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Identification and cloning of DNA affecting production of the adhesive holdfast of Caulobacter crescentus… Mitchell, David Walter 1989

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IDENTIFICATION AND CLONING OF DNA AFFECTING PRODUCTION OF THE ADHESIVE HOLDFAST OF CAULQBACTER CRESCENTUS CB2 By DAVID WALTER MITCHELL B.Sc. ( B i o l o g y ) , U n i v e r s i t y of B r i t i s h Columbia, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n The F a c u l t y of Graduate S t u d i e s (Department of M i c r o b i o l o g y ) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August 1989 © D a v i d Walter M i t c h e l l , 1989 € ln 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 M i c r o b i o l g y The University of British Columbia Vancouver, Canada D a t e September 30, 198 9 DE-6 (2/88) ABSTRACT As p a r t of a p r e l i m i n a r y i n v e s t i g a t i o n i n t o the b i o l o g i c a l and chemical p r o p e r t i e s of the freshwater Caulobacter c r e s c e n t u s s t r a i n CB2 h o l d f a s t ( H f ) , t h i s r e s e a r c h e n t a i l e d the i d e n t i f i c a t i o n , phenotypic c h a r a c t e r i z a t i o n , and c l o n i n g of genomic DNA t h a t i n some way c o n t r i b u t e to the p r o d u c t i o n or r e g u l a t i o n of the Hf. Random transposon (Tn5) mutagenesis was used to i d e n t i f y genomic DNA encoding H f - r e l a t e d f u n c t i o n s . A l i b r a r y of approximately 16,000 independently s e l e c t e d mutants (P > 98%) was prepared from which approximately 12,000 c o l o n i e s (P = 95%) were screened f o r 77 Hf-mutants t h a t were aberrant i n the a b i l i t y to form l a r g e c e l l u l a r masses ad j o i n e d at the Hf (termed r o s e t t e s ) and/or the a b i l i t y of c o l o n i e s to adhere to c e l l u l o s e - a c e t a t e . Ten unique s i t e s of Tn5_-insertion were i d e n t i f i e d from Southern b l o t s of chromosomal DNA from a l l screened mutants u s i n g r e s t r i c t i o n enzymes that cut once or twice w i t h i n the Tn5_ element. Subsequently, u s i n g r e s t r i c t i o n enzymes t h a t cut f r e q u e n t l y or i n f r e q u e n t l y o u t s i d e of the Tn5_ element, the ten s i t e s of T n 5 - i n s e r t i o n were found to be c l u s t e r e d i n f o u r genomic r e g i o n s . R e s t r i c t i o n fragments of Tn5_-mutated genomic DNA t h a t were shown to c o n t a i n a l l of the i d e n t i f i e d s i t e s of Tn5_-i n s e r t i o n w i t h i n a given c l u s t e r were cloned from one r e p r e s e n t a t i v e mutant of each of the f o u r r e g i o n s . In attempting to a s c r i b e f u n c t i o n s to the regions of DNA t h a t encode H f - r e l a t e d f u n c t i o n s , the phenotype of Hf-mutants was i n v e s t i g a t e d on the b a s i s of Hf-adhesiveness to g l a s s and c e l l u l o s e - a c e t a t e and on the b a s i s of l e c t i n - b i n d i n g to Hf. Four p h e n o t y p e s w e r e i d e n t i f i e d : n o H f p r o d u c e d , a l o w a m o u n t o f H f p r o d u c e d , H f p r o d u c e d a n d s h e d i n t o t h e m e d i u m , a n d a f o u r t h c a t e g o r y w h e r e a p o s s i b l e a l t e r a t i o n o f s t a l k w a s r e l a t e d t o i m p a i r e d a d h e s i o n o f t h e H f m a t e r i a l . T h e s h e d d i n g p h e n o t y p e i s o f p a r t i c u l a r i n t e r e s t a s a p o s s i b l e m e a n s o f i s o l a t i n g b o t h w i l d t y p e a n d m u t a n t H f m a t e r i a l f o r c h e m i c a l a n a l y s i s . T o t h i s e n d , a g e n e r e p l a c e m e n t s t r a t e g y w a s u s e d t o t r a n s f e r a T n J i - m u t a t e d DNA f r a g m e n t c l o n e d f r o m o n e H f - m u t a n t i n t o a p r e v i o u s l y p r e p a r e d u l t r a v i o l e t l i g h t - i n d u c e d H f - s h e d d i n g m u t a n t . D o u b l e m u t a n t s c o n f i r m e d f o r b o t h s h e d d i n g a n d T n 5 - i n d u c e d H f c h a r a c t e r i s t i c s w e r e i s o l a t e d . i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i INTRODUCTION 1. Ca u l o b a c t e r s and the adhesive h o l d f a s t 1 2. B a c t e r i a l adhesion 3 3. The transposon Tn5_ 5 MATERIALS AND METHODS 1. S t r a i n s , plasmids, and growth c o n d i t i o n s 8 2. The Tn5-mutant l i b r a r y i . P r o d u c t i o n of l i b r a r y 11 i i . A n a l y s i s of l i b r a r y 12 3. A n a l y s i s of h o l d f a s t - d e f e c t i v e mutants i . I d e n t i f y i n g the s i t e s of Tn5_-insert ion 15 i i . T e s t i n g f o r p l e i o t r o p i c mutants 18 i i i . Determining phenotype 20 4. C l o n i n g of h o l d f a s t - r e l a t e d and Tn5_-mutated DNA 22 5. Pr o d u c t i o n of double holdfast-mutants 25 RESULTS 1. The Tn5-mutant l i b r a r y i . P r o d u c t i o n of l i b r a r y 27 i i . A n a l y s i s of l i b r a r y 27 2. A n a l y s i s of h o l d f a s t - d e f e c t i v e mutants i . I d e n t i f y i n g the s i t e s of T n 5 - i n s e r t i o n 31 i i . T e s t i n g f o r p l e i o t r o p i c mutants 35 i i i . Determining phenotype 39 3. C l o n i n g of h o l d f a s t - r e l a t e d and Tn5_-mutated DNA 41 4. Pro d u c t i o n of double holdfast-mutants 42 DISCUSSION 47 REFERENCES 51 V LIST OF TABLES TABLE TITLE PAGE 1. B a c t e r i a , bacteriophage, and plasmids 9 2. Carbohydrate s p e c i f i c i t y of l e c t i n s 21 3. Phenotype of holdfast-mutants 32 4. Average s i z e of Tn5_-containing fragments produced by d i g e s t i o n of holdfast-mutant chromosomal DNA with r e s t r i c t i o n enzymes that cut o u t s i d e of the Tn5_ element...36 5. Phenotype of double mutants ....45 v i LIST OF FIGURES FIGURE TITLE PAGE 1. The Tn5_ element and prob i n g of Tn5_-mutated DNA 13 2. Southern b l o t of Tn5_-mutated chromosomal DNA: prob i n g f o r Tn5_ and PSUP2021 28 3. Southern b l o t comparison of Tn5_-containing fragments among double mutants: B_ajaHI d i g e s t i o n of chromosomal DNA 29 4. Southern b l o t comparison of Tn5_-containing fragments among h o l d f a s t - d e f e c t i v e mutant groups: BamHI d i g e s t i o n of chromosomal DNA 30 5. Southern b l o t comparison of Tn5 - c o n t a i n i n g fragments among h o l d f a s t - d e f e c t i v e mutant groups: B g l l l d i g e s t i o n of chromosomal DNA 34 6. Southern b l o t comparison of cloned h o l d f a s t - r e l a t e d DNA fragments with chromosomal DNA d i g e s t s of h o l d f a s t -d e f e c t i v e mutant groups t h a t c o n t a i n l i n k e d s i t e s of Tn5_-insertion : p r o b i n g f o r TnJ5_ 37 7. P o s i t i o n i n g of Tn5_-mutated and h o l d f a s t - r e l a t e d DNA in the CB2 genome: p u l s e d - f i e l d g r a d i e n t g e l e l e c t r o p h o r e s i s 38 8. F l u o r e s c e i n - c o n j u g a t e d l e c t i n l a b e l l i n g of the c a u l o b a c t e r h o l d f a s t 40 9. Southern b l o t comparison of cloned h o l d f a s t - r e l a t e d DNA fragments with chromosomal DNA d i g e s t s of h o l d f a s t -d e f e c t i v e mutant groups t h a t c o n t a i n l i n k e d s i t e s of Tn5_-insertion : p r o b i n g with cloned DNA fragments 43 ACKNOWLEDGEMENTS I g r a t e f u l l y acknowledge the U.S. O f f i c e of Naval Research and Dr. J . Smit f o r f i n a n c i a l a s s i s t a n c e . I am s i n c e r e l y t h a n k f u l to Dr. J . Smit f o r generously p r o v i d i n g d i r e c t i o n and f o r c r i t i c a l r e a d i n g of t h i s t h e s i s , Dr. W. B i n g l e f o r p a t i e n t l y and generously p r o v i d i n g e x c e l l e n t s c i e n t i f i c and t e c h n i c a l advice, Dr. B. E l y f o r the PFGE analyses of mutant DNA, and Ms. P. Edwards f o r p r e p a r a t i o n of the e l e c t r o n microscope sample g r i d s . F i n a l l y , I would l i k e to thank my f a m i l y and f r i e n d s f o r t h e i r unwavering support. T h i s t h e s i s i s de d i c a t e d to those who have encouraged me to th i n k . 