r o p . I f a b r o w n p r e c i p i t a t e s t a r t e d t o f o r m l e s s s i l v e r n i t r a t e w a s a d d e d (e.g. 3.5 m i s w a s o f t e n s u f f i c i e n t ) . T h e g e l s w e r e t h e n w a s h e d f o u r t i m e s i n m i l l i Q w a t e r f o r 10 m i n u t e s e a c h . D e v e l o p m e n t o f t h e g e l s w a s d o n e i n a w a t e r b a t h at 2 5 ° C t o r e d u c e s t a i n i n g o f p r o t e i n s . T h i s w a s d o n e f o r 10-20 m i n u t e s i n 5 0 m g c i t r i c a c i d , 0.5 m l 3 7 . 0 % f o r m a l d e h y d e ( f r e s h ) , m i l l i Q w a t e r a d d e d t o g e t 1.0 L i t r e s . T h e r e a c t i o n w a s s t o p p e d j u s t b e f o r e t h e b a n d s w e r e t h e c o r r e c t i n t e n s i t y b e c a u s e d a r k e n i n g s t i l l o c c u r r e d i n t h e S t o p b a t h . T h e S t o p b a t h w a s a d d e d q u i c k l y a n d c o n t a i n e d 1.0 m l 7 . 0 % a c e t i c a c i d a n d 2 0 0 m l m i l l i Q w a t e r a n d a l l o w e d t o s i t f o r o n e h o u r . T h e g e l w a s t h e n s t o r e d i n m i l l i Q w a t e r . T h e s a m e s t a n d a r d s w e r e u s e d as f o r t h e m e m b r a n e p r o t e i n g e l s . 2.21 Extraction of Lipids T h i s m e t h o d w a s o r i g i n a l l y d e s c r i b e d b y K a w a i et a l , 1 9 78. 10.0 L i t e r s o f b a c t e r i a w e r e g r o w n t o l o g p h a s e (OD600 0.600). C e l l s w e r e h a r v e s t e d b y c e n t r i f u g a t i o n at 7 , 0 0 0 x g f o r 10 m i n u t e s a n d r e s u s p e n d e d i n 0 . 9 % s o d i u m c h l o r i d e s o l u t i o n . C e l l s w e r e w a s h e d t h r e e t i m e s i n 0 . 9 % s o d i u m c h l o r i d e . P e l l e t s w e r e f r o z e n o v e r n i g h t at - 7 0 ° C a n d f r e e z e d r i e d . O n e g r a m o f c e l l s w a s h o m o g e n i z e d b y s t i r r i n g f o r 3 0 m i n u t e s w i t h 2 0 m l o f 0 . 9 % s o d i u m c h l o r i d e , 5 0 m l o f m e t h a n o l a n d 2 5 m l c h l o r o f o r m ( T L C g r a d e c h e m i c a l s w e r e u s e d ) . A f t e r a n o t h e r 2 5 m i s o f c h l o r o f o r m w a s a d d e d t h e s o l u t i o n w a s s t i r r e d o v e r n i g h t . 2 5 m i s o f s o d i u m c h l o r i d e w a s t h e n a d d e d a n d s t i r r e d 10 m i n u t e s . T h e s u s p e n s i o n w a s c e n t r i f u g e d at 7 , 0 0 0 x g f o r 10 m i n u t e s t o s e p a r a t e o u t t h e c h l o r o f o r m l a y e r ( b o t t o m p h a s e ) . T h e c h l o r o f o r m l a y e r w a s r e m o v e d a n d e v a p o r a t e d u n d e r n i t r o g e n f o r o n e h o u r . T h e l i p i d e x t r a c t w a s r e s u s p e n d e d i n c h l o r o f o r m -41 Chapter 2 - Materials and Methods m e t h a n o l (2:1 v / v ) a n d t h e n p a s s e d t h r o u g h a g l a s s f i l t e r t o r e m o v e i n s o l u b l e m a t e r i a l s . T h i s f i l t r a t e w a s e v a p o r a t e d , w e i g h e d a n d r e s u s p e n d e d i n 2:1 c h l o r o f o r m - m e t h a n o l . 2.22 Thin-Layer Chromatography (TLC) T w o d i m e n s i o n a l t h i n - l a y e r c h r o m a t o g r a p h y o f t o t a l e x t r a c t a b l e c e l l u l a r l i p i d s w a s p e r f o r m e d o n a n a l u m i n u m b a c k e d T L C p l a t e ( M e r c k 5 5 5 4 ) w i t h a t w o s o l v e n t s y s t e m . I n t h e v e r t i c a l d i r e c t i o n , c h l o r o f o r m / m e t h a n o l / w a t e r (65:25:4 b y v o l u m e ) a n d i n t h e h o r i z o n t a l d i r e c t i o n c h l o r o f o r m / m e t h a n o l / a c e t i c a c i d (65:25:10 b y v o l u m e ) . T h e d e v e l o p e d p l a t e s w e r e a l l o w e d t o a i r d r y f o r 3 0 m i n u t e s . F o r v i s u a l i z a t i o n o f a l l l i p i d s , p l a t e s w e r e s p r a y e d w i t h m o l y b d o p h o s p h o r i c a c i d ( 5 . 0 % i n 9 5 % e t h a n o l ) . C o n t r o l l i p i d c a r d i o l i p i n , p h o s p h a t i d y l e t h a n o l a m i n e a n d p h o s p h a t i d y l g l y c e r o l w e r e p u r c h a s e d f r o m S i g m a C h e m i c a l Co., St. L o u i s , M o . 4 2 Chapter 3 - Results Chapter 3 Isolation and Partial Characterization of a Tn5-751S Insertion Mutant of B. multivorans with Reduced Resistance to Polymyxin B. 3.1 Objective T o i d e n t i f y a m u t a n t o f B. multivorans r e n d e r e d s e n s i t i v e t o k i l l i n g b y p o l y m y x i n B a n d c a t i o n i c p e p t i d e s , a r a n d o m t r a n s p o s o n i n s e r t i o n l i b r a r y o f t h e B. multivorans s t r a i n A T C C 1 7 6 1 6 w a s c o n s t r u c t e d u s i n g t h e t e m p e r a t u r e s e n s i t i v e p l a s m i d c a r r y i n g Tn5-751S. T h i s l i b r a r y w a s t h e n s c r e e n e d f o r m u t a n t s s u s c e p t i b l e t o p o l y m y x i n B . M u t a n t 2 6 D 7 w a s i s o l a t e d . 3.2 Transposition of Tn5- 75IS in B. multivorans W h i l e c o n s t r u c t i n g t h e r a n d o m t r a n s p o s o n i n s e r t i o n l i b r a r y , i t w a s d e t e r m i n e d t h a t m a n y s t r a i n s o f t h e B. cepacia c o m p l e x w e r e p o o r r e c i p i e n t s f o r p l a s m i d D N A . T h e r e f o r e , w h e n s u i c i d e p l a s m i d s w e r e u n s u c c e s s f u l i n d e l i v e r i n g t r a n s p o s o n s t o t h e s e s t r a i n s , i t w a s d i f f i c u l t t o k n o w w h e t h e r t h e c o n j u g a l t r a n s f e r o r t h e t r a n s p o s i t i o n h a d f a i l e d . T h e u s e o f a t e m p e r a t u r e s e n s i t i v e p l a s m i d a l l o w e d f o r s e p a r a t i o n o f t h e s e t w o s t e p s . A s e c o n d i m p o r t a n t c o n s i d e r a t i o n w i t h B. multivorans w a s i t s n a t u r a l r e s i s t a n c e t o m o s t a n t i b i o t i c s a n d s u b s t a n c e s ( i n c l u d i n g m e r c u r y ) u s e d f o r s e l e c t i o n o f g e n e t i c c a s s e t t e s . B. multivorans A T C C 1 7 6 1 6 s h o w e d s o m e s u s c e p t i b i l i t y t o t r i m e t h o p r i m a n d t e t r a c y c l i n e , a n d t h e t e m p e r a t u r e s e n s i t i v e p l a s m i d p T G L 1 6 6 s h o w n i n F i g u r e 3 h a s s h o w n p r e v i o u s s u c c e s s i n t r a n s p o s i t i o n o f Tn5-751S i n B. multivorans ( C h e n g et a l . 1 9 9 4 ) a n d t h i s s y s t e m w a s t h u s e m p l o y e d h e r e . T h i s p l a s m i d c a r r i e s a t e t r a c y c l i n e r e s i s t a n c e c a s s e t t e a n d t h e t r a n s p o s o n i s T n 5 w i t h t h e t r i m e t h o p r i m c a s s e t t e i n s e r t e d i n t o t h e Bam H I s i t e o n t h e t r a n s p o s o n . C o n j u g a l t r a n s f e r o f t h e p l a s m i d f r o m E. coli D H 5 a t o B. multivorans A T C C 1 7 6 1 6 w a s a c h i e v e d o n s o l i d m e d i a at l o w t e m p e r a t u r e ( 3 0 ° C ) w i t h a d o n o r t o r e c i p i e n t r a t i o o f 1:1. R e c i p i e n t B. multivorans c e l l s , i m m e d i a t e l y f o l l o w i n g c o n j u g a l t r a n s f e r , w e r e p l a c e d i n L B b r o t h at 4 7 ° C f o r 6 0 h o u r s . A t t e m p e r a t u r e s l o w e r t h a n 4 7 ° C B. multivorans 4 3 Chapter 3 - Results EcoR\ Sma\ Sma\ p M E 9 (carries tn5-751) BamHl BamHl I Pstl BamHl I I • 1 JSatl EcoRl Pst\ Psfl 1 1 Tn1 Trimethoprim p T G L 1 6 6 - Spe\ site within Tn5-751S replaced with Swa\ Figure 3. Transposon-donor plasmid pTGL166. pTGL166 is a pME9 derivative in which the Spe I site within Tn5-75/ was replaced by a Swa I site to make Tn-75/5 (Cheng et al. 1994). The vector pME9 is pME305::Tn5-757. The vector pME305 is a derivative of RP1 (Thomas, 1981) with a temperature sensitive mutation in the trfA gene. The primase genes (Lanka et al. 1984), the kanamycin resistance gene and IS2J of RP1 were removed by a single 12 kb deletion between the transfer gene regions tra-1 and tra-2. Transposition of TnJ-757 into pME305 is described in Rella et al. 1985. 44 Chapter 3 - Results n e v e r l o s t t h e p l a s m i d . T h i s m a y b e d u e t o t h e f a c t t h a t t h e t e m p e r a t u r e s e n s i t i v e trfA m u t a n t p l a s m i d p T G L 1 6 6 w a s s e l e c t e d f o r i n E. coli ( T h o m a s et a l , 1 9 8 1 ) a n d t e m p e r a t u r e s e n s i t i v i t y w a s p r e s u m a b l y d i f f e r e n t l y e x p r e s s e d i n B. multivorans. P u t a t i v e m u t a n t s w e r e r a n d o m l y s e l e c t e d a n d s u b j e c t e d t o P C R w i t h p r i m e r s s p e c i f i c f o r t h e k a n a m y c i n c a s s e t t e f o u n d i n Tn5-751S. T h e r e s u l t i n g P C R p r o d u c t o f 3 7 5 b p c o r r e s p o n d e d t o t h e p r e s e n c e o f t h e t r a n s p o s o n w i t h i n t h e s e l e c t e d p u t a t i v e m u t a n t c l o n e s as s h o w n i n F i g u r e 4. T h e s e s a m e m u t a n t s w e r e r e p l i c a p l a t e d o n t o t e t r a c y c l i n e a n d a b s e n c e o f g r o w t h s h o w e d t h a t t h e v e c t o r h a d b e e n l o s t . S o u t h e r n a n a l y s i s o f r a n d o m l y s e l e c t e d m u t a n t s u s i n g a p r o b e s p e c i f i c f o r t h e t r a n s p o s o n i n d i c a t e d t h a t s e l e c t e d c l o n e s f r o m t h e B. multivorans i n s e r t i o n l i b r a r y c a r r i e d Tn5-751S i n s e r t e d i n t o d i f f e r e n t s i t e s i n t h e c h r o m o s o m e . T h i s i s d e m o n s t r a t e d i n F i g u r e 5 w h e r e t h e t r a n s p o s o n s p e c i f i c p r o b e h y b r i d i z e s w i t h a d i f f e r e n t s i z e d Sal I D N A fragment f o r e a c h m u t a n t . T o e n s u r e t h a t t h e t r a n s p o s o n m u t a n t s w e r e B. multivorans, R a n d o m A m p l i f i e d P o l y m o r p h i c D N A ( R A P D ) P C R w a s d o n e o n t h e p a r e n t s t r a i n a n d r a n d o m l y s e l e c t e d m u t a n t s . R A P D - P C R u s e s r a n d o m p r i m i n g o f D N A t o c r e a t e P C R p r o d u c t s o f v a r y i n g s i z e s a n d w h e n r e s o l v e d o n a g a r o s e g e l s , r e s u l t s i n a p a t t e r n t h a t i s u n i q u e a n d s t r a i n d e p e n d a n t . T h e p a t t e r n d e p i c t e d b y B. multivorans A T C C 1 7 6 1 6 i s t r u e t o R A P D t y p e 3 0 f o u n d a m o n g s t t h e B. multivorans s t r a i n s ( g e n o m o v a r II o f t h e B. cepacia c o m p l e x ) a n d t h e r a n d o m m u t a n t s s h o w e d t h e s a m e p a t t e r n a s s h o w n i n f i g u r e 6. 3.3 Isolation of Mutant 26D7 T r a n s p o s o n m u t a n t s w e r e r e p l i c a p l a t e d o n s u p p l e m e n t e d m i n i m a l s a l t s m e d i a w i t h e i t h e r 6 0 0 o r 1 2 0 0 U n i t s o f p o l y m y x i n . M i n i m a l s a l t s m e d i a w a s u s e d b e c a u s e i t r e d u c e d t h e b a c k g r o u n d g r o w t h o f s p o n t a n e o u s l y r e s i s t a n t c l o n e s o f B. multivorans. S i x t h o u s a n d c o l o n i e s w e r e s c r e e n e d a n d c l o n e s t h a t d i d n o t g r o w o n e i t h e r 6 0 0 o r 1 2 0 0 U n i t s o f p o l y m y x i n w e r e 4 5 Chapter 3 - Results A , eg s- i ^ S ^ Randomly selected mutants from library 1 2 3 4 5 6 344bp 296bp «* *m «*» Figure 4. PCR of putative mutants using primers specific for the transposon. Tetracycline sensitive mutants were selected from the Tn5-751S insertion library of B. multivorans strain A T C C 17616. PCR was performed on D N A extracted from these strains using primers N725-5 and N727-3 (see appendix B). These primers are specific for the kanamycin cassette within the transposon Tw5-751S. A PCR product of 375 bp indicated the presence of the transposon within the chromosome of the selected putative mutant. PCR products were resolved on a 1.0% agarose gel buffer and D N A was stained with EtBr (represented by dark bands). A T C C 17616 was used as a negative control and 17616 pTGL166 as a positive control. 4 6 Chapter 3 - Results 4 i 4 5 7 8 *ty V 9 10 11 12 13 14 15 16 17 18 4kh •I Figure 5. Southern Blot Analysis of Putative Mutants Using a 32P-labeIed probe specific for the transposon. C h r o m o s o m a l D N A w a s i s o l a t e d f r o m 18 r a n d o m t r a n s p o s o n i n s e r t i o n m u t a n t s , t h e p a r e n t s t r a i n A T C C 1 7 6 1 6 , A T C C 1 7 6 1 6 p T G L 1 6 6 a n d E. coli p T G L 1 6 6 . T h e s e w e r e r e s t r i c t i o n d i g e s t e d u s i n g t h e r e s t r i c t i o n e n d o n u c l e a s e Sail. D i g e s t e d D N A w a s r e s o l v e d o n a 1 . 0 % a g a r o s e g e l a n d t r a n s f e r r e d b y S o u t h e r n b l o t t o a n y l o n m e m b r a n e . A 3 2 P - l a b e l e d p r o b e , s p e c i f i c f o r t h e k a n a m y c i n r e s i s t a n c e g e n e o n Tn5-751S, w a s u s e d t o d e t e r m i n e t h e s i z e o f t h e Sal 1 f r a g m e n t c a r r y i n g p a r t o f t h e t r a n s p o s o n i n s e r t i o n f o r e a c h m u t a n t . H y b r i d i z a t i o n w a s d o n e at h i g h s t r i n g e n c y a s d e s c r i b e d i n s e c t i o n 2.4 o f t h e M a t e r i a l s a n d M e t h o d s . 47 Chapter 3 -1 2 3 4 5 6 7 8 9 10 11 k b — Figure 6. RAPD-PCR of mutants compared with the parent B. multivorans strain ATCC 17616. R A P D - P C R w a s p e r f o r m e d o n r a n d o m l y s e l e c t e d m u t a n t c o l o n i e s f r o m t h e Tn5-751S B. multivorans A T C C 1 7 6 1 6 i n s e r t i o n l i b r a r y u s i n g t h e r a n d o m p r i m e r ( a p p e n d i x B ) . P C R p r o d u c t s w e r e r e s o l v e d o n a 1 . 0 % a g a r o s e g e l f o l l o w e d b y s t a i n i n g w i t h E t B r . T h e R A P D - P C R p r o f i l e s f o r a l l m u t a n t s t e s t e d w e r e t h e s a m e a s t h e p a r e n t s t r a i n p r o f i l e . L a n e 1, 1.0 K b l a d d e r ; L a n e 2, B. multivorans A T C C 1 7 6 1 6 ; L a n e s 3-11, r a n d o m l y s e l e c t e d m u t a n t s . Chapter 3 - Results s e l e c t e d o f f t h e m a s t e r p l a t e as p u t a t i v e p o l y m y x i n s u s c e p t i b l e m u t a n t s . T h e s e m u t a n t s w e r e t h e n t e s t e d t h r e e s e p a r a t e t i m e s i n a s t a n d a r d M I C e x p e r i m e n t t o d e t e r m i n e i f a r e d u c t i o n i n t h e M I C t o p o l y m y x i n o f a n y o f t h e m u t a n t s h a d o c c u r r e d . S o u t h e r n a n a l y s i s o f Sal I d i g e s t e d D N A o f t h e s e s e l e c t e d m u t a n t s , u s i n g t h e p r o b e s p e c i f i c f o r t h e t r a n s p o s o n k a n a m y c i n c a s s e t t e , s h o w e d t h a t a s i n g l e c o p y o f t h e t r a n s p o s o n w a s p r e s e n t i n e a c h m u t a n t c h r o m o s o m e ( F i g u r e 7). F i g u r e 7 a l s o d e m o n s t r a t e d t h a t s o m e o f t h e s e m u t a n t s m i g h t h a v e i n s e r t i o n s i n t h e s a m e s i t e o n t h e c h r o m o s o m e o r t h a t s i b l i n g s w e r e p r e s e n t i n t h e i n s e r t i o n l i b r a r y . F o r e x a m p l e , 4 6 C 8 , 4 6 D 3 a n d 4 6 G 2 h a v e t h e s a m e M I C s t o p o l y m y x i n B i n T a b l e 2 a n d t h e p r o b e h y b r i d i z e s t o a Sal I f r a g m e n t t h a t a p p e a r s t o b e a p p r o x i m a t e l y 1 2 k b i n s i z e . T h e p r o b e h y b r i d i z e s t o a 4.2 k b f r a g m e n t f o r b o t h 4 5 D 6 a n d 4 5 G 3 ( F i g u r e 7), h o w e v e r t h e i r M I C s t o p o l y m y x i n B w e r e d i f f e r e n t as s h o w n i n T a b l e 2. M u t a n t 2 6 D 7 h a d t h e l a r g e s t r e d u c t i o n i n M I C o f 8 - f o l d w h e n c o m p a r e d w i t h t h e p a r e n t s t r a i n a s s h o w n i n t a b l e 2 a n d t h e t r a n s p o s o n s p e c i f i c p r o b e h y b r i d i z e d t o a 3.75 k b Sal I f r a g m e n t ( F i g u r e 7). M u t a n t 2 6 D 7 w a s s e l e c t e d f o r f u r t h e r c h a r a c t e r i z a t i o n . 3.4 Initial Characterization of Mutant 26D7 M u t a n t 2 6 D 7 w a s s h o w n t o h a v e a r e d u c e d M I C t o p o l y m y x i n B . S u s c e p t i b i l i t y t o o t h e r c a t i o n i c p e p t i d e s w a s a l s o t e s t e d a n d t h e r e s u l t s a r e s h o w n i n t a b l e 3. T h e s e p e p t i d e s w e r e C P 2 6 a n d C E M E , t w o s y n t h e t i c c e c r o p i n - m e l i t t i n h y b r i d a - h e l i c a l p e p t i d e s , a n d c o l i s t i n ( p o l y m y x i n E ) . A s i g n i f i c a n t r e d u c t i o n i n M I C f r o m > 5 0 0 u g / m l t o 31 u g / m l w a s o b s e r v e d f o r C P 2 6 . H o w e v e r , s u r p r i s i n g l y , n o d i f f e r e n c e i n M I C w a s s e e n f o r C E M E . T h e M I C t o c o l i s t i n w a s r e d u c e d 8 - f o l d from 6 4 u,g/ml t o 8 |ag/ml. T o e n s u r e t h a t t h e m u t a t i o n i n m u t a n t 2 6 D 7 w a s n o t a g e n e r a l p e r m e a b i l i t y m u t a t i o n , M I C s o f o t h e r f a m i l i e s o f a n t i b i o t i c s w e r e a l s o d e t e r m i n e d f o r t h e p a r e n t a n d m u t a n t s t r a i n . N o d i f f e r e n c e s w e r e o b s e r v e d f o r r i f a m p i n , t e t r a c y c l i n e , e r y t h r o m y c i n , c i p r o f l o x a c i n , c e f t a z i d i m e , t i c a r c i l l i n a n d a m p i c i l l i n ( T a b l e 4 ) . 4 9 Chapter 3 - Results Figure 7. Southern blot analysis of selected putative polymyxin B sensitive mutants using a probe specific for the transposon. C h r o m o s o m a l D N A f o r B. multivorans p a r e n t s t r a i n A T C C 1 7 6 1 6 a n d t w e l v e m u t a n t s w a s d i g e s t e d w i t h t h e r e s t r i c t i o n e n z y m e Sal I. D i g e s t e d D N A w a s s e p a r a t e d b y g e l e l e c t r o p h o r e s i s a n d t r a n s f e r r e d t o a n y l o n m e m b r a n e . A 3 2 P - l a b e l e d P C R p r o d u c t s p e c i f i c f o r t h e k a n a m y c i n r e s i s t a n c e c a s s e t t e i n t h e t r a n s p o s o n , Tn5-751S, w a s u s e d f o r S o u t h e r n b l o t a n a l y s i s . T h e p r o b e h y b r i d i z e d t o a 3.75 k b Sal 1 f r a g m e n t i n m u t a n t 2 6 D 7 . 5 0 Chapter 3 - Results Table 2. MICs of polymyxin B and gentamicin for selected mutants. The parent strain ATCC 17616 and the putative mutants isolated were incubated at 37°C for 24 and 48 hours in the presence of increasing concentrations of gentamicin and polymyxin. The lowest concentration of antibiotic that inhibited growth of the strains tested at 48 hours was recorded. MICs experiments were done by broth dilution as described in the National Committee for Clinical Standards Manual. Results from one of three replicate experiments are shown. B. multivorans Gentamicin (u,g/ml) Polymyxin (u.g/ml) parent strain 256 64 26D7 112E11 310C2 45D6 45G3 46C8 46D3 46G2 47A7 47B4 47B10 47C1 128 100 200 256 256 256 256 256 256 256 >256 150 8 16 16 16 64 32 32 32 64 64 16 32 * 7640 Units per ml 51 Chapter 3 - Results Table 3. MICs of cationic antimicrobial agents for the parent B. multivorans ATCC 17616 and mutant 26D7. T h e p a r e n t s t r a i n ( A T C C 1 7 6 1 6 ) a n d m u t a n t 2 6 D 7 w e r e i n c u b a t e d f o r 2 4 h o u r s at 3 7 ° C i n t h e p r e s e n c e o f i n c r e a s i n g c o n c e n t r a t i o n s o f t h e c a t i o n i c a n t i m i c r o b i a l s : p o l y m y x i n B , c o l i s t i n , a c e c r o p i n - m e l i t t i n h y b r i d ( C E M E ) a n d a m o d i f i e d c e c r o p i n - m e l i t t i n h y b r i d ( C P 2 6 ) . M I C s w e r e d o n e b y b r o t h d i l u t i o n a s d e s c r i b e d i n t h e N a t i o n a l C o m m i t t e e f o r C l i n i c a l L a b o r a t o r y S t a n d a r d s M a n u a l . R e s u l t s f r o m o n e o f t h r e e r e p l i c a t e e x p e r i m e n t s a r e s h o w n . S t r a i n p o l y m y x i n B c o l i s t i n C P 2 6 C E M E ( u g / m l ) (Mg/ml) ( u g / m l ) ( u g / m l ) B . m u l t i v o r a n s 1 7 6 1 6 6 4 6 4 > 5 0 0 6 4 2 6 D 7 8 8 31 6 4 52 Chapter 3 - Results Table 4. MICs of different families of antibiotics for the parent B. multivorans ATCC 17616 and mutant 26D7. T h e p a r e n t s t r a i n ( A T C C 1 7 6 1 6 ) a n d m u t a n t 2 6 D 7 w e r e i n c u b a t e d at 3 7 ° C f o r 2 4 h o u r s i n t h e p r e s e n c e o f i n c r e a s i n g c o n c e n t r a t i o n s o f t h e a n t i b i o t i c i n d i c a t e d . T h e l o w e s t c o n c e n t r a t i o n o f a n t i b i o t i c t h a t i n h i b i t e d g r o w t h o f t h e s t r a i n s t e s t e d w a s r e c o r d e d . R e s u l t s i n d i c a t e d t h a t t h e t r a n s p o s o n i n s e r t i o n i n m u t a n t 2 6 D 7 h a d n o e f f e c t o n M I C s t o o t h e r a n t i b i o t i c s . M I C s w e r e d o n e b y b r o t h d i l u t i o n as d e s c r i b e d i n t h e N a t i o n a l C o m m i t t e e f o r C l i n i c a l L a b o r a t o r y S t a n d a r d s M a n u a l . R e s u l t s f r o m o n e o f t h r e e r e p l i c a t e e x p e r i m e n t s a r e s h o w n . A n t i b i o t i c t e s t e d P a r e n t (u,g/ml) M u t a n t 2 6 D 7 ( u g / m l ) R i f a m p i n 8 8 T e t r a c y c l i n e 0.25 0.25 E r y t h r o m y c i n 3 2 3 2 C i p r o f l o x a c i n 2 2 C e f t a z i d i m e 2 2 T i c a r c i l l i n 1 2 8 1 2 8 A m p i c i l l i n > 2 5 6 > 2 5 6 53 Chapter 3 - Discussion 3.5 Discussion B. multivorans and other members of the family Burkholderia are known to have an extremely high level of resistance to cationic peptides and resistance to polymyxin is often used in clinical laboratories to aid in identification of the B. cepacia complex. A transposon insertion mutant of B. multivorans strain A T C C 17616 was isolated and showed reduced resistance to polymyxin B, polymyxin E and the cecropin-melittin hybrid CP26. The mutant identified (26D7) had MICs for polymyxin B and E of 8 ug/ml, reduced by 8-fold when compared to the MIC of the parent strain as shown in table 3. In E. coli, Salmonella typhi or P. aeruginosa an MIC of 8 ug/ml to polymyxin is considered fully resistant. This suggested that B. multivorans may have more than one mechanism or phenotype that causes resistance to cationic antimicrobial agents and a second mutation would be required for mutant 26D7 to become fully susceptible to polymyxin B and colistin. Since aminoglycosides and cationic peptides enter Gram-negative bacterial cells by the same 'self promoted uptake' pathway, (Hancock et al, 1981, Nicas et al, 1983), MICs to gentamicin for the various mutants identified were also determined. None of these mutants showed a significant (i.e. greater than two-fold) reduction in MIC to gentamicin. The most likely explanation for this is the above mentioned possibility that more than one mechanism is involved in B. multivorans resistance to cationic antimicrobial agents. However, the presence of aminoglycoside modifying enzymes or ribosomes resistant to aminoglycoside binding in B. multivorans cannot be ignored, although neither has been identified to date. Cecropin and melittin are alpha helical cationic peptides that take on a helix-turn-helix configuration upon interaction with membranes. Both peptides have one amphipathic cc-helix and one hydrophobic a-helix and the order of these two helices is reversed in the two peptides. Cecropin-melittin hybrids consist of the amphipathic N-terminal a-helix of cecropin A and the 54 Chapter 3 - Discussion h y d r o p h o b i c N - t e r m i n a l a - h e l i x o f m e l i t t i n ( P i e r s et a l . 