1 INTRODUCTION CAULOBACTERS AND THE ADHESIVE HOLDFAST C a u l o b a c t e r s are a e r o b i c , gram ne g a t i v e , chemoheterotrophic b a c t e r i a t h a t d u r i n g t h e i r growth c y c l e express two d i s t i n c t m orphological c e l l types: the d i s p e r s i v e f l a g e l l a t e d swarmer c e l l and the p r o l i f e r a t i v e s e s s i l e s t a l k e d c e l l (41, 45). Swarmer c e l l s g e n e r a l l y express f o u r of the f i v e temporally and s p a t i a l l y c o n t r o l l e d p o l a r o r g a n e l l e s ; a c l u s t e r of p i l i , caulophage r e c e p t o r and attachment s i t e s , a h o l d f a s t ( H f ) , and a f l a g e l l u m i s l o c a l i z e d at one p o l e of the c e l l . D i f f e r e n t i a t i o n i n t o the s t a l k e d c e l l i n v o l v e s l o s s of the f l a g e l l u m and extension of the c e l l envelope to form a s t a l k , on the end of which i s l o c a t e d the adhesive Hf m a t e r i a l (16, 45, 57). C a u l o b a c t e r s p e c i e s and s e v e r a l other adhesive b a c t e r i a c o n t r i b u t e to the i n i t i a l stages of b i o f o u l i n g of submerged o b j e c t s i n both freshwater and marine environments. The attachment to s u r f a c e s , where both or g a n i c and i n o r g a n i c n u t r i e n t s tend to concentrate, by c a u l o b a c t e r s appears to be promoted by swarmer c e l l m o t i l i t y , p o s s i b l y a s s i s t e d by p i l i , and s t a b i l i z e d by Hf. These p o l a r f u n c t i o n s , i n combination with the subsequently developed s t a l k t h a t i s capable of lengthening i n response to n u t r i e n t d e f i c i e n t c o n d i t i o n s , are thought to c o n t r i b u t e to c a u l o b a c t e r ' s competitiveness t h a t i s e x e m p l i f i e d by i t s near u b i q u i t o u s r e p r e s e n t a t i o n i n o l i g o t r o p h i c aqueous environments (16, 39, 41, 45). None of these p o l a r o r g a n e l l e s are e s s e n t i a l to c e l l v i a b i l i t y s i n c e mutants d e f e c t i v e i n p r o d u c t i o n of each of the f i v e o r g a n e l l e s can be i s o l a t e d . And the frequent occurence of 2 both spontaneous and u l t r a v i o l e t l i g h t - or c h e m i c a l l y - i n d u c e d mutants t h a t are p l e i o t r o p i c f o r p o l a r - r e l a t e d f u n c t i o n s when i s o l a t e d a c c o r d i n g to a b e r r a t i o n s of any of the f i v e p o l a r s t r u c t u r e s a l s o suggests some degree of s t r u c t u r a l and/or g e n e t i c interdependence (16, 45). Although some of the observed p l e i o t r o p y i s due to m u l t i p l e mutations which on l y p a r t i a l l y r e v e r t or by s u p p r e s s i o n r e v e r t to w i l d t y p e , the v a r i e t y and frequency of p l e i o t r o p i c mutants i s o l a t e d suggest t h a t s e v e r a l d i f f e r e n t t a r g e t s i n the chromosome are r e s p o n s i b l e f o r s i n g l e - s i t e induced p l e i o t r o p y . These l o c i are thought to c o n t r o l e i t h e r the c o o r d i n a t e r e g u l a t i o n of p o l a r -r e l a t e d f u n c t i o n s or the p r o d u c t i o n of s t r u c t u r a l components t h a t are r e q u i r e d f o r the e x p r e s s i o n of more than one p o l a r o r g a n e l l e (21, 45, 57). C aulobacter Hf seems only to f u n c t i o n i n adhesion and mediates c e l l attachment to a v a r i e t y of b i o l o g i c a l and n o n b i o l o g i c a l s u b s t r a t a (39, 44, 45, 46). A n a l y s i s of Hf-mutants suggests that there i s a s p e c i f i c s i t e at which the Hf m a t e r i a l i s anchored to the c a u l o b a c t e r s t a l k and t h a t there may be a l i m i t e d p e r i o d of time d u r i n g which the Hf m a t e r i a l remains adhesive (44). That bound Hf can remain i n t a c t under c o n d i t i o n s of high shear f o r c e and f o r many c e l l g e n e r a t i o n s suggests that the Hf mediates s t r o n g and s t a b l e adhesion (39, 46). Although very l i t t l e i s known about the a c t u a l composition or s t r u c t u r e of the Hf m a t e r i a l or any other adhesive e x t r a c e l l u l a r polymer (10, 11, 25, 45, 55, 56), l e c t i n - b i n d i n g and enzyme d i g e s t s t u d i e s suggest that s t r e t c h e s of three or more u n i t s of N-acetylglucosamine are a component of the exposed 3 s u r f a c e of the Hf m a t e r i a l and that u r o n i c a c i d s - which are suggested to be important components of adhesive polymers - are, l i k e l i p i d and p r o t e i n , minor components of the Hf m a t e r i a l i f they are present at a l l (39). BACTERIAL ADHESION P h y s i o l o g i c a l and p h y s i c o c h e m i c a l s t u d i e s of adhesive b a c t e r i a suggest that the p h y s i o l o g i c a l s t a t e of the bacterium and the c h a r a c t e r s of the adhesive polymers, n u t r i e n t medium, and substratum are a l l important f a c t o r s i n c o n t r o l l i n g or i n f l u e n c i n g adhesion. In g e n e r a l , these s t u d i e s suggest t h a t b a c t e r i a adhere by a v a r i e t y of as yet mostly unresolved mechanisms (19, 37, 56). The most popular g e n e r a l theory to d e s c r i b e e x t r a c e l l u l a r polymer-mediated adhesion i s known as polymeric b r i d g i n g and proposes adhesion as a two stage process. F i r s t i s envisaged a r e v e r s i b l e and time-independent a s s o c i a t i o n mediated by s h o r t range f o r c e s (hydrogen bonding, hydrophobic bonding, and d i p o l e -d i p o l e i n t e r a c t i o n s ) between the c e l l s u r f a c e and a l a y e r of c o u n t e r - i o n s s i t u a t e d next to what predominantly are n e g a t i v e l y charged s u r f a c e s . Second, extensions of the b a c t e r i a l c e l l s u r f a c e such as f l a g e l l a , p i l i , f i m b r i a e , s t a l k , or Hf i n i t i a t e a time-dependent phase of i r r e v e r s i b l e adhesion t h a t i s thought to be mediated by complex e x t r a c e l l u l a r polymers b r i d g i n g across the r e p u l s i o n b a r r i e r between c e l l and the substratum s u r f a c e (10, 11, 19, 37, 56, 57). The time-dependence of polymeric b r i d g i n g i n d i c a t e s the importance of b a c t e r i a l p h y s i o l o g y to the process of adhesion. P h y s i o l o g i c a l s t u d i e s on adhesive b a c t e r i a show that environmental c o n d i t i o n s - from p h y s i c o c h e m i c a l parameters such 4 as pH and e l e c t r o l y t e c o n c e n t r a t i o n to n u t r i t i o n a l s t r e s s - do a f f e c t the chemistry, r e g u l a t i o n , and hence the f u n c t i o n of adhesive e x t r a c e l l u l a r polymers. I t i s f o r t h i s reason that there i s g e n e r a l l y both d i r e c t evidence and c o n t r a d i c t o r y evidence f o r most t h e o r i e s of adhesion t e s t e d on e i t h e r a p u r e l y p h y s i c o c h e m i c a l or p h y s i o l o g i c a l b a s i s (19, 37). Enzyme d i g e s t i o n and h i s t o c h e m i c a l s t a i n i n g of p u t a t i v e adhesive polymers suggest t h a t they are predominantly p o l y s a c c h a r i d e or g l y c o p r o t e i n i n nature. No complete secondary or t e r t i a r y s t r u c t u r a l s t u d i e s have been done on polymers thought to be i n v o l v e d i n the attachmen.t process. Few have been s t u d i e d even i n primary composition. Of those t h a t have, i n a d d i t i o n to some p r o t e i n and l i p i d , the predominant carbohydrates i d e n t i f i e d are g l u c u r o n i c a c i d , glucosamine, glucose, mannose, g a l a c t o s e , rhamnose, and fucose (19, 56). There are three major problems a s s o c i a t e d with the i s o l a t i o n of adhesive m a t e r i a l f o r chemical a n a l y s i s : to o b t a i n enough m a t e r i a l , to o b t a i n pure m a t e r i a l , and to show t h a t the i s o l a t e d polymers are r e s p o n s i b l e , i n p a r t or whole, f o r the observed adhesion (19, 37, 56). In gram negative b a c t e r i a , any of the e x t r a c e l l u l a r p o l y s a c c h a r i d e s , l i p o p o l y s a c c h a r i d e , and outer membrane p r o t e i n s have the p o t e n t i a l of a c t i n g as adhesives e i t h e r i n d i v i d u a l l y or i n combination under the c o r r e c t environmental c o n d i t i o n s (19). T h i s complex and v a r i a b l e s t r u c t u r e and f u n c t i o n of adhesive c e l l s u r f a c e polymers makes t h e i r i s o l a t i o n d i f f i c u l t . That Hf seems only to f u n c t i o n i n adhesion, i s l o c a l i z e d to a s i n g l e p o l e of the c e l l , i s demonstrated as adhesive m a t e r i a l 5 by mutants that shed the Hf i n t o the medium, and i s demonstrated to produce a v a r i e t y of d i f f e r e n t mutant phenotypes suggests t h a t Caulobacter s p e c i e s are u s e f u l organisms f o r r e s e a r c h on the molecular b a s i s of adhesion and f o r i s o l a t i o n of adhesive m a t e r i a l f r e e from contamination of other s u r f a c e polymers f o r chemical a n a l y s i s . (39, 44). THE TRANSPOSON Tn5_ As p a r t of our l a b o r a t o r y ' s p r e l i m i n a r y i n v e s t i g a t i o n i n t o the g e n e t i c b a s i s of e x p r e s s i o n and r e g u l a t i o n of the c a u l o b a c t e r Hf, H f - r e l a t e d genomic DNA was i n v e s t i g a t e d with the use of the transposon Tn5 (9, 13, 17, 41, 47, 51). Tn5_ i s a u s e f u l t o o l with which to i d e n t i f y the number and l o c a t i o n of genomic DNA regions that encode f o r H f - r e l a t e d f u n c t i o n s . As w e l l , i t p r o v i d e s a means of d e t e r m i n i n g whether or not these r e g i o n s are d i s p e r s e d i n the genome or c l u s t e r e d w i t h i n operons as i s the case f o r most of the f l a genes r e s p o n s i b l e f o r e x p r e s s i o n of the p o l a r f l a g e l l u m i n C.crescentus (41, 47) and as i s the case f o r genes i n v o l v e d i n the p r o d u c t i o n of s e v e r a l e x o p o l y s a c c h a r i d e s so f a r i n v e s t i g a t e d (9, 12, 13, 35, 58). In a d d i t i o n to c a r r y i n g a n t i b i o t i c - r e s i s t a n c e genes and unique r e s t r i c t i o n endonuclease cleavage s i t e s t h a t g r e a t l y f a c i l i t a t e molecular manipulations of DNA, transposons have the advantage of s t a b l y and completely e l i m i n a t i n g t a r g e t gene f u n c t i o n . They are u s e f u l f o r i s o l a t i n g v i r t u a l l y any gene with an i d e n t i f i a b l e n o n l e t h a l mutant phenotype and have been s u c c e s s f u l l y employed i n both i n v i v o and in. v i t r o mutagenesis of both p r o k a r y o t i c and e u k a r y o t i c DNA (5, 6, 42, 47). The 5.8kb Tn5 element transposes by a n o n r e p l i c a t i v e mechanism. That i s , when Tn5 transposes from donor to r e c i p i e n t DNA i t does not leave a copy of i t s e l f i n the donor DNA (26). The element c o n t a i n s s t r u c t u r a l genes that encode f o r enzymes t h a t confer r e s i s t a n c e upon the host to c e r t a i n aminoglycoside a n t i b i o t i c s : kanamycin i n E . c o l i . kanamycin and streptomycin i n C.crescentus (43). F l a n k i n g the c e n t r a l gene are two n e a r l y i d e n t i c a l i n v e r t e d repeats (IS50L and IS50R) t h a t c o n t a i n the genes r e s p o n s i b l e f o r both t r a n s p o s i t i o n ( p i p r o t e i n ) and r e g u l a t i o n (p2 p r o t e i n ) (6, 62). Transposons have evolved v a r i o u s mechanisms to l i m i t t h e i r t r a n s p o s i t i o n f r e q u e n c i e s and have used two g e n e r a l s t r a t e g i e s to do so: mechanisms to l i m i t the s y n t h e s i s of the transposase and mechanisms to i n h i b i t the t r a n s p o s i t i o n event i t s e l f . At the t r a n s c r i p t i o n a l l e v e l , the Tn5_ transposase promoter i s weak and su b j e c t to dam methylat ion-dependent i n h i b i t i o n . And Tn5_ r e g u l a t e s i t s own t r a n s p o s i t i o n with the p2 p r o t e i n , an e f f i c i e n t t r a n s - a c t i n g n e g a t i v e r e g u l a t o r of both r e s i d e n t and incoming Tn5_. As a r e s u l t , Tn_. g e n e r a l l y e x h i b i t s only a s l i g h t tendency to transpose to a d i f f e r e n t s i t e once i t i s s i t u a t e d w i t h i n the t a r g e t DNA (29, 33, 61, 62). Although Tn5 shows some hotspots f o r t r a n s p o s i t i o n , i t i s considered to g e n e r a l l y e x h i b i t low i n s e r t i o n a l s p e c i f i c i t y . The reason f o r Tn5_ s i t e - s p e c i f i c i t y i s not known but i s suspected to be due to i n t e r a c t i o n between the d e l i v e r y v e c t o r , the transposon, and the t a r g e t DNA (5, 6, 17). When i n s e r t e d i n t o an operon, Tn5 p a r t i a l l y or completely e l i m i n a t e s e x p r e s s i o n of p r o m o t e r - d i s t a l genes r e g a r d l e s s of i n s e r t i o n a l o r i e n t a t i o n . Complete p o l a r i t y i s presumed to be due to t r a n s c r i p t i o n a l stop s i g n a l s c a r r i e d by the Tn5_ element 7 thereby h a l t i n g t r a n s c r i p t i o n which ent e r s Tn5 from o u t s i d e of the element - and p a r t i a l p o l a r i t y i s a s c r i b e d to p r o m o t e r - l i k e DNA sequences at the ends of the Tn5 element that can i n i t i a t e a low l e v e l of c o n s t i t u t i v e t r a n s c r i p t i o n (3, 6, 32, 33). L i k e most transposons, Tn5_ t r a n s p o s i t i o n can induce some nonhomologous, recA-independent ( i l l e g i t i m a t e ) recombination events at the t a r g e t s i t e : d e l e t i o n s , d u p l i c a t i o n s , and i n v e r s i o n s . These DNA rearrangements are not e x t e n s i v e l y produced with the use of Tn5 but tend to e x h i b i t a c h a r a c t e r i s t i c s t r u c t u r e when they do occur: one endpoint i s at or w i t h i n the Tn5_ element and extends i n one or the other, but not both, d i r e c t i o n i n t o adjacent DNA (6, 32). In g e n e r a l , Tn5_ transposes randomly enough and with few enough secondary rearrangements to produce mutants t h a t are s u f f i c i e n t l y s t a b l e to f a c i l i t a t e the i d e n t i f i c a t i o n and c l o n i n g of T n 5 - c o n t a i n i n g DNA, plasmid gene-replacement experiments, t r a n s d u c t i o n a l mapping i n s e v e r a l b a c t e r i a l s p e c i e s , and d i r e c t e d DNA sequencing of cloned genes (5, 6, 17, 47, 62). 8 MATERIALS AND METHODS STRAINS, PLASMIDS, AND GROWTH CONDITIONS. A l l s t r a i n s and plasmids are l i s t e d i n Table 1. Unless otherwise noted, a l l Caulobacter cresoentus s t r a i n s were grown at 30C to mi d - l a t e l o g a r i t h m i c phase (0D600~0.4) i n peptone yeast e x t r a c t complex medium (PYE: 16g/l Bactoagar f o r s o l i d media ( D i f c o L a b o r a t o r i e s ) , 2 g / l peptone, lg/1 yeast e x t r a c t ) supplemented with 0.1g/l CaC12.2H20, 0.2g/l MgS04.7H20 (28), and a p p r o p r i a t e a n t i b i o t i c s at the f o l l o w i n g c o n c e n t r a t i o n s : chloramphenicol 2ug/ml (Cm2), kanamycin 50ug/ml (Km50), r i f a m p i c i n 5ug/ml ( R f 5 ) , streptomycin 50ug/ml (Sm50), and trimethoprim 150ug/ml ( T p l 5 0 ) . A d e f i n e d medium, used f o r the i d e n t i f i c a t i o n of auxotrophic mutants, was Hutner's m i n e r a l base supplemented with 2 g / l glucose as the s o l e carbon source (HMG) (45). A l l E s c h e r i c h i a c o l j s t r a i n s were grown at 37C to mi d - l a t e l o g a r i t h m i c phase (0D600~0.8) i n L u r i a b r o t h (LB: 16g/l bactoagar ( f o r p l a t e s ) , 10g/l tryptone, 5 g / l yeast e x t r a c t , 5 g / l NaCl) with host and plasmid a n t i b i o t i c s e l e c t i o n as r e q u i r e d at the f o l l o w i n g c o n c e n t r a t i o n s : a m p i c i l l i n 50ug/ml (Ap50), Cm30, and Km50. Phage T7 was propagated i n l i q u i d c u l t u r e (50) as f o l l o w s ; an E . c o l i C600 c u l t u r e i n m i d - l o g a r i t h m i c growth phase was i n o c u l a t e d at a m u l t i p l i c i t y of i n f e c t i o n (MOI) of 5 and the supernatant harvested (~12,000xg, lOmin) when o p t i c a l d e n s i t y of the c u l t u r e was reduced to 0D600~0.02. T e n - f o l d d i l u t i o n s of phage were s p o t - t i t r e d on host lawn. g TABLE 1: BACTERIA, BACTERIOPHAGE, AND PLASMIDS ID . DESCRIPTION REFERENCE C.crescentus : B9(G5) 9.5kb Tn5_-containing ECJIRI fragment cloned from pG5 i n t o CB2A-B9,double Hf-mutant, Tp-r,Km-r,Sm-r TW CB2A wi ldtype CB2, Tp-r, R f - r , SA-minus NA CB15BE wil d t y p e CB15,Tp-r BE CB2A-B5 UV-induced Hf-minus mutant, Tp-r 44 CB2A-B6 UV-induced phage CBK-r,Hf-minus mutant,Tp-r..44 CB2A-B9 UV-induced Hf-shedding mutant,Tp-r 44 CB2A-B9: :Tn5_. . .UV-induced Hf-shedding and Tn5_-induced double mutants,Tp-r ,Km-r, Sm-r TW CB2A: :Tn5_ • . Tn5_-library mutants,Tp-r , R f - r , Km-r, Sm-r TW,43 g l to glO Tn5_-induced H f - d e f e c t i v e mutant groups, Tp-r, R f - r , Km-r, Sm-r TW E . c o l i : C600 F-, t h i - 1 , t h r - 1 , leuB6, l a c Y l , tonA21, supE44, lambda-minus, g e n e r a l host 24 DH5-alpha F-,recAl,hsdR17(r-,m-),supE44,thi-1, endAl,gyrA96,relAl,phage-80dlacZdM15, d(lacZYA-argF)U169,lambda-minus, pUC plasmid host 23 LE392 F-, hsdR514( r-,m- )SupE44, SupF58,dlacIZY galK2,galT22,metBl,TrpR55,lambda-minus, phage-T7 host 40 RB404 F-,dam-3,dam-6,metBl,galK2,galT22, l a c Y l , t h i - 1 , tonA31, tsx-78,mt 1-1, supE44 4 SM10 F-, t h i - 1 , t h r - 1 , leuB6,suIII, recA, r+m+, RP4-2-Tc::Mu t r a n s f e r (tra+) f u n c t i o n s cloned i n t o chromosome 51 Phage: CBK caulophage that a ttaches p r e f e r e n t i a l l y to the Hf s i t e of swarmer c e l l s 1,34 T7 s t r i c t l y v i r u l e n t c o l i p h a g e 7 Plasmids: PBR322. . Ap-r,Tc-r pBR322-Neo-r. . . 1.8kb Hindlll/BamHI fragment of Tn5_ cloned i n t o pBR322,Ap-r,Km-r pGl 14.5kb Tn5_-containing S s t I fragment cloned from mutant group g l i n t o the MCS of pPR510, Cm-r,Km-r TW pG3 . ...12.5kb T n 5 - c o n t a i n i n g C l a l / K p n l fragment cloned from mutant group g3 i n t o the C l a l / K p n l s i t e of the E1F2 SA gene of pPR510ElF2,Cm-r,Km-r TW pG5 ..9.5kb Tn5_-containing EcoRI fragment cloned 10 PSUP2021 PPR510.... PPR510E1F2 pG9 from mutant group g5 i n t o the MCS of pPR510, Cm-r,Km-r 12.0kb Tn5_-containing Sstl/EcoRI fragment cloned from mutant group g9 i n t o the MCS of pPR510,Cm-r,Km-r pUC19,Cm-r SA gene (E1F2) cloned from the assembly d e f e c t i v e mutant CB15ACa-10 i n t o the MCS of pPR510,Cm-r Tn5_ i n s e r t e d i n t o Tc gene of pBR325,S_&u3A fragment c o n t a i n i n g Mob s i t e of RP4 cloned i n t o pBR325,Ap-r,Cm-r,Km-r,Mob+ TW TW 48 JS 51 Ap-r = a m p i c i l l i n - r e s i s t a n c e , BE = B. E l y ( B i o l o g y Department, U n i v e r s i t y of South C a r o l i n a ) , Cm = chloramphenicol, Hf = h o l d f a s t , JS = J . Smit ( M i c r o b i o l o g y Department, U n i v e r s i t y of B r i t i s h Columbia), kb = k i l o b a s e , Km = kanamycin, MCS = m u l t i p l e c l o n i n g s i t e , NA = N. Agabian ( B i o c h e m i s t r y Department, U n i v e r s i t y of Washington), Rf = r i f a m p i c i n , SA = s u r f a c e a r r a y , Sm = streptomycin, Tp = trimethoprim, TW = t h i s work, and UV = u l t r a v i o l e t l i g h t . 