1 9 9 4 ) . T h e C E M E p e p t i d e c o n s i s t s o f r e s i d u e s 1 t o 8 o f c e c r o p i n f o l l o w e d b y r e s i d u e s 1 t o 18 o f m e l i t t i n (KWKLFKKIGIG A V L K V L T T G L P A L I S ) . C P 2 6 i s a m o d i f i e d f o r m o f C E M E ( K W K S F I K K L T S A A K K V V T T A K P L I S S ) s u c h t h a t a l l t h e h y d r o p h o b i c r e s i d u e s a r e f o u n d o n o n e f a c e o f t h e a - h e l i x a n d a l l t h e h y d r o p h i l i c r e s i d u e s a r e f o u n d o n t h e o p p o s i t e s i d e o f t h e h e l i x ( H a n c o c k et a l . 1 9 9 8 ) . T h e s e s t r u c t u r a l d i f f e r e n c e s b e t w e e n C P 2 6 a n d C E M E a r e i m p o r t a n t f o r t h e a c t i v i t y o f t h e s e p e p t i d e s o n G r a m - n e g a t i v e b a c t e r i a . D e s p i t e t h e i r s i m i l a r i t i e s i n s e q u e n c e a n d s t r u c t u r e , t h e s e p e p t i d e s h a v e d i f f e r e n t s p e c t r a o f a c t i v i t y (e.g. C P 2 6 i s G r a m -n e g a t i v e s e l e c t i v e , w h e r e a s C E M E i s m o r e b r o a d s p e c t r u m ) , d i f f e r e n t s u s c e p t i b i l i t i e s t o s a l t a n d d i f f e r e n t m e c h a n i s m s o f i n t e r a c t i o n w i t h m e m b r a n e s ( F r i e d r i c h et a l , 1 9 9 9 , S c o t t et a l , 1 9 9 9 ) . B. multivorans s t r a i n A T C C 1 7 6 1 6 w a s r e s i s t a n t t o b o t h C P 2 6 a n d C E M E a l t h o u g h , C E M E c o u l d b e c o n s i d e r e d m a r g i n a l l y m o r e a c t i v e a g a i n s t B. multivorans t h a n C P 2 6 i n t h a t a n M I C c o u l d n o t b e d e t e r m i n e d f o r t h e f o r m e r a s s h o w n i n t a b l e 3. T h e m u t a n t 2 6 D 7 s h o w e d a > 1 6 f o l d r e d u c e d M I C t o C P 2 6 a n d n o c h a n g e i n M I C t o C E M E . T h u s t h e s e q u e n c e d i f f e r e n c e s b e t w e e n C P 2 6 a n d C E M E a p p e a r e d t o b e s i g n i f i c a n t i n t e r m s o f t h e a c t i v i t y o f t h e s e t w o p e p t i d e s o n B. multivorans a n d m u t a n t 2 6 D 7 . O n c e i t h a d b e e n e s t a b l i s h e d t h a t a m u t a n t s u s c e p t i b l e t o c a t i o n i c p e p t i d e s h a d b e e n i s o l a t e d i t w a s i m p o r t a n t t o e n s u r e t h a t t h i s w a s a m u t a t i o n s p e c i f i c t o c a t i o n i c p e p t i d e a c t i v i t y a n d t h a t a g e n e r a l o u t e r m e m b r a n e p e r m e a b i l i t y b a r r i e r m u t a n t h a d n o t b e e n i s o l a t e d . D e t e r m i n a t i o n o f t h e M I C s f o r d i f f e r e n t f a m i l i e s o f a n t i b i o t i c s t h a t u s e d i f f e r e n t p a t h w a y s t o e n t e r i n t o b a c t e r i a l c e l l s w a s p e r f o r m e d a n d t h e m u t a n t a n d p a r e n t s t r a i n c o m p a r e d . R i f a m p i n a n d t h e m a c r o l i d e , e r y t h r o m y c i n s h o w e d n o c h a n g e i n M I C c o n f i r m i n g t h a t p e r m e a b i l i t y t o h y d r o p h o b i c a n t i b i o t i c s a n d e f f l u x o f t h e s e s u b s t a n c e s w a s n o t a f f e c t e d b y t h i s m u t a t i o n . T e t r a c y c l i n e , t h e p e n i c i l l i n s , t i c a r c i l l i n a n d a m p i c i l l i n , t h e c e p h a l o s p o r i n , c e f t a z i d i m e a n d t h e 55 Chapter 3 - Discussion f l u o r o q u i n o l o n e , c i p r o f l o x a c i n , a l s o s h o w e d n o c h a n g e i n M I C c o n f i r m i n g t h a t t h e h y d r o p h i l i c ( p o r i n m e d i a t e d ) u p t a k e p a t h w a y h a d n o t b e e n a f f e c t e d b y t h i s m u t a t i o n . O v e r a l l , i t w a s c o n f i r m e d t h a t a B. multivorans m u t a n t w a s c r e a t e d u s i n g t r a n s p o s o n i n s e r t i o n a l m u t a g e n e s i s . T h i s m u t a n t w a s p a r t i a l l y c h a r a c t e r i z e d a s h a v i n g a r e d u c e d r e s i s t a n c e t o s p e c i f i c c a t i o n i c p e p t i d e s a n d n o c h a n g e i n t h e M I C f o r t h e a m i n o g l y c o s i d e , g e n t a m i c i n o r o t h e r f a m i l i e s o f a n t i b i o t i c s . T h i s a b s e n c e o f c h a n g e i n M I C t o g e n t a m i c i n , e v e n t h o u g h a m i n o g l y c o s i d e s a r e k n o w n t o u s e t h e s a m e s e l f - p r o m o t e d - u p t a k e p a t h w a y a s p o l y m y x i n B , m a y b e d u e t o d i f f e r e n c e s i n t h e p r e f e r r e d i n t e r a c t i o n s i t e s o f p o l y m y x i n v e r s u s a m i n o g l y c o s i d e s ( H a n c o c k et al . , 1 9 9 4 ) . P e r h a p s t h e m u t a t i o n i n m u t a n t 2 6 D 7 h a d n o t a f f e c t e d t h e s e l f - p r o m o t e d - u p t a k e p a t h w a y b u t r a t h e r a n o t h e r s t e p i n t h e m e c h a n i s m o f a c t i o n o f c a t i o n i c p e p t i d e s . 5 6 Chapter 4 - Results Chapter 4 Identification of Putative Genes Involved in Resistance to Polymyxin 4.1 Objective T o i d e n t i f y t h e g e n e s r e s p o n s i b l e f o r t h e p h e n o t y p i c c h a n g e i n m u t a n t 2 6 D 7 , t h e D N A a d j a c e n t t o t h e t r a n s p o s o n i n s e r t i o n s i t e w a s c l o n e d f r o m t h e p a r e n t s t r a i n , B. multivorans A T C C 1 7 6 1 6 . D N A s e q u e n c e a n a l y s i s o f b o t h s t r a n d s i d e n t i f i e d f o u r c o m p l e t e o p e n r e a d i n g f r a m e s ( O R F s ) a n d o n e t r u n c a t e d O R F . 4.2 Cloning of the Locus at the Transposon Insertion Site T o i d e n t i f y t h e g e n e s r e s p o n s i b l e f o r t h e p h e n o t y p e o f m u t a n t 2 6 D 7 , i t w a s n e c e s s a r y t o c l o n e t h e D N A f r o m w i l d - t y p e B. multivorans A T C C 1 7 6 1 6 c o r r e s p o n d i n g t o t h e s i t e o f t h e T n 5 -7575* i n s e r t i o n i n m u t a n t 2 6 D 7 . A Sal I m i n i l i b r a r y o f t h e c h r o m o s o m a l D N A f r o m m u t a n t 2 6 D 7 w a s c o n s t r u c t e d . Sal I d i g e s t e d Tn5-751S s o t h a t t h e k a n a m y c i n r e s i s t a n c e c a s s e t t e f o u n d o n t h e t r a n s p o s o n w a s l e f t i n t a c t . A k a n a m y c i n r e s i s t a n t c l o n e , p B S l , w a s i s o l a t e d from t h i s l i b r a r y b y s e l e c t i o n o n a g a r p l a t e s c o n t a i n i n g k a n a m y c i n . p B S l c o n t a i n e d a 3.75 K b f r a g m e n t o f D N A . A s s h o w n i n f i g u r e 8 t h i s D N A c o r r e s p o n d e d t o 2.3 K b o f t r a n s p o s o n ( w i t h t h e k a n a m y c i n c a s s e t t e ) p l u s 1.45 K b o f c h r o m o s o m a l D N A f l a n k i n g t h e t r a n s p o s o n at t h e 5' e n d . D N A s e q u e n c e d a t a f r o m e i t h e r e n d o f t h i s 1.45 K b f r a g m e n t o f c h r o m o s o m a l D N A w a s o b t a i n e d ( d e s c r i b e d i n s e c t i o n 2.72). P C R p r i m e r s w e r e d e s i g n e d f r o m t h i s s e q u e n c e d a t a , S e q l L a n d S e q l R . T h e l o c a t i o n o f t h e p r i m i n g s i t e s f o r S e q l L a n d S e q l R a r e s h o w n i n f i g u r e 8. T h e r e s u l t i n g P C R p r o d u c t w a s l a b e l e d a n d u s e d as a r a d i o a c t i v e p r o b e t o i d e n t i f y a n y h y b r i d i z i n g c l o n e s from a B. multivorans A T C C 1 7 6 1 6 c o s m i d l i b r a r y m a d e u s i n g t h e c o s m i d v e c t o r p S u p e r C o s 1. T h e c o s m i d l i b r a r y , m a i n t a i n e d i n E. coli X L - 1 B l u e M R , w a s g r i d d e d o n t o n y l o n m e m b r a n e s a s d e s c r i b e d i n s e c t i o n 2.73. F i v e c l o n e s 5 G 6 , 5 H 1 1 , 2 F 6 , 2 F 9 a n d 1 G 3 w e r e i d e n t i f i e d b y c o l o n y h y b r i d i z a t i o n as s h o w n a n d c i r c l e d i n F i g u r e 9. T h e s a m e P C R p r o d u c t 5 7 Chapter 4 - Results Figure 8. Restriction enzyme map of the 3.75 Kb Sal I fragment from mutant 26D7 that hybridized the probe specific for the transposon. Chromosomal DNA from mutant strain 26D7 was restriction digested completely with the restriction enzyme Sal I. The entire chromosomal digest from this mutant was cloned into the Sal I cloning site on the vector pBluescript SK+ and clones were selected for kanamycin resistance. The restriction enzyme Sal I digested Tn5-751S such that the kanamycin resistance cassette was left intact. Only those clones carrying the kanamycin cassette plus flanking DNA were able to grow. The Sal I fragment that contained this piece of Tn5-75JS had the second Sal I site approximately 1.45 Kb 5' to the transposon insertion in mutant 26D7. 58 Chapter 4 - Results 5 9 Chapter 4 - Results * 5 G 6 2 F 6 2 F 9 O 0 '. •** * • " , t- i" * 1 5 H 1 1 ^ r o # 1 G 3 » • • «. — * Figure 9. Southern blot analysis of colony dot blots of the B. multivorans strain ATCC 17616 cosmid library. 8 0 0 E. coli X L - 1 B l u e M R c o s m i d c l o n e s f r o m t h e B. multivorans c o s m i d l i b r a r y w e r e s e l e c t e d a n d g r i d d e d i n t o 9 6 w e l l E L I S A p l a t e s . T h e s e c l o n e s w e r e r e p l i c a p l a t e d o n t o a n y l o n m e m b r a n e p l a c e d o n L - a g a r . A f t e r o v e r n i g h t g r o w t h , S o u t h e r n b l o t a n a l y s i s w a s p e r f o r m e d u n d e r s t r i n g e n t c o n d i t i o n s u s i n g a r a n d o m l a b e l e d 3 2 P - p r o b e s p e c i f i c f o r t h e D N A a d j a c e n t t o t h e t r a n s p o s o n i n s e r t i o n s i t e i n m u t a n t 2 6 D 7 . P o s i t i v e c l o n e s a r e c i r c l e d a n d l a b e l e d . 6 0 Chapter 4 - Results f r o m p r i m e r S e q l R a n d S e q l L w a s u s e d as a p r o b e f o r S o u t h e r n h y b r i d i z a t i o n o f t h e Bam H I , Sal I a n d Pst I d i g e s t e d c o s m i d c l o n e s i d e n t i f i e d . Pst I a n d Bam I (Sal I n o t s h o w n ) d i g e s t s o f t h e c o s m i d c l o n e s i d e n t i f i e d a r e s h o w n i n f i g u r e 1 0 A . S o u t h e r n h y b r i d i z a t i o n ( F i g u r e 1 0 B ) r e s u l t e d i n t h e i d e n t i f i c a t i o n o f 4.0 K b Bam H I , 3.8 K b Sal I a n d 8.0 K b Pst I f r a g m e n t s t h a t h y b r i d i z e d t o t h e P C R p r o d u c t m a d e from p r i m e r s S e q l R a n d S e q 2 R . A l l t h r e e f r a g m e n t s w e r e c l o n e d from t h e c o s m i d c l o n e 2 F 6 i n t o p B l u e s c r i p t II K S + . T h e c l o n e p B S 1 5 c o n t a i n e d t h e 3.6 K b Sal I fragment, p B S 4 c o n t a i n e d t h e 4.0 K b Bam H I f r a g m e n t a n d p B S P l c o n t a i n e d t h e 8.0 K b Pst I fragment. 4.3 Sequence Analysis of the region of DNA near the transposon insertion site D N A s e q u e n c e o f b o t h s t r a n d s o f D N A o f t h e 4.0 K b Bam H I a n d t h e o v e r l a p p i n g 3.6 K b Sal I fragment f o r a n e n t i r e r e g i o n o f 6,029-bp w a s d e t e r m i n e d a s d e s c r i b e d i n t h e m a t e r i a l s a n d m e t h o d s . T h e D N A s e q u e n c e o f t h i s r e g i o n i s s h o w n i n F i g u r e 11. T h e o v e r a l l G C c o n t e n t f o r t h i s r e g i o n o f D N A w a s 7 2 % . F o u r c o m p l e t e o p e n r e a d i n g f r a m e s ( O R F s ) a n d o n e t r u n c a t e d O R F w e r e i d e n t i f i e d . T w o O R F s w e r e i d e n t i f i e d o n t h e 4.0 K b Bam H I , n e i t h e r o f w h i c h w a s t h e s i t e o f t h e t r a n s p o s o n i n s e r t i o n f o u n d i n m u t a n t 2 6 D 7 . T h e f i r s t O R F ( O R F 1 ) s t a r t e d at b a s e 1 1 3 8 a n d e n d e d a t b a s e 139. O R F 1 w a s i n t h e o p p o s i n g o r i e n t a t i o n a n d 6 3 2 b a s e p a i r s u p s t r e a m o f t h e st a r t s i t e o f O R F 2 . T h e s e c o n d O R F ( O R F 2 ) s t a r t e d at b a s e 1 7 7 0 a n d e n d e d at b a s e 2 9 4 8 . T h e l a r g e s t O R F i d e n t i f i e d i n t h e i n t e r g e n i c r e g i o n b e t w e e n O R F 1 a n d O R F 2 c o n t a i n e d o n l y 1 7 7 bp. T h e r e m i g h t n o t b e a f u n c t i o n a l O R F b e t w e e n O R F 1 a n d O R F 2 a n d t h i s l a r g e i n t e r g e n i c d i s t a n c e m a y b e r e q u i r e d t o a c c o m m o d a t e f o r t h e p r e s e n c e o f t w o p r o m o t e r s . A p u t a t i v e p r o m o t e r r e g i o n f o r O R F 2 w a s i d e n t i f i e d a n d i s u n d e r l i n e d w i t h t h e - 3 5 a n d - 1 0 r e g i o n s l a b e l e d i n f i g u r e 11. S e q u e n c i n g o f t h e o v e r l a p p i n g Sal I f r a g m e n t i d e n t i f i e d t w o m o r e c o m p l e t e O R F s a n d o n e t r u n c a t e d O R F . T h e f i r s t O R F o n t h i s fragment ( O R F 3 ) s t a r t e d at b a s e 3 1 2 0 a n d e n d e d 61 Chapter 4 - Results / A b U^(r '^ B^ ^^ ^^  4kb 1.0 kb Figure 10. Identification of restriction enzyme digested cosmid fragments that hybridized to the DNA probe specific for the DNA adjacent to the transposon insertion in mutant 26D7. The five clones selected from the cosmid library were digested with Pst I (shown as the first 5 digests in panel A), Bam HI (shown as the last five lanes in panel A) and Sal I (not shown). The fragments were resolved on a 1.0% agarose gel and stained with EtBr (panel A). Southern analysis was performed using the probe specific for the D N A at the transposon insertion site (panel B). 8.0 Kb Pst I, a 4.0 Kb Bam HI and a 3.8 Kb Sal I fragments were identified as hybridizing to the probe specific to the transposon insertion site. 62 Chapter 4 - Results Figure 11. Nucleotide sequence of the 6,029 bp DNA region from B. multivorans ATCC 17616 near the transposon insertion site in mutant 26D7. T h e d e d u c e d a m i n o a c i d s e q u e n c e f o r t h e O R F s b e l o n g i n g t o t h e p u t a t i v e o p e r o n i s l i s t e d b e l o w t h e f i r s t n u c l e o t i d e o f t h e c o r r e s p o n d i n g c o d o n . T h e p o t e n t i a l r i b o s o m a l b i n d i n g r e g i o n ( u n d e r l i n e d a n d l a b e l e d as R B ) f o r t h e p u t a t i v e O R F s a r e i n d i c a t e d . O R F s a r e m a r k e d at t h e i r s t a r t c o d o n , w h i c h i s i n d i c a t e d i n , b o l d . T h e Sal I s i t e l a b e l e d w i t h i n O R F 2 s h o w s w h e r e t h e c u t s i t e i s f o r t h e Sal I f r a g m e n t t h a t d i d n o t c o m p l e m e n t t h e m u t a n t p h e n o t y p e . T h e Bam H I c u t s i t e i s l a b e l e d w i t h i n O R F 3 c o r r e s p o n d i n g t o t h e 3' e n d o f t h e Bam H I f r a g m e n t t h a t p h e n o t y p i c a l l y s u p p r e s s e d t h e m u t a n t p h e n o t y p e . T h e t r a n s p o s o n i n s e r t e d b e t w e e n t h e b o l d f a c e d C a n d T b a s e s a n d i s m a r k e d as a d o w n w a r d a r r o w . A p u t a t i v e p r o m o t e r s e q u e n c e w a s i d e n t i f i e d u p s t r e a m o f O R F 2 a n d t h e - 3 5 a n d - 1 0 r e g i o n s a r e l a b e l e d . A p o t e n t i a l r h o - i n d e p e n d e n t t e r m i n a t o r i s m a r k e d a s i n v e r t e d a r r o w s b e t w e e n O R F 2 a n d O R F 3 . T h e d e d u c e d a m i n o a c i d s e q u e n c e f o r O R F 1 i s p r e s e n t e d i n F i g u r e 12. 6 3 Chapter 4 - Results Bam HI 1 GGATCCGGACGCCTTCCACAAGGCGCTGCTCGACGGGCTCGACGCGGTGCGCACGCCGTAACGCGCAGCCGGGCGCGCGCGGCATGCGGC 91 GGCGCGCATTGGGCCGCGCGCATCTCCGCAGCGGCGCCCGGCGCCGTTGCTATCGCCGCTCGCTCGCGAGCTCGTCGCGGATCTGCGCGG 181 CCAGTTCGAACGAGCGCAGCCGCGCCGCGTGATCGTAGATCTGCGCGGTGATGATCAGCTCGTCCGCGCCCGTCTGCGCGATCCGGTCGC 271 GCAGCTTGTCGCGCACCGTGTCGCGCGAGCCGACCGCCGCGAACGACAGCGAATGCGCGACGTTCGCGAGTTCGAGTTCGTTCGCCTCGA 361 GCACGTCGACGGGCGGCGGCAGCTTGCCCGGCGTGCCGCGCCGCAGGTTGATGAACTGCTGCTGCAGCGACGTGAAGAGGCGCCGCGCCT 451 CGTCGTCGGTGTCGGCCGCGAACACGTTGACACCTACCATCGCATACGGCTTCGGCCACGCGGCCGACGGCCGGTACTGCGCGCGGTACA 540 CCTCGAGCGCCCGCATCAGATAGTCGGGCGCGAAGTGCGACGCGAACGCGAACGGCAGGCCGAGCATCGCGGCGAGCTGCGCGCTGAACA 631 GGCTCGAGCCGAGCAGCCATACGGGCACGTCGAGCCCCGCGCCGGGCACCGCACGCACGCGCTGGCCGGGCGCCGGCTCGGCGAAGTAGC 721 GCAGCAGTTCGGCGACGTCGTCGGGGAATGAATCGGCGCTGCCGATCAGGTCGCGGCGCAGCGCACGCGACGTCGTCTGATCGGTGCCCG 811 GCGCGCGGCCGAGGCCGAGATCGATGCGCCCCGGATACAGTGACGCCAGCGTGCCGAACTGCTCTGCGATCACGAGCGGCGCGTGGTTCG 901 GCAGCATGATGCCGCCGGAGCCGACGCGGATCGTCTTCGTGCCGCCCGCGACGTGGCCGATCACGACCGCGGTGGCCGCGCTCGCGATGC 991 CGGGCATGTTGTGGTGTTCGGCGAGCCAGTAACGTCGATAGCCCCAGCGCTCCGCATGTTGCGCGAGATCGAGCGTATTGCGGAAGGCCT 1080 GCGCGGCGTCGGCGCCGGCGGGAATGGGGGCGAGGTCAAGAACGGAAAACGGTGTCATCGGGTGCCTTCGAAGGGGGAGGGGCGCGGTGA - o r f l RB 1171 TGTGCGCACGCAACAGATCCGTCGATTTTGCCAAAGGGTTCCGGAACGCGCTGGCGCCGCCGCGATACCCATGCGTGTAGCAGGGCCGCG 12 61 CGCGACGTGCTCGCGATTGTTGCAAAAATCAATCCAATCGTCGTTTTGTTACGAATCTTTACCGCGATTGGCGGCGAATCTGCATCGGCA 1351 TACAAATCTGCACGAAAGGTTTGATTTTCAAGACTGCAATACGCCTGCAAAGCGCTTACGCTAACGTGAACGTGGCTGCGCCGGATCGTT 1441 TCGTCGGCTGCCCGGCTTTGCGGGAATCGCACCAGTCCGTTTCGCGCGGTGCCACGCGCGGGCGCGTGCAGACTGTTAAAATCCGGCGCC 1531 TGTCCATCTCGTGCCGAAGGCAATCATCCGATTTTTCCGCGCCGCACGGCATGCGGGAAACGCCACGCTGAACGGGGCACGGCCGCCTTG 1621 CGCGGACCGGGCCGTGCCGCCCCAAATACTGAGGTCGCTGGAACAAGGAGTCGGGTCGTGCCCGCGTTTGTCGCTGTCGGAATATCCCAT -35 1711 CATCTGCCGGGCGGCCGGATCGGCCGCCACGCGGCCGGCCTCGTCGGAGGGCGCCGCCAATGACGACGCCCGCCGTGTCGCTGCTCGACT -10 RB m t t p a v s l l d w orf2 1801 GGCTGCTGATCGCGTTCGCCGTGGCGGCGGCCGCTTACGCGCTGCTCGCGGCATTCGCGCCGCGTCCGCGCGTGCCGCGCACGGCCGCGC l l i a f a v a a a a y a l l a a f a p r p r v p r t a a r 1891 GCGACGGCGCCGAACCGGTCAGCGTGCTCAAGCCGCTGTGCGGCGCCGAGCCGCATCTGTACGAGAACCTCGCGACTTTCTGCGAACAGC d g a e p v s v l k p l c g a e p h l y e n l a t f c e q h 1980 ATCATCCGCGTTACGAGGTGCTGTTCGGCGTCGCGTCGGCCGGAGATCCCGCGATTGCGGTCGTCGAGCGGCTGCGCGCCGCGTATCCGG h p r y e v l f g v a s a g d p a i a v v e r l r a a y p d 2071 ATCGCGATATCGCGCTCGTCGTCGACGCGCGCGTGCACGGCACGAATCTGAAGGTCAGCAATCTGATCAATCTCGCCGACCGCGCGAAAT r d i a l v v d a r v h g t n l k v s n l l n l a d r a k y 2161 ACGATCGGATCGTGATCGCGGACAGCGACATCGCGGTCGCGCCCGACTACCTCGAGCGCGTGACGGCGCCGCTGGCCGACGCGTCGGTCG d r i v i a d s d i a v a p d y l e r v t a p l a d a s v g 2251 GCGTCGTCACGTGTCTGTATCATGCGCGCAGCGTCGGCGGGTTCTGGACGCGCATCGGCGCGCAGTTCGTCGACGCATGGTTCGCGCCGT v v t c l y h a r s v g g f w t r i g a q f v d a w f a p s Sal I 2341 CGGTGCGGATCACGCATCTCGGCGGCTCGAGCCGCTTCGGCTTCGGCGCGACGCTCGCGCTCACGCGCGACACGCTCGAGCGGATCGGCG v r l t h l g g s s r f g f g a t l a l t r d t l e r i g g 2431 GCCTGCGCGTGCTGAGGGACGAGCTCGCCGACGATTTCTGGCTCGCCGAGCTGCCGCGCCGCCTCGGGCGGCGCACCGTGCTGTCCGAAG l r v l r d e l a d d f w l a e l p r r l g r r t v l s e v 2521 TCGAGGTCGCAACCGACGTGATCGAGGCGTCGTTCGGCCCGCTCTGGCATCGCGAGACGCGCTGGCTGCGCACGATCCGGTCGCTGAATC e v a t d v i e a s f g p l w h r e t r w l r t i r s l n p 2611 CGGCCGGCTTCGCGTTCCTGTTCATCACGTTCACCGCGCCGTGGCTCGCGATCGCCGCGGCGCTCGCGCTGAAGCTCGACGGCACCGTCG a g f a f l f i t f t a p w l a i a a a l a l k l d g t v a 2701 CCGGCACGGTGGCCGGCGTGGCGGCGGCCGCGGGCACGTTCGGCCGGCTCGTGCTGCACGCGCGCGGCGAAGACGGCTGGCGCGCGTACT g t v a g v a a a a g t f g r l v l h a r g e d g w r a y w 2791 GGCGCGACCTGCCGCTCGTCGCGGTGCGCGACACGCTGCTCGCGCTCGAATGGCTCGTCGCCGCGTTCGGTACGCAGGTCGTCTGGCGCG r d l p l v a v r d t l l a l e w l v a a f g t q v v w r g 2881 GCGCACGCATGACGGTGGTCGGCGGCGAACGCGCGGCGGCGGCCGTGGAAGGCGGCGACGGTCGCTAGCGCCGCCTCGGACACTGGCAGG a r m t v v g g e r a a a a v e g g d g r end 2971 CGCGCGCGACCCGCGTGCCGACGGAATACGGCCCGGCAGCGGGCCCATGCGCGGCGAGCGAAGCTGCCGCCGCGCGACATCACGGAGTAG 3061 CGGGCAGCCGGTACGGCGGCCCGCCGTTTTTTTGACTGAATCGTTGAGACGAATCGAACTATGCAGGCTACCGGAGCATTCATGAAAACG < RB m q a t g a f m k t orf3 3151 CTGTTCTTGCAGGCCCCGTCCTACGACGGCTTCGACGGCGGAGCCGGCTCGCGCTATCAGGCCAAGCGTGAAATCCGTTCCTTCTGGTAT l f l q a p s y d g f d g g a g s r y q a k r e i r s f w y 3241 CCGACCTGGCTCGCGCAGCCGGCCGCGCTCGTGCCGGGCAGCCGCGTCGTCGATGCGCCGGCCGACGGCCTGTCGGTCGAGGAAACGCTG p t w l a q p a a l v p g s r v v d a p a d g l s v e e t l 64 Chapter 4 - Results 3331 AAGATCGCGAACGACTACGACCTCGTGATCATCCATACGAGCACGCCGTCGTTCCCGACCGACGCGATGTTCGCGCAGGACCTGAAGAAG k i a n d y d l v i i h t s t p s f p t d a m f a q d l k k 3421 ATGAAGCCGTCGATGCTGGTCGGCATGGTGGGTGCGAAGGTGATGGTCGATCCGCACAACTCGCTGACGGCGAGCGACGCGATCGACTTC m k p s m l v g m v g a k v m v d p h n s l t a s d a i d f 3510 GTCTGCCGCGAGGAATTCGACTACACCTGCAAGGAACTCGCCGAAGGCAAGCCGTTCGCCGAGATCAAGGGCTTGAGCTGGCGCGCGAAG v c r e e f d y t c k e l a e g k p f a e i k g l s w r a k 3601 GACGGCTCGATCGAGCATAACGAAGCGCGTCCGATCCTCGAGAACATGGACGAACTGCCGTTCGTCGCGCCCGTCTACAAGCGAGATCTG d g s i e h n e a r p i l e n m d e l p f v a p v y k r d l 3691 AAGATCGAGAACTACTTCATCGGCTATCTGAACTATCCGTACGTGTCGATCTACACGGGCCGCGGCTGCAAGTCGCGCTGCACGTTCTGC k i e n y f i g y l n y p y v s i y t g r g c k s r c t f c 3781 CTGTGGCCGCAGACGGTCAGCGGCCACCGCTATCGCACGCGCTCGGTCGAGAACGTGCTCGCGGAAGCGAAGTGGATCCGCGACAACATG l w p q t v s g h r y r t r s v e n v l a e a k w i r d n m Bam HI 3871 CCGGAAGTGAAGGAACTGATGTTCGACGACGACACCTTCACCGACGATCTGCCGCGCGCGGAAGCGATCGCCATCGGCCTCGGCAAGCTC p e v k e l m . f d d d t f t d d l p r a e a i a i g l g k l 3961 GGCATCACGTGGTCGTGCAACGCGAAGGCGAACGTGCCGTACAAGACGCTGAAGGTGATGAAGGAAAACGGGCTGCGCCTGCTGCTCGTC