11 Phage CBK was propagated i n s o f t PYE f i r s t to a high t i t r e l y s a t e on the n o n r e s t r i c t i v e C.crescentus host CB15BE and then on the r e s t r i c t i v e host CB2A as f o l l o w s ; lOOul of t e n - f o l d s e r i a l d i l u t i o n s of phage and lOOul of host (approximately 10E+7 c e l l s ) were mixed and incubated f o r l/2h at room temperature a f t e r which 3.5ml of s o f t PYE medium (0.6% agar, 46C) was added. T h i s mixture was poured over a PYE medium p l a t e and incubated o v e r n i g h t . P l a t e s with n e a r l y c o n f l u e n t plaques were e x t r a c t e d and t i t r e d as d e s c r i b e d above. THE TN5-MUTANT LIBRARY. i . PRODUCTION OF LIBRARY. The equation N = l n ( 1 - P ) / l n ( 1 -f ) (where N = number of mutants, P = p r o b a b i l i t y , and f = p r o p o r t i o n of the genome represented by a s i n g l e event) was used to c a l c u l a t e the number of mutants r e q u i r e d (N = 16,000) to make a l i b r a r y of Tn5_ mutants with P > 98% (36). For t h i s c a l c u l a t i o n , i t was assumed t h a t t r a n s p o s i t i o n was completely random, t h a t one "event' was the i n s e r t i o n of the Tn5_ element i n t o one k i l o b a s e (kb) of DNA, t h a t lkb was the average gene s i z e , and t h a t f = 1/4000 f o r C.crescentus s t r a i n s (18). The d e l i v e r y v e h i c l e used f o r Tn5-mutagenesis was the narrow host range ( C o l E l ) plasmid pSUP2021 which i s not maintained i n C. cre s c e n t u s s t r a i n s . I n t e g r a t e d i n t o t h i s plasmid are the Tn5_ element and the m o b i l i z a t i o n (mob) s i t e d e r i v e d from an RP4 plasmid (51). The plasmid pSUP2021 was pro v i d e d i n t r a n s with t r a n s f e r f u n c t i o n s from the E_.c_g_li host SM10, a s t r a i n designed to reduce s e l f - t r a n s f e r and v i a b i l i t y of RP4 DNA by i n t e g r a t i o n 12 of RP4 t r a n s f e r f u n c t i o n s i n t o the chromosome (6, 17, 20, 51, 54). The plasmid pSUP2021 was t r a n s f e r r e d to c a u l o b a c t e r by con j u g a l mating of CB2A(Rf-r) with SM10(pSUP2021). Approximately 10E+8 c e l l s of each (as c o n t r o l s ) or of both were suspended i n 500ul PYE, c e n t r i f u g e d to a p e l l e t , resuspended i n 25ul of PYE, and then incubated overnight on s t e r i l e n i t r o c e l l u l o s e f i l t e r s ( M i l l i p o r e ) . The c e l l s were then resuspended i n 750ul PYE to which approximately 10E+8 phage-T7 were added. T h i s mixture was incubated f o r 2h at 37C and then lOOul a l i q u o t s were p l a t e d onto c o n t r o l medium (PYE), c o n t r o l medium s e l e c t i v e a g a i n s t the SM10 donor c e l l s ( Tpl50/Rf5), and medium a l s o s e l e c t i v e f o r a n t i b i o t i c r e s i s t a n c e c o n f e r r e d to c e l l s by the Tn5_ element (Tpl50/Rf5/Km50/Sm50) (43). A f t e r seven days of growth, approximately 16,000 independently s e l e c t e d c o l o n i e s were pooled (Tn5_- l i b r a r y ) and f r o z e n at -70C. i i . ANALYSIS OF LIBRARY. In the Tn5_-library, . the presence or absence of spontaneously d e r i v e d mutants r e s i s t a n t to Km50/Sm50 s e l e c t i o n was determined by colony h y b r i d i z a t i o n with the 1.8kb H_iniiIII/aam.HI fragment of the Tn5_ element ( F i g u r e 1). One hundred twenty s e l e c t e d c o l o n i e s , w i l d t y p e CB2A ( n e g a t i v e c o n t r o l ) , and SM10(pSUP2021) ( p o s i t i v e c o n t r o l ) c o l o n i e s were t r a n s f e r r e d to s t e r i l e hardened a s h l e s s f i l t e r paper (Whatman). These " l i f t s " were t r e a t e d f o r lOmin i n 0.5M NaOH, n e u t r a l i z e d twice f o r lOmin i n 1M T r i s (pH7), soaked f o r 5min i n 0.5M Tris/1.5M NaCl, r i n s e d with 95% EtOH, and baked f o r 2h at 80C. The l i f t s were then incubated i n 10ml p r e h y b r i d i z a t i o n s o l u t i o n (5X SSC (36), 200ug/ml salmon sperm DNA (sheared by Tn5_ element: k i l o b a s e s : 0.1 H. B. 1.5 .Ba 3.1 . B. H 4.3 5.8 PROBE: BAM: • ..I.-*..>..J>, , t , I „ , f , t BGL: FIGURE 1. THE TN5 ELEMENT AND PROBING OF TN5-MUTATED DNA. The purpose of t h i s f i g u r e i s to e x p l a i n the number and r e l a t i v e i n t e n s i t y of h y b r i d i z i n g bands observed on autoradiographs of Tn5-mutated chromosomal DNA t h a t i s d i g e s t e d with enzymes that cut w i t h i n the Tn5_ element. Tn5_ = a p h y s i c a l map ( B g i l fragment) of the Tn5 element and shows the i n v e r t e d repeats ( u n d e r l i n e d ) and r e s t r i c t i o n s i t e s f o r B = B g l l l . Ba = BjauHI, and H = H i n d l l l PROBE = 1.8kb Hjjidlll/BamHI fragment ( u n d e r l i n e d ) used f o r a l l Tn5_-probing of Southern b l o t s . BAM = h y b r i d i z a t i o n ( u n d e r l i n e d ) of the Tn5_ probe to B&mHT-digested Tn5_-mutant chromosomal DNA (*****): produces one l i g h t and one dark band of v a r i a b l e s i z e (depending on the s i z e of the r e s t r i c t i o n fragment produced upon d i g e s t i o n with BjuaHI) on an autoradiograph, BGL = h y b r i d i z a t i o n ( u n d e r l i n e d ) of Tn5_-probe to B g l I I - d i g e s t e d Tn5_-mutant chromosomal DNA (*****)-. produces two l i g h t bands of v a r i a b l e s i z e and one 3kb dark band. (30). 14 s o n i c a t i o n , b o i l e d , and i c e - c o o l e d ) , and 0.1% s a r c o s y l ) f o r l h at 65C. A f t e r the a d d i t i o n of approximately 10E+6 cpm of b o i l e d and i c e - c o o l e d probe, the l i f t s were incubated o v e r n i g h t f o r high s t r i n g e n c y h y b r i d i z a t i o n at 65C. Probe p r e p a r a t i o n was as d e s c r i b e d below. The next day, l i f t s were r i n s e d i n IX b l o t wash (3X SSC, 5mM EDTA, 0.02% s a r c o s y l ) , incubated f o r 30min at 65C i n IX b l o t wash, incubated twice f o r 30min at 65C i n 0.IX b l o t wash, d r i e d , and autoradiographed with X-Omat Xray f i l m (Kodak) at -70C f o r approximately 24h. Since i t i s known that Tn5 does not transpose i n t o genomic DNA i n a p e r f e c t l y random f a s h i o n i n other b a c t e r i a (5, 6, 17), the degree of randomness i n the p r o d u c t i o n of the Tn5_-library was i n v e s t i g a t e d . F i r s t , the frequency of auxotrophic mutation was determined f o r 400 randomly i s o l a t e d Tn5_-library i s o l a t e s . These were r e p l i c a p l a t e d onto PYE and HMG media p l a t e s . C o l o n i e s unable to grow on HMG medium were r e t e s t e d to c o n f i r m auxotrophy. Second, Southern b l o t s of Tn5_-library chromosomal DNA d i g e s t e d with 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 enzymes were probed f o r Tn5_. DNA p r e p a r a t i o n and b l o t t i n g was as d e s c r i b e d below. During p r e p a r a t i o n of the Tn5_-library, i t was expected that Tn5_ would transpose from the narrow host range plasmid pSUP2021 i n t o the CB2A genome before the plasmid was l o s t by s e g r e g a t i o n . A l t e r n a t e l y , p a r t of or a l l of the v e c t o r and transposon could have i n t e g r a t e d i n t o the r e c i p i e n t chromosome by a s i n g l e recombination event (5, 17, 51). Three experiments were designed to t e s t f o r t h i s l a t t e r event. F i r s t , the c h l o r a m p h e n i c o l - r e s i s t a n c e c h a r a c t e r i s t i c of plasmid pSUP2021 was t e s t e d f o r i n 263 randomly i s o l a t e d Tn5_-15 l i b r a r y mutants and wildtype CB2A. C o l o n i e s grown on n o n s e l e c t i v e medium were r e p l i c a p l a t e d onto Cm2, Km50/Sm50, Km50/Sm50/Cm2, and PYE medium. Second, s i n c e pSUP2021 c o n t a i n s a pBR-replicon, a Southern b l o t of genomic DNA from w i l d t y p e CB2A and the Tn5_-l i b r a r y was probed with pBR322. F i n a l l y , a colony l i f t of 92 Cm2-r e s i s t a n t Tn5_-library i s o l a t e s , w i l d t y p e CB2A (n e g a t i v e c o n t r o l ) , and C600(pSUP2021) ( p o s i t i v e c o n t r o l ) was probed with the e n t i r e pBR322 plasmid. To i s o l a t e Hf-mutants, approximately 12,000 T n 5 - l i b r a r y i s o l a t e s were assayed f o r aberrant colony-adhesiveness to c e l l u l o s e - a c e t a t e (44). Ethanol-washed and oven-dried (37C) c e l l u l o s e - a c e t a t e d i s c s were pressed onto p l a t e s of c o l o n i e s and l e f t f o r 3min. D i s c s were then washed with high-pressure c o o l tap water to remove v i s i b l e c e l l m a t e r i a l , s t a i n e d f o r 3min (0.01% Coomassie b r i l l i a n t blue, 10% i s o p r o p a n o l , 10% g l a c i a l a c e t i c a c i d ) , r i n s e d i n water f o r 3min, a i r - d r i e d , and then compared to photocopies of the master p l a t e s . Suspected c e l l u l o s e - a c e t a t e -minus (acetate-minus) c o l o n i e s were reassayed and then checked on a Z e i s s phase-contrast microscope f o r the a b i l i t y to form r o s e t t e s (46). I s o l a t e d c o l o n i e s of each a c e t a t e - m i n u s / r o s e t t e -minus and a c e t a t e - m i n u s / r o s e t t e - p l u s mutant (Hf-mutant) were then s t o r e d f r o z e n at -70C. ANALYSIS OF HOLDFAST-DEFECTIVE MUTANTS. i . IDENTIFYING THE SITES OF TN5-INSERTI0N. Chromosomal DNA e x t r a c t i o n s from each Hf-mutant were performed i n microfuge tubes and produced approximately 50ug of n u c l e i c a c i d (2, 15, 60). Approximately 10E+9 c e l l s were harvested at ~12,000xg f o r lOmin 16 and suspended f i r s t i n 0.6ml lOmM T r i s (pH8) and ImM EDTA (TE(10/1)) and then i n 0.6ml TE(100/10). Lysozyme was added to £&• 300ug/ml, and the mixture was incubated f o r 5min at 37C. SDS was then added to 1.0%, and the mixture was incubated at 65C f o r lOmin. F i n a l l y , p r o t e i n a s e K was added to c_&. 300ug/ml, and the mixture was incubated at 65C f o r l h . The n u c l e i c a c i d s were then p u r i f i e d by c o n s e c u t i v e e x t r a c t i o n s with TE(10/1)-saturated phenol, phenol/chloroform/isoamyl a l c o h o l (25:24:1), and chloroform, each time r e t a i n i n g the aqueous phase. N u c l e i c a c i d s were then p r e c i p i t a t e d by the a d d i t i o n f i r s t of sodium-acetate (pH7) to a c o n c e n t r a t i o n of 300mM and second of approximately two volumes of 95% e t h a n o l . N u c l e i c a c i d s were p e l l e t e d by c e n t r i f u g a t i o n , suspended i n 150ul TE(10/1), and incubated f o r 2h at 37C a f t e r the a d d i t i o n of h e a t - t r e a t e d (DNase-free) p a n c r e a t i c RNase to ca. 200ug/ml. R e s t r i c t i o n enzyme d i g e s t i o n s were g e n e r a l l y performed on l O u l (~3ug) of the above n u c l e i c a c i d e x t r a c t s i n a p p r o p r i a t e b u f f e r s with 2 u l (10 to 20 u n i t s ) of enzyme (Bethesda Research Labs (BRL)) i n a t o t a l volume of 20ul. Tn5_-mutated chromosomal DNA was d i g e s t e d with r e s t r i c t i o n enzymes t h a t cut once (BamHI) or twice (BgJJI) w i t h i n the Tn5_ element ( F i g u r e 1) and with enzymes t h a t do not cut w i t h i n the Tn5_ element (*ClaI . E_C_Q_RI , Kpjil, and S_s_£.I) (6, 30). R e s t r i c t i o n fragments were separated by e l e c t r o p h o r e s i s i n a 0.45 to 0.50% agarose g e l (BRL) prepared with TBE b u f f e r (50mM T r i s , 50mM b o r i c a c i d , 2.5mM EDTA) except when fragments were to be g e l - p u r i f i e d i n which case they were separated i n TAE-prepared 17 agarose (40mM T r i s , g l a c i a l a c e t i c a c i d to pH7.9, 2.OmM EDTA). Gels were run at 40 to 60 v o l t s on a 25cm g e l bed f o r 20 to 30h. L i n e a r i z e d double-stranded DNA markers ranging i n s i z e from 1 to 30kb were run with each g e l (BRL). F i n a l l y , the g e l s were s t a i n e d f o r 15min i n lug/ml ethidium bromide ( E t B r ) and then photographed. S e l e c t e d n u c l e i c a c i d p r e p a r a t i o n s were a l s o d i g e s t e d with Ase_I, a r e s t r i c t i o n enzyme that cuts i n f r e q u e n t l y i n the c a u l o b a c t e r genome by Dr. B. E l y ( U n i v e r s i t y of South C a r o l i n a ) . R e s t r i c t i o n fragments were separated by Dr. E l y u s i n g p u l s e d -f i e l d g r a d i e n t g e l e l e c t r o p h o r e s i s (PFGE) (Geneline apparatus, 45sec p u l s e , 140mA, 16h) a c c o r d i n g to p u b l i s h e d procedures (18). When Southern-type b l o t s were r e q u i r e d , DNA was t r a n s f e r r e d to Hybond-N nylon membrane (Amersham) (49, 53). Ge l s were soaked f o r l/2h i n 0.25M HC1, r i n s e d with water, soaked f o r l/2h i n d e n a t u r a t i o n s o l u t i o n (0.5M NaOH, 1.5M NaCl), soaked f o r lOmin i n t r a n s f e r s o l u t i o n (0.25M NaOH, 1.5M NaCl), and then b l o t t e d o v e r n i g h t with t r a n s f e r s o l u t i o n . The next day, the b l o t was r i n s e d i n 2X SSC and baked f o r lOmin at 80C. The Southern b l o t s were incubated ( a t approximately lml/15 sq.cm.) f o r l h at 65C i n p r e h y b r i d i z a t i o n s o l u t i o n : 5X SSPE, 5X Denhardt's s o l u t i o n (36), 0.5% SDS, and 20ug/ml of salmon sperm DNA. B l o t s were then h y b r i d i z e d overnight at 65C a f t e r the a d d i t i o n of b o i l e d and i c e - c o o l e d probe (~50,000 cpm/ml, ~10E+7 cpm/ug probe DNA, " l n g probe DNA/ml). Nick t r a n s l a t i o n s of probe DNA (38) were c a r r i e d out i n a t o t a l volume of 25ul. B u f f e r (50mM T r i s (pH7.5), 15mM MgC12, ImM d i t h i o t h r e i t o l , 50ug/ml bovine serum albumin), approximately 75ng 18 of DNA, a mixture of dGTP/dTTP/dATP <40mM), 50uCi alpha-32P-dCTP (ICN Radiochemicals), and 1 u n i t DNasel/DNA polymerase (BRL) were combined to g i v e the f i n a l c o n c e n t r a t i o n s noted i n br a c k e t s and incubated at 14C f o r approximately 2h. A l l probes were p u r i f i e d u s i n g Geneclean ( B i o 101 Inc.) ac c o r d i n g to manufacturers i n s t r u c t i o n s . H y b r i d i z e d b l o t s were washed at high s t r i n g e n c y (twice i n 2X SSPE (36) and 0.1% SDS at room temperature f o r lOmin, once i n IX SSPE and 0.1% SDS at 65C f o r 15min, and twice i n 0.IX SSPE and 0.1% SDS at 65C f o r lOmin), a i r - d r i e d , and then autoradiographed. When r e q u i r e d , probe DNA was removed by soaking the b l o t s at 45C f o r l/2h f i r s t i n 0.4M NaOH and then i n a mixture of 0.IX SSC, 0.1% SDS, and 0.2M T r i s (pH7.5). Hf-mutants t h a t were found by Southern b l o t a n a l y s i s to have the same s i t e of Tn5_-insert ion were c o l l e c t i v e l y r e f e r r e d to as a mutant group (gX, where X = 1 to 10). Unless otherwise noted, a l l subsequently d e s c r i b e d experiments on "mutant groups" were performed on one r e p r e s e n t a t i v e Hf-mutant from each group. Two t e s t s were performed to show t h a t the i n s e r t i o n of Tn5_ i n t o Hf-mutant genomic DNA was a r e s u l t of a t r a n s p o s i t i o n event and was not a r e s u l t of a s i n g l e recombination event between the d e l i v e r y plasmid pSUP2021 (Cm-r, pBR-replicon) and r e c i p i e n t genomic DNA. A l l 10 mutant groups and a l l nine g6 Hf-mutants were t e s t e d f o r Cm-resistance as d e s c r i b e d above and a Southern b l o t of chromosomal DNA from a pool of 71 Hf-mutants and from each of seven mutant groups was probed with the e n t i r e pBR322 plasmid. i i . TESTING FOR PLEIOTROPIC MUTANTS. To determine the presence or absence of mutants p l e i o t r o p i c f o r p o l a r - r e l a t e d 19 f u n c t i o n s , Hf-mutants were t e s t e d f o r three other p o l a r - r e l a t e d c h a r a c t e r i s t i c s : m o t i l i t y , p o l a r phage attachment, and s t a l k presence. M o t i l i t y was t e s t e d i n two ways. F i r s t , l i q u i d c u l t u r e s of a l l Hf-mutant i s o l a t e s were checked m i c r o s c o p i c a l l y d u r i n g l a t e l o g a r i t h m i c growth phase (0D600 "0.9). Second, a l l i s o l a t e s were t e s t e d f o r the a b i l i t y of c e l l s to swarm through s e m i - s o l i d (0.3% agar) PYE medium (27). P l a t e s were stabbed i n d u p l i c a t e with an inoculum of each i s o l a t e and incubated at room temperature f o r f o u r days. To a s c e r t a i n whether or not any v a r i a b i l i t y i n these r e s u l t s was simply due to d i f f e r e n c e s i n growth, s p e c i f i c growth r a t e s were a l s o determined. A l l ten mutant groups were grown f o r approximately twenty g e n e r a t i o n s (OD600~0.0001 to 0.5, LKB Biochrom U l t r a s p e c II u l t r a v i o l e t spectrophotometer) at 30C i n 50ml PYE shaken at 200rpm. L o g a r i t h m i c r e g r e s s i o n s were used to c a l c u l a t e s p e c i f i c growth r a t e s . S e n s i t i v i t y to the s w a rmer-specific p o l a r phage-CBK was t e s t e d on a l l Hf-mutants. In a 1ml volume, approximately 10E+6 mutant c e l l s were incubated ov e r n i g h t a f t e r i n f e c t i o n at 0.1 MOI with phage-CBK p r e v i o u s l y propagated on CB2A. C o n t r o l s i n c l u d e d each experimental and c o n t r o l sample without phage a d d i t i o n and the p h a g e - r e s i s t a n t CB2A-B6 mutant. The s t a l k c h a r a c t e r of a l l ten mutant groups and w i l d t y p e CB2A was checked by n e g a t i v e s t a i n whole mount e l e c t r o n microscopy (52). C u l t u r e samples i n O.lmg/ml b a c i t r a c i n were a p p l i e d to c a r b o n - s t a b i l i z e d and P a r l o d i o n - c o a t e d copper g r i d s (400 mesh) rendered h y d r o p h i l i c with a s i l i c o n monoxide l a y e r . G r i d s were then n e g a t i v e - s t a i n e d with 2% ammonium-molybdate 20 (pH7.5) and viewed at 60kV under a Siemens Elmiskop model 101A t r a n s m i s s i o n e l e c t r o n microscope. i i i . DETERMINING PHENOTYPE. H f - d e f e c t i v e mutant groups were d i f f e r e n t i a t e d p h e n o t y p i c a l l y on the absence or r e l a t i v e presence of both l e c t i n b i n d i n g to Hf and Hf-adhesiveness to g l a s s , to c e l l u l o s e - a c e t a t e , and to Hf m a t e r i a l . Late l o g a r i t h m i c growth phase (OD600 ~0.9) c u l t u r e s of mutant groups were examined by phase-contrast microscopy f o r the a b i l i t y of c e l l s to form r o s e t t e s : the a b i l i t y to adhere to other c e l l s at the Hf r e g i o n (46). The presence of Hf i n a l l mutant groups and c o n t r o l s was t e s t e d by a s s a y i n g f o r the b i n d i n g of seven f l u o r e s c e i n isothyocyanate-conjugated (FITC) l e c t i n s (Table 2) ( F l u o r e s c e i n l e c t i n k i t I, Vector Labs Inc.) to the Hf m a t e r i a l (39, 60). F I T C - l e c t i n ( 2 u l at 5mg/ml) was added to 200ul of c e l l c u l t u r e and incubated f o r l/2h on i c e . C e l l s were then c e n t r i f u g e d to a p e l l e t , r i n s e d with 1ml of water, and then suspended i n a 20ul s o l u t i o n of 50% g l y c e r o l and 2% N-propyl g a l l a t e i n 20mM potassium-phosphate (pH7). Samples were examined by f l u o r e s c e n c e and p h a s e - c o n t r a s t microscopy. For a l l ten mutant groups, the r e l a t i v e adhesiveness of Hf m a t e r i a l to g l a s s and c e l l u l o s e - a c e t a t e c o v e r s l i p s was t e s t e d . G l a s s c u l t u r e f l a s k s (250ml) were b r i e f l y r i n s e d with 2% d i m e t h y l - d i c h l o r o s i l a n e s o l u t i o n i n 1 , 1,1-thrichloroethane to reduce Hf-mediated b i n d i n g of c e l l s to the f l a s k w a l l s and then r i n s e d s e v e r a l times with water to remove the r e s i d u a l s i l a n e s o l u t i o n . Ethanol-washed and autoclaved g l a s s c o v e r s l i p s and ethanol-washed and oven-dried (37C) c e l l u l o s e - a c e t a t e c o v e r s l i p s 21 TABLE 2: CARBOHYDRATE SPECIFICITY OF LECTINS LECTIN PREDOMINANT RESIDUE SPECIFICITY ConA alpha-D-mannosy1-, a l p h a - D - g l u c o s y l - r e s i d u e s DBA N - a c e t y l g a l a c t o s a m i n y l - isomers PNA D-galactosyl-beta-(1,3)-N-acety1-galactosaminy1-RCA1 b e t a - D - g a l a c t o s y l - r e s i d u e s SBA N - a c e t y l g a l a c t o s a m i n y l - isomers UEA1 a l p h a - L - f u c o s y l - r e s i d u e s WGA (b e t a - N - A c e t y l g l u c o s a m i n y l - ) n ConA = Concanavalin A, DBA = D o l i c h o s b i f l o r u s a g g l u t i n i n , PNA = Peanut a g g l u t i n i n , RCA1 = R i c i n u s communis a g g l u t i n i n , SBA = Soybean a g g l u t i n i n , UEA1 = Ulex europaeus a g g l u t i n i n , WGA = Wheat germ a g g l u t i n i n . 22 were added to 50mls PYE and c e l l s were then i n o c u l a t e d to OD600~0.005. C u l t u r e s were grown to 0D600~0.5 (lOOrpm, 30C). C o v e r s l i p s were then rescued, r i n s e d thoroughly w i t h PYE, incubated f o r l/2h on i c e i n 2.5ml PYE with 4 u l F I T C - l a b e l l e d Wheat germ a g g l u t i n i n (WGA, Table 2), r i n s e d i n a l a r g e volume of water, and then mounted on g l a s s s l i d e s with the standard g l y c e r o l mixture f o r e p i f l u o r e s c e n t m i c r o s c o p i c examination. CLONING OF HOLDFAST-RELATED AND TN5-MUTATED DNA. From each of the fo u r genomic r e g i o n s t h a t were found to c o n t a i n c l u s t e r e d s i t e s of T n 5 - i n s e r t i o n , a Tn5-mutated genomic DNA r e s t r i c t i o n fragment was cloned i n t o a pPR510 v e c t o r plasmid and propagated i n E . c o l i DH5-alpha c e l l s . R e s t r i c t i o n fragments t h a t were shown to c o n t a i n a l l of the i d e n t i f i e d s i t e s of Tn5_-insertion w i t h i n a g i v e n c l u s t e r and t h a t were approximately 10 to 15kb i n s i z e were chosen f o r c l o n i n g . From each r e p r e s e n t a t i v e mutant of groups g l , g3, g5, and g9, 350ug of n u c l e i c a c i d was cut with the a p p r o p r i a t e r e s t r i c t i o n enzyme(s), phenol-chloroform e x t r a c t e d , p r e c i p i t a t e d , suspended i n lOOul TE(10/1), and then l a y e r e d onto a 12ml sucrose g r a d i e n t (10 to 40% s t e r i l e sucrose) prepared i n p o l y a l l o m e r tubes ( P o l y c l e a r , Beckman). Tubes were u l t r a c e n t r i f u g e d ("198,OOOxg, 18h, 20C) i n a Beckman SW41 swinging bucket r o t o r . F r a c t i o n s (500ul) were c o l l e c t e d and l O u l from each f r a c t i o n were e l e c t r o p h o r e s c e d , b l o t t e d , and probed f o r presence of Tn5_. DNA from the f r a c t i o n with the most h y b r i d i z a t i o n was p r e c i p i t a t e d and suspended i n TE(10/1) (36). 23 The v e c t o r plasmid used f o r c l o n i n g from mutant groups g l , g5, and g9 was e x t r a c t e d by a l k a l i n e l y s i s (36) from E . c o l i C600(pPR510) and then c e s i u m - c h l o r i d e g r a d i e n t - p u r i f i e d . The crude plasmid DNA p r e p a r a t i o n was suspended i n 8ml TE(10/1) to which C s C l ( l . l g / m l ) , EtBr (0.25mg/ml), and m i n e r a l o i l were added. The sample was u l t r a c e n t r i f u g e d i n a Beckman 50Ti f i x e d angle r o t o r f o r 36h at ~130,OOOxg. The plasmid DNA band was c o l l e c t e d , twice e x t r a c t e d from EtBr with an equal volume of n-bu t a n o l , p r e c i p i t a t e d away from C s C l with 2.5 volumes of 70% EtOH, and c e n t r i f u g e d at ~12,000xg f o r l/2h. The DNA p e l l e t was suspended i n TE(10/1) and then d i g e s t e d with the a p p r o p r i a t e r e s t r i c t i o n enzymes f o r c l o n i n g . F i n a l l y , the d i g e s t i o n mixture was phenol-chloroform e x t r a c t e d , p r e c i p i t a t e d , and suspended i n TE(10/1). Since the m u l t i p l e c l o n i n g s i t e of the pPR510 plasmid does not c o n t a i n a C l a l s i t e , the plasmid pPR510ElF2 was used f o r c l o n i n g of Tn5_-mutated DNA from mutant group g3. A crude a l k a l i n e plasmid p r e p a r a t i o n of t h i s v e c t o r was d i g e s t e d with the r e s t r i c t i o n enzymes C l a l / K p n l to remove a l l but 1.2kb of the E1F2 i n s e r t . The v e c t o r DNA was then g e l - p u r i f i e d from t h i s d i g e s t i o n mixture. Vector DNA (0.5ug) was l i g a t e d o vernight to one-half of the i n s e r t DNA prepared from a s i n g l e sucrose g r a d i e n t f r a c t i o n . L i g a t i o n s were performed i n a t o t a l volume of 15ul at 14C i n l i g a s e b u f f e r (BRL) and with l u l (1 u n i t ) of T4 DNA l i g a s e (BRL). The l i g a t i o n mixture was d i l u t e d f i v e - f o l d with water f o r use i n e l e c t r o p o r a t i o n . 24 Electrocompetent c e l l s were prepared by c h i l l i n g mid-l o g a r i t h m i c growth phase c e l l s on i c e f o r l/2h, c e n t r i f u g i n g f o r lOmin at ~12,000xg, and suspending f i r s t i n 1.0 volume and then i n 0.5 volume of i c e - c o l d s t e r i l e water. F i n a l l y , the c e l l s were supended at approximately 10E+10 c e l l s / m l i n 0.02 volumes of i c e -c o l d s t e r i l e 10% g l y c e r o l , f r o z e n on dry i c e , and s t o r e d at -70C (8, 14, 22). In a d d i t i o n to experimental samples, f o r each e l e c t r o p o r a t i o n a no DNA ( n e g a t i v e ) c o n t r o l and v e c t o r only ( p o s i t i v e ) c o n t r o l were run. In a s t e r i l e and i c e - c o l d e l e c t r o p o r a t i o n c u v e t t e , 3ul of d i l u t e d l i g a t i o n mix and 40ul electrocompetent c e l l s were mixed and e l e c t r o p o r a t e d i n a Bio-Rad Gene p u l s e r at 200 ohms, 25 uFD, and 2.5 kV. A f t e r the a d d i t i o n of 1ml of n o n s e l e c t i v e medium, the mixture was incubated at growth temperature f o r three to four c e l l g e n e r a t i o n s . C e l l s were then suspended i n 400ul of n o n s e l e c t i v e media from which lOOul a l i q u o t s were p l a t e d onto medium s e l e c t i v e f o r v e c t o r DNA <Cm20) and medium s e l e c t i v e f o r v e c t o r DNA p l u s Tn5.-containing DNA (Cm20/Km50). To c o n f i r m t h a t a Tn5_-mutated fragment of DNA was cloned, a colony l i f t of s e l e c t e d c o l o n i e s was probed f o r presence of Tn5_. Crude plasmid p r e p a r a t i o n s of f i v e clones t h a t probed p o s i t i v e were then d i g e s t e d with the a p p r o p r i a t e r e s t r i c t i o n enzyme(s) to e x c i s e the cloned i n s e r t : pGl(£s_tI), pG3(ClaI/KpnI). pG5(EcoRI), and p G 9 ( S s t l / E c o R I ) . Since plasmids propagated i n DH5-alpha c e l l s are methylated at C l a l s i t e s which, i n t u r n , are not recognized by the C l a l enzyme, then to e x c i s e the i n s e r t cloned from mutant group g3, a crude p r e p a r a t i o n of pG3 was f i r s t e l e c t r o p o r a t e d 25 i n t o the dam-minus E . c o l i s t r a i n RB404 and then t r e a t e d as d e s c r i b e d above. On a Southern b l o t probed f o r Tn5_, these cloned i n s e r t s were compared i n molecular weight with the Tn5_-containing genomic DNA fragments of mutants that were suspected to have l i n k e d s i t e s of Tn5_-insert i o n . To prove t h a t the s i t e s of Tn5_-insert ion i n c e r t a i n mutant groups were l i n k e d and that chromosomal DNA c o n t a i n i n g a l l of these l o c i was cloned, a Southern b l o t of each cloned plasmid and chromosomal DNA from each mutant group suspected to c o n t a i n l i n k e d s i t e s of T n 5 - i n s e r t i o n was probed with the corresponding g e l - p u r i f i e d and n i c k - t r a n s l a t e d cloned i n s e r t (Tn5 element p l u s f l a n k i n g chromosomal DNA). Samples were d i g e s t e d with the a p p r o p r i a t e enzymes used f o r c l o n i n g from each of the four genomic r e g i o n s . With two of these four Southern b l o t s , samples of w i l d t y p e CB2A and of mutants that contained a s i t e of Tn5_-i n s e r t i o n t h at was not l i n k e d to the probe DNA were i n c l u d e d as n e g a t i v e c o n t r o l s . PRODUCTION OF DOUBLE HOLDFAST-MUTANT. A double r e c i p r o c a l homologous recombination (gene replacement) event was f o r c e d between the genomic DNA of CB2A-B9 (UV-induced Hf-shedding mutant) and the cloned i n s e r t of plasmid pG5 to produce a double Hf-mutant. A crude a l k a l i n e plasmid p r e p a r a t i o n of the narrow host range plasmid pG5 was e l e c t r o p o r a t e d i n t o CB2A-B9 and gene t r a n s f e r events were s e l e c t e d on PYE p l a t e s supplemented with Km50/Sm50. 