g i t w s c n a k a n v p y k t l k v m k e n g l r l l l v 4051 GGCTTCGAATCCGGCGACGACCAGATCCTCGTGAACATCAAGAAGGGCGTGCGCACCGATTTCGCGCGCCGCTTCAGCGCGGACTGCAAG g f e s g d d q i l v n l k k g v r t d f a r r f s a d c k 4141 AAGCTCGGCATCAAGATCCACGGCACCTTCATCCTCGGCCTGCCGGGCGAGACGAAAGAGACGATCAAGAAGACGATCCAGTACGCGAAG k l g i k i h g t f i l g l p g e t k e t i k k t i q y a k 4231 GAAATCAATCCGCACACGATCCAGGTGTCGCTCGCCGCGCCGTATCCGGGCACGACGCTCTACAAGCAGGCCATCGAAAACGGCTGGATG e i n p h t i q v s l a a p y p g t t l y k q a i e n g w m 4321 GAAGAGAACAAGACGATCAACCTCGTCAGCAAGGAAGGCGTGCAGCTCGCCGCGATCGGCTATCCGCACCTGTCGCGCGACGAGATCTAT e e n k t i n l v s k e g v q l a a i g y p h l s r d e i y 4410 CACCATCTCGAGCAGTTCTATCGCGAGTTCTACTTCCGTCCGTCGAAGATCTGGGAGATCGTGCGCGAGATGCTGACGAGCTGGGACATG h h l e q f y r e f y f r p s k i w e i v r e m l t s w d m 4501 ATGAAGCGCCGTCTGCGCGAGGGCGTCGAATTCTTCCGTTTCCTGCGCGCGCACGAGGCCTGATTCGTGACGACGCAGCGGGCGGCGCGG m k r r l r e g v e f f r f l r a h e a end v t t q r a a r RB orf4 4591 GCGCTCATCTTCACCGCGGACGACTTCGGGCTGCACGAGCGTGTGAATGCGGCGGTCGAGCGCGCGCATCGCGACGGCGTGCTGAACGCC a l i f t a d d f g l h e r v n a a v e r a h r d g v l n a 4 681 GCGAGCCTGATGGTCGGCGCGCCGGCCGCGCAGGATGCGATCGCACGCGCGCGGCGGCTGCCGTCGCTCGCGGTCGGGTTGCATCTGGTG a s l m v g a p a a q d a i a r a r r l p s l a v g l h l v 4771 CTCGCGGACGGCCCCGCGACGCTGCCCGCGCGCGAGATTCCGGCGCTGGTCGGCGCCGACGGCCGCTTCGGCGGCGCGATGGCGAAGGAC l a d g p a t l p a r e i p a l v g a d g r f g g a m a k d 4 8 61 GGCTGCCGGTTCTTCTTTCTGCCGCACGTGCGCGCGCAACTGCGGCGCGAGATCCGTGCGCAGTTCGAAGCGTTCGCGGCGACGGGCCTG g c r f f f l p h v r a q l r r e i r a q f e a f a a t g l 4950 CCGCTCGATCACGTGAACGCGCACAAGCACTTCCATCTGCATCCGACCGTGCTGTCGATGATCGTCGAGATCGGGCGCGACTACGGTCTG p l d h v n a h k h f h l h p t v l s m i v e i g r d y g l 5041 CGCGCGGTGCGGCTGCCGTACGAGACGACCGCGCCCGTGTGGCTGCGGCCGTGGATCGCGCTCGTGCGCGCGCAGCTCGACCGCGCGGGG r a v r l p y e t t a p v w l r p w i a l v r a q l d r a g 5131 CTCGCGCATAACGACTACGTGGTCGGCATCGCGCAGACGGGCCGGATGGACGAGGCCGTGCTGCTCGATGCGCTCGCGGCGCTGCCGCCC l a h n d y v v g i a q t g r m d e a v l l d a l a a l p p 5221 GGTGTCGGCGAGATCTACTGTCATCCGGCCGAAGCGGGCGACGGGCCGATCACGCCGACGATGGCCGACTACCGTCCCGCCGACGAACTC g v g e l y c h p a e a g d g p l t p t m a d y r p a d e l 5311 GACGCGCTGCTGTCGCCGCGCGTCGCCGCGGCGCTCGCGCGCGCGGGCGTCGCGACCGGCGGCTTCGCGGCCGTGTTCGGCACGCGCGAC d a l l s p r v a a a l a r a g v a t g g f a a v f g t r d 5401 GCGCAGCGCGGCGCACCCGCGGCGCGCAGACCGGGAGCGCAGCCGTCATGAGCAAATGGTTCAAATGGCTCGGCTGGCCGATCGGCATCG a q r g a p a a r r p g a q p s end RB m s k w f k w l g w p i g i g orf5 54 91 GCATCCTGCTCGCGCTGGGGCTGCACGAGGGCATCGGCGACGTGTCGCAGATGCTCGCGCGAGCCGGTTACGCGCTGCTGTGGCTCGTGC i l l a l g l h e g i g d v s q m l a r a g y a l l w l v p 6 5 Chapter 4 - Results 5581 CGTTCCACGCGCTGCCGCTGCTGCTCGATGCGTACGCATGGCACGTGCTGCTCGACCGGCGCGGCTCGCTGCCGTTTCTGTGGTGGATCG f h a l p l l l d a y a w h v l l d r r g s l p f l w w i a 5 671 CAACCGTGCGCGAGGCCGTGAACCGGCTGCTGCCGGTCGTCGGGATCGGCGGCGAGCTCGTCGGGATCCGGCTCGCGCGCTGGCAGGTCG t v r e a v n r l l p v v g i g g e l v g i r l a r w q v a 5761 CCGATGCGAGTCGCGTGACGGCTTCGGTGATCGTCGAGGTGCTGGTGACGATCGTCGTCCAGTACGCGTTTGCCGCGCTCGGCCTCGTGC d a s r v t a s v i v e v l v t i v v q y a f a a l g l v l 5851 TGCTGCTCGCGATGACGAACGCGATGGGCGGCGGCACGATCGGCGTCGCGCTGCTGCTGACGCTGCCGTTGCCCGTGCTCGGCGTCGTGC l l a m t n a m g g g t l g v a l l l t l p l p v l g v v l 5941 TGATGCGGCGCGGCGGGATCTTCCATGCGATCGAGCGCTTCGCCGGGCGGCTGCTCGGCGATTCGCACCGGCTGTTGCAAGGCGTCGAC m r r g g l f h a l e r f a g r l l g d s h r l l q g v d Sal I 66 Chapter 4 - Results at b a s e 4 5 6 3 a n d c o n t a i n e d t h e t r a n s p o s o n i n s e r t i o n i n m u t a n t 2 6 D 7 w h i c h i s m a r k e d as a d o w n w a r d a r r o w i n f i g u r e 11. A t e r m i n a t o r s e q u e n c e w a s i d e n t i f i e d i m m e d i a t e l y u p s t r e a m o f O R F 3 . T h e s e c o n d O R F ( O R F 4 ) o n t h i s f r a g m e n t s t a r t e d at b a s e 4 5 6 6 ( o n l y 3 b a s e p a i r s a r e b e t w e e n O R F 3 a n d O R F 4 ) a n d e n d e d at b a s e 5 4 5 3 . A f i f t h O R F ( O R F 5 ) s t a r t e d at b a s e 5 4 4 8 a n d n o s t o p c o d o n w a s i d e n t i f i e d b e c a u s e O R F 5 w a s t r u n c a t e d b y t h e Sal I s i t e at b a s e 6 0 2 9 . T h e c o d i n g r e g i o n s f o r O R F 1 , O R F 2 , O R F 3 a n d O R F 5 a l l u s e d A T G c o d o n s t o i n i t i a t e t r a n s c r i p t i o n , O R F 4 u s e d G T G . 4.4 Identification of Putative Genes on the Complementing Fragment 4.4.1 ORF 1 T h e n u c l e o t i d e s e q u e n c e a n d c o r r e s p o n d i n g a m i n o a c i d s e q u e n c e o f O R F 1 a r e f o u n d i n f i g u r e 12. P S O R T a n d P r e d i c t P r o t e i n p r o g r a m s s u g g e s t e d t h a t t h i s p r o t e i n m i g h t b e l o c a t e d i n t h e i n n e r m e m b r a n e , h o w e v e r f u r t h e r s t u d i e s w o u l d b e r e q u i r e d t o p r o v e t h i s . O R F 1 c o n t a i n e d 9 9 9 b a s e a n d t h e G C c o n t e n t o f t h i s s e q u e n c e w a s 7 2 % as e x p e c t e d f o r Burkholderia sp. T h e O R F 1 st a r t c o d o n i s l a b e l e d i n F i g u r e 11 a n d w a s d o w n s t r e a m o f a p u t a t i v e r i b o s o m e b i n d i n g s i t e ( R B S ) . T h i s s t a r t s i t e w a s f o u n d 6 3 2 b a s e p a i r s u p s t r e a m a n d i n t h e o p p o s i t e o r i e n t a t i o n f r o m t h e s t a r t s i t e o f t h e O R F 2 . O R F 1 e n c o d e d a p r e d i c t e d p r o t e i n o f 3 3 2 a m i n o a c i d 3 6 . 0 - k D a p r o t e i n w i t h a p r e d i c t e d p i o f 6.46. A g a p p e d - B L A S T P s e a r c h o f t h e n o n r e d u n d a n t d a t a b a s e o f N C B I w a s p e r f o r m e d u s i n g O R F 1 a s t h e q u e r y s e q u e n c e . A B L A S T P s e a r c h u s e s p r o t e i n s e q u e n c e as t h e q u e r y s e q u e n c e a n d p r o t e i n s e q u e n c e as t h e s u b j e c t s e q u e n c e . T h e t h r e e s e q u e n c e s from t h i s s e a r c h t h a t g a v e t h e l o w e s t e x p e c t v a l u e s a r e l i s t e d i n T a b l e 5. T h e l o w e s t e x p e c t v a l u e o f l e - 1 0 1 b e l o n g e d t o a h y p o t h e t i c a l p r o t e i n o f E. coli f o u n d i n t h e yhbW-mtr i n t e r g e n i c r e g i o n . T h e n e x t t w o g e n e s i d e n t i f i e d w e r e f r o m B. subtilis, o n e s i m i l a r t o a l k a n a l m o n o o x y g e n a s e a n d o n e 6 7 Chapter 4 - Results 5' -ATGACACCGTTTTCCGTTCTTGACCTCGCCCCCATTCCCGCCGGCGCCGACGCCGCGCAGGCCTTCCGCAATACGCTCGATCTCGCGCAA 9 0 m t p f s v l d l a p i p a g a d a a q a f r n t l d l a q 30 91 CATGCGGAGCGCTGGGGCTATCGACGTTACTGGCTCGCCGAACACCACAACATGCCCGGCATCGCGAGCGCGGCCACCGCGGTCGTGATC 180 3 1 h a e r w g y r r y w l a e h h n m p g i a s a a t a v v l 60 181 GGCCACGTCGCGGGCGGCACGAAGACGATCCGCGTCGGCTCCGGCGGCATCATGCTGCCGAACCACGCGCCGCTCGTGATCGCAGAGCAG 270 6 1 g h v a g g t k t i r v g s g g i m l p n h a p l v i a e q 90 271 TTCGGCACGCTGGCGTCACTGTATCCGGGGCGCATCGATCTCGGCCTCGGCCGCGCGCCGGGCACCGATCAGACGACGTCGCGTGCGCTG 3 60 9 1 f g t l a s l y p g r i d l g l g r a p g t d q t t s r a l 120 361 CGCCGCGACCTGATCGGCAGCGCCGATTCATTCCCCGACGACGTCGCCGAACTGCTGCGCTACTTCGCCGAGCCGGCGCCCGGCCAGCGC 450 121 r r d l i g s a d s f p d d v a e l l r y f a e p a p g q r 150 451 GTGCGTGCGGTGCCCGGCGCGGGGCTCGACGTGCCCGTATGGCTGCTCGGCTCGAGCCTGTTCAGCGCGCAGCTCGCCGCGATGCTCGGC 540 151 v r a v p g a g l d v p v w l l g s s l f s a q l a a m l g 180 541 CTGCCGTTCGCGTTCGCGTCGCACTTCGCGCCCGACTATCTGATGCGGGCGCTCGAGGTGTACCGCGCGCAGTACCGGCCGTCGGCCGCG 630 181 l p f a f a s h f a p d y l m r a l e v y r a q y r p s a a 210 631 TGGCCGAAGCCGTATGCGATGGTAGGTGTCAACGTGTTCGCGGCCGACACCGACGACGAGGCGCGGCGCCTCTTCACGTCGCTGCAGCAG 720 211 w p k p y a m v g v n v f a a d t d d e a r r l f t s l q q 240 721 CAGTTCATCAACCTGCGGCGCGGCACGCCGGGCAAGCTGCCGCCGCCCGTCGACGTGCTCGAGGCGAACGAACTCGAACTCGCGAACGTC 810 241 q f i n l r r g t p g k l p p p v d v l e a n e l e l a n v 270 811 GCGCATTCGCTGTCGTTCGCGGCGGTCGGCTCGCGCGACACGGTGCGCGACAAGCTGCGCGACCGGATCGCGCAGACGGGCGCGGACGAG 900 271 a h s l s f a a v g s r d t v r d k l r d r i a q t g a d e 300 901 CTGATCATCACCGCGCAGATCTACGATCACGCGGCGCGGCTGCGCTCGTTCGAACTGGCCGCGCAGATCCGCGACGAGCTCGCGAGCGAG 990 301 l i i t a q i y d h a a r l r s f e l a a q i r d e l a s e 330 991 CGGCGATAG -3' 331 r r end Figure 12. DNA and corresponding amino acid sequence for the identified B. multivorans putative ORF1. T h i s p u t a t i v e O R F 1 c o n t a i n e d 9 9 9 b a s e p a i r s , e n c o d e d f o r 3 3 2 a m i n o a c i d s , a 3 6 . 0 - k D a p r o t e i n . T h e G C c o n t e n t f o r t h i s O R F w a s 7 2 % . W i t h i n t h e 6 0 2 9 b a s e p a i r r e g i o n t h a t w a s s e q u e n c e d , t h i s g e n e w a s f o u n d t o st a r t at b a s e 1 1 3 8 a n d e n d at b a s e 139. O R F 1 w a s f o u n d 6 3 2 b a s e p a i r s u p s t r e a m o f t h e p u t a t i v e O R F 2 sta r t s i t e i n t h e o p p o s i t e o r i e n t a t i o n a s s h o w n i n f i g u r e 11. 6 8 Chapter 4 - Results Table 5. Genes and expect values from the results of a gapped-BLASTP search using B. multivorans ORF1 predicted protein sequence as the query sequence. The first three genes or hits from a gapped-BLASTP search and their respective expect values are listed. The last gene listed was the most closely related sequence that has been functionally studied. The expect values is the number of hits one can "expect" to see by chance when doing a BLAST search on a database of a particular size (in this case it is the nonredundant database of NCBI). % Similarity is the % of amino acid residues that are identical or have similar properties to the residues in the query sequence. Similar properties refer to polarity, acidic or basic properties or similar charges at the same pH and amino acid substitutions like these do not change the property of the protein. Organism Esherichia coli; Eubacteria; Proteobacter; gamma subdivision; Enterobacteriaceae; Escherichia Description hypothetical 37.1 kDa protein, ORF found in yhbW-mtr intergenic region %similarity Expect Value 75% le-101 Bacillus subtilus; Eubacteria; Similar to alkanal 75% 2e-97 firmicutes; Bacillaceae; monooxygenase Bacillus Bacillus subtilus; Eubacteria; Similar to alkanal 63% 5e-69 firmicutes; Bacillaceae; monooxygenase alpha chain Bacillus Most closely related functionally studied sequence Vibrio harveyi; Eubacteria; Alkanal monoxygenase alpha 69% 5e-06 Proteobacter; gamma chain subdivision; Vibrionaceae; Vibrio 69 Chapter 4 - Results s i m i l a r t o t h e a l p h a c h a i n o f a l k a n a l m o n o o x y g e n a s e . T h e s e w e r e t w o d i f f e r e n t p r o t e i n s e q u e n c e s . N o n e o f t h e s e g e n e s h a v e b e e n s t u d i e d f u n c t i o n a l l y a n d w e r e n a m e d d u e t o t h e i r h i g h s i m i l a r i t y w i t h a l k a n a l m o n o o x y g e n a s e s f r o m o t h e r s p e c i e s . T h e g e n e w i t h t h e h i g h e s t s i m i l a r i t y t o t h e O R F T o f B. multivorans t h a t h a s b e e n f u n c t i o n a l l y s t u d i e d w a s t h e a l p h a c h a i n o f a l k a n a l m o n o o x y g e n a s e e n c o d e d b y t h e luxA g e n e o f t h e lux o p e r o n f o u n d i n Vibrio harveyi. T h e s i m i l a r i t y b e t w e e n t h e s e t w o p r o t e i n s e q u e n c e s g a v e a n e x p e c t v a l u e o f 5e-6 a n d s i m i l a r i t y o f 4 1 % f r o m a m i n o a c i d s 2 3 t o 182 f o r O R F 1 a n d 2 2 t o 1 8 9 f o r L u x A , t h e N t e r m i n a l h a l f o f t h e p r o t e i n . T h e lux o p e r o n i s r e s p o n s i b l e f o r p r o d u c t i o n o f l u c i f e r a s e i n b i o l u m i n e s c e n t o r g a n i s m . T h e s e q u e n c e a l i g n m e n t i n f i g u r e 13 s h o w s t h a t t h e s i m i l a r i t i e s l i s t e d i n t a b l e 5 w e r e a c r o s s t h e e n t i r e p r o t e i n s e q u e n c e s . 4.4.2 ORF 2 T h e n u c l e o t i d e s e q u e n c e a n d c o r r e s p o n d i n g a m i n o a c i d s e q u e n c e o f O R F 2 a r e f o u n d i n f i g u r e 11. T h e s e c o n d Sal I s i t e c o r r e s p o n d i n g t o t h e 5' e n d o f t h e Sal I f r a g m e n t , as l a b e l e d i n f i g u r e 11, w a s f o u n d w i t h i n t h e O R F 2 c o d i n g r e g i o n . O R F 2 c o n t a i n e d 1 1 7 9 a n d t h e G C c o n t e n t f o r t h i s s e q u e n c e w a s 7 2 % . T h e s t a r t c o d o n i s l a b e l e d i n F i g u r e 11 d o w n s t r e a m o f a p u t a t i v e R B S . A s t o p c o d o n o f t h i s O R F w a s f o u n d 173 b a s e p a i r s u p s t r e a m o f O R F 3 . O R F 2 e n c o d e d f o r a p r e d i c t e d p r o t e i n o f 3 9 2 a m i n o a c i d s , a 4 2 . 3 - k D a p r o t e i n w i t h a p r e d i c t e d p i o f 7.11. A g a p p e d - B L A S T P s e a r c h o f t h e N C B I n o n r e d u n d a n t s e q u e n c e d a t a b a s e w a s p e r f o r m e d u s i n g t h e a m i n o a c i d s e q u e n c e o f O R F 2 a s t h e q u e r y s e q u e n c e . T h e f i v e p r o t e i n s t h a t g a v e t h e l o w e s t e x p e c t v a l u e s i n t h e r e s u l t s o f t h i s B L A S T P s e a r c h a r e l i s t e d i n T a b l e 6. T h e l o w e s t e x p e c t v a l u e o f 2 e - 3 2 b e l o n g e d t o a p u t a t i v e c e r a m i d e g l u c o s y l t r a n s f e r a s e o f Synechocystis sp. T h e t h r e e o t h e r p r o t e i n s i n t h i s t a b l e c o r r e s p o n d t o t h e c e r a m i d e g l u c o s y l t r a n s f e r a s e s o f t h e m o u s e , rat a n d h u m a n T h e l a s t p r o t e i n s e q u e n c e l i s t e d i n T a b l e 6 i s 7 0 Chapter 4 - Results B m u l t i o r f 1 E c o l i h y p Bsubalkan Bsubccchain B m u l t i o r f 1 E c o l i h y p Bsubalkan Bsubachain 54 58 61 56 \TAVVIGfiVAGlTKTIRVGSGGIMLPNri|jri Jvxnc J v^^J-^M^^i^"- L" j-'"-"^""'-'-' j-^ \T|VLI _ i • * : _ / [ G ) ILPNHSPLVIAEQFGTLSTLYPGRIDLGLGRAPGJJD TAVLIGHIAG1T™IRVGSGGIMLPNHS|LVIAEQFGTLETLYPGRIDLGLGRAPGTD ""HLL|AITKSLRVGSGGIMLPNHSPLSVAEQFGTLETLYP_R|DL|LGRAPGTD IPLVIAEQFGTLBSLYPGRIDLGLGRAPGTD B m u l t i o r f 1 E c o l i h y p Bsubalkan Bsubachain B m u l t i o r f 1 173 E c o l i h y p 177 ggQ Bsubalkan 180 | _ B E Bsubachain 175 iSraiiK LGLPFAFA LGLPFAFA LGLPFAFA LGLPIAFI B m u l t i o r f 1 E c o l i h y p Bsubalkan Bsubachain B m u l t i o r f 1 E c o l i h y p Bsubalkan Bsubachain WOBWMASEMR— I_C_WKNR LK QJV__JFKK_MEQ L Figure 13. Amino acid sequence alignment for the ORF1 predicted protein and similar sequences identified in the gapped-BLASTP search using the ORF1 predicted protein sequence as the query sequence. Protein sequence alignment for B. multivorans ORF1 (Bmultiorf 1) and the E. coli 37.1 kDa hypothetical protein found in the Y h b W - M t r intergenic region (Ecolihyp), B. subtilis similar to alkanal monooxygenases (Bsubalkan) and the B. subtilis similar to alkanal monooxygenase alpha chain sequence (Bsubachain). Shading is black for identical amino acids and grey is for amino acids with similar properties. Similar properties refers to polarity, acidic or basic properties or similar charges at the same p H such that amino acid substitutions like these do not change the property of the protein. 71 Chapter 4 - Results Table 6. Genes and expect values from the results of a gapped-BLASTP search using B. multivorans putative ORF2 as the query sequence. T h e first f o u r g e n e s o r h i t s f r o m a g a p p e d -B L A S T P s e a r c h u s i n g O R F 2 as t h e q u e r y s e q u e n c e a r e l i s t e d h e r e w i t h t h e i r r e s p e c t i v e e x p e c t v a l u e s a n d p e r c e n t s i m i l a r i t y . E x p e c t v a l u e i s t h e n u m b e r o f h i t s o n e c a n " e x p e c t " t o s e e b y c h a n c e w h e n d o i n g a B L A S T s e a r c h o n a d a t a b a s e o f a p a r t i c u l a r s i z e ( i n t h i s c a s e i t i s t h e n o n r e d u n d a n t d a t a b a s e o f N C B I ) . % S i m i l a r i t y i s t h e % o f a m i n o a c i d s r e s i d u e s t h a t a r e i d e n t i c a l o r h a v e s i m i l a r p r o p e r t i e s t o t h e r e s i d u e s i n t h e q u e r y s e q u e n c e . T h e u n f i n i s h e d f r a g m e n t f r o m T. ferrooxidans w a s i d e n t i f i e d i n a T B L A S T N s e a r c h o f t h e u n f i n i s h e d g e n o m e s . O r g a n i s m D e s c r i p t i o n % s i m i l a r i t y E x p e c t V a l u e Nonredundant database Synechocystis sp.; B a c t e r i a ; c e r a m i d e g l u c o s y l t r a n s f e r a s e 4 5 % 2 e - 3 2 Cyanobacteria; Chroococcales; Synechocystis Mus musculus c e r a m i d e g l u c o s y l t r a n s f e r a s e 4 8 % 4 e - 2 2 Rattus norvegicus c e r a m i d e g l u c o s y l t r a n s f e r a s e 4 9 % 9e-22 Homo sapiens c e r a m i d e g l u c o s y l t r a n s f e r a s e 4 8 % l e - 2 1 Incomplete genome databases Thiobacillus ferrooxidans; u n f i n i s h e d f r a g m e n t 6 1 % 6e-54 B a c t e r i a ; Proteobacter; g a m m a s u b d i v i s i o n ; Thiobacillus 7 2 Chapter 4 - Results f r o m a n u n f i n i s h e d f r a g m e n t o f Thiobacillus ferrooxidans w h i c h w a s i d e n t i f i e d i n a T B L A S T N s e a r c h o f t h e u n f i n i s h e d g e n o m e s d a t a b a s e a n d s h o w e d 6 1 % s i m i l a r i t y t o O R F 2 o f B. multivorans. G a p p e d - B L A S T P s e a r c h e s o f t h e c e r a m i d e g l u c o s y l t r a n s f e r a s e s l i s t e d i n T a b l e 6 g a v e t h e s a m e r e s u l t s a s s e e n i n T a b l e 6 s u g g e s t i n g t h a t t h e s e q u e n c e s i m i l a r i t i e s i n t h i s t a b l e a r e r e a l . T h e a l i g n m e n t s f o r t h e s e g e n e s a r e f o u n d i n F i g u r e 14. T h e h u m a n a n d r a t p r o t e i n s e q u e n c e s w e r e n o t l i s t e d i n t h e a l i g n m e n t b e c a u s e t h e y a r e 9 6 % s i m i l a r t o t h e m o u s e s e q u e n c e . T h e s e q u e n c e s i m i l a r i t y f o r t h e p r o t e i n s e q u e n c e s w a s o b s e r v e d t h r o u g h o u t t h e e n t i r e p r o t e i n . M a n y o t h e r p r o t e i n s w e r e i d e n t i f i e d i n t h i s B L A S T P s e a r c h , a n d , a l t h o u g h t h e e x p e c t v a l u e s w e r e r e l a t i v e l y h i g h r a n g i n g f r o m 5e-6 t o 5.7, t h e y a l l a p p e a r e d t o h a v e a g l u c o s y l t r a n s f e r a s e f u n c t i o n ( d a t a n o t s h o w n ) . O n e o f t h e s e p r o t e i n s w a s P. aeruginosa M i g A ( E x p e c t v a l u e 0.66) k n o w n t o e n c o d e a g l u c o s y l t r a n s f e r a s e ( W a n g , J. et a l . , 1 9 9 6 ) . B e c a u s e o f t h e s i m i l a r i t y o f B. multivorans O R F 2 w i t h t h e m a m m a l i a n c e r a m i d e g l u c o s y l t r a n s f e r a s e s a p h y l o g e n e t i c t r e e w a s c o n s t r u c t e d u s i n g t h e n e i g h b o r j o i n i n g d i s t a n c e m a t r i x m e t h o d o f t h e P H Y L I P p a c k a g e ( F e l s e n t e i n , J., 1 9 9 3 ) i n c o n s u l t a t i o n w i t h D r . F i o n a B r i n k m a n , U n i v e r s i t y o f B r i t i s h C o l u m b i a a n d i s s h o w n i n f i g u r e 15. T h e t r e e s h o w s t h e r e l a t e d n e s s o f t h e d i f f e r e n t p r o t e i n s e q u e n c e s as p r o p o r t i o n a l t o t h e l e n g t h o f t h e t r e e b r a n c h e s . T h e c e r a m i d e g l u c o s y l t r a n s f e r a s e o f h u m a n a n d m o u s e a r e t h e o n l y f u n c t i o n a l l y s t u d i e d c e r a m i d e g l u c o s y l t r a n s f e r a s e s e q u e n c e s a v a i l a b l e . T h e o n l y c o m p l e t e g e n o m e s e q u e n c e a v a i l a b l e f r o m a m u l t i c e l l u l a r o r g a n i s m (Caenorhabditis elegans), h a d t h r e e c o p i e s o f g e n e s d e s c r i b e d a s p u t a t i v e c e r a m i d e g l u c o s y l t r a n s f e r a s e s . Synechocystis sp. i s a c y a n o b a c t e r i a a n d t h e c o m p l e t e g e n o m e s e q u e n c e w a s a l s o a v a i l a b l e f o r t h i s o r g a n i s m s h o w e d t w o c o p i e s o f g e n e s w i t h s e q u e n c e s i m i l a r i t y t o c e r a m i d e g l u c o s y l t r a n s f e r a s e ( h t t p : / / w w w . k a z u s a . o r . j p : 8 0 8 0 / c y a n o ) . Myxococcus xanthus, a m e m b e r o f t h e d e l t a s u b d i v i s i o n o f Proteobacteria a n d Lactococcus 7 3 Chapter 4 - Results B m u l t i o r f 2 T f e r r o r f s y n e c e r e B m u l t i o r f 2 T f e r r o r f s y n e c e r e B m u l t i o r f 2 T f e r r o r f s y n e c e r e m o u s e c e r 8cBjTPg|LTQA SHG IIPYVADR B m u l t i o r f 2 1 7 3 GG T f e r r o r f 1 7 8 A T s y n e c e r e 1 7 5 ST m o u s e c e r 1 7 8 B m u l t i o r f 2 T f e r r o r f s y n e c e r e m o u s e c e r IGPLiHfflETg L D N | QNSGSYSIfj lY^DQNjiYLARj|LE J L R I N | L 3 A T I | C E P | j'T | P IIRA S E C B m u l t i o r f 2 T f e r r o r f s y n e c e r e m o u s e c e r 2 9 3 2 9 8 2 9 5 2 9 7 FVASLl JpWMGBWgGfflP rFCEwSEMAWi I F R H D I < J R A § - -WgDjj — SFGB - E T — iGVQGHTLCjjSKjjDHAVAWI B m u l t i o r f 2 T f e r r o r f s y n e c e r e m o u s e c e r Figure 14. Amino acid sequence alignment for the ORF2 predicted protein sequence and similar sequences identified in the gapped-BLASTP search using the ORF2 predicted protein sequence as the query sequence. Protein sequence alignment for B. multivorans putative O R F 2 (Bmultiorf?) and the Thiobacillus ferrooxidans predicted amino acid sequence from the identified O R F (Tferrorf), Synechocystis sp. putative ceramide glucosyltransferase (synecere) and the mouse ceramide glucosyltransferase (mousecer). The rat and human sequences were not presented because they are 96% similar to the mouse sequence. Shading is black for identical amino acids and gray for amino acids with similar properties. Similar properties refers to polarity, acidic or basic properties or similar charges at the same p H such that amino acid substitutions like these do not change the property of the protein. 74 Chapter 4 - Results Figure 15. Phylogenetic tree of protein sequences similar to the ORF2 predicted protein sequence of B. multivorans. T h i s t r e e w a s c o n s t r u c t e d u s i n g t h e n e i g h b o r j o i n i n g d i s t a n c e m a t r i x m e t h o d ( F e l s e n t e i n , 1 9 9 3 ) . T h e l e n g t h o f t h e t r e e a r m s i s p r o p o r t i o n a l t o t h e e v o l u t i o n a r y d i s t a n c e o f t h e p r o t e i n s e q u e n c e s . T h i o C e r , T. ferrooxidans p u t a t i v e O R F ; M o u s e C e r , m o u s e c e r a m i d e g l u c o s y l t r a n s f e r a s e ; H u m a n C e r , h u m a n c e r a m i d e g l u c o s y l t r a n s f e r a s e ; C e l e g a n s l , 2 a n d 3; C. elegans h y p o t h e t i c a l c e r a m i d e g l u c o s y l t r a n s f e r a s e s c o p y 1, 2 a n d 3, r e s p e c t i v e l y ; B m u l t i o r f 2 , B. multivorans O R F 2 p r e d i c t e d p r o t e i n s e q u e n c e ; M x a n t h u s , M. xanthus h y p o t h e t i c a l p r o t e i n ; S y n e c h o l C e r a n d S y n e c h o 2 C e r , Synechocystis sp. p u t a t i v e c e r a m i d e g l u c o s y l t r a n s f e r a s e c o p i e s 1 a n d 2, r e s p e c t i v e l y ; L l a c t i s , L. lactis h y p o t h e t i c a l p r o t e i n ; R h i z N o d C , R. legunimosarum N o d C p r o t e i n . B o o t s t r a p a n a l y s i s f o r e a c h b r a n c h i s i n d i c a t e d as t h e n u m b e r o f t i m e s o u t o f 1 0 0 t h a t t h i s b r a n c h o c c u r r e d w h e n t h i s t r e e w a s c o n s t r u c t e d 1 0 0 t i m e s u s i n g a r a n d o m i n p u t o r d e r o f s e q u e n c e s . A p p e n d i x E s h o w s t h e p r o t e i n s e q u e n c e a l i g n m e n t s f o r t h e p r o t e i n s p r e s e n t e d i n t h i s t r e e a n d t h e r e s u l t s o f t h e b o o t s t r a p a n a l y s i s a r e a l s o p r e s e n t e d t h e r e i n . 7 5 Chapter 4 - Results 76 Chapter 4 - Results lactus, a G r a m - p o s i t i v e o r g a n i s m , b o t h h a d h y p o t h e t i c a l p r o t e i n s w i t h s i m i l a r i t y t o O R F 2 t h a t w e r e i n c l u d e d i n t h i s t r e e . Rhizobium leguminosarum ( a m e m b e r o f t h e a l p h a s u b d i v i s i o n o f Proteobacter) N o d C p r o t e i n w a s a l s o i n c l u d e d . T h e d i s t a n c e b e t w e e n t h e B. multivorans a n d h u m a n / m o u s e s e q u e n c e s w a s l e s s t h a n t h e d i s t a n c e b e t w e e n t h e B. multivorans a n d C. elegans s e q u e n c e s a n d t h e s e d i s t a n c e s i n t h i s t r e e w e r e t h e r e f o r e n o t c o n s i s t e n t w i t h r e c e n t h o r i z o n t a l g e n e t r a n s f e r b e t w e e n m a m m a l s a n d B. cepacia. A p p e n d i x E s h o w s t h e s e q u e n c e a l i g n m e n t s f o r a l l t h e p r o t e i n s s h o w n i n t h e p h y l o g e n e t i c t r e e o f f i g u r e 15. 4.4.3 ORF 3 T h e n u c l e o t i d e s e q u e n c e a n d p r e d i c t e d a m i n o a c i d s e q u e n c e o f O R F 3 a r e f o u n d i n f i g u r e 11. O R F 3 c o n t a i n e d 1 4 4 3 b a s e s a n d t h e G C c o n t e n t f o r t h i s g e n e w a s l o w f o r B. multivorans at 6 2 % . O R F 3 w a s f o u n d 173 b a s e p a i r s d o w n s t r e a m o f O R F 2 a n d t h e s t o p c o d o n o f t h i s O R F 3 w a s 3 b a s e p a i r s u p s t r e a m o f t h e O R F 4 . T h e t r a n s p o s o n , d e p i c t e d i n F i g u r e 11 as a d o w n w a r d a r r o w , w a s i n s e r t e d i n t h e m u t a n t g e n o m e at a l o c a t i o n t h a t c o r r e s p o n d e d t o b e t w e e n b a s e s 3 7 5 9 a n d 3 7 6 0 o n t h i s D N A f r a g m e n t . T h e Bam H I s i t e t h a t c o r r e s p o n d e d t o t h e 3' e n d o f t h e Bam H I f r a g m e n t i s i n d i c a t e d i n u n d e r l i n e d b o l d c h a r a c t e r s . O R F 3 e n c o d e d f o r a p r e d i c t e d p r o t e i n o f 4 8 1 a m i n o a c i d s , a 5 4 . 0 - k D a p r o t e i n w i t h a p r e d i c t e d p i o f 7.59. P S O R T p r o g r a m s h o w e d t h a t t h e m o s t l i k e l y l o c a t i o n f o r B. multivorans O R F 3 p r o t e i n w a s c y t o p l a s m i c h o w e v e r , f u r t h e r s t u d i e s w o u l d b e r e q u i r e d t o c o n f i r m t h i s . T h e p r e d i c t e d p r o t e i n s e q u e n c e o f O R F 3 w a s r u n i n a g a p p e d - B L A S T P s e a r c h a n d t h e p r o t e i n s e q u e n c e s w i t h t h e l o w e s t e x p e c t v a l u e s a r e l i s t e d i n T a b l e 7. T h e f i r s t t h r e e g e n e s l i s t e d i n t a b l e 7 a r e f r o m Pyrococcus horikoshii, Methanobacterium thermoautotrphicum a n d Thermotoga maritama h a v e n o t b e e n s t u d i e d f u n c t i o n a l l y a n d a r e n a m e d as a r e s u l t o f s i m i l a r i t y w i t h o t h e r M g - p r o t o p h o r p h y r i n I X m o n o m e t h y l e s t e r o x i d a t i v e c y c l a s e s . G a p p e d - B L A S T P s e a r c h e s o f 7 7 Chapter 4 - Results Table 7. Genes and expect values from the results of a gapped-BLASTP search using the B. multivorans ORF3 predicted protein sequence as the query sequence. T h e f i r s t t h r e e p r o t e i n s a r e h i t s from a g a p p e d - B L A S T P s e a r c h a n d t h e i r r e s p e c t i v e e x p e c t v a l u e s a r e l i s t e d . E x p e c t v a l u e i s t h e n u m b e r o f h i t s o n e c a n " e x p e c t " t o s e e b y c h a n c e w h e n d o i n g a B L A S T s e a r c h o n a d a t a b a s e o f a p a r t i c u l a r s i z e ( i n t h i s c a s e i t i s t h e n o n r e d u n d a n t d a t a b a s e o f N C B I ) . % s i m i l a r i t y i s t h e % o f a m i n o a c i d r e s i d u e s t h a t a r e i d e n t i c a l o r h a v e s i m i l a r p r o p e r t i e s t o t h e r e s i d u e s i n t h e q u e r y s e q u e n c e . T h e n e x t t w o g e n e s w e r e a l s o h i t s from s a m e B L A S T s e a r c h b u t r e p r e s e n t t w o g e n e s t h a t h a v e b e e n s t u d i e d f u n c t i o n a l l y . T h e l a s t g e n e s l i s t e d a r e u n f i n i s h e d fragments a n d t h e f i r s t t w o w e r e f o u n d i n a T B L A S T N s e a r c h o f t h e u n f i n i s h e d g e n o m e s a n d t h e l a s t f r a g m e n t w a s i d e n t i f i e d f r o m a T B L A S T N s e a r c h o f t h e T. ferrooxidans g e n o m e . O r g a n i s m D e s c r i p t i o n % S i m i l a r i t y E x p e c t V a l u e Nonredundant database Pyrococcus horikoshii 4 9 8 a m i n o a c i d l o n g 4 8 % 3 e - 3 6 A r c h a e a ; h y p o t h e t i c a l m e t h y l Euryarchaeota; t r a n s f e r a s e Thermococcales; Thermococcaceae Methanobacterium M g - p r o t o p o r p h y r i n I X 4 8 % 2 e -28 thermoautotrophicum m o n o m e t h y l e s t e r A r c h e a ; Euryarchaeota; o x i d a t i v e c y c l a s e Methanobaeriales; Methanobacteriaceae Thermotoga maritima M g - p r o t o p h o r p h y r i n I X 4 6 % 5 e-24 B a c t e r i a ; m o n o m e t h y l e s t e r Thermotogales; o x i d a t i v e c y c l a s e T h e r m o t o g a Functionally studied genes Streptomyces p h o s p h o n o a c e t a l d e h y d e 4 8 % l e - 1 8 wedmorensis m e t h y l a s e F O M 3 E u b a c t e r i a ; F i r m i c u t e s ; Actinomycetes; Streptomycetes; Streptomyccetaceae; Streptomyces Streptomyces P - m e t h y l t r a n s f e r a s e 4 6 % l e - 1 0 hygroscopicus E u b a c t e r i a ; F i r m i c u t e s ; Actinomycetes; Streptomycetes; Streptomycetaceae; Streptomyces Unfinished genomes database Thiobacillus T w o u n f i n i s h e d fragments F r a g m e n t A ferrooxidans; B a c t e r i a ; i d e n t i f i e d 9 9 % a a 8-43 7e-14 Proteobacter; g a m m a F r a g m e n t B s u b d i v i s i o n ; 7 2 % a a 4 3 3 - 4 7 6 2e-08 Thiobacillus F r a g m e n t C 4 5 % a a 2 1 2 - 3 6 7 0.003 78 Chapter 4 - Results t h e s e t h r e e g e n e s s h o w e d t h e s a m e r e s u l t s as t h e B. multivorans O R F 3 s u g g e s t i n g t h a t t h e s e s i m i l a r i t i e s w e r e r e a l . T h e f u n c t i o n a l l y s t u d i e d g e n e s t h a t s h o w e d t h e h i g h e s t s i m i l a r i t y t o t h e B. multivorans O R F 3 w e r e Streptomyces wedmorensis p h o s p h o a c e t a l d e h y d e m e t h y l a s e F O M 3 a n d Streptomyces hygroscopicus P - m e t h y l a s e . S e q u e n c e a l i g n m e n t s i n f i g u r e 16 o f a l l f i v e p r o t e i n s e q u e n c e s w i t h t h e B. multivorans O R F 3 s h o w e d p r o t e i n s e q u e n c e s i m i l a r i t y t h r o u g h o u t t h e p r o t e i n s . W h e n a T B L A S T N s e a r c h w i t h t h e O R F 3 p r o t e i n s e q u e n c e w a s p e r f o r m e d o n t h e u n f i n i s h e d g e n o m e s d a t a b a s e t w o u n f i n i s h e d f r a g m e n t s f r o m t h e T. ferrooxidans g e n o m e w e r e i d e n t i f i e d . T h e f i r s t f r a g m e n t s h o w e d 9 9 % s i m i l a r i t y w i t h a m i n o a c i d r e s i d u e s 8 t o 4 3 o f B. multivorans O R F 3 , t h e s e c o n d f r a g m e n t s h o w e d 7 2 % s i m i l a r i t y w i t h a m i n o a c i d r e s i d u e s 2 1 2 t o 3 6 7 . T h i s s e c o n d c o n t i g f r o m T. ferrooxidans w a s t h e s a m e c o n t i g t h a t w a s i d e n t i f i e d a s h a v i n g s i m i l a r i t y t o p a r t o f B. multivorans O R F 2 . T h e s t o p c o d o n o f T. ferrooxidans O R F t h a t h a d s i m i l a r i t y t o t h e B. multivorans O R F 2 , w a s f o u n d 2 4 b a s e p a i r s u p s t r e a m o f t h e p u t a t i v e s t a r t s i t e o f t h e T. ferrooxidans O R F t h a t h a d s i m i l a r i t y t o B. multivorans O R F 3 . T h i s s u g g e s t e d t h a t O R F 2 a n d O R F 3 l i k e s e q u e n c e s e x i s t e d i n T. ferrooxidans a n d m a y b e l i n k e d i n t h i s o r g a n i s m as w e l l as i n B. multivorans. T h e t h i r d f r a g m e n t i d e n t i f i e d f o r T. ferrooxidans a n d l i s t e d i n t a b l e 7 w a s f o u n d a f t e r a T B L A S T N s e a r c h o f t h e T. ferrooxidans g e n o m e . 4.4.4 ORF 4 T h e n u c l e o t i d e s e q u e n c e a n d t h e c o r r e s p o n d i n g a m i n o a c i d s e q u e n c e o f t h e f o u r t h o p e n r e a d i n g f r a m e ( O R F 4 ) a r e f o u n d i n F i g u r e 11. T h i s O R F c o n t a i n e d 8 8 6 b a s e p a i r s a n d t h e G C c o n t e n t f o r t h i s g e n e w a s 7 4 % . O R F 4 w a s f o u n d 3 b a s e p a i r s d o w n s t r e a m o f t h e O R F 3 . O R F 4 e n c o d e d f o r a p r e d i c t e d p r o t e i n o f 2 9 4 a m i n o a c i d s , 3 1 . 2 - k D a p r o t e i n w i t h a p r e d i c t e d p i o f 8.73. T h e p r e d i c t e d a m i n o a c i d s e q u e n c e o f O R F 4 w a s e n t e r e d i n t o a g a p p e d -B L A S T P s e a r c h o f t h e n o n r e d u n d a n t d a t a b a s e o f N C B I a n d T a b l e 8 l i s t s t h e t h r e e g e n e s , w h i c h 7 9 Chapter 4 - Results Figure 16. Amino acid sequence alignment for the ORF3 predicted protein sequence and similar sequences identified in the gapped-BLASTP similarity search. P r o t e i n s e q u e n c e a l i g n m e n t f o r t h e B. multivorans p u t a t i v e O R F 3 ( B m u l t i O R F 3 ) a n d P y r o m e t h y l , Pyrococcus horikoshii 4 9 8 a m i n o a c i d l o n g h y p o t h e t i c a l m e t h y l t r a n s f e r a s e ; M e t h M g o x , Methanobacterium thermoautotrophicum M g - p r o t o p o r p h y r i n I X m o n o m e t h y l e s t e r o x i d a t i v e c y c l a s e ; T h e r m M g o x , Thermotoga maritima M g - p r o t o p o r p h y r i n I X m o n o m e t h y l e s t e r o x i d a t i v e c y c l a s e ; S w e d m e t h , Streptococcus wedmorensis p h o s p h o n o a c e t a l d e h y d e m e t h y l a s e F O M 3 ; S h y g m e t h , Streptococcus hygroscopi P - m e t h y l t r a n s f e r a s e . S h a d i n g i s b l a c k f o r i d e n t i c a l a m i n o a c i d s a n d g r a y f o r a m i n o a c i d s w i t h s i m i l a r p r o p e r t i e s . S i m i l a r p r o p e r t i e s r e f e r s t o p o l a r i t y , a c i d i c o r b a s i c p r o p e r t i e s o r s i m i l a r c h a r g e s at t h e s a m e p H s u c h t h a t a m i n o a c i d s u b s t i t u t i o n s l i k e t h e s e d o n o t c h a n g e t h e p r o p e r t y o f t h e p r o t e i n . 8 0 Chapter 4 - Results BmultiORF3 Pyrmethyl MethMgox TherMgox Swedmeth Shygmeth BmultiORF3 Pyrmethyl MethMgox TherMgox Swedmeth Shygmeth BmultiORF3 Pyrmethyl MethMgox TherMgox Swedmeth Shygmeth BmultiORF3 116 TASD. Pyrmethyl 114 R§|cPCj MethMgox TherMgox Swedmeth Shygmeth BmultiORF3 163 Pyrmethyl 164 MethMgox TherMgox Swedmeth Shygmeth BmultiORF3 206 Pyrmethyl 202 MethMgox TherMgox Swedmeth Shygmeth BmultiORF3 266 Pyrmethyl 2 60 MethMgox TherMgox Swedmeth Shygmeth BmultiORF3 320 Pyrmethyl 317 MethMgox 315 TherMgox 307 Swedmeth 398 Shygmeth 369 BmultiORF3 Pyrmethyl MethMgox TherMgox Swedmeth Shygmeth BmultiORF3 417 J Pyrmethyl 410 F MethMgox TherMgox Swedmeth Shygmeth 374 371 369 361 458 423 HWYYMQSTPIHj 407 BJGL 398 F--|_ 4 98 |w -Di _ 483 WGVPYLLGKGJSHAEIBJRSLDLA3SLTSANVSRGSAEE|ATAERRF|DF|TGLDLAPARY iRRjgEGVEFFRFL Fl I|RA]|i|JGL§fivflYQA SYMl|VFFETLYgTHlEPKIPF iPFLVR|LFYREAHF PQ|QWNANQY-BmultiORF3 4 69 gjAHEA Pyrmethyl MethMgox 458 |KDR TherMgox Swedmeth Shygmeth 543 |TAGSEFR 81 Chapter 4 - Results Table 8. Genes and expect values from the results of a gapped-BLASTP search with B. multivorans ORF4 predicted protein sequence as the query sequence. The first four genes that were the result of a gapped-BLASTP search of the nonredundant database using the putative ORF 4 from B. multivorans with expect values less that 1.0 are listed. The fifth gene listed was identified in a gapped-BLAST search of the unfinished genomes. Expect value is the number of hits one can "expect" to see by chance when doing a B L A S T search of a database of a particular size (in this case it is the nonredundant database of NCBI). % similarity is the % of amino acid residues that are identical or have similar properties to the residues in the query sequence. Organism Description % Similarity Expect Value Bacillus stearothermophilus; Eubacteria; Firmicutes; Low GC content gram-positive bacteria; Bacillaceae; Bacillus Bacillus cereus; Bacteria; Firmicutes; Bacillus/Clostridium group; Bacillaceae Escherichia coli; Eubacteria; Proteobacteria; gamma subdivision; Enterobacteriaceae; Escherichia Thiobacillus ferrooxidans; Bacteria; Proteobacter; gamma subdivision; Thiobacillus Cellobiose 42% phosphotransferase system CelC YdjC-like protein 48% Hypothetical 27.8kDa 46% protein, ORF found in celF-katE intergenic region Unfinished fragment 62% 5e-15 8e-10 4e-8 8e-54 82 Chapter 4 - Results gave the lowest, expect values. These were Bacillus stearothermophilus celC, Bacillus cereus ydjC-like and E. coli hypothetical protein in the ceW-katE region. Protein sequence alignments in figure 17 showed that the sequence similarity to those genes listed in Table 8 was throughout the amino acid sequence. The celC gene of Bacillus stearothemophilus has not been studied functionally. The B. multivorans predicted amino acid sequence from ORF4 showed similarity to a predicted protein from an ORF called ydjC found in the celF-kaiE region of E. coli at 46%. A similarity of 48%) was exhibited with the Bacillus cereus YdjC-like protein, which was named as a result of its similarity with this same region in E. coli. A gapped-BLASTP search was performed on the unfinished genomes database and 61% similarity was found with a fragment from the T. ferrooxidans genome. The T. ferrooxidans contig identified did not overlap with previous two identified contigs with similarity to the ORF2 and ORF3 and it could not be concluded whether or not these contigs were adjacent in T. ferrooxidans. 4.4.5 ORF 5 The nucleotide sequence of the fifth open reading frame is found in figure 11 however further sequencing is required of this region. ORF5 was truncated at the Sal I site and a stop codon was not identified. A gapped-BLASTP search of the nonredundant database of NCBI of this incomplete amino acid sequence showed no sequence similarity with any known genes. A B L A S T search of the unfinished genomes database showed 69% sequence similarity with a contig of T. ferrooxidans at the N-terminal. This suggested that these four genes may also be adjacent to each other and closely linked in T. ferrooxidans as was seen for B. multivorans however, no conclusions can be drawn until the genome sequencing project of T. ferrooxidans is finished. 83 Chapter 4 - Results B m u l t i O R F 4 T f e r r o r f B s t e c e l C B c e r y d j C E c o l i h y p 1 V T T Q R A A _ ? _ _ F T H I 1 ^ B m u l t i O R F 4 6 1 T f e r r o r f 3 2 B s t e c e l C 5 6 B c e r y d j C 5 5 E c o l i h y p 5 6 J'DgCRFlFLPHVRA R B V R Y _ F L P Q V R K |E-ALVGARRG|-BE - - _ 2 S V - V § E G K I N P | E | E | K W I W Q L A E E D A L P L _ E _ T B m u l t i O R F 4 T f e r r o r f B s t e c e l C B c e r y d j C E c o l i h y p 1 2 1 9 2 108 B 9 L S - H _ T 1 0 9 L f f l l E L f f l R K J P Y H T T P W g P L R A A R C S G A K G V T A R P H R L ^ E E _ R A V R D R Q M A F D L P B m u l t i O R F 4 T f e r r o r f B s t e c e l C B c e r y d j C E c o l i h y p 1 6 9 A P | W L R 1 5 1 A P T F W S j j F L A 1 5 8 1 5 8 I _ V R A Q L D : ARMRRQLDRR: rBDBfflFPDl SDSBYS 1 6 1 - L R T T Q G H S S Q T g M g g A V L L D A L A A L P P - GVGI T|J§M H E R L L R M L R C L P A T G \ DABSRGSR § B m u l t i O R F 4 T f e r r o r f B s t e c e l C B c e r y d j C E c o l i h y p 'EAGDGPBTPSMADP ~|QTAA{J_ALMPN_E P PLASGISHCC; P E L ^ R B S H V M D B m u l t i O R F 4 2 8 4 P A A R R P G A Q P S T f e r r o r f B s t e c e l C B c e r y d j C E c o l i h y p Figure 17. Amino acid sequence alignments for the ORF4 predicted protein sequence and similar sequences identified in the gapped-BLASTP search using the ORF4 predicted protein sequence as the query sequence. Protein sequence alignment for the B. multivorans putative O R F 4 and the putative O R F (BmultiORF4) from Thiobacillus ferrooxidans (Tferrorf), Bacillus stearothermophilus Ce lC (BstecelC), Bacillus cereus Yd jC-like protein (BcerydjC) and the E. coli hypothetical 27.8-kDa protein in CelF-KatE intergenic region. Note that the T. ferrooxidans sequence is truncated at the start. Shading is black for identical amino acids and gray for amino acids with similar properties. Similar properties refers to polarity, acidic or basic properties or similar charges at the same p H such that amino acid substitutions like these do not change the property of the protein. 84 Chapter 4 - Results .4.5 Subcloning of Bam HI, Sal I and Pst I fragments into pDN18 S i n c e p B l u e s c r i p t d o e s n o t r e p l i c a t e i n B. multivorans, t h e Bam H I , Sal I a n d Pst I f r a g m e n t s w e r e s u b c l o n e d i n t o t h e b r o a d - h o s t - r a n g e v e c t o r p D N 1 8 . p D N 1 8 w a s t r a n s f e r r e d b y t r i p a r e n t a l m a t i n g , u s i n g t h e h e l p e r p l a s m i d p R K 2 0 1 3 i n t o B. multivorans A T C C 1 7 6 1 6 . T h e p l a s m i d p D N 1 8 r e p l i c a t e d i n B. multivorans a n d w a s m a i n t a i n e d at a l o w c o p y n u m b e r . T h e s e f r a g m e n t s w e r e s u b c l o n e d i n b o t h d i r e c t i o n s i n t o p D N 1 8 . T h e 8.0 K b Pst I f r a g m e n t , a f t e r l i g a t i o n w i t h Pst I d i g e s t e d p D N 1 8 , c o u l d n o t b e e l e c t r o p o r a t e d o r t r a n s f o r m e d i n t o E. coli D H 5 a o r X L 1 - B l u e M R . B e c a u s e O R F 2 , O R F 3 , O R F 4 , O R F 5 a n d p o s s i b l y m o r e a r e a l l f o u n d o n t h i s 8.0 k b Pst I f r a g m e n t t h i s r e s u l t s u g g e s t e d t h a t e x p r e s s i o n o f a l l t h e s e g e n e s o n a m u l t i -c o p y n u m b e r v e c t o r m a y b e l e t h a l t o E. coli. 4.6 Complementation of Mutant 26D7 p D B S 6 , p D B S 9 , p D B S l , p D B S 2 ( d e t a i l e d d e s c r i p t i o n o f c l o n e s i n t a b l e 1, C h a p t e r 2 ) a n d p D N 1 8 w e r e c o n j u g a t e d b y t r i p a r e n t a l m a t i n g s i n t o m u t a n t 2 6 D 7 . F i g u r e 1 8 A s h o w s t h e Bam H I f r a g m e n t c l o n e d i n o p p o s i t e o r i e n t a t i o n s i n p D B S 6 a n d p D B S 9 a n d t h e Sal I f r a g m e n t c l o n e d i n o p p o s i t e o r i e n t a t i o n s i n p D B S l a n d p D B S 2 . T h e a r r o w b e n e a t h t h e v e c t o r n a m e i n f i g u r e 1 8 A i n d i c a t e s t h e d i r e c t i o n o f t h e lac p r o m o t e r o n t h e v e c t o r f o r e a c h c l o n e . F i g u r e 1 8 B s h o w s O R F 1 a n d O R F 2 a r e f o u n d o n t h e Bam H I f r a g m e n t a n d O R F 3 , O R F 4 a n d p a r t o f O R F 5 a r e f o u n d o n t h e Sal I f r a g m e n t . T o d e t e r m i n e w h i c h c l o n e c o m p l e m e n t e d t h e m u t a t i o n , M I C s f o r c a t i o n i c a n t i m i c r o b i a l s w e r e a s s a y e d . T h e s e M I C e x p e r i m e n t s w e r e e x e c u t e d i n t h e a b s e n c e o f t e t r a c y c l i n e s e l e c t i o n ( r e q u i r e d f o r m a i n t e n a n c e o f t h e v e c t o r p D N 1 8 ) , b e c a u s e i n t h e p r e s e n c e o f t e t r a c y c l i n e , a l l t h e c l o n e s , as w e l l as t h e v e c t o r p D N 1 8 a p p e a r e d t o c o m p l e m e n t t h e m u t a t i o n . U n d e r c o n t r o l l e d c o n d i t i o n s , w h e r e e a c h w e l l w a s i n o c u l a t e d w i t h e x a c t l y 1 0 5 c e l l s / m l , o n l y t h e p D B S 9 m u t a n t c l o n e a p p e a r e d t o s h o w c o m p l e m e n t a t i o n . T h e M I C f o r t h e m u t a n t c a r r y i n g 85 Chapter 4 - Results Figure 18. Restriction map of the Bam HI and Sal I fragments and their respective orientations when cloned into pDN18. P a n e l A : a r r o w s d e p i c t t h e o r i e n t a t i o n o f O R F 2 i n c l o n e s p D B S 6 a n d p D B S 9 a n d t h e o r i e n t a t i o n o f O R F 3 , 4 a n d 5 i n c l o n e s p D B S l a n d p D B S 2 w i t h r e s p e c t t o t h e lac p r o m o t e r o n t h e v e c t o r . P a n e l B : r e s t r i c t i o n e n z y m e m a p o f t h e Bam H I a n d Sal I f r a g m e n t s . C l o n e p B S 6 c a r r i e d t h e Bam H I f r a g m e n t o f D N A t h a t a p p e a r e d t o c o m p l e m e n t t h e k n o c k o u t m u t a t i o n i n m u t a n t 2 6 D 7 . T h e Bam H I fragment c o n t a i n e d t w o c o m p l e t e p u t a t i v e O R F s , O R F 1 a n d O R F 2. T h e Sal I fragment a l s o c o n t a i n e d t w o c o m p l e t e O R F s , O R F 3 a n d O R F 4 a n d t h e i n c o m p l e t e O R F 5 a n d d i d n o t c o m p l e m e n t t h e m u t a t i o n . T h e s i t e o f t h e t r a n s p o s o n i n s e r t i o n i n m u t a n t 2 6 D 7 i s i n d i c a t e d ( " - ) . 