26 To screen out i s o l a t e s t h a t had i n t e g r a t e d pG5 i n t o the r e c i p i e n t chromosome by a s i n g l e c r o s s over event, s e l e c t e d i s o l a t e s (CB2A-B9::Tn5) were t e s t e d f o r p l a s m i d - c o n f e r r e d Cm-r e s i s t a n c e by r e p l i c a p l a t i n g c o l o n i e s onto Cm2 p l a t e s . In a d d i t i o n , s i n c e the plasmid pG5 contained a p B R - r e p l i c o n , the i n t e g r a t i o n of pG5 i n t o r e c i p i e n t genomic DNA was checked by p r o b i n g a colony l i f t of Cm2-resistant i s o l a t e s with the e n t i r e pBR322 plasmid. To determine i f the plasmid pG5 was maintained w i t h i n s e l e c t e d i s o l a t e s , plasmid i s o l a t i o n procedures (31) were a p p l i e d to both Cm-resistant and Cm-sensitive i s o l a t e s . Double Hf-mutants (B9(G5)) were expected to express both mutant group g5 ( l e s s Hf) and CB2A-B9 (shed Hf) c h a r a c t e r i s t i c s . The presence or absence of the double mutant phenotype was determined f o r 25 Cm2-sensitive i s o l a t e s by t e s t i n g f o r the r e l a t i v e amount of Hf produced (FITC-WGA p o s i t i v i t y ) and the r e l a t i v e adhesiveness of Hf m a t e r i a l to c e l l u l o s e - a c e t a t e c o v e r s l i p s as d e s c r i b e d above. The mutant group g5 c h a r a c t e r was confirmed by p r o b i n g f o r Tn5_ a Southern b l o t of chromosomal DNA d i g e s t e d with ESJIHI from mutants t h a t expressed the B9(G5) phenotype. As c o n t r o l s , mutant group g5 ( p o s i t i v e c o n t r o l ) , mutant group g3 ( n e g a t i v e c o n t r o l ) , three Cm2-resistant i s o l a t e s , and three screened mutants t h a t expressed only the CB2A-B9 phenotype were a l s o probed. 27 RESULTS THE TN5-MUTANT LIBRARY. i . PRODUCTION OF LIBRARY. The frequency of p o s i t i v e s e l e c t i o n f o r CB2A: :Tn5_ mutants was 10E-5 per i n i t i a l r e c i p i e n t i n the c o n j u g a t i o n . No SM10 donor c e l l s s u r v i v e d the s e l e c t i o n process. A l l 120 s e l e c t e d c o l o n i e s chosen f o r colony h y b r i d i z a t i o n probed p o s i t i v e f o r Tn5_, i n d i c a t i n g l i t t l e or no spontaneous r e s i s t a n c e of CB2A to the double a n t i b i o t i c (Km50/Sm50) s e l e c t i o n . i i . ANALYSIS OF LIBRARY. The frequency of auxotrophy i n the Tn5_-library was 3.3% (13/400). T h i s was somewhat higher than the f r e q u e n c i e s of 1 to 2% re p o r t e d i n the l i t e r a t u r e (17, 51), but was s t i l l s u p p o r t i v e of the argument that Tn5_ t r a n s p o s i t i o n i n CB2A was s u f f i c i e n t l y random to make i t a u s e f u l method f o r mutagenesis. Southern b l o t s of T n 5 - l i b r a r y genomic DNA probed with Tn5_ produced a smear and one p a r t i c u l a r l y n o t i c e a b l e hotspot r e g i o n on autoradiographs ( F i g u r e s 2 and 3). T h i s a p p a r e n t l y p r e f e r e n t i a l s i t e of t r a n s p o s i t i o n was not the same as any of the H f - r e l a t e d s i t e s of Tn5_-insertion ( F i g u r e s 2, 3, and 4 ) . Since the Southern b l o t of chromosomal DNA e x t r a c t s from the Tn5_-l i b r a r y and wi l d t y p e CB2A probed negative with pBR322 ( F i g u r e 2), then the hotspot r e g i o n could not be e x p l a i n e d by pBR-replicon i n t e g r a t i o n i n t o genomic DNA by a s i n g l e recombination event and t h e r e f o r e supported the argument f o r some degree of non-random t r a n s p o s i t i o n . 28 PROBE: Tn5 pBR322 ID: L H C L H C P g5 g7 g8 g1 g2 g4 g10 FIGURE 2. SOUTHERN BLOT OF TN5-MUTATED CHROMOSOMAL DNA: PROBING FOR TN5 AND pSUP2021. The same b l o t i s probed f i r s t w ith the e n t i r e pBR322 plasmid and then with the 1.8kb tLiMIII/BajiHI fragment of the Tn5_ element. A l l chromosomal DNA i s d i g e s t e d with S s t l / K p n l . ID.: C = C.crescentus CB2A ( w i l d t y p e ) , gX = h o l d f a s t -d e f e c t i v e mutant group (where X = 1 to 10), H = a po o l of 71 holdfast-mutants (approximately 85% of the h o l d f a s t mutants (65/77) are represented by two S s t l / K p n l bands: T a b l e s 3 and 4 ) , L = l i b r a r y of CB2A: :Tn5_ mutants, and P = pBR322 cut with ECJIRI . 2 9 CONTROL: Li g3 g5 g5 g5 ISOLATE: a1 a2 a3 a4 a5 a6 T1 bi T2 d T3 FIGURE 3. SOUTHERN BLOT COMPARISON OF TN5-CONTAINING SITES AMONG DOUBLE MUTANTS: BamHI DIGESTION OF CHROMOSOMAL DNA. Southern b l o t probed with the 1.8kb Hindlll/BamHI Tn5_-f ragment. CONTROLS: L i = l i b r a r y of CB2A::Tn5 mutants, gX = h o l d f a s t - d e f e c t i v e mutant group (where X = 1 to 6). ISOLATE: aX = c h l o r a m p h e n i c o l - s e n s i t i v e (2ug/ml) CB2A-B9: :Tn5_ i s o l a t e s that express both mutant group g5 and CB2A-B9 phenotypic c h a r a c t e r i s t i c s (B9(G5) double h o l d f a s t -mutants), T l = mixture of a l to a6, bX = chl o r a m p h e n i c o l -s e n s i t i v e CB2A-B9: :Tn5_ i s o l a t e s that express CB2A or CB2A-B9 phenotypic c h a r a c t e r i s t i c s , T2 s mixture of b l to b3, cX = c h l o r a m p h e n i c o l - r e s i s t a n t CB2A-B9: :Tn5_ i s o l a t e s , T3 = mixture of c l to c3. The expected number of h y b r i d i z i n g fragments per sample i s shown i n F i g u r e 1. 30 GROUP: g5 g7 g9 g8 g1 g2 g3 g4 g10g6 FIGURE 4. SOUTHERN BLOT COMPARISON OF TN5-CONTAINING FRAGMENTS AMONG HOLDFAST-DEFECTIVE MUTANT GROUPS: B_am.HI DIGESTION OF CHROMOSOMAL DNA. Southern b l o t probed with the 1.8kb Hjjidlll/BamHI Tn5-fragment. The expected number of h y b r i d i z i n g fragments per sample i s shown i n F i g u r e 1. gX = h o l d f a s t -d e f e c t i v e mutant group (where X = 1 to 10). 31 Screening of randomly chosen T n 5 - l i b r a r y i s o l a t e s on Cm2 p l a t e s showed t h a t 4.2% (11/263) were Cm- r e s i s t a n t . However, on l y 56% (52/92) of s e l e c t e d Cm2-resistant T n 5 - l i b r a r y i s o l a t e s probed p o s i t i v e f o r pBR322 which suggested that about h a l f of the Cm-r e s i s t a n c e frequency (2%) could be e x p l a i n e d by pBR-replicon i n t e g r a t i o n by a s i n g l e c r o s s o v e r event between v e c t o r and chromosomal DNA. That the observed frequency of Cm-resistance was too low to be" r e s o l v e d by p r o b i n g of T n 5 - l i b r a r y chromosomal DNA with pBR322 ( F i g u r e 2) suggested that p B R-replicon i n t e g r a t i o n was random. Since a l l ten mutant groups and a l l nine mutants of group g6 probed negative with pBR322, then p B R - i n t e g r a t i o n d i d not occur i n any of the Hf-mutants. R e s u l t s from Cm2-resistance t e s t s on both l i b r a r y i s o l a t e s and on double mutant i s o l a t e s (below) suggested t h a t the l e v e l of chloramphenicol used was not s t r i n g e n t enough to p r o p e r l y screen f o r Cm-resistance c o n f e r r e d upon i s o l a t e s by the v e c t o r . Hf-mutants were screened f o r aberrant Hf-adhesiveness from the Tn5_-library as colony-acetate-minus/rosette-minus c h a r a c t e r at a frequency of 0.6% (66/11,958) and as c o l o n y - a c e t a t e -m i n u s / r o s e t t e - p l u s c h a r a c t e r at a frequency of 0.2% (6/2580). A t o t a l of 77 mutants were i s o l a t e d and these were twice confirmed f o r the colony-acetate-minus c h a r a c t e r i s t i c ( T able 3). ANALYSIS OF HOLDFAST-MUTANTS. i . IDENTIFYING THE SITES OF TN5-INSERTI0N. The Tn5_-c o n t a i n i n g r e s t r i c t i o n fragments i n each of the 77 Hf-mutants were i d e n t i f i e d i n Southern b l o t s of chromosomal DNA d i g e s t e d with enzymes t h a t cut once (BamHI: F i g u r e s 4) or twice (Bgl.II: 32 TABLE 3: PHENOTYPE OF HOLDFAST-MUTANTS STRAIN COLONY ROSETTE LECTIN BINDING HOLDFAST HOLDFAST HOLDFAST ADHESION FORMATION JffiA. DBA SBA ILEA ADHESION ADHESION PHENOTYPE TO cAc TO GLASS TQ cAc SUMMARY CB2A yes +++ +++ ++ ++ ++ +++ +++ wildtype (£. crescentus wildtype) CB2A::Tn5 yes +++ +++ ++ ++ ++ +++ +++ wildtype (Tn5.