8 6 Chapter 4 - Results B A Sal I fragment ORF3,4,5 pDBSl <-ORF3,4,5 pDBS2 • Bam HI fragment 0RF2 pDBS6 < 0RF2 pDBS9 • 3 «5 3 <3 3 «5 C £ cd a 0Q £ cd I T ; O _ jo 4i c H OH o o o 87 Chapter 4 - Results p D B S 9 r e t u r n e d t o t h e p a r e n t M I C o f 6 4 u g / m l f o r b o t h p o l y m y x i n B a n d c o l i s t i n a s s h o w n i n T a b l e 9. p D B S 6 d i d n o t c o m p l e m e n t t h e m u t a t i o n i n 2 6 D 7 a n d c a r r i e d t h e s a m e Bam H I fragment as p D B S 9 i n t h e o p p o s i n g o r i e n t a t i o n ( f i g u r e 1 8 A ) . T h i s s u g g e s t e d t h a t t h e lac p r o m o t e r m i g h t b e d r i v i n g g e n e e x p r e s s i o n . M u t a n t 2 6 D 7 c a r r y i n g t h e c o m p l e m e n t i n g v e c t o r , p D B S 9 s h o w e d r e d u c e d v i a b i l i t y o n L B p l a t e s a n d i n b o t h L B a n d M u e l l e r - H i n t o n b r o t h m e d i a w i t h o r w i t h o u t t e t r a c y c l i n e f o r s e l e c t i o n . I n t h e M I C e x p e r i m e n t s t h i s r e d u c e d v i a b i l i t y o f 2 6 D 7 + p D B S 9 w a s o b s e r v e d as r e d u c e d g r o w t h i n a l l w e l l s . T h e c o m p l e m e n t e d m u t a n t , 2 6 D 7 + p D B S 9 a n d t h e m u t a n t 2 6 D 7 c a r r y i n g t h e v e c t o r p D N 1 8 b o t h s h o w e d a n i n c r e a s e i n M I C o f 31 u g / m l t o 1 2 5 u g / m l t o C P 2 6 as s h o w n i n t a b l e 9. T h u s , t h e p r e s e n c e o f t h e v e c t o r c l o n e d i n t o m u t a n t 2 6 D 7 c a u s e d a n i n c r e a s e i n M I C t o C P 2 6 . F i n a l l y , a l l c l o n e s s h o w e d a n M I C o f 6 4 u g / m l f o r C E M E . T h e r e f o r e t h e c o m p l e m e n t i n g v e c t o r p D B S 9 h a d n o e f f e c t o n t h e M I C f o r t h i s p e p t i d e a s s h o w n i n T a b l e 9. 88 Chapter 4 - Results Table 9. MICs of cationic antimicrobial agents for the parent B. multivorans ATCC 17616, mutant 26D7 and 26D7+pDBS9. T h e p a r e n t s t r a i n ( A T C C 1 7 6 1 6 ) , m u t a n t 2 6 D 7 a n d 2 6 D 7 p D N 1 8 a n d 2 6 D 7 p D B S 9 ( c o m p l e m e n t i n g m u t a n t c l o n e ) w e r e i n c u b a t e d f o r 2 4 h o u r s at 3 7 ° C i n t h e p r e s e n c e o f i n c r e a s i n g c o n c e n t r a t i o n s o f t h e c a t i o n i c a n t i m i c r o b i a l s : p o l y m y x i n B , c o l i s t i n , a c e c r o p i n - m e l i t t i n h y b r i d ( C P - 2 7 ) a n d a m o d i f i e d c e c r o p i n - m e l i t t i n h y b r i d ( C P 2 6 ) . T h e l o w e s t c o n c e n t r a t i o n o f a n t i b i o t i c t h a t i n h i b i t e d g r o w t h o f t h e s t r a i n s t e s t e d w a s r e c o r d e d . R e s u l t s from o n e o f t h r e e r e p l i c a t e d e x p e r i m e n t s a r e s h o w n . S t r a i n p o l y m y x i n c o l i s t i n C P 2 6 C E M E ( U g / m l ) ( u g / m l ) ( u g / m l ) ( u g / m l ) B. multivorans 1 7 6 1 6 6 4 6 4 > 5 0 0 6 4 2 6 D 7 8 8 31 6 4 2 6 D 7 p D N 1 8 8 8 125 6 4 2 6 D 7 p D B S 9 6 4 6 4 125 6 4 E. c o / / H B 1 0 0 5 0.5 0.5 - 15 15 8 9 Chapter 4 - Discussion 4.7 Discussion O R F 1 p r e d i c t e d p r o t e i n s e q u e n c e s h o w e d 7 8 % s i m i l a r i t y t o a l k a n a l m o n o o x y g e n a s e s o f E. coli a n d Bacillus subtilis. S i n c e t h i s O R F w a s t r a n s c r i b e d i n t h e o p p o s i t e o r i e n t a t i o n , w a s l o c a t e d d i s t a n t l y u p s t r e a m o f O R F 2 , O R F 3 , O R F 4 a n d O R F 5 a n d t h e t r a n s p o s o n w a s u p s t r e a m f r o m t h i s O R F b y 1,770 b a s e p a i r s i t w a s u n l i k e l y t h a t t h i s g e n e c o u l d h a v e b e e n a f f e c t e d b y t h e t r a n s p o s o n i n s e r t i o n . T h e s e q u e n c e s i m i l a r i t y s e a r c h e s s h o w e d a h i g h d e g r e e o f s i m i l a r i t y f o r s e v e r a l g e n e s t h a t h a d n o t b e e n s t u d i e d f u n c t i o n a l l y b u t w e r e n a m e d b a s e d o n t h e i r s i m i l a r i t y t o o t h e r a l k a n a l m o n o o x y g e n a s e s . T h e g a p p e d - B L A S T P s e a r c h o f O R F 1 s h o w e d s i m i l a r i t y t o t h e s e a l k a n a l m o n o o x y g e n a s e s h o w e v e r t h e c l o s e s t s i m i l a r i t y t o a f u n c t i o n a l l y s t u d i e d a l k a n a l m o n o o x y g e n a s e w a s r e l a t i v e l y l o w a n d o n l y a c r o s s t h e N - t e r m i n a l h a l f o f t h e p r o t e i n . T h i s a l k a n a l m o n o o x y g e n a s e g e n e (luxA) f r o m Vibrio harveyi e n c o d e s f o r t h e a l p h a c h a i n o f l u c i f e r a s e ( a n a l k a n a l m o n o o x y g e n a s e ) a n d i n c l u d e s o n e o f t h e t w o a c t i v e c e n t e r s o f l u c i f e r a s e at r e s i d u e s 1 0 0 - 1 1 5 . L u c i f e r a s e i s a n a l p h a - b e t a h e t e r o d i m e r m o n o o x y g e n a s e t h a t c a t a l y z e s t h e o x i d a t i o n o f a l o n g c h a i n a l d e h y d e a n d r e l e a s e s e n e r g y i n t h e f o r m a v i s i b l e l i g h t . F r o m t h e r e s u l t s p r e s e n t e d h e r e i t w a s n o t p o s s i b l e t o c o n c l u d e t h e e x a c t f u n c t i o n o f O R F 1 f r o m B. multivorans. O R F 2 s h o w e d 4 5 % p r o t e i n s i m i l a r i t y a n d 3 0 % p r o t e i n i d e n t i t y t o a p u t a t i v e c e r a m i d e g l u c o s y l t r a n s f e r a s e o f Synechocystis sp. T h i s e n z y m e h a s n o t b e e n s t u d i e d f u n c t i o n a l l y i n Synechocystis a n d w a s i d e n t i f i e d as a p r e d i c t e d p r o t e i n u p o n s e q u e n c i n g o f i t s e n t i r e g e n o m e . T h e m o u s e , rat, a n d h u m a n c e r a m i d e g l u c o s y l t r a n s f e r a s e s w e r e a l l s i m i l a r t o t h e B. multivorans O R F 2 . T h e h u m a n c e r a m i d e g l u c o s y l t r a n s f e r a s e h a s b e e n s h o w n t o c a t a l y z e t h e f i r s t g l y c o s y l a t i o n s t e p i n t h e s y n t h e s i s o f g l y c o s p h i n g o l i p i d s ( I c h i k a w a , S et a l , 1 9 9 8 ) . G l y c o s p h i n g o l i p i d s a r e n o t f o u n d i n p r o k a r y o t e s . T h e s e a r e a g r o u p o f m e m b r a n e c o m p o n e n t s i n e u k a r y o t e s t h a t h a v e t h e l i p i d p o r t i o n e m b e d d e d i n t h e o u t e r p l a s m a m e m b r a n e l e a f l e t a n d t h e 9 0 Chapter 4 - Discussion c a r b o h y d r a t e c h a i n s e x t e n d e d t o t h e o u t e r e n v i r o n m e n t . O t h e r p r o t e i n s t h a t w e r e i d e n t i f i e d i n t h e g a p p e d - B L A S T P s e a r c h w i t h l o w l e v e l s i m i l a r i t y w e r e t h e M i g A p r o t e i n o f P. aeruginosa, w h i c h h a s a g l u c o s y l t r a n s f e r a s e f u n c t i o n ( W a n g et a l , 1 9 9 6 ) a n d N o d C p r o t e i n o f Rhizobium sp. N o d C a c t s a s a n A f - a c e t y l g l u c o s a m i n y l t r a n s f e r a s e , w h i c h i s i n v o l v e d i n t h e f o r m a t i o n o f t h e N o d f a c t o r b a c k b o n e . N o d f a c t o r s a r e l i p o o l i g o s a c c h a r i d e s t h a t c o n t a i n a c h i t o o l i g o s a c c h a r i d e c h a i n w i t h t h r e e t o f i v e N - a c e t y l g l u c o s a m i n e r e s i d u e s . T h i s s i m i l a r i t y d a t a s u g g e s t e d t h a t O R F 2 e n c o d e d f o r a p r o t e i n p r o d u c t t h a t i s i n v o l v e d i n s u g a r t r a n s f e r . T h e s i m i l a r i t y t h a t O R F 2 s h a r e d w i t h c e r a m i d e g l u c o s y l t r a n s f e r a s e s f r o m e u k a r y o t e s c o u l d b e e x p l a i n e d i f t h e p r o t e i n e n c o d e d b y O R F 2 c a t a l y z e s t h e t r a n s f e r o f a s u g a r m o l e c u l e t o a l i p i d m o l e c u l e . W h e t h e r t h e s u b s t r a t e l i p i d m o l e c u l e i s l i p i d A o f L P S o r a p h o s p h o l i p i d f o u n d i n e i t h e r t h e o u t e r o r i n n e r m e m b r a n e r e m a i n s t o b e d e t e r m i n e d . P e r h a p s t h e a d d i t i o n o f a s u g a r m o i e t y t o t h i s m e m b r a n e l i p i d b y a p h o s p h o d i e s t e r l i n k w o u l d r e d u c e t h e n e g a t i v e c h a r g e o f t h e l i p i d , r e d u c i n g t h e a b i l i t y o f t h e c a t i o n i c p e p t i d e t o a c t o n t h e m e m b r a n e . T h e a d d i t i o n o f a c a r b o h y d r a t e c h a i n t o a l i p i d m a y a l s o h i n d e r t h e a b i l i t y o f c a t i o n i c p e p t i d e s t o a c c e s s t h e n e g a t i v e c h a r g e s f o u n d o n b a c t e r i a l l i p i d s . T h e p h y l o g e n e t i c t r e e f o r r e l a t e d n e s s o f t h e s e q u e n c e s i d e n t i f i e d a s s i m i l a r t o B. multivorans O R F 2 s h o w e d t h a t t h e s e q u e n c e f r o m B. multivorans i s r e l a t i v e l y c l o s e l y r e l a t e d t o m a m m a l i a n s e q u e n c e s . T h e f a c t t h a t t h e B. multivorans O R F 2 s e q u e n c e s e e m s t o b e m o r e c l o s e l y r e l a t e d t o t h e m o u s e a n d h u m a n s e q u e n c e s t h a n t o t h e t h r e e C. elegans s e q u e n c e s s u g g e s t e d t h a t t h i s O R F m a y h a v e h a d s i m i l a r c o n s t r a i n t s o n e v o l u t i o n a r y d i v e r g e n c e as m a m m a l i a n s e q u e n c e s . H o w e v e r , i t i s d i f f i c u l t t o d r a w a n y r e a l c o n c l u s i o n s b e c a u s e o f t h e i n a d e q u a t e n u m b e r o f o r g a n i s m s i n t h e t r e e w i t h w h i c h t o b a s e c o n c l u s i o n s . T h e i d e n t i f i c a t i o n o f a p o t e n t i a l c e r a m i d e g l u c o s y l t r a n s f e r a s e i n v o l v e d i n c a t i o n i c p e p t i d e r e s i s t a n c e f o r B. multivorans c o u l d i n d i c a t e t h e p o s s i b i l i t y t h a t a l i p i d e x i s t s i n t h e m e m b r a n e o f 91 Chapter 4 - Discussion t h i s o r g a n i s m t h a t i s r e l a t e d t o e u k a r y o t i c c e r a m i d e . O n e o f t h e r e a s o n s t h a t c a t i o n i c p e p t i d e s h a v e n o e f f e c t o n e u k a r y o t i c m e m b r a n e s h a s t o d o w i t h t h e i r l i p i d c o m p o s i t i o n . E u k a r y o t i c m e m b r a n e s c o n s i s t o f z w i t t e r i o n i c l i p i d s l i k e c e r a m i d e , n o t t h e a n i o n i c l i p i d s t h a t a r e s e e n i n p r o k a r y o t e s . It i s i n t e r e s t i n g t h a t B. cepacia h a s b e e n s h o w n t o h a v e a u n i q u e o r n i t h i n e -c o n t a i n i n g l i p i d a s w e l l a s a h i g h e r a b u n d a n c e o f o r n i t h i n e - c o n t a i n i n g l i p i d s i n g e n e r a l w h e n c o m p a r e d w i t h P. aeruginosa ( K a w a i et a l . 1 9 8 8 ) . O r n i t h i n e i s p o s i t i v e l y c h a r g e d a n d as a r e s u l t o r n i t h i n e - c o n t a i n i n g l i p i d s a r e z w i t t e r i o n i c w i t h a n o v e r a l l p o s i t i v e c h a r g e . O R F 3 o f B. multivorans s h o w e d t h e h i g h e s t s i m i l a r i t y ( a l t h o u g h o n l y 4 8 % ) t o t h e Pyrococcus horikooshii h y p o t h e t i c a l m e t h y l t r a n s f e r a s e . T h e p u t a t i v e M g - p r o t o p o r p h y r i n I X m o n o m e t h y l e s t e r o x i d a t i v e c y c l a s e e n z y m e s o f b o t h Methanobacterium thermoautotrophicum a n d Thermotoga maritima w e r e a l s o i d e n t i f i e d . N o n e o f t h e s e h a v e b e e n s t u d i e d f u n c t i o n a l l y a n d a l l w e r e i d e n t i f i e d a s a r e s u l t o f g e n o m e s e q u e n c i n g p r o j e c t s a n d n a m e d b e c a u s e o f t h e i r h i g h s e q u e n c e s i m i l a r i t y w i t h m a n y m e t h y l t r a n s f e r a s e s a n d , i n t h e c a s e s o f M thermoautotrophicum a n d T. mariima, o t h e r M g - p r o t o p o r p h y r i n I X m o n o m e t h y l e s t e r o x i d a t i v e c y c l a s e e n z y m e s . T h e m o r e d i s t a n t l y r e l a t e d s e q u e n c e s f r o m S. wedmorensis a n d S. hygroscopicus w e r e a l s o i d e n t i f i e d f r o m t h i s g a p p e d - B L A S T P s e a r c h a n d t h e s e t w o m e t h y l t r a n s f e r a s e h a v e b e e n s t u d i e d f u n c t i o n a l l y . B o t h o f t h e s e e n z y m e s h a v e b e e n s h o w n t o u s e m e t h y l c o b a l a m i n a s t h e m e t h y l d o n o r a n d t h e p o s i t i v e c h a r g e s o n t h e m e t h y l g r o u p a l l o w e l e c t r o p h i l i c a t t a c k o f t h e n e g a t i v e c h a r g e s o n t h e p h o s p h a t e r e s i d u e . T h e s e q u e n c e s i m i l a r i t y o f t h e s e e n z y m e s f r o m Streptomyces w i t h t h e M g - p r o t o p o r p h y r i n I X m o n o m e t h y l e s t e r o x i d a t i o n m a y b e d u e t o t h e s t r u c t u r a l s i m i l a r i t y o f m e t h y l c o b a l a m i n e a n d p r o t o p o r p h y r i n ( H i d a k a et a l . , 1 9 9 5 ) . T h u s t h e s i m i l a r i t i e s a r e c o n s i s t e n t w i t h t h e s u g g e s t i o n t h a t t h e B. multivorans O R F 3 m a y f u n c t i o n a s a m e t h y l t r a n s f e r a s e ; h o w e v e r f u n c t i o n a l s t u d i e s w o u l d b e r e q u i r e d t o c o n f i r m t h a t t h e p r e d i c t e d p r o t e i n o f O R F 3 i s t r u l y a m e t h y l t r a n s f e r a s e . It i s p o s s i b l e t h a t s u c h a 92 Chapter 4 - Discussion m e t h y l t r a n s f e r a s e m i g h t f u n c t i o n t o r e d u c e t h e n e g a t i v e c h a r g e o f p h o s p h a t e r e s i d u e s f o u n d o n t h e l i p i d s o r L P S o f B. multivorans. O R F 4 a n d O R F 5 s h o w e d n o s i g n i f i c a n t s e q u e n c e s i m i l a r i t y w i t h a n y k n o w n s e q u e n c e s i n t h e g a p p e d - B L A S T P s e a r c h o f t h e N C B I n o n r e d u n d a n t d a t a b a s e . T h e o r i e n t a t i o n a n d c l o s e p r o x i m i t y o f O R F 2 , O R F 3 , O R F 4 a n d O R F 5 a n d t h e i d e n t i f i c a t i o n o f t h e p r o m o t e r s e q u e n c e u p s t r e a m o f O R F 2 s u g g e s t e d t h e s e g e n e s m a y b e m e m b e r s o f a n o v e l B. multivorans o p e r o n . T h i s w a s s u p p o r t e d b y t h e i d e n t i f i c a t i o n o f s e q u e n c e s i m i l a r i t y o f a l l f o u r g e n e s w i t h t h e s e q u e n c e s f r o m Thiobacillus ferrooxidans. T h e c o n t i g s i d e n t i f i e d from T. ferrooxidans u n f i n i s h e d g e n o m e p r o j e c t , w e r e a l i g n e d t o d e t e r m i n e w h e t h e r o r n o t t h e y o v e r l a p p e d . T h e t w o c o n t i g s o f t h e T. ferrooxidans g e n o m e s e q u e n c e i d e n t i f i e d b y O R F 2 a n d O R F 3 o f B. multivorans d i d o v e r l a p . U n f o r t u n a t e l y o n l y t h e first 3 5 a m i n o a c i d s o f t h e O R F 3 o f T. ferrooxidans, w h i c h s h o w e d 9 9 % s i m i l a r i t y w i t h t h e B. multivorans O R F 3 s e q u e n c e , w a s u s e d i n t h e s e a l i g n m e n t s b e c a u s e t h e c o n t i g w a s t r u n c a t e d at b a s e 105. T h e d i s t a n c e b e t w e e n t h e O R F 2 s t o p c o d o n a n d O R F 3 sta r t c o d o n o f T. ferrooxidans w a s 2 4 b a s e p a i r s . I n t h i s r e g i o n a p u t a t i v e r h o - i n d e p e n d e n t t e r m i n a t o r w a s i d e n t i f i e d w h i c h i n d i c a t e s t h a t O R F 2 a n d O R F 3 m a y b e t r a n s c r i b e d a s s e p a r a t e g e n e s . It i s p o s s i b l e t h a t a p r o m o t e r s e q u e n c e m a y b e f o u n d b e t w e e n t h e s e t w o O R F s b u t f u r t h e r s t u d i e s w o u l d b e r e q u i r e d t o d e t e r m i n e t h i s . T h e f a c t t h a t T. ferrooxidans a n d B. multivorans a r e b o t h m e m b e r s o f t h e b e t a s u b d i v i s i o n o f Proteobacter m a y b e s i g n i f i c a n t s u g g e s t i n g t h a t g r o u p i n g o f t h e s e g e n e s m a y b e c o n s e r v e d w i t h i n t h i s s u b d i v i s i o n . T h e T. ferrooxidans g e n o m e p r o j e c t i s s t i l l i n p r o g r e s s a n d m o s t o f t h e c o n t i g s i d e n t i f i e d w e r e q u i t e s h o r t i n d i c a t i n g t h e p r o j e c t i s s t i l l i n i t s e a r l y s t a g e s . It w i l l b e i n t e r e s t i n g t o d e t e r m i n e w h e t h e r o r n o t t h e s e f o u r g e n e s a r e t r u l y l i n k e d a n d w h e t h e r o r n o t a s e c o n d c o p y o f O R F 2 e x i s t s i n t h i s o r g a n i s m as w a s s e e n i n t h e c o m p l e t e g e n o m e s e q u e n c e s o f b o t h Synechocystis sp. a n d C. elegans. T h e Synechocystis sp. g e n o m e p r o j e c t i s 93 Chapter 4 - Discussion f i n i s h e d a n d n e i t h e r o f t h e t w o p u t a t i v e c e r a m i d e g l u c o s y l t r a n s f e r a s e e n z y m e s i d e n t i f i e d w e r e l i n k e d t o a m e t h y l t r a n s f e r a s e , a l t h o u g h t w o m e t h y l t r a n s f e r a s e s w i t h l o w s e q u e n c e s i m i l a r i t y t o t h e O R F 3 f r o m B. multivorans w e r e f o u n d i n t h e g e n o m e o f Synechocystis sp. T h e Bam H I f r a g m e n t i n c l o n e p D B S 9 a p p e a r e d t o c o m p l e m e n t t h e i n s e r t i o n m u t a n t 2 6 D 7 a n d c a r r i e d i n t a c t O R F 1 a n d O R F 2 as w e l l a s o n l y p a r t o f O R F 3 . T h i s o b s e r v a t i o n w a s s u r p r i s i n g s i n c e O R F 3 w a s t h e s i t e o f t h e t r a n s p o s o n i n s e r t i o n i n m u t a n t 2 6 D 7 . B e c a u s e O R F 1 w a s p r e s e n t o n t h e c o m p l e m e n t i n g f r a g m e n t , i t w a s n o t p o s s i b l e t o c o m p l e t e l y e l i m i n a t e t h e i n v o l v e m e n t o f O R F l . H o w e v e r , i t s g e n e t i c d i s t a n c e f r o m t h e t r a n s p o s o n i n s e r t i o n s i t e a n d i t s o r i e n t a t i o n m a k e i t u n l i k e l y t h a t O R F l i s i n v o l v e d i n r e s i s t a n c e t o c a t i o n i c p e p t i d e s . O R F 2 , O R F 3 , O R F 4 a n d O R F 5 w e r e a l l f o u n d i n t h e s a m e o r i e n t a t i o n a n d i n c l o s e p r o x i m i t y o f e a c h o t h e r a s s h o w n i n f i g u r e 18. F u r t h e r s e q u e n c i n g d o w n s t r e a m o f t h e Sal I s i t e i s r e q u i r e d t o i d e n t i f y t h e s t o p c o d o n f o r O R F 5 a n d w h e t h e r o r n o t m o r e O R F s e x i s t d o w n s t r e a m o f O R F 5 . T h e i n t e r g e n i c r e g i o n b e t w e e n O R F 2 a n d O R F 3 w a s c l o n e d i n p D B S l a n d p D B S 2 a n d c o n t a i n e d a r h o - i n d e p e n d e n t t e r m i n a t o r s e q u e n c e d i r e c t l y u p s t r e a m o f t h e O R F 3 s t a r t c o d o n as s h o w n i n f i g u r e 11. T h i s m a y e x p l a i n w h y p D B S l a n d p D B S 2 c o u l d n o t c o m p l e m e n t t h e m u t a n t p h e n o t y p e i n t h a t t h i s t e r m i n a t o r p r o b a b l y i n h i b i t e d e x p r e s s i o n o f t h e s e g e n e s o f f t h e lac p r o m o t e r . T h e i n a b i l i t y t o c l o n e a l a r g e f r a g m e n t o f t h i s e n t i r e r e g i o n i n t o p D N 1 8 (t h e Pst I f r a g m e n t ) m a y b e d u e t o e x p r e s s i o n o f t h e s e g e n e s o n a m u l t i - c o p y v e c t o r m a y h a v e a l e t h a l e f f e c t o n t h e b a c t e r i a l c e l l . T h e r e d u c t i o n i n v i a b i l i t y c a u s e d b y t h e v e c t o r , p D B S 9 , w h e n c l o n e d i n t o m u t a n t 2 6 D 7 s u g g e s t e d t h a t t h e f r a g m e n t c l o n e d i n p D B S 9 e x p r e s s e d O R F 2 o f f t h e lac p r o m o t e r c a u s e d a l e t h a l e f f e c t o n t h e b a c t e r i a a s w e l l . T h e r e s u l t s p r e s e n t e d h e r e s u g g e s t t h a t e x p r e s s i o n o f t h e g e n e s m a y b e t i g h t l y c o n t r o l l e d a n d p r o b a b l y m a i n t a i n e d at a l o w l e v e l . T h e c o m p l e m e n t a t i o n s t u d i e s s u g g e s t e d t h a t O R F 2 a l o n e w a s c a p a b l e o f r e t u r n i n g t h e m u t a n t p h e n o t y p e b a c k t o t h e p a r e n t p h e n o t y p e . T h e a b i l i t y o f O R F 2 t o c o m p l e m e n t t h e m u t a n t 9 4 Chapter 4 - Discussion p h e n o t y p e b y i t s e l f s u g g e s t e d t h a t o v e r - e x p r e s s i o n o f O R F 2 i n t h e m u t a n t m a y c a u s e p h e n o t y p i c s u p p r e s s i o n , o v e r c o m p e n s a t i n g f o r t h e a b s e n c e o f O R F 3 e x p r e s s i o n , O R F 3 b e i n g t h e s i t e o f t h e t r a n s p o s o n i n s e r t i o n . C o m p l e m e n t a t i o n s t u d i e s w i t h O R F 3 a n d O R F 4 f a i l e d a n d i t w a s n o t p o s s i b l e t o d e t e r m i n e w h e t h e r O R F 3 a n d O R F 4 w e r e e x p r e s s e d i n t h e c o m p l e m e n t a t i o n e x p e r i m e n t . H o w e v e r , s e q u e n c e a n a l y s i s s h o w e d t h e p r e s e n c e o f a t e r m i n a t o r u p s t r e a m o f O R F 3 , w h i c h m o s t l i k e l y i n h i b i t e d e x p r e s s i o n o f O R F 3 a n d O R F 4 o f f t h e lac p r o m o t e r i n c l o n e p D B S 2 . A n i n c r e a s e i n M I C f o r b o t h t h e m u t a n t c a r r y i n g t h e v e c t o r p D N 1 8 a n d t h e m u t a n t c a r r y i n g t h e c l o n e p D B S 9 w a s s e e n f o r C P 2 6 . I n t h i s c a s e e x p r e s s i o n o f t h e t e t r a c y c l i n e p u m p ( T e t A ) i n t h e m e m b r a n e o f t h e c e l l m a y h a v e a l l o w e d t h e m u t a n t t o b e c o m e m o r e r e s i s t a n t t o C P 2 6 . A n o v e l g r o u p B. multivorans g e n e s t h a t m a y b e i n v o l v e d i n m a i n t a i n i n g h i g h l e v e l s o f r e s i s t a n c e t o c a t i o n i c p e p t i d e s w a s i d e n t i f i e d . T h e p o s s i b i l i t y O R F 2 m a y h a v e a g l u c o s y l t r a n s f e r a s e f u n c t i o n w a s c o n s i s t e n t w i t h t h e i d e n t i f i c a t i o n o f s u g a r t r a n s f e r t o L P S i n S. typhimurium r e s u l t i n g i n r e s i s t a n c e t o c a t i o n i c p e p t i d e s ( H e l a n d e r et a l . 1 9 9 4 ) . T h e t r a n s f e r o f a n A r a 4 N t o a 4 ' - p h o s p h a t e r e s i d u e o n l i p i d A r e d u c e s t h e n e g a t i v e c h a r g e o f t h e L P S i n S. typhi r e s u l t i n g i n r e d u c e d b i n d i n g o f c a t i o n i c p e p t i d e s t o t h e c e l l ( G u o et a l . 1 9 9 4 ) . H o w e v e r , t h e g e n e s from t h e s e S. typhi s t u d i e s a r e n o t t h e s a m e as t h e o n e s f o u n d i n t h i s s t u d y . It i s p o s s i b l e t h a t t h e p u t a t i v e g l u c o s y l t r a n s f e r a s e o f O R F 2 i n B. multivorans c o u l d b e i n v o l v e d i n t r a n s f e r r i n g a s u g a r r e s i d u e t o a n i n n e r o r o u t e r m e m b r a n e l i p i d . O R F 3 , t h e s i t e o f t h e t r a n s p o s o n i n s e r t i o n i n m u t a n t 2 6 D 7 , w a s s u g g e s t e d t o b e a m e t h y l t r a n s f e r a s e b a s e d o n s e q u e n c e s i m i l a r i t y w i t h o t h e r m e t h y l t r a n s f e r a s e . T h e f u n c t i o n a l l y s t u d i e d m e t h y l t r a n s f e r a s e s t h a t s h o w e d s e q u e n c e s i m i l a r i t y w i t h O R F 3 a r e a l l i n v o l v e d i n t r a n s f e r o f a m e t h y l g r o u p d i r e c t l y t o a p h o s p h a t e r e s i d u e as i n t e r m e d i a t e s t e p s i n b i o s y n t h e s i s p a t h w a y s ( H i d a k a et a l . 1 9 9 5 , K a m i g i r i et a l . 1 9 9 2 ) . O R F 4 a n d O R F 5 h a d n o s i m i l a r i t y t o a n y s t u d i e d p r o t e i n s b u t t h e i r c l o s e p r o x i m i t y t o e a c h o t h e r a n d O R F 3 s u g g e s t s t h a t t h e s e t h r e e g e n e s m a y b e c o - e x p r e s s e d o f f a c o m m o n p r o m o t e r u p s t r e a m o f 9 5 Chapter 4 - Discussion 0RF3. Knock out mutants directed in the four ORFs by allelic exchange would help identify which of the identified genes is important for resistance to cationic peptides or whether all of the genes found downstream of the transposon insertion are important for this phenotype. It would also be interesting to do PCR cloning of these ORFs, promoter regions and terminator to study where the promoter for these genes may be and whether the rho-independent terminator functions as a terminator between ORF2 and ORF3 such that they are expressed as separate genes or whether or not it functions as an attenuator and these genes are actually members of an operon. 96 Chapter 5 - Results Chapter 5 Phenotypic Characterization of Mutant 26D7 5.1 Basic Biochemistry and Microbiology Gram stains of B. multivorans ATCC 17616, mutant 26D7 and randomly selected mutant 41E1 indicated a short Gram-negative rod (not shown). Biochemical tests were also done on B. multivorans ATCC 17616 and compared with mutant 26D7, the vector containing mutant, 26D7+ pDN18 and 26D7+pDBS9. Carbon assimilation tests were done. Al l 12 carbon-containing compounds listed in table 10 were assimilated by B. multivorans. Oxidation of glucose, maltose, lactose, adonitol and sucrose were tested and only sucrose could not be oxidized by B. multivorans ATCC 17616. Lack of sucrose oxidation is typical of genomovar II and genomovar IV strains of the B. cepacia complex. Lysine decarboxylase is found in all genomovars of the B. cepacia complex however, unlike the other genomovars, in general only half of the members of B. multivorans have this enzyme. B. multivorans ATCC 17616 strain was positive for lysine decarboxylase. Nitrate reduction to nitrites was observed and is typical for B. multivorans (Deborah Henry, University of B.C., personal communications). B. multivorans ATCC 17616 was negative for indole production, glucose acidification, urease production, P-galactosidase (esculin and PNPG reactions were negative) and protease (gelitine reaction was negative). No change in biochemistry was shown as a result of the transposon insertion or complementation with pDN18 as the vector as shown by comparison of the parent strain, mutant 26D7, 26D7+pDN18 and 26D7+pDBS9. Al l biochemical results were consistent with those for RAPD type 30 of B. multivorans or genomovar II of the B. cepacia complex (Mahenthiralingam, unpublished results). In particular genomovar II strains do not oxidize or ferment sucrose (Henry et al., 1997). 97 Chapter 5 - Results Table 10. Carbon assimilation and biochemical tests for B. multivorans. A T C C 1 7 6 1 6 M u t a n t 2 6 D 7 2 6 D 7 p D N 1 8 2 6 D 7 p D B S 9 A s s i m i l a t i o n o f : G l u c o s e + + + + A r a b i n o s e + + + + M a n n o s e + + + + M a n n i t o l + + + + N - a c t e y l - + + + + g l u c o s a m i n e M a l t o s e + + + + G l u c o n a t e + + + + + + + + + + C a p r a t e A d i p a t e + + M a l a t e + + + + C i t r a t e + + + + + P h e n y l - a c e t a t e + + + O x i d a t i o n o f : G l u c o s e + + + + M a l t o s e + + + + L a c t o s e + + + + A d o n i t o l + + + + S u c r o s e . . . . L y s i n e d e c a r b o x y l a t i o n + + + + N i t r a t e r e d u c t i o n + + + + I n d o l e p r o d u c t i o n -G l u c o s e a c i d i f i c a t i o n -A r g i n i n e d i h y d r o l a s e -U r e a - - - -E s c u l i n . . . . G e l a t i n e . . . -P N P G * + + + + o x i d a s e + s l o w + s l o w + s l o w + s l o w * j ? - n i t r o p h e n y l - p - D - g a l a c t o p y r a n o s i d e 9 8 Chapter 5 - Results 5.2 Neutrophil Killing Assay T h e in vitro c h r o n i c g r a n u l o m a t o u s d i s e a s e ( C G D ) n e u t r o p h i l k i l l i n g a s s a y w a s p e r f o r m e d f o r b o t h t h e m u t a n t a n d p a r e n t s t r a i n i n t h e p r e s e n c e o f R 5 s e r u m ( c o m p l e m e n t c o m p o n e n t 5 d e f i c i e n t ) . C G D n e u t r o p h i l s w e r e i n c u b a t e d at a r a t i o o f 1:1 w i t h b a c t e r i a . C G D n e u t r o p h i l s w e r e a b l e t o k i l l P. aeruginosa M 2 b y r e d u c i n g t h e l o g c o l o n y f o r m i n g u n i t s b y o n e l o g (6.29 t o 5.25) i n t w o h o u r s . T h e p o s i t i v e c o n t r o l B. multivorans J T C , t h e p a r e n t s t r a i n B. multivorans A T C C 1 7 6 1 6 a n d t h e m u t a n t 2 6 D 7 w e r e a b l e t o g r o w i n t h e p r e s e n c e o f C G D n e u t r o p h i l s as s h o w n i n f i g u r e 19. T h u s m u t a n t 2 6 D 7 r e m a i n e d r e s i s t a n t t o k i l l i n g b y C G D n e u t r o p h i l s . A l l s t r a i n s o f b a c t e r i a w e r e k i l l e d e q u a l l y w e l l b y n o r m a l n e u t r o p h i l s . 5.3 Outer Membrane Permeability N P N i s a h y d r o p h o b i c c o m p o u n d t h a t f l u o r e s c e s w e a k l y i n a h y d r o p h i l i c e n v i r o n m e n t b u t s t r o n g l y i n a h y d r o p h o b i c e n v i r o n m e n t . T h e o u t e r m e m b r a n e o f G r a m - n e g a t i v e b a c t e r i a a c t s as a b a r r i e r t o u p t a k e o f s u c h h y d r o p h o b i c c o m p o u n d s as N P N . P o l y m y x i n B a n d o t h e r p o l y c a t i o n i c c o m p o u n d s c a n i n t e r a c t w i t h t h e d i v a l e n t c a t i o n L P S b i n d i n g s i t e s o f t h e o u t e r m e m b r a n e r e s u l t i n g i n w e a k e n i n g o f t h i s b a r r i e r , a l l o w i n g u p t a k e o f N P N i n t o t h e b a c t e r i a l c e l l m e m b r a n e w h i c h i s m e a s u r e d b y a n i n c r e a s e i n N P N f l u o r e s c e n c e . T h i s h a s b e e n d e m o n s t r a t e d f o r b o t h P. aeruginosa a n d E. coli ( H a n c o c k , 198 4 , L o h , 1 9 8 4 ) . B. cepacia i s r e s i s t a n t t o m e m b r a n e p e r m e a b i l i z a t i o n b y p o l y m y x i n B a n d o t h e r p o l y c a t i o n s ( M o o r e , 1 9 8 6 ) . S i n c e m u t a n t 2 6 D 7 h a d a n 8 - f o l d r e d u c t i o n i n M I C t o p o l y m y x i n c o m p a r e d t o t h e w i l d - t y p e p a r e n t a l s t r a i n i t w a s i m p o r t a n t t o d e t e r m i n e w h e t h e r t h e o u t e r m e m b r a n e p e r m e a b i l i t y o f m u t a n t 2 6 D 7 h a d c h a n g e d . T h e m u t a n t a n d t h e p a r e n t s t r a i n w e r e e x p o s e d t o i n c r e a s i n g c o n c e n t r a t i o n s o f p o l y m y x i n i n t h e p r e s e n c e o f N P N . T h e r e s u l t s i n f i g u r e 2 0 s h o w t h a t t h e p a r e n t s t r a i n w a s r e s i s t a n t t o p e r m e a b i l i z a t i o n b y N P N e v e n i n t h e p r e s e n c e o f h i g h c o n c e n t r a t i o n s o f p o l y m y x i n 9 9 Chapter 5 - Results CGD Neutrophil Killing Assay c • JTC M2 • ATCC17616 —V • 26D7 20 40 60 80 100 120 140 Time in minutes Figure 19. CGD neutrophil killing assay for B. multivorans ATCC 17616 and mutant 26D7. C G D n e u t r o p h i l s w e r e i n c u b a t e d at 3 7 ° C 1:1 w i t h b a c t e r i a a n d s a m p l e s w e r e t a k e n a t 0, 3 0 a n d 1 2 0 m i n u t e s . V i a b l e c o l o n y c o u n t s w e r e d e t e r m i n e d f o r e a c h t i m e p o i n t a n d l o g t o t h e b a s e t e n r e c o r d e d . J T C , B. multivorans s t r a i n J T C ; M 2 , P. aeruginosa s t r a i n M 2 ; A T C C 1 7 6 1 6 , B. multivorans p a r e n t s t r a i n ; 2 6 D 7 , B. multivorans t r a n s p o s o n m u t a n t . J T C i s r e s i s t a n t t o k i l l i n g b y C G D n e u t r o p h i l s a n d w a s u s e d a s t h e p o s i t i v e c o n t r o l . M 2 i s s e n s i t i v e t o k i l l i n g b y C G D n e u t r o p h i l s a n d w a s u s e d a s a n e g a t i v e c o n t r o l . N o d i f f e r e n c e i n k i l l i n g b y C G D n e u t r o p h i l s w a s o b s e r v e d f o r t h e p a r e n t a n d t h e m u t a n t s t r a i n s . 1 0 0 Chapter 5 - Results 100 80 60 40 20 -•— mutant 26D7 O - • parent 17616 o--o- •o •o- •o 10 20 30 P o l y m y x i n (ug/ml) 40 50 Figure 20. The effect of increasing concentrations of polymyxin B on the outer membrane permeability of B. multivorans ATCC 17616 and mutant 26D7. P o l y m y x i n B w a s t i t r a t e d i n t o a c u v e t t e c o n t a i n i n g 1.0 m l o f i n t a c t b a c t e r i a l c e l l s O D 6 0 0 0.5 i n H E P E S b u f f e r ( p H 7.2) c o n t a i n i n g 5 u M C C C P a n d l O u M N P N . N P N f l u o r e s c e s i n a h y d r o p h o b i c e n v i r o n m e n t . A n i n c r e a s e i n c h a n g e i n f l u o r e s c e n c e c o r r e s p o n d s d i r e c t l y t o a n i n c r e a s e i n t h e u p t a k e o f N P N i n t o t h e o u t e r m e m b r a n e . M u t a n t 2 6 D 7 s h o w e d a n i n c r e a s e d p e r m e a b i l i t y t o N P N i n t h e p r e s e n c e o f 1.6 u g / m l p o l y m y x i n w h i l e t h e p a r e n t a l s t r a i n r e m a i n e d i m p e r m e a b l e a t 5 0 u g / m l ( o n e o f t h r e e r e p l i c a t e e x p e r i m e n t s s h o w n ) . 101 Chapter 5 - Results since no increase in fluorescence was observed. In contrast, the mutant was permeable to NPN in the presence of 1.6 ug/ml of polymyxin as shown by the large increase in fluorescence at concentrations of 1.6 u,g/ml and higher. The transposon insertion in mutant 26D7, therefore, had changed the outer membrane permeability such that in the presence of polymyxin, NPN was able to penetrate the bacterial cell. 5.4 Dansyl-polymyxin binding to the Outer Membrane Interaction of dansylated polymyxin (DPX) with LPS has been shown to enhance fluorescence of the fluorescent antibiotic. DPX has been shown to bind purified LPS but not whole cells of B. cepacia (Moore et al., 1986). Since mutant 26D7 is more susceptible to lysis by polymyxin B and more permeable in an NPN assay, it was important to determine whether the mutant was better at binding DPX than the wild-type parent strain. As expected, there was no increase in fluorescence when whole cells of the parent strain ATCC 17616 were exposed to DPX when compared with buffer and DPX alone as shown in figure 21. The mutant 26D7, like the parent strain also showed no increase in fluorescence when exposed to DPX. This suggested that intact cells of the polymyxin susceptible mutant, 26D7 remained incapable of binding DPX. Since DPX is an LPS-binding probe, it could be tentatively concluded that no obvious alteration in LPS had occurred. 5.5 Membrane Proteins Membrane proteins were isolated as described in section 2.17 and separation into inner and outer membrane fractions was attempted on a sucrose gradient. Separation of inner and outer membrane proteins was crude and a clear separation was not seen for the parent and mutant strain. The parent strain ATCC 17616 and mutant 26D7 had a total of five membrane fractions loaded in lanes 2 to 7 and 10, 11 on the gel in figure 22. Samples were alternated for easy 102 Chapter 5 - Results Figure 21. Binding of dansyl-polymyxin (DPX) to intact parent and mutant cells. DPX was titrated into a cuvette containing 1.0 ml of 5 mM HEPES buffer (pH 7.35), 5.0 mM CCCP and cells with an OD 6 0 0 of 0.250. The whole cells of both the mutant and parent B. multivorans did not bind DPX as indicated by the absence of increase in fluorescence by DPX upon addition to cells when compared with the cuvette containing buffer alone. 103 Chapter 5 - Results Figure 22. Outer and inner membrane protein profiles of the parent strain, mutant 26D7, 26D7pDBS9 and 26D7pDN18. O u t e r a n d i n n e r m e m b r a n e p r o t e i n s w e r e p u r i f i e d a s d e s c r i b e d i n s e c t i o n 2.17. T h e m a j o r O M P s o f B. multivorans a r e m a r k e d o n t h e r i g h t . L a n e s a l t e r n a t e b e t w e e n t h e p a r e n t a n d m u t a n t s t r a i n s f o r d i r e c t c o m p a r i s o n . S a m p l e s w e r e n o t e q u a l l y l o a d e d a n d t h e g e l w a s c o o m a s s i e b l u e s t a i n e d . 2 6 D 7 p D N 1 8 a n d 2 6 D 7 p D B S 9 a r e r u n s i d e b y s i d e t o f o r c o m p a r i s o n b e t w e e n c o m p l e m e n t e d m u t a n t a n d m u t a n t w i t h v e c t o r . L a n e 1 c o r r e s p o n d s t o t h e b i o r a d b r o a d r a n g e m a r k e r s d e s c r i b e d i n s e c t i o n 2.19. L a n e 2, 4, 6, 10 a r e t h e p a r e n t A T C C 1 7 6 1 6 ; L a n e 3, 5, 7 a r e m u t a n t 2 6 D 7 ; L a n e 8, 12 a r e 2 6 D 7 p D N 1 8 , L a n e 9, 13 a r e 2 6 D 7 p D B S 9 . A r r o w s p o i n t t o t h e r e g i o n i n t h e g e l w h e r e d i f f e r e n c e s a r e o b s e r v e d . 1 0 4 Chapter 5 - Results 105 Chapter 5 - Results c o m p a r i s o n b e t w e e n m u t a n t a n d p a r e n t s t r a i n s a n d b e t w e e n t h e c o m p l e m e n t e d m u t a n t ( 2 6 D 7 p D B S 9 ) a n d t h e m u t a n t t h a t c a r r i e d t h e v e c t o r a l o n e ( 2 6 D 7 p D N 1 8 ) . C a r e f u l i n s p e c t i o n s u g g e s t e d t h a t t h e r e w a s a m i n o r m e m b r a n e p r o t e i n o f a p p r o x i m a t e l y 5 5 - k D a m i s s i n g i n m u t a n t 2 6 D 7 ( l a n e s 3, 5, 7 a n d 11) a n d p r e s e n t i n t h e p a r e n t ( L a n e s 2, 4, 6 a n d 10) i n d i c a t e d b y a n a r r o w i n f i g u r e 2 2 . T h i s 5 5 - k D a p r o t e i n w a s a b s e n t i n t h e m e m b r a n e f r a c t i o n s o f 2 6 D 7 p D B S 9 ( L a n e 9 a n d 13) a n d 2 6 D 7 p D N 1 8 ( L a n e 8 a n d 12). T h i s m i n o r m e m b r a n e p r o t e i n w a s t h e r i g h t s i z e f o r t h e p u t a t i v e g e n e p r o d u c t o f t h e p u t a t i v e O R F 3 , p r e d i c t e d t o b e 5 4 . 7 - k D a h o w e v e r , f u r t h e r s t u d i e s w o u l d b e r e q u i r e d t o c o n f i r m w h e t h e r t h i s p r o t e i n a c t u a l l y c o r r e s p o n d s t o t h e O R F 3 p r e d i c t e d p r o t e i n p r o d u c t . T h e s e f i n d i n g s a l s o c o n t r a d i c t t h e P S O R T a n d P r o t e i n P r e d i c t r e s u l t s t h a t s u g g e s t e d t h a t O R F 3 e n c o d e d f o r a c y t o p l a s m i c p r o t e i n . T h e c o m p l e m e n t i n g f r a g m e n t i n p D B S 9 d i d n o t c a r r y O R F 3 e x p l a i n i n g w h y b o t h t h e m u t a n t c a r r y i n g t h e v e c t o r p D N 1 8 a n d t h e m u t a n t c a r r y i n g t h e c o m p l e m e n t i n g c l o n e , p D B S 9 b o t h l a c k e d t h e 5 4 . 7 k D a p r o t e i n b a n d i n f i g u r e 22. B e l o w t h e 5 4 . 7 - k D a b a n d t h e r e a p p e a r e d t o b e a m o r e s i g n i f i c a n t l y e x p r e s s e d p r o t e i n f o u n d o n l y i n l a n e s 3, 5, a n d 7 c o r r e s p o n d i n g t o t h e m u t a n t a n d n o t f o u n d i n l a n e s 2, 4 a n d 6 c o r r e s p o n d i n g t o t h e p a r e n t s t r a i n . T h i s w a s i n t h e c o r r e c t s i z e r a n g e f o r f l a g e l l i n p r o t e i n , w h i c h i s 5 0 - k D a i n s i z e . E l e c t r o n m i c r o g r a p h s o f t h e m u t a n t a n d t h e p a r e n t s t r a i n s h o w n i n f i g u r e 2 3 i n d i c a t e d t h a t t h e m u t a n t d i d e x p r e s s f l a g e l l a b u t t h a t t h e p a r e n t s t r a i n d i d n o t . T h i s c o r r e l a t e d w e l l w i t h t h e o b s e r v a t i o n s i n t h e p r o t e i n g e l i n f i g u r e 22. 5.6 Lipopolysaccharide Ladders L P S p r o f i l e s o f t h e p a r e n t s t r a i n , m u t a n t 2 6 D 7 , 2 6 D 7 + p D N 1 8 , 2 6 D 7 + p D B S 9 , r a n d o m m u t a n t 4 1 E 1 , A T C C 1 7 6 1 6 + p T G L 1 6 6 s h o w e d t h a t t h e p r o f i l e s f o r a l l s t r a i n s w e r e t h e s a m e ( f i g u r e 2 4 ) . T h e p a r e n t s t r a i n B. multivorans A T C C 1 7 6 1 6 i s s u s c e p t i b l e t o h u m a n s e r u m a n d d o e s n o t h a v e O - a n t i g e n p r e s e n t o n i t s L P S s o a s e r u m r e s i s t a n t P. aeruginosa P A K w a s i n c l u d e d i n l a n e 7 a n d s h o w e d a c o m p l e t e L P S l a d d e r . T h e c o r e r e g i o n o f t h e B. multivorans s t r a i n s s h o w s a d o u b l e t 1 0 6 Chapter 5 - Results A B Figure 23. Electron microscopy of B. multivorans parent strain and mutant 26D7. Panel A is mutant 26D7 showing the presence of a flagellum as seen on the mutant. One half of the mutant bacteria did carry a flagellum. Panel B is the parent strain showing no flagellum, and none of the parent strain seen had flagella. 107 Chapter 5 - Results ^j^HlfallililliK :^BniiBJi^^ L ^i i l i l i l i l i l i lH^ Wtoffe 66kDa 45kDa 31 kDa 21.5kDa 14.5kDa Figure 24. Lipopolysaccharide profiles for parent strain B. multivorans A T C C 17616 and mutant strains. Whole cell lysates were prepared as described in section 2.18 and silver stained specifically for polysaccharides as described in section 2.19. 1.0 ul of the whole cell lysate for each sample was loaded on a 12.5% polyacrylamide gel except for the P. aeruginosa sample for which 5.0 ul of the whole cell lysate was loaded. L a n e l , 26D7pDBS9; Lane 2, 26D7pDN18; Lane 3, 26D7; Lane 4, 41E1, Lane 5, A T C C 17616pTGL166; Lane 6, A T C C 17616; Lane 7, P. aeruginosa P A K ; Lane 8, broad range protein standards (BioRad). 108 Chapter 5 - Results b a n d as s h o w n i n f i g u r e 24, q u i t e d i f f e r e n t f r o m t h a t o f P. aeruginosa P A K w h i c h s h o w e d t h e t y p i c a l t h r e e b a n d i n g p a t t e r n ( l a n e 7 i n f i g u r e 2 4 ) . 5.7 Thin Layer Chromatography E x t r a c t a b l e l i p i d s w e r e p u r i f i e d f r o m B. multivorans A T C C 1 7 6 1 6 a n d m u t a n t s 2 6 D 7 a n d 4 1 E 1 as d e s c r i b e d i n s e c t i o n 2.21. T w o - d i m e n s i o n a l T L C w a s p e r f o r m e d o n 10 u,g o f e x t r a c t e d l i p i d s a s d e s c r i b e d i n s e c t i o n 2.22. M u t a n t 4 1 E 1 , a r a n d o m m u t a n t s e l e c t e d from t h e t r a n s p o s o n i n s e r t i o n l i b r a r y , d i d n o t h a v e r e d u c e d r e s i s t a n c e t o p o l y m y x i n . P a n e l C w e r e t h e c o n t r o l l i p i d s , c a r d i o l i p i n ( C L ) , p h o s p h a t i d y l g l y c e r o l ( P G ) a n d p h o s p h a t i d y l e t h a n o l a m i n e ( P E ) . M u t a n t 41 E l a n d t h e p a r e n t s t r a i n s h o w e d i d e n t i c a l 2 D T L C p a t t e r n s c o n f i r m i n g t h a t t h e t r a n s p o s o n i n s e r t i o n a l o n e d i d n o t e f f e c t t h e e x t r a c t a b l e l i p i d c o m p o s i t i o n o f B. multivorans ( p a n e l s B a n d D, f i g u r e 25). M u t a n t 2 6 D 7 a p p e a r e d t o h a v e a p o s s i b l e d i f f e r e n t o r c h a n g e d l i p i d b e n e a t h P E - 1 ( m a r k e d as A i n f i g u r e 2 5 ) . S p o t A w a s c o n s i s t e n t l y p r e s e n t i n r e p e a t e d 2 D T L C s o f t h e m u t a n t as s h o w n i n p a n e l A . S p o t A w o u l d o c c a s i o n a l l y a p p e a r i n t h e p a r e n t 2 D T L C a s s h o w n i n p a n e l D, h o w e v e r , i t w a s a l w a y s m u c h l e s s a b u n d a n t w h e n c o m p a r e d t o t h e m u t a n t 2 D T L C . It w a s a l s o i n t e r e s t i n g t h a t t h e P E - 1 s p o t i n t h e m u t a n t a l w a y s a p p e a r e d t o b e l e s s t h a n t h a t f o r t h e p a r e n t s t r a i n ( c o m p a r e p a n e l A a n d B ) e v e n t h o u g h t h e p l a t e s w e r e a l w a y s l o a d e d e q u a l l y w i t h 10 u,g o f t o t a l l i p i d . A l s o , m a r k e d B i n t h e s a m e figure, m u t a n t 2 6 D 7 l a c k e d a n u n k n o w n g l y c o l i p i d . A l l th e l i p i d s i n t h e s e c h r o m a t o g r a m s w e r e i d e n t i f i e d b y c o m p a r i s o n w i t h t h e c o n t r o l l i p i d s ( p a n e l C ) as w e l l a s c o m p a r i s o n w i t h p u b l i s h e d 2 D c h r o m a t o g r a m s ( Y a b u u c h i et a l . , 1 9 9 2 a n d K a w a i et a l . , 1 9 8 8 ) . 