-library i s o l a t e ) CB2A-B9 no + ++++ ++ + ++ ++++ +++ shed ( h o l d f a s t shedding mutant) CB2A-B5 no - _ _ _ _ _ _ none ( h o l d f a s t minus mutant) ____ _5 6* no ++ ++ - - - ++ ++ l e s s g7 2 no ++ ++++ ++ + + ++++ ++++ shed g9 2 no + +++ ++ + + +++ +++ shed g8 2 no - ++++ +++ + ++ ++++ ++++ shed g l 6 no - - none g2 16 no - - none g3 30 no - - none g- 3 no - - none gio 1 no - - none g6 9 no - ++ - - - + l e s s / curved s t a l k gX = H o l d f a s t - d e f e c t i v e mutant groups ( g l to g l O ) . No = number of mutant c o l o n i e s screened from approximately 12,000 Tn5_-l i b r a r y i s o l a t e s as unable to bind to c e l l u l o s e - a c e t a t e d i s c s (cAc) and unable to form r o s e t t e s . * = number of mutants screened from approximately 2,600 Tn5_-library i s o l a t e s as unable to bind to c e l l u l o s e - a c e t a t e and able to form r o s e t t e s . P l u s (+) va l u e s are r e l a t i v e amount (compared to wild t y p e Caulobacter CB2A) of r o s e t t e formation or r e l a t i v e amount of fl u o r e s c e n c e produced by FITC-conjugated l e c t i n s bound to h o l d f a s t : DBA = Do l i c h o s b i f l o r u s a g g l u t i n i n , SBA = Soybean a g g l u t i n i n , UEA = Ulex europaeus 1 a g g l u t i n i n , WGA = Wheat germ a g g l u t i n i n . H o l d f a s t adhesion to cAc ( c e l l u l o s e - a c e t a t e c o v e r s l i p s ) and g l a s s c o v e r s l i p s are v i s u a l i z e d by l a b e l l i n g of bound h o l d f a s t m a t e r i a l with WGA. 33 F i g u r e 5) w i t h i n the Tn5_ element. Using these enzymes, d i f f e r e n c e s of l e s s than O.lkb could be r e s o l v e d between most of the Tn5_-containing fragments. Only 10 unique s i t e s of Tn5_-insertion were i d e n t i f i e d among the 77 Hf-mutants. A l l mutants were c l a s s i f i e d as belonging to one of these 10 mutant groups ( g l to g l O ) . That no d i f f e r e n c e s i n s i z e were seen between Tn 5 - c o n t a i n i n g fragments of any Hf-mutants w i t h i n a given group suggested t h a t Tn5_ t r a n s p o s i t i o n i n CB2A was not completely random. The number of mutants that f i t i n t o the groups are given i n Table 3. Approximately 40% of the 77 Hf-mutants showed a s i n g l e s i t e of Tn5 i n s e r t i o n (g3, Table 3). T h i s c o u l d not be e x p l a i n e d by d i f f e r e n c e s i n growth r a t e among mutant groups and probably i s another i n d i c a t i o n that Tn5_ t r a n s p o s i t i o n i n CB2A was not a completely random process. Except f o r group g6 mutants, a l l B_ajaHI-digested samples produced two bands and a l l BgJLI I - d i g e s t e d samples produced three bands on autoradiographs ( F i g u r e 1). In F i g u r e 4, it.was assumed f o r mutant group g6 that both l i g h t and dark bands represented DNA fragments t h a t were n e a r l y the same s i z e . And i n F i g u r e 5, the t h i r d band i n sample g6 - which appeared to be m i s s i n g -seemed r a t h e r to p a r t i a l l y o v e r l a p the 2.9kb i n t e r n a l T n 5 ( B g l I I ) fragment ( F i g u r e 1). C e r t a i n H f - r e l a t e d s i t e s of Tn5_-insertion were c l u s t e r e d : gl/g2, g3/g4/g6/gl0, and g7/g8/g9. T h i s l e f t g5 as a separate l o c u s f o r a t o t a l of f o u r d i s t i n c t r e g i o n s . These r e g i o n s were r e s o l v e d by standard g e l e l e c t r o p h o r e t i c s e p a r a t i o n of genomic fragments produced by r e s t r i c t i o n enzyme d i g e s t i o n o u t s i d e of the 3 4 GROUP: g5 g7 g9 g8 g1 g2 g3 g4 g10g6 kb 13.7 -10.0 8.1 -5.1 -2.9 1.6 FIGURE 5. SOUTHERN BLOT COMPARISON OF TN5-CONTAINING FRAGMENTS AMONG HOLDFAST-DEFECTIVE MUTANT GROUPS: B_gJJI DIGESTION OF CHROMOSOMAL DNA. Southern b l o t probed w i t h the 1.8kb HindLIII/BamHI Tn5-fragment. The expected number of h y b r i d i z i n g fragments per sample i s shown i n F i g u r e 1. gX = h o l d f a s t -d e f e c t i v e mutant group (where X = 1 to 10). 35 Tn5 element. The s i m i l a r i t y i n s i z e between l a r g e Tn5-eontaining fragments (10 to 25kb) of c e r t a i n mutant groups i s shown i n Table 4 and F i g u r e 6. Probing f o r Tn5 of the PFGE-separated genomic fragments (Table 4, F i g u r e 7) produced r e s u l t s which agreed with the above i n t e r p r e t a t i o n . The l a r g e s i z e of these fragments (90 to 950kb) a l s o suggested that the four r e g i o n s of H f - r e l a t e d DNA were s c a t t e r e d i n the CB2 genome. i i . TESTING FOR PLEIOTROPIC MUTANTS. A l l 77 Hf-mutants appeared to be e q u a l l y m o t i l e as determined i n m o t i l i t y p l a t e assays and as assessed m i c r o s c o p i c a l l y i n l a t e - l o g a r i t h m i c growth phase l i q u i d c u l t u r e . The only d i f f e r e n c e s noted were between mutant and w i l d t y p e a b i l i t i e s to swarm, and these d i f f e r e n c e s appeared to be d i r e c t l y r e l a t e d to d i f f e r e n c e s i n s p e c i f i c growth r a t e s : 0.3/h compared to 0.4/h, r e s p e c t i v e l y . A l l Hf-mutants and w i l d t y p e CB2A were e q u a l l y phage-CBK-s e n s i t i v e : growth from 0D600~0.003 to ~0.01 compared with a f i n a l 0D600~1.0 f o r both phage-CBK-resistant (CB2A-B6) and a l l no-phage added c o n t r o l s . A l l ten mutant groups d i d produce s t a l k s . There were no n o t i c e a b l e d i f f e r e n c e s observed on the e l e c t r o n microscope between mutant groups. However, i n group g6 c u l t u r e s l a b e l l e d with F I T C - l e c t i n , predominantly curved s t a l k s were observed. That no mutants were observed to be p l e i o t r o p i c f o r any of the three p o l a r - r e l a t e d f u n c t i o n s t e s t e d - other, perhaps, than those of mutant group g6 - again suggested t h a t t r a n s p o s i t i o n of Tn5 i n CB2A was not a t o t a l l y random pro c e s s . A l t e r n a t e l y , e x p r e s s i o n of p l e i o t r o p i c phenotypes that are f r e q u e n t l y observed i n c h emical- or u l t r a v i o l e t l i g h t - t r e a t e d c e l l s may r e q u i r e 36 TABLE 4: AVERAGE SIZE OF TN5-CONTAINING FRAGMENTS PRODUCED BY DIGESTION OF HOLDFAST-MUTANT CHROMOSOMAL DNA WITH RESTRICTION ENZYMES THAT CUT OUTSIDE OF THE TN5 ELEMENT GRP: TN5-el ET9 e8 e l g2 S3 g4 elO *6 EN 7. -CONTAINING FRAGMENTS (KB 1 A 350 310 310 950 950 90 90 C 12 22 22 22 14 14 20 20 S 11 15 15 15 IS. 16 27 27 E 10 14 HM HM 17 7 S/E 10 13 13 13 16 16 16 7 S/K 11 15 15 15 16 16 22 22 22 22 C/K 12 22 22 22 14 14 14 14 14 14 E/K 10 14 HM HM 11 7 1. g5 g9 g l g3 2. E S/E S C/K 3. pG5 pG9 pGl pG3 4. 9.5 12.0 14.5 12.5 5. 3.5 6.0 8.5 6.5 A l l Southern b l o t s of chromosomal DNA are probed with the 1.8kb Hindlll/BamHI fragment of the Tn5_ element. Fragment s i z e s ( k i l o b a s e s ) presented are an amalgamation of r e s u l t s from s e v e r a l Southern b l o t s and are meant to repr e s e n t only r e l a t i v e d i f f e r e n c e s between Tn5_-containing fragments. A = AsfiJ, C = CLLaJ., E = EcoRI. K = Kpnl. S - S s t I , HM = u n r e s o l v a b l e l a r g e s i z e fragment, gX = H f - d e f e c t i v e mutant groups (where X = 1 to 10). The Southern b l o t of Aae.1 d i g e s t s was prepared by Dr. B. E l y ( U n i v e r s i t y of South C a r o l i n a ) . 1. = mutant group from which T n 5 - c o n t a i n i n g r e s t r i c t i o n fragment i s cloned ( u n d e r l i n e d fragments i n t a b l e ) , 2. = enzyme(s) used f o r c l o n i n g , 3. = plasmid clone name, 4. = approximate s i z e (kb) of fragment cloned, 5. = approximate s i z e (kb) of cloned chromosomal DNA f l a n k i n g the Tn5_ element. 3 7 ENZYME: E S S /E S C C/K FIGURE 6. SOUTHERN BLOT COMPARISON OF CLONED HOLDFAST-RELATED DNA FRAGMENTS WITH CHROMOSOMAL DNA DIGESTS OF HOLDFAST-DEFECTIVE MUTANT GROUPS THAT CONTAIN LINKED SITES OF TN5-INSERTION: PROBING FOR TN5. C = C l a l . E = E__Q_RI, K = K j D n J, S = S_sjtl, GX = the cloned plasmid pGX t h a t c o n t a i n s H f - r e l a t e d and Tn5_-mutated chromosomal DNA cloned from mutant group gX (where X = 1 to 10). Note that the overloaded plasmid lanes (GX) make i t appear th a t ( f o r example) G5 and g5 samples do not migrate the same d i s t a n c e . These d i s c r e p a n c i e s are shown to be due s o l e l y to the la r g e amount of DNA run on t h i s g e l by the r e s u l t s presented i n Fi g u r e 9 which show that the plasmid and chromosomal DNA do migrate s i m i l a r d i s t a n c e s . 38 CB g3 g6 g9 g7 g1 g2 g5* CB g3 g6 g9 g7 g1 g2 g5' kb 950 820 750 400 350,330 310 140 90 40 20 fit S S S B B ? 950 350 • 310 • 90 • FIGURE 7. POSITIONING OF TN5-MUTATED AND HOLDFAST-RELATED DNA IN THE CB2 GENOME: PULSED-FIELD GRADIENT GEL ELECTROPHORESIS. A l l samples are d i g e s t e d with AaeJ. CB = C.crescentus CB2A ( w i l d t y p e ) and gX = h o l d f a s t - d e f e c t i v e mutant group (where X = 1 to 9). The ethidium-bromide s t a i n e d g e l (*) and Southern b l o t (**) probed f o r Tn5_ were prepared by Dr. B. E l y ( U n i v e r s i t y of South C a r o l i n a ) . 39 s i n g l e p o i n t mutations i n the r e g u l a t o r y r e g i o n s of the p o l a r o r g a n e l l e s . That i s , i f p o l a r o r g a n e l l e r e g u l a t i o n i s a h i e r a r c h y of r e g u l a t o r y r e g i o n s , then i n s e r t i o n of the Tn5_ element may simply have been too l a r g e of an i n t e r r u p t i o n i n the DNA, completely i n t e r