1 0 9 Chapter 5 - Results A . M u t a n t 26D7 «>CL P G O i P E - l o ? OOL-2 QPE -2 G. Control lipids 3. Mutant 4IE 1 CL PG 0 GL-X O B O? OL-1 3 » PE-1 « A QPE -2 c?L"2 D. A multivorans ATCC 1761 <3> g L-X O? CL PGV7 Q O L - 1 WPE-1 0 L - 2 O ^ A OPE-2 Figure 25. Two-dimensional thin-layer chromatograms of total extractable lipids. Arrow A points to the new lipid or shifted lipid in mutant 26D7 but not the parent or mutant 41 E l . Arrow B points to the glycolipid that is absent in mutant 26D7. CL, cardiolipin (diphosphotidylglycerol); PG, phosphotidylglycerol; OL-1 and -2, ornithine containing lipids 1 and 2, PE-1 and -2, phosphotidylethanolamine; GL-X, unknown glycolipid; ? Unknown lipid. The solvent in the horizontal direction was chloroform/methanol/water (65:25:4) and in the vertical direction chloroform/methanol/acetic acid (65:25:10). 110 Chapter 5 - Discussion 5.8 Discussion C h r o n i c g r a n u l o m a t o u s d i s e a s e ( C G D ) n e u t r o p h i l s a r e d e p e n d a n t o n n o n o x i d a t i v e k i l l i n g m e c h a n i s m s a n d r e s i s t a n c e o f B. multivorans a n d o t h e r m e m b e r s o f t h e B. cepacia c o m p l e x t o C G D n e u t r o p h i l s w a s c o n s i d e r e d a p o s s i b l e m e c h a n i s m o f p a t h o g e n e s i s . T h e m u t a n t i d e n t i f i e d h e r e w i t h e n h a n c e d s u s c e p t i b i l i t y t o c a t i o n i c p e p t i d e s w a s t e s t e d i n a n e u t r o p h i l k i l l i n g a s s a y t o d e t e r m i n e w h e t h e r C G D n e u t r o p h i l s w e r e c a p a b l e o f k i l l i n g t h i s r e l a t i v e l y s u s c e p t i b l e m u t a n t 2 6 D 7 . T h i s m u t a n t w a s f o u n d t o b e e q u a l l y r e s i s t a n t t o k i l l i n g b y C G D n e u t r o p h i l s a s t h e p a r e n t s t r a i n a n d t h e c o n t r o l B. multivorans J T C s t r a i n . T h i s s u p p o r t e d t h a t t h e M I C o f 8 u,g/ml t o p o l y m y x i n B f o r t h i s m u t a n t w a s s t i l l r e s i s t a n t i n c o m p a r i s o n w i t h o t h e r b a c t e r i a l s t r a i n s s u c h as P. aeruginosa. T h i s c a n b e c o m p a r e d t o t h e 12 s t r a i n s o f P. aeruginosa t e s t e d b y S p e e r t e t a l , 1994, f o r w h i c h a l l s t r a i n s w e r e s u s c e p t i b l e t o k i l l i n g b y C G D n e u t r o p h i l s . S t u d i e s o f t h e e f f e c t o f p o l y m y x i n B o n t h e p e r m e a b i l i t y o f t h e o u t e r m e m b r a n e o f B. multivorans a n d B. cepacia s h o w t h a t t h e s e s t r a i n s o f b a c t e r i a w e r e r e s i s t a n t t o t h e e f f e c t s o f p o l y m y x i n B o n N P N u p t a k e . E v e n at e x t r e m e l y h i g h c o n c e n t r a t i o n s o f p o l y m y x i n B , t h e s e b a c t e r i a l c e l l s ( M o o r e e t a l . 1 9 8 4 ) n e v e r t o o k u p N P N . W h e n t h e m u t a n t s t r a i n w a s t e s t e d i n t h i s s a m e a s s a y f o r o u t e r m e m b r a n e p e r m e a b i l i t y o n l y l o w c o n c e n t r a t i o n s o f p o l y m y x i n B w e r e r e q u i r e d f o r t h e m u t a n t c e l l s t o b e c o m e p e r m e a b i l i z e d b y N P N . T h i s p e r m e a b i l i t y c h a n g e w a s n o t a c c o m p a n i e d b y a n i n c r e a s e d a b i l i t y o f d a n s y l a t e d p o l y m y x i n B t o b i n d i n t a c t m u t a n t c e l l s as i s s e e n i n o t h e r o r g a n i s m s s u c h as P. aeruginosa ( L o h et a l . , 1 9 8 4 ) . N e i t h e r t h e p a r e n t n o r t h e m u t a n t s t r a i n w a s a b l e t o b i n d d a n s y l a t e d p o l y m y x i n B . T h i s s u g g e s t e d t h a t t h e p h e n o t y p i c i n c r e a s e i n p e r m e a b i l i t y o f t h e m u t a n t w a s n o t a c c o m p a n i e d b y a c h a n g e i n L P S t h a t m i g h t a f f e c t b i n d i n g t o p o l y m y x i n B . L P S l a d d e r s w e r e i d e n t i c a l f o r t h e m u t a n t a n d p a r e n t s t r a i n . A m i n o r L P S m o d i f i c a t i o n s u c h as a s i n g l e s u g a r m o d i f i c a t i o n m i g h t n o t b e e a s i l y o b s e r v e d o n t h i s k i n d o f a g e l n o r w o u l d I e x p e c t t o s e e t h e e f f e c t s o f a c h a n g e i n L P S c h a r g e . I l l Chapter 5 - Discussion M e m b r a n e p r o t e i n s w e r e c o m p a r e d f o r t h e m u t a n t a n d p a r e n t s t r a i n s a s w e l l a s t h e c o m p l e m e n t e d m u t a n t . A m i n o r m e m b r a n e p r o t e i n t h a t w a s m i s s i n g i n t h e m u t a n t s t r a i n c o r r e s p o n d e d t o t h e p r e d i c t e d s i z e o f t h e p u t a t i v e p r o t e i n p r o d u c t f r o m O R F 3 o f 54 . 7 - k D a . T h i s c o n t r a d i c t e d t h e P S O R T d a t a a n d P r o t e i n P r e d i c t d a t a t h a t s u g g e s t e d O R F 3 i s a c y t o p l a s m i c p r o t e i n . T h e t r a n s p o s o n h a d i n s e r t e d i n t o t h e c o d i n g r e g i o n o f t h i s O R F s u p p o r t i n g i t s a b s e n c e i n t h e m u t a n t s t r a i n . T h e c o m p l e m e n t e d m u t a n t w a s m i s s i n g t h i s s a m e p r o t e i n w h i c h c o r r e s p o n d s w i t h t h e f a c t t h a t t h i s O R F w a s n o t f o u n d o n t h e D N A c l o n e d i n p D B S 9 . F u r t h e r s t u d i e s a r e r e q u i r e d t o d e t e r m i n e w h e t h e r o r n o t O R F 3 c o r r e s p o n d s t o a m e m b r a n e p r o t e i n a n d w h e t h e r o r n o t i t e x i s t s a s a n i n n e r m e m b r a n e p r o t e i n o r o u t e r m e m b r a n e p r o t e i n . S i m i l a r s t u d i e s w o u l d b e i n t e r e s t i n g f o r t h e O R F 2 g e n e p r o d u c t s i n c e t h i s w a s p r e d i c t e d t o b e a n i n n e r m e m b r a n e p r o t e i n . G o o d s e p a r a t i o n o f i n n e r a n d o u t e r m e m b r a n e p r o t e i n f r a c t i o n s o f B. multivorans w o u l d b e r e q u i r e d . T h i s c o u l d b e d o n e b y s u c r o s e - g r a d i e n t i s o p y c n i c c e n t r i f u g a t i o n , f o l l o w e d b y c o l l e c t i o n o f s e v e r a l f r a c t i o n s f r o m t h a t g r a d i e n t . T o d e t e r m i n e w h i c h s u c r o s e g r a d i e n t f r a c t i o n c o n t a i n s i n n e r o r o u t e r m e m b r a n e p r o t e i n s , t h e f o l l o w i n g e x p e r i m e n t s c o u l d b e d o n e . E a c h f r a c t i o n i s o l a t e d c a n b e t e s t e d f o r N A D H o x i d a s e a c t i v i t y , w h i c h i s a n i n n e r m e m b r a n e m a r k e r . S e p a r a t i o n o f e a c h f r a c t i o n b y S D S - P A G E f o l l o w e d b y t r a n s f e r t o a m e m b r a n e f o r i m m u n o b l o t i n g w i t h a n a n t i b o d y a g a i n s t a n y o n e o f t h e f i v e O M P s i d e n t i f i e d f o r B. mutivorans ( A n w a r et a l . 1 9 9 3 ) c o u l d b e d o n e . T h i s a n d K D O a s s a y s ( s p e c i f i c f o r t h e f r a c t i o n s c o n t a i n i n g L P S ) c o u l d a c t a s o u t e r m e m b r a n e m a r k e r s . D e t e r m i n a t i o n o f w h i c h m a r k e r i s a s s o c i a t e d w i t h w h i c h f r a c t i o n a n d t h e n i d e n t i f i c a t i o n o f w h i c h f r a c t i o n s t h e O R F 2 a n d O R F 3 p r o t e i n s a r e a s s o c i a t e d w o u l d h e l p i d e n t i f y t h e c e l l u l a r l o c a l i z a t i o n o f O R F 2 a n d O R F 3 . T h e e x p r e s s i o n o f f l a g e l l a i n t h e m u t a n t b u t n o t t h e p a r e n t s t r a i n w a s p r o b a b l y d u e t o t h e p r e s e n c e o f t h e t r a n s p o s o n a n d t h e m u t a g e n e s i s m e t h o d s b e c a u s e a r a n d o m l y c h o s e n m u t a n t a l s o e x p r e s s e d f l a g e l l a . R e g u l a t i o n o f p r o m o t e r t r a n s c r i p t i o n o f f l a g e l l a b i o s y n t h e s i s i s c o m p l e x a n d 112 Chapter 5 - Discussion i s o r g a n i z e d i n t o a t r a n s c r i p t i o n a l h i e r a r c h y o f t h r e e c l a s s e s o f g e n e s a n d s e v e r a l s i g m a f a c t o r s a r e i n v o l v e d . E a c h l a t e r c l a s s o f g e n e s r e q u i r e s a l t h e g e n e s o f t h e p r e v i o u s c l a s s t o b e e x p r e s s e d . C l a s s 1 g e n e s a r e r e g u l a t e d i n r e s p o n s e t o n u m e r o u s e n v i r o n m e n t a l s t i m u l i s u c h a s c A M P l e v e l s a n d t e m p e r a t u r e . T h e s e g e n e p r o d u c t s i n t e r a c t t o a c t i v a t e c l a s s 2 p r o m o t e r s , w h i c h e n c o d e t h e p r o t e i n s n e e d e d f o r a s s e m b l y o f t h e b a s a l b o d y - h o o k p l u s a n o t h e r r e g u l a t o r y g e n e jliA. T h i s g e n e p r o d u c t e n c o d e s f o r a n a l t e r n a t i v e a f a c t o r r e q u i r e d f o r t r a n s c r i p t i o n from C l a s s 3 p r o m o t e r s . T h e s e a r e t h e g e n e s r e q u i r e d f o r t h e f i n a l s t a g e s o f f l a g e l l a r a s s e m b l y a n d t h e c h e m o t a c t i c s i g n a l t r a n s d u c t i o n p a t h w a y ( H u g h e s et a l . 1 9 9 8 ) . T h e m o s t l i k e l y e x p l a n a t i o n f o r flagellin e x p r e s s i o n i n m u t a n t 2 6 D 7 a n d t h e r a n d o m l y s e l e c t e d m u t a n t , i s t h a t t h e s e m u t a n t s u n d e r w e n t a g e n e t i c m o d i f i c a t i o n at e l e v a t e d t e m p e r a t u r e s s i n c e d u r i n g t h e m u t a g e n e s i s p r o c e d u r e t h i s s t r a i n w a s i n c u b a t e d at 4 7 ° C f o r 6 0 h o u r s . It a p p e a r e d t h a t w h e n t h e 2 D T L C s o f t h e p a r e n t a n d m u t a n t s t r a i n w e r e c o m p a r e d , t h e r e m i g h t b e a s h i f t i n P E - 1 i n t h e m u t a n t o r p o s s i b l y a d i f f e r e n t l i p i d s u c h t h a t i t d o e s n o t t r a v e l as f a r i n t h e s e c o n d s o l v e n t . I f a s u g a r c o u l d n o t b e t r a n s f e r r e d o n t o e i t h e r P E - 1 o r a l i p i d t h a t t r a v e l s t o t h e s a m e s p o t , t h e m o b i l i t y o f t h i s l i p i d w o u l d c h a n g e . A s w e l l , t h e r e w a s a n u n k n o w n g l y c o l i p i d m i s s i n g i n t h e m u t a n t T L C w h e n c o m p a r e d w i t h t h e p a r e n t T L C . T h i s l i p i d w a s i d e n t i f i e d a s a n u n k n o w n g l y c o l i p i d b y Y a b u u c h i et a l , 1 9 9 2 u s i n g 2 D T L C a n d w a s n o t s e e n i n B. mallei, B. gladioli o r B. solanacearum. I n a n o t h e r s t u d y , a n u n k n o w n g l y c o l i p i d w a s a l s o s e e n i n 2 D T L C u n i q u e t o B. cepacia w h e n c o m p a r e d w i t h P. aeruginosa, P. Fluorescens a n d P. stutzeri ( K a w a i e t a l , 1 9 8 8 ) . T h e s e o b s e r v a t i o n s s u g g e s t t h a t a u n i q u e l i p i d m a y e x i s t i n t h e m e m b r a n e o f B. cepacia t h a t i s g l y c o s y l a t e d a n d m a y b e a b s e n t i n t h e m u t a n t i d e n t i f i e d i n t h i s s t u d y . O t h e r s t u d i e s h a v e s h o w n t h a t B. cepacia m e m b r a n e s c o n t a i n a p p r o x i m a t e l y 4 t i m e s l e s s p h o s p h a t i d y l g l y c e r o l t h a n P. aeruginosa a n d t h a t B. cepacia c o n t a i n s a n ' o r n i t h i n e c o n t a i n i n g ' l i p i d a n d a g l y c o l i p i d n o t f o u n d i n P. aeruginosa ( K a w a i et a l . 1 9 9 1 ) . M a s s s p e c t r o p h o t o m e t r y 113 Chapter 5 - Discussion o f a l l t h e s p o t s o n t h e T L C p l a t e s w o u l d g i v e a m o r e d e f i n i t i v e a n s w e r a s t o w h e t h e r t h e s e l i p i d d i f f e r e n c e s i n t h e m u t a n t a r e s i g n i f i c a n t a n d h o w t h e y r e l a t e t o t h e p h e n o t y p e o f r e d u c e d r e s i s t a n c e t o c a t i o n i c p e p t i d e s . M u t a n t 2 6 D 7 h a s a r e d u c e d M I C t o p o l y m y x i n B b u t w a s s t i l l r e s i s t a n t i n t h e C G D n e u t r o p h i l a s s a y , s h o w e d n o c h a n g e i n L P S l a d d e r a n d n o c h a n g e i n L P S b i n d i n g o f D P X . T h e m u t a n t d i d s h o w a c h a n g e i n m e m b r a n e p e r m e a b i l i t y a n d a c h a n g e i n l i p i d p r o f i l e o n a T L C p l a t e . T h i s s u g g e s t e d t h a t t h e p e r m e a b i l i t y c h a n g e o b s e r v e d i n t h e N P N a s s a y a n d t h e r e d u c e d M I C t o p o l y m y x i n B m a y b e d u e t o a l i p i d c h a n g e o n t h e i n n e r o r o u t e r m e m b r a n e , p e r h a p s i n d e p e n d e n t o f L P S . T h e p r o p o s e d m e c h a n i s m o f a c t i o n o f c a t i o n i c p e p t i d e s o n G r a m - n e g a t i v e b a c t e r i a h a s b e e n p r o p o s e d t o i n v o l v e , i n o r d e r , L P S b i n d i n g , o u t e r m e m b r a n e p e r m e a b i l i z a t i o n a n d t h e n c y t o p l a s m i c m e m b r a n e p e r m e a b i l i z a t i o n . T h e o b s e r v a t i o n s h e r e a r e c o n s i s t e n t w i t h t h e c o n c l u s i o n t h a t t h e m u t a t i o n i n m u t a n t 2 6 D 7 h a s a f f e c t e d t h e m e m b r a n e p e r m e a b i l i z a t i o n step. A s e c o n d m u t a t i o n i n t h e L P S o f B. multivorans t o i n c r e a s e t h e b i n d i n g a b i l i t y o f c a t i o n i c p e p t i d e s m a y b e r e q u i r e d f o r f u l l s u s c e p t i b i l i t y . P e r h a p s t h e a b i l i t y o f C E M E t o e n t e r t h e b a c t e r i a l c e l l i s m o r e d e p e n d e n t o n i t s e n h a n c e d a b i l i t y t o b i n d t o t h e L P S o f i n t a c t c e l l s t h a n C P 2 6 . P e r h a p s , a c h a n g e i n b i n d i n g t o L P S w o u l d h a v e b e e n r e q u i r e d t o s e e a c h a n g e i n M I C f o r C E M E a n d C P 2 6 , r a t h e r t h a n o n l y C P 2 6 . 114 Chapter 6 - Discussion Chapter 6 General Discussion T r a n s p o s o n i n s e r t i o n a l m u t a g e n e s i s w a s e m p l o y e d h e r e t o c o n s t r u c t a m u t a n t o f Burkholderia multivorans r e n d e r e d s u s c e p t i b l e t o k i l l i n g b y c a t i o n i c p e p t i d e s . O n l y t h r e e t r a n s p o s o n i n s e r t i o n s y s t e m s h a d b e e n s h o w n t o b e s u c c e s s f u l i n p r o d u c i n g m u t a n t l i b r a r i e s o f B. cepacia c o m p l e x o r g a n i s m s at t h a t t i m e ( J a y a s w a l et a l . , 1 9 9 1 , P i d c o c k e t a l . , 1 988, C h e n g , et a l . , 1 9 9 4 ) . A l l o f t h e B. cepacia s t r a i n s t h a t w e r e t e s t e d as c a n d i d a t e s f o r t r a n s p o s o n m u t a g e n e s i s s h o w e d h i g h l e v e l s o f r e s i s t a n c e t o a m i n o g l y c o s i d e s . B o t h T n 5 ( P i d c o c k et a l . , 1 9 8 8 ) a n d T n 5 -2 5 9 ( J a y a s w a l et a l . , 1 9 9 1 ) , c a r r y t h e k a n a m y c i n r e s i s t a n c e c a s s e t t e a s t h e s e l e c t a b l e m a r k e r a n d w e r e t h e r e f o r e n o t u s e f u l f o r t h e s t r a i n s i n t h i s s t u d y . T h e m e t h o d o f C h e n g et a l , 1 9 9 4 w a s e m p l o y e d h e r e b e c a u s e t h e v e c t o r c a r r i e d a t e t r a c y c l i n e c a s s e t t e a n d t h e t r a n s p o s o n c a r r i e d a t r i m e t h o p r i m c a s s e t t e b o t h a n t i b i o t i c s o f w h i c h A T C C 1 7 6 1 6 , t h e s t r a i n c h o s e n i n t h i s s t u d y , w a s s u s c e p t i b l e t o . O t h e r t r a n s p o s o n m u t a g e n e s i s s y s t e m s t r i e d w e r e t h e m i n i - t n 5 t r a n s p o s o n d e r i v a t i v e s o f d e L o r e n z o et a l . , 1 9 9 0. A f t e r s e v e r a l d i f f e r e n t a t t e m p t s at m u t a g e n e s i s o n e l i b r a r y w a s c o n s t r u c t e d i n B. multivorans s t r a i n A T C C 1 7 6 1 6 . T w o o t h e r c l i n i c a l s t r a i n s a l s o h a d s u s c e p t i b i l i t y t o t r i m e t h o p r i m a n d t e t r a c y c l i n e b u t t r a n s p o s o n i n s e r t i o n n e v e r w o r k e d . T r a n s p o s o n m u t a g e n e s i s i s k n o w n t o b e e x t r e m e l y d i f f i c u l t i n B. cepacia c o m p l e x o r g a n i s m s a n d s u c c e s s f u l a t t e m p t s h a v e o n l y b e e n d o c u m e n t e d t w i c e s i n c e t h e s t a r t o f t h i s p r o j e c t ( A b e et a l . , 1 9 9 6 a n d L e w e n z a et a l . , 1 9 9 9 ) . S c r e e n i n g o f 6,000 m u t a n t s f r o m t h e B. multivorans A T C C 1 7 6 1 6 i n s e r t i o n l i b r a r y r e s u l t e d i n t h e i d e n t i f i c a t i o n o f 4 0 p u t a t i v e p o l y m y x i n s u s c e p t i b l e m u t a n t . M u t a n t s i d e n t i f i e d i n t h e s c r e e n w e r e t e s t e d i n a s t a n d a r d M I C a s s a y . O n l y 8 s h o w e d a s i g n i f i c a n t r e d u c t i o n i n M I C t o p o l y m y x i n ( g r e a t e r t h a n 2 - f o l d ) w h e n c o m p a r e d t o t h e p a r e n t s t r a i n a n d o n l y o n e m u t a n t ( 2 6 D 7 ) w a s s e l e c t e d f o r f u r t h e r c h a r a c t e r i z a t i o n . It w o u l d b e i n t e r e s t i n g t o s e q u e n c e t h e D N A at t h e 115 Chapter 6 - Discussion t r a n s p o s o n i n s e r t i o n s i t e i n t h o s e o t h e r m u t a n t s i d e n t i f i e d i n F i g u r e 7. T h i s w o u l d c o n f i r m w h e t h e r t h o s e m u t a n t s t h a t a p p e a r e d t o h a v e t h e t r a n s p o s o n i n s e r t e d i n t o t h e s a m e s i t e as 2 6 D 7 b y S o u t h e r n a n a l y s i s w e r e s i b l i n g s o f 2 6 D 7 , n o v e l i n s e r t i o n s i n t h e s a m e r e g i o n o f D N A o r n o v e l i n s e r t i o n s i n c o m p l e t e l y d i f f e r e n t s i t e s o n t h e c h r o m o s o m e o f B. multivorans. A t t h e t i m e o f i n s e r t i o n a l m u t a g e n e s i s , t h e c e l l s w e r e g r o w n f o r 6 0 h o u r s at 4 7 ° C i n o r d e r t o s e e l o s s o f t h e p l a s m i d p T G L 1 6 6 . T h i s a l l o w e d f o r t h e f o r m a t i o n o f s i b l i n g s t o b e a l i k e l y e v e n t . S i n c e t h e r a t e o f s p o n t a n e o u s m u t a g e n e s i s i n B. cepacia c o m p l e x o r g a n i s m s i s 2 i n l x l 0 6 c e l l s g r o w n i n t h e p r e s e n c e o f s e l e c t i o n p r e s s u r e i t i s n o t l i k e l y t h a t t h e m u t a n t s i n t h i s s t u d y a r e a r e s u l t o f s p o n t a n e o u s m u t a g e n e s i s ( v a n W a a s b e r g e n et a l . 1 9 9 8 ) . T h e m u t a n t s i n t h i s s t u d y w e r e s e l e c t e d b y s c r e e n i n g a n d n e v e r s a w s e l e c t i o n p r e s s u r e . O n e m u t a n t o u t o f 6,000 w a s s e l e c t e d f o r s t u d y a n d i t i s u n l i k e l y t h a t t h i s m u t a n t i s a r e s u l t o f a s p o n t a n e o u s m u t a t i o n at a n o t h e r s i t e i n t h e g e n o m e . I n o r d e r t o t r y a n d l i n k t h e p h e n o t y p i c c h a n g e i n m u t a n t 2 6 D 7 t o t h e t r a n s p o s o n i n s e r t i o n , c o m p l e m e n t a t i o n o f t h e m u t a n t p h e n o t y p e w a s a t t e m p t e d . S i n c e e l e c t r o p o r a t i o n e x p e r i m e n t s f a i l e d w i t h s t r a i n A T C C 1 7 6 1 6 a m o b i l i z a b l e v e c t o r w a s r e q u i r e d . V e c t o r p D N 1 8 w a s c h o s e n b e c a u s e i t c a r r i e d a t e t r a c y c l i n e c a s s e t t e , t h e o n l y a n t i b i o t i c t o w h i c h 2 6 D 7 w a s s e n s i t i v e . T w o s e p a r a t e f r a g m e n t s w e r e c l o n e d i n t o p D N 1 8 , o n e c a r r y i n g O R F 1 a n d O R F 2 a n d t h e o t h e r f r a g m e n t c a r r i e d p a r t o f O R F 2 , O R F 3 , O R F 4 a n d p a r t o f O R F 5 . O R F 1 a n d O R F 2 a p p e a r e d t o c o m p l e m e n t t h e m u t a n t i n a n M I C a s s a y w i t h p o l y m y x i n h o w e v e r t h e t r a n s p o s o n i n s e r t i o n w a s w i t h i n O R F 3 a n d t h e f r a g m e n t c o n t a i n i n g t h i s O R F d i d n o t c o m p l e m e n t t h e m u t a t i o n i n 2 6 D 7 . S i n c e i t w a s n o t p o s s i b l e t o m a k e a d i r e c t c o n c l u s i o n from t h e s e c o m p l e m e n t a t i o n e x p e r i m e n t s o t h e r m e t h o d s w i l l h a v e t o b e e m p l o y e d t o t r y a n d c o n f i r m t h a t t h e t r a n s p o s o n i n s e r t i o n c a u s e d t h e p h e n o t y p i c c h a n g e i n m u t a n t 2 6 D 7 . A l l e l i c e x c h a n g e i s k n o w n t o b e d i f f i c u l t i n B. cepacia c o m p l e x o r g a n i s m s a n d h a s o n l y j u s t b e e n r e p o r t e d i n t h e l i t e r a t u r e f o r t h e f i r s t t i m e ( L e w e n z a , 1 1 6 Chapter 6 - Discussion S. et a l . 1 9 9 9 ) u s i n g t h e s u i c i d e v e c t o r p E X 1 8 T c . W i t h a v a i l a b i l i t y o f t h i s v e c t o r , i t m a y b e p o s s i b l e t o t r y a l l e l i c e x c h a n g e i n 1 7 6 1 6 t o k n o c k o u t t h e p u t a t i v e c e r a m i d e g l u c o s y l t r a n s f e r a s e , p u t a t i v e m e t h y l t r a n s f e r a s e o r a n y o f t h e p u t a t i v e g e n e s d o w n s t r e a m o f t h e t r a n s p o s o n i n s e r t i o n . K n o c k o u t o f t h e s e d i f f e r e n t g e n e s w o u l d a i d i n i d e n t i f y i n g e x a c t l y w h i c h o f t h e O R F s i d e n t i f i e d n e a r t h e t r a n s p o s o n i n s e r t i o n w a s r e q u i r e d f o r c a t i o n i c p e p t i d e r e s i s t a n c e , N P N p e r m e a b i l i t y a n d c h a n g e s i n T L C p a t t e r n s . T h e t r a n s p o s o n h a d i n s e r t e d i t s e l f i n t o O R F 3 , w h i c h w a s u p s t r e a m o f s e v e r a l c l o s e l y l i n k e d g e n e s w i t h t h e p u t a t i v e r i b o s o m e b i n d i n g s i t e s f o r t h e s e O R F s o v e r l a p p i n g w i t h t h e u p s t r e a m c o d i n g r e g i o n s . F u r t h e r s t u d i e s t o d e t e r m i n e w h e t h e r o r n o t a p r o m o t e r r e g i o n i s u p s t r e a m o f O R F 3 w o u l d b e r e q u i r e d . T h e s e c o n c e r n s c o u l d b e a d d r e s s e d b y c l o n i n g p o t e n t i a l p r o m o t e r r e g i o n s n e x t t o a r e p o r t e r g e n e , s u c h as lacZ, h o w e v e r t h e s e e x p e r i m e n t s w o r k b e s t w i t h c o n s t i t u t i v e l y a c t i v e p r o m o t e r s w h i c h m a y n o t b e t h e c a s e h e r e . It w o u l d a l s o b e i n t e r e s t i n g t o d o N o r t h e r n b l o t a n a l y s i s t o d e t e r m i n e t h e l e n g t h o f t h e m R N A t r a n s c r i p t t h a t t h e O R F 3 g e n e i s o n a n d w h i c h o f t h e g e n e s ( u s i n g a d i f f e r e n t p r o b e s p e c i f i c f o r e a c h g e n e ) n e a r t h e t r a n s p o s o n i n s e r t i o n a r e f o u n d o n t h i s t r a n s c r i p t . T o d e t e r m i n e i f O R F 2 i s c o - e x p r e s s e d w i t h O R F 3 a n d t h e d o w n s t r e a m g e n e s , o n e c o u l d d e l e t e o r k n o c k o u t b y a l l e l i c e x c h a n g e t h e p r o m o t e r r e g i o n u p s t r e a m o f O R F 2 a n d l o o k f o r c h a n g e s i n O R F 3 e x p r e s s i o n o n a p r o t e i n g e l . S i n c e c l o n i n g o f t h e g e n e s i n t h i s r e g i o n p r o v e d t o b e e x t r e m e l y d i f f i c u l t a n d t h e r e s u l t s h e r e s u g g e s t e d t h a t t h e s e g e n e s e x p r e s s e d o f f a v e c t o r m a y b e l e t h a l , t h e s e c l o n i n g e x p e r i m e n t s c o u l d b e d i f f i c u l t t o p e r f o r m . T h e G C c o n t e n t o f O R F 3 at 6 2 % w a s c o n s i d e r a b l y l o w e r t h a n t h a t e x p e c t e d f o r B. multivorans, w h i c h h a s a n a v e r a g e G C c o n t e n t o f 7 2 % . T h i s s u g g e s t s p o s s i b l e g e n e t r a n s f e r o f t h i s m e t h y l t r a n s f e r a s e f r o m a n o t h e r o r g a n i s m w i t h a l o w e r G C c o n t e n t , h o w e v e r t h e c o d o n u s a g e i n t h i s O R F w a s f o u n d t o b e v e r y s i m i l a r t o t h a t e x p e c t e d B. cepacia. I S e l e m e n t s e q u e n c e s a r e 11 7 Chapter 6 - Discussion u s u a l l y a s s o c i a t e d w i t h r e c e n t l y t r a n s f e r r e d g e n e s o n t h e o u t s i d e e n d s o f t h e g e n e s e q u e n c e . It w a s o b s e r v e d t h a t a m e t h y l t r a n s f e r a s e i s f o u n d d o w n s t r e a m o f a c e r a m i d e g l u c o s y l t r a n s f e r a s e i n T. ferroxidans b u t n o n e o f t h e o t h e r o r g a n i s m s i n t h e p h y l o g e n e t i c t r e e ( F i g . 15). W i t h t h e c o m p l e t i o n o f m o r e g e n o m e s e q u e n c i n g p r o j e c t s i t w i l l b e i n t e r e s t i n g t o s e e w h e t h e r i d e n t i f i e d c e r a m i d e g l u c o s y l t r a n s f e r a s e s a r e f o u n d i n c l o s e p r o x i m i t y t o m e t h y l t r a n s f e r a s e s . A t t h i s p o i n t t h e m o s t c o m p e l l i n g e v i d e n c e t o s u p p o r t t h a t t h e t r a n s p o s o n i n s e r t i o n i s t h e c a u s e o f t h e p h e n o t y p i c c h a n g e i n m u t a n t 2 6 D 7 i s t h a t t h e i d e n t i f i e d g e n e s e n c o d e f o r p r o t e i n s t h a t p r o b a b l y p l a y a r o l e i n l i p i d m o d i f i c a t i o n . O R F 2 e n c o d e d f o r a p r e d i c t e d p r o t e i n w i t h s e q u e n c e s i m i l a r i t y t o c e r a m i d e g l u c o s y l t r a n s f e r a s e , w h i c h i s k n o w n t o b e i m p o r t a n t f o r t h e t r a n s f e r o f a g l u c o s e r e s i d u e t o a c e r a m i d e , a l i p i d f o u n d o n l y i n e u k a r y o t e s . O R F 3 , a p u t a t i v e p h o s p h o m e t h y l t r a n s f e r a s e c o u l d p o t e n t i a l l y b e i n v o l v e d i n t r a n s f e r r i n g a m e t h y l g r o u p t o p h o s p h a t e r e s i d u e o n a l i p i d . T h e m o d i f i c a t i o n o f l i p i d s , s p e c i f i c a l l y L P S , h a s a l r e a d y b e e n s h o w n t o b e i m p o r t a n t f o r r e s i s t a n c e t o c a t i o n i c p e p t i d e s . T h e t r a n s f e r o f a s u g a r r e s i d u e t o a p h o s p h a t e r e s i d u e i n L P S c a u s e s r e s i s t a n c e t o c a t i o n i c p e p t i d e s i n S. typhimurium ( G u o et a l . 1 9 9 8 ) . A d d i t i o n o f a p a l m i t a t e t o L P S i n b o t h & typhimurium ( G u o et a l . 1 9 9 7 ) a n d P. aeruginosa ( E r n s t et l a . 1 9 9 9 ) r e s u l t i n g i n a h e p t a - a c y l a t e d f o r m o f L P S , i n c r e a s e s t h e r e s i s t a n c e o f b o t h t h e s e o r g a n i s m s t o c a t i o n i c p e p t i d e s . I n t w o c l o s e l y r e l a t e d o r g a n i s m s , k n o c k o u t o f L P S s t r u c t u r a l g e n e s h a s b e e n a s s o c i a t e d w i t h l o s s o f r e s i s t a n c e t o c a t i o n i c p e p t i d e s . I n R. solanocearum a h e p t o s y l t r a n s f e r a s e (rfaA) w a s i d e n t i f i e d a s b e i n g i n v o l v e d i n r e s i s t a n c e t o c a t i o n i c p e p t i d e s f r o m p l a n t s . T h e rfaA g e n e i s i n v o l v e d i n L P S c o r e b i o s y n t h e s i s . I n B. pseudomallei i t h a s a l s o b e e n s h o w n t h a t g e n e s i n v o l v e d i n L P S c o r e o l i g o s a c c h a r i d e b i o s y n t h e s i s a r e i m p o r t a n t f o r r e s i s t a n c e t o c a t i o n i c p e p t i d e s ( B u r t n i c k et a l . , 1 9 9 9 ) . T h i s g r o u p a l s o i d e n t i f i e d a s e c o n d g r o u p o f g e n e s t h a t a f f e c t t h e lytB g e n e , t h e p r o d u c t o f w h i c h i s k n o w n t o a f f e c t p h o s p h o l i p i d a n d p e p t i d o g l y c a n s y n t h e s i s . T h e d a t a c o l l e c t e d h e r e s u g g e s t s t h a t t h e 1 1 8 Chapter 6 - Discussion a r c h i t e c t u r e o f t h e B. multivorans c e l l p r e v e n t e d c a t i o n i c p e p t i d e s f r o m i n t e r a c t i n g w i t h t a r g e t g r o u p s o f e i t h e r L P S o r p h o s p h o l i p i d s . T h i s w a s s e e n b y t h e c h a n g e i n o u t e r m e m b r a n e p e r m e a b i l i t y o f t h e m u t a n t a s s e e n i n t h e N P N a s s a y a n d t h e c h a n g e s s e e n i n t h e l i p i d p r o f i l e s o f t h e m u t a n t w h e n c o m p a r e w i t h t h e p a r e n t b y 2 D T L C . F u r t h e r c h a r a c t e r i z a t i o n o f t h e s e l i p i d c h a n g e s w i l l b e r e q u i r e d t o d e t e r m i n e t h e e x a c t l i p i d c h a n g e i n t h e m u t a n t . M a s s s p e c t r o p h o t o m e t r y o f t h e s p o t s o n t h e T L C p l a t e s w i l l a i d i n i d e n t i f y i n g t h e e x a c t c h a n g e t h a t h a s o c c u r r e d i n t h e m u t a n t . 1 1 9 References References A b e , M., a n d N a k a z a w a , T. ( 1 9 9 6 ) T h e dsbB g e n e p r o d u c t i s r e q u i r e d f o r p r o t e a s e p r o d u c t i o n b y Burkholderia cepacia. I n f e c t , a n d I m m u n . 64:4378-4380. 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( 1 9 9 4 ) I n t e r a c t i o n s b e t w e e n h u m a n d e f e n s i n s a n d l i p i d b i l a y e r s : e v i d e n c e f o r f o r m a t i o n o f m u l t i m e r i c p o r e s . P r o t e i n S c i . 3:1362-1373 W i s e , M., S h i m k e t s , L . J., a n d M c A r t h u r , J.V. ( 1 9 9 5 ) G e n e t i c s t r u c t u r e o f a l o t i c p o p u l a t i o n o f Burkholderia (Pseudomonas) cepacia. A p p l i e d a n d E n v i r o n . M i c r o b i o l . 61:1791-1798. W o o d , M.S., L o r y , C , a n d L e s s i e , T .G. ( 1 9 9 0 ) A c t i v a t i o n o f t h e lac g e n e s o f T n P 5 7 b y i n s e r t i o n s e q u e n c e s f r o m Pseudomonas cepacia. J. B a c t e r i o l . 172:1719-1724. 135 References Y a b u u c h i , E., K o s a k o , Y., O y a i z u , H., Y a n o , I., H o t t a , H., H a s h i m o t o , Y., E z a k i , T., a n d A r a k a w a , M . 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U S A 84:5449-5453. Z h a o , C , W a n g , I., a n d L e h r e r , R.I. ( 1 9 9 6 ) W i d e s p r e a d e x p r e s s i o n o f b e t a - d e f e n s i n h B D - 1 i n h u m a n s e c r e t o r y g l a n d s a n d e p i t h e l i a l c e l l s . F E B S L e t t . 396:319-322. Z h a o , C , L i a w , L., L e e , I.H., a n d L e h r e r , R.I. ( 1 9 9 7 ) c D N A c l o n i n g o f t h r e e c e c r o p i n - l i k e a n t i m i c r o b i a l p e p t i d e s ( S t y e l i n s ) f r o m t h e t u n i c a t e , Styela clava. F E B S L e t t . 412:144-148. Z u g h a i e r , S.M., R y l e y , H . C , a n d J a c k s o n , S.K. ( 1 9 9 9 a ) L i p o p o l y s a c c h a r i d e ( L P S ) f r o m Burkholderia cepacia i s m o r e a c t i v e t h a n L P S f r o m Pseudomonas aeruginosa a n d Stenotrophomonas maltophilia i n s t i m u l a t i n g t u m o r n e c r o s i s f a c t o r a l p h a from h u m a n m o n o c y t e s . I n f e c t , a n d I m m u n . 67:1505-1507. Z u g h a i e r , S.M., R y l e y , H.C. a n d J a c k s o n , S.K. ( 1 9 9 9 b ) A m e l a n i n p i g m e n t p u r i f i e d from a n e p i d e m i c s t r a i n o f Burkholderia cepacia a t t e n u a t e s m o n o c y t e r e s p i r a t o r y b u r s t a c t i v i t y b y s c a v e n g i n g s u p e r o x i d e a n i o n . I n f e c t , a n d I m m u n . 67: 9 0 8 - 9 1 3 . 13 6 Appendix A - Media APPENDIX A: MEDIA Luria Britani Broth and Agar NaCl lO.Og Bacto-yeast extract 5.0g Bacto-tryptone lO.Og make up to 1.0 Litre with distilled water add 15.0 g granulated agar for solid media sterilize by autoclaving Mueller-Hinton Broth soluble starch 1.5g casamino acids 17.5g Beef Infusion 300.0g make up to 1.0 Litre with distilled water sterilize by autoclaving SOB Bacto-tryptone 20.0g Bacto-yeast extract 5.0g NaCl 0.5g make up to 1.0 Litre with distilled water sterilize by autoclaving Minimal Salts (10X) dipotassium orthophosphate (K 2 HP0 4 ) 105.0g potassium phosphate (KH 2 P0 4 ) 45.Og ammonium suphate lO.Og sodium citrate 5.0g make up to 1.0 Litre with distilled water sterilize by autoclaving Supplemented Minimal Salts Media Minimal Salts 100.0ml Casamino Acids (2.0%) 10.0ml Mannitol (5.0%) 100.0ml Distilled water 780.0ml Agar 15.0g mix agar and distilled water sterilize by autoclaving cool to 56°C add sterile minimal salts, casamino acids and mannitol warmed to 56°C to the sterilized water-agar. 137 Appendix B - Table of Primers APPENDIX B: TABLE OF PRIMERS Table 11. Primer sequences. PRIMER SEQUENCE Primers specific for sequencing off the end of Tn5- 751 Tn5-1 5' -GAG-GTC-AC A-TGG-AAG-TC A-G-3' DS-1 5' -GTC-AC A-TGG-AAG-TC A-GAT-C-3' Neomycin (kanamycin) cassette primers N727-3 N725-5 5' - ATC-GAC-AAG-ACC-GGC-TTC-C AT-CCG-A-3' 5' -TC A-GCG-C AG-GGG-CGC-CCG-GTT-CTT-T-3' Tetracycline cassette primers Tet-1 Tet-2 5' -C AC-C AA-CTT-ATC-AGT-G AT-A AA-G A-3' 5' - AGT-TGC-ATG-ATG-AAG-A AG-AC-3' Sequencing primers SeqlR SeqlL Seq2L Seq2R RI R2 R3 R4 R5 Bam-1 R6 R7 LR-1 LR-2 LR-3 LR-4 RL-1 RL-2 RL-5 Bam-2 RL-6 RL-7 B2 LR-5 LR-6 5'-GCG-CTT-CTT-ATG-CTC-GAT-3' 5'-ACT-AGC-TCG-CCG-ACG-ATT-3' 5' -GTT-CGG-TAC-GC A-GGT-CGT-CT-3' 5 '-GCC-TGC-ATA-GTT-CGA-TTC-GT-3' 5'-ATC-GAA-CTA-TGC-AGG-CTA-CC-3' 5' -GCG-TCC-GAT-CCT-CGA-G AA-C A-3' 5' - AGA-CGC-TGA-AGG-TGA-TGA-AG-3' 5' - ATC-ACC-ATC-TCG-AGC-AGT-TC-3' 5' -GTT-GCG-ATC-C AC-C AC-AG A-3' 5' - AAG-TAG-CGC-AGC-AGT-TCG-3' 5' - AAC-GCC-GCG-AGC-CTG-ATG-3' 5'-ACA-GCA-GCG-CGT-CGA-GTT-3' 5 '-ATC-CGC-ACA-ACT-CGC-TGA-CG-3' 5 '-ACC-GTG-CTG-TCG-ATG-ATC-GT-3' 5' -G AA-CTG-CTC-G AG-ATG-GTG-AT-3' 5' -GAT-GAA-GGT-GCC-GTG-GAT-CT-3' 5'-TTC-GAA-CTG-CGC-ACG-GAT-CT-3' 5 '-ATT-CAC-ACG-CTC-GTG-CAG-3' 5 '-CAG-CTC-GGC-GAG-CCA-GAA-3' 5' -GCG-TGG-TTC-GGC-AGC-ATG-AT-3' 5' -CTT-GAG-C AC-GCT-GAC-CGG-TT-3' 5' - AAG-TCG-TCC-GCG-GTG-AAG-TA-3' 5' -TGT-TGT-GGT-GTT-CGG-CGA-GC-3' 5' -CTG-CTC-G AC-TGG-CTG-CTG-AT-3' 5' - ACG-GC A-CGA-ATC-TGA-AGG-TC-3' 138 Appendix C - Partial digests APPENDIX C: PARTIAL DIGESTS OF CHROMOSOMAL DNA Purified chromosomal D N A was partially digested with Sau3A. In order to obtain large Chromosomal fragments of between 35 and 40 kb, five reactions were set up as follows: On ice, 50 u.g of chromosomal D N A (quantitated by Absorbance readings at OD260) was mixed with the amount of reaction buffer and B S A required for a final volume of 500 u l . 5.0 ul was removed and added to 1.0 ul of 6x D N A loading buffer with E D T A . In a 2.5 ul volume, 0.075 Units of Sau3A were added to the reaction mix. The reaction mix was then aliquoted into 5 tubes. These tubes were incubated at 37°C for 5, 10, 15, 20 and 25 minutes. A t each time point 5 ul was removed from the sample, added to D N A loading buffer with E D T A and placed on ice. The remaining 95 ul was phenolxhloroform extracted, ethanol precipitated, resuspended in sterile distilled water and frozen at -20°C . A l l the 5.0 ul samples mixed with the D N A loading buffer were resolved on a 0.8% agarose gel. The 5 minute sample was chosen as the sample to be used for construction of the cosmid library (see figure 11). 1 2 3 4 5 6 7 Figure 26. B. multivorans strain A T C C 17616 D N A partially digested with Sau 3A. B. multivorans strain A T C C 17616 D N A was partially digested with the restriction enzyme Sau3A. Lane 1 is the one-kilobase ladder. Lanes 2 to 7 represent the samples that were digested for 0, 5, 10, 15, 20 and 25 minutes respectively in the presence 0.015 Units of Sau 3A. Restricted D N A was resolved on a 0.8% agarose gel and stained with EtBr. D N A from the 5 minute digest was used for construction of the cosmid library. 139 Appendix D - Cosmid Library APPENDIX D: QUALITY OF B. multivorans ATCC 17616 COSMID BANK After packaging of the ligated insert and cosmid into X phage as per the manufacturer's instructions (Stratagene catalogue #251301), transfection of E. coli X L 1 Blue M R was performed. The transfection titre was low at 4 .46xl0 5 cfu/ml of undiluted packaged library. 19 colonies were randomly selected from this library and restriction digested with Bam HI (see Figure 28). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Figure 27 Restriction digests of random clones from the B. multivorans ATCC 17616 cosmid bank. Random cosmid clones were selected from the SuperCos 1 library and restriction digested with Bam HI. The resulting D N A fragments were resolved on a 1.0% agarose gel and stained with EtBr. Each clone tested exhibited a different pattern suggesting that each cosmid contains a different random chromosomal D N A insert. Insert sizes ranged from approximately 26.6 kb to 42.6 kb and the average size was 29.0 kb. Lane 1 is the 1.0 kb ladder and lanes 2-18 represent randomly selected clones. 140 Appendix E - Phylogeny APPENDIX E: SEQUENCE ALIGNMENT FOR THE PHYLOGENETIC TREE B c e p o r f 2 1 T h i o C e r 1 S y n e c h l C e r 1 M o u s e C e r 1 H u m a n c e r 1 C e l e g a n s l 1 C e l e g a n s 3 1 C e l e g a n s 2 1 M x a n t h u s 1 R h i z N o d C 1 s y n e c h o 2 C e r 1 L l a c t i s 1 B c e p o r f 2 1 T h i o C e r 1 S y n e c h l C e r 1 M o u s e C e r 1 H u m a n c e r 1 C e l e g a n s l 6 1 C e l e g a n s 3 4 4 C e l e g a n s 2 5 3 M x a n t h u s 1 R h i z N o d C 1 s y n e c h o 2 C e r 4 2 L l a c t i s 2 B c e p o r f 2 4 3 T h i o C e r 4 3 S y n e c h l C e r 4 5 M o u s e C e r 4 9 H u m a n c e r 4 9 C e l e g a n s l 1 1 3 C e l e g a n s 3 9 6 C e l e g a n s 2 1 0 5 M x a n t h u s 3 9 R h i z N o d C 4 7 s y n e c h o 2 C e r 9 9 L l a c t i s 6 2 B c e p o r f 2 9 3 T h i o C e r 9 8 S y n e c h l C e r 9 5 M o u s e C e r 9 9 H u m a n c e r 9 9 C e l e g a n s l 1 6 3 C e l e g a n s 3 1 4 6 C e l e g a n s 2 1 5 5 M x a n t h u s 8 7 R h i z N o d C 9 7 s y n e c h o 2 C e r 1 5 8 L l a c t i s 1 1 6 M C K Y G K S N L A A V A S S T S S I I G A A A A A V A E A Q P S A S P S T S S S F L L L E V P F R H L L R L Q P P P Y M G D K I T G L E V E I I P E I S E V Q P E S L Q E Q N A S S L Y Y P L F S Q Y I P L M E A A N E V V N L F A S Q A T T P S S L D A V T T L E T V S T P T F I F P E V S D S Q I L Q L M P A T - M P Q F P W K D N D A E L S P L E A F L A E W D D P E A E E E D F R N D F F R G S M - M T T P A V f J L - j j D W L L I - M C W W I G G PAAL0S L A A M N M A L M A L F I A G T R R M A A L L T G T K K I L E D I M T E G R R K K A A V M L T I Y I V W R I L Y T R D D I L C K Q D Q P Y S Q P Y S E D S P N P P D P S R A R A G A A P Q P G T T I S T T P A K D I E T N T P E E A P P L L F W K i N T K E D K E L V E L H P G A E P l D G l _PTPR--CFNE@PIV-|QVC[|MVAAKSEEA FI A TYNESSS--CEQHH(1R C V Q D IAQQN F E L D F E L D F T T F T T FTS QSIDYAGBL LAEQDHAG G K i g Q Q L C S L D f IASKKV: I A J j V V D g J R V H J LSWVCTEJ V H Q I E ( F I C F I C F F G i FFG|E| LFEJEI T . T P N R J i B i - | E j g F N F T I L P K N | | K W R R G A § A S G g K S GI N N E N . ~ B c e p o r f 2 T h i o C e r S y n e c h l C e r M o u s e C e r H u m a n c e r C e l e g a n s l C e l e g a n s 3 C e l e g a n s 2 M x a n t h u s R h i z N o d C s y n e c h o 2 C e r L l a c t i s 1 5 3 A P W A D - A S 1 5 8 A S | Q N - R D 1 5 5 S P M G D - R L 1 5 9 NQ|T--E 2 1 5 P Y F A S - P T 1 6 7 G Y F E D - S K i C L Y H A R S V G g CLYRARPVAT TPFKLTQAQT IHGLPYVADBQH HGLPYVAD9Q| QTPYCKDSEH QTPYCKDIKJ Q I P Y C K D g Q g -A G P P E H R G L P V G P g D f -- P A B f f l A A M G Q M K A S N Q A D T Q V R K A I A N E A V N Q A P Q T F F N N D P -- F W T R I G A Q F V D K S K N C A P I Q I L K R L T L I F I V V N V L A C L A G ELL EQ EQ DAAFEQ IA|SISVWA H A A F E Q A S R A i A G |LTR|ID HWTRGQG YQFNHFSEKNLN 141 Appendix E - Phytogeny Bceporf2 186 ThioCer 188 SynechlCer 185 MouseCer 187 Humancer 187 Celegansl 253 Celegans3 266 Celegans2 245 Mxanthus 163 RhizNodC 184 synecho2Cer 245 L l a c t i s 201 Bceporf2 235 ThioCer 240 SynechlCer 237 MouseCer 239 Humancer 239 Celegansl 305 Celegans3 320 Celegans2 297 Mxanthus 212 RhizNodC 243 synecho2Cer 295 L l a c t i s 251 Bceporf2 280 ThioCer 285 SynechlCer 282 MouseCer 284 Humancer 284 Celegansl 350 Celegans3 365 Celegans2 342 Mxanthus 256 RhizNodC 288 synecho2Cer 350 L l a c t i s 311 Bceporf2 310 ThioCer 314 SynechlCer 310 MouseCer 313 Humancer 313 Celegansl 379 Celegans3 394 Celegans2 376 Mxanthus 288 RhizNodC 322 synecho2Cer 387 L l a c t i s 371 Bceporf2 344 ThioCer 347 SynechlCer 344 MouseCer 347 Humancer 347 Celegansl 413 Celegans3 Celegans2 409 Mxanthus 322 RhizNodC 362 synecho2Cer 424 L l a c t i s 431 [THfflGl SSRFG J!_&_ELGPNIFCG |DFMPSJ|LFAEVTG|SKACL SSYBS INVTIFK IVT 1S§NVT§F CMD CLQFNHPT FL|VV_SS SAIAKAVC -ME_|LACNEERAAQ_RFGivMJCC --AEM_LDAY FQQQRIVTGglGEL! --NDQDFFMR|HENQKDIYNSVMYI EVEBATDVIEAgFGFLWHHET DYVfflDTEVREAN !LPYV_DAGVNLDNWi QVAIQNSISY QVA|QNs|sY SHPAHQNSSS THPA|QNS|A THPA|QNS|S _ _PAV|PLD-|EVLWAAPLS_FT EYVPSAJJAATVVPDB|GV_LRQQL G|LVNPP|EEE|VT_AIALWHQRN RFVNKN_ASGL|PENF_DLIKQRD LjTIRS-LHS|TTRS-QLYHDQNTYL-H H R | 1Q2LAS H |STFR D \EGGYQRY LDYWRWI CT 3S|GN|QVARKWLPLKIKGLNK_ R_A-TLL-T F TA_WLA_A_A_ALK_DGT-TY PLBLVWW5PW1GLWGG-IR AVPFAFLITFCHWSEP--KLD|FSFLLIQY SE— SE— QD-QD-QNGKFNFTgFL LFTP--TL_L, IGLDRYLTLDAIGQN! FVL FG FYSLNARFSGj |FR-|FR-/G-re_AN-|FRE FT SP-SS PBL AG-|LA|SVL-VLWQRR-?FWLPSFIAS QLAFNR i|GRLV|HAR GEDG-RQIMHKLSA DGSF-—GTAAlSlAQE LKDG-IFDHJQLR-IFD||QLR-SMDgSLMN--ITMDC_VMC--RNNKfKLSW-—RLAGLHAPV--GVQG--GVQG--SMQN--KMQV--GGRI--ADAG--WRAY|RD -GVALLG--ETIPJLP -GTLC |SK -GTLC jSK -GKLSjjSP R T S K N S -RAYAVTD - -|T I LVgGSMT LVRCS VAAY RARELRF S C | G M Y § J G L K R LTPSE GESP|QQ ETVMAPfflSYSVISDAVLKSKKGFTVTNKGYNTMKKYYNWRLSLPL LALE|LV_A|GT GBWIIFHSMAR ifflVFHALSFSQR DP DP HQFMLIWL_R§SPLlSSLSKL QFNGET--ARMTHVG—GERAAA -SQBAIG ADGRMD -IT|R|TK--GGKMIP -GRHRIR CGGTAE -GR«R_R CGGTAE -MFHIWHG--VDRYFH W -QKVPEAVSLS WLLG |A|lflAA_LRSBRQG |GFALHTL|NIF ILMPLKAY^CTLS IPALLARTHGGT ]YSFHS.LI iiaPAVTA QTyVMRP—GGRMIR GSVAjjAPTVGQQGAT KRLKWVKTVHGAATE |lLLFFSlj|ALCKS|FVIFNl|PFESGKDAIYINAF|LLYNVFILIFAVgvPFERPRFRK 142 Appendix E - Phylogeny B c e p o r f 2 T h i o C e r S y n e c h l C e r M o u s e C e r H u m a n c e r C e l e g a n s l C e l e g a n s 3 C e l e g a n s 2 M x a n t h u s R h i z N o d C s y n e c h o 2 C e r L l a c t i s 3 8 5 AVEGGDGR 3 8 0 GHDGAKR • 383 A S P I P K S 3 9 0 E I L D V 3 9 0 E I L D V 457 Q N V D C F I Y S F Y F Q I S L T P N S S F L S V I L F L N I I V N P L T T S S E S L K S G L L S L H Q L H C 3 6 1 VAPELSGGPG 4 1 0 KMPGRATSEIAYSGE 472 DALELKQS 4 91 S E R F L S S K E A Q L L D K E S H L I I D C K V M D W N E L G A G I T I E C N N K I E L K E E Q K I I L S V N G Y 143 Appendix E -Phylogeny M a j o r i t y - r u l e and s t r i c t consensus tre e program, v e r s i o n 3.572c Species i n order: SynechlCer Synech2Cer L l a c t i s MouseCer Humancer Celecgansl Celegans2 Celegans3 RhizNodC Mxanthus Bceporf2 Thiocerglu Sets i n c l u d e d i n the consensus t r e e Set (species i n order) * * * * * * * * * * * * * * * * * * * * * ** * * * * How many times out of 100.00 100.00 99.00 99.00 79.00 72.00 58.00 55.00 37.00 33.00 Sets NOT inc l u d e d i n consensus t r e e : Set (species i n order) How many times out of 100.00 * * 28 00 * * . * 23 00 * * * 18 00 * * 17 00 * * * 16 00 ******** 15 00 ******* 14 00 __***** * * * 13 00 ********* 11 00 ****** * * 10 00 ********* * 10 00 * _ ******* * * 9 00 * * * * * * 8 00 * * 6 00 * * * * 6 00 _******* 6 00 ***** * * 5 00 ******** * * 4 00 ****** 4 00 * * * * * 4 00 * * * 3 00 * * * 3 00 * * * * * * 3 00 144 Appendix E - Phylogeny * * * * * * * * * 2 00 * + 2 00 * * * * * * 2 00 * * * * * * * * 2 00 * + 2 00 * * * * * * * * * * 2 00 + + * * 1 00 * * * * * * * * * 1 00 * * * + * 1 00 * * 1 00 * * * * * * 1 00 * * * * * * * * * 1 00 * * * 1 00 * * * 1 00 * * * * 1 00 * * * * * * 1 00 * * * * * 1 00 * * 1 00 * * * * * * * * * 1 00 * * 1 00 * * * * * * * * * 1 00 * * * * * * * 1 00 * * * * * * * 1 00 * * * 1 00 * * * * 1 00 * * * * * * * * 1 00 CONSENSUS TREE: the numbers at the forks i n d i c a t e the number of times the group c o n s i s t i n g of the species which are to the r i g h t of that f o r k occurred among the t r e e s , out of 100.00 tre e s + I +-99.0 i + -37 . 0 +-73.0 +--99.0 + -+-! +-72.0 +100.0 +-i + + +-58.0 i -33.0 i +-+ -+ -55.0 +-+ --79.0 +-Humancer MouseCer — C e l e c g a n s l Celegans3 — C e l e g a n s 2 — T h i o c e r g l u — B c e p o r f 2 — M x anthus RhizNodC L l a c t i s — S y n e c h 2 C e r SynechlCer remember: t h i s i s an unrooted t r e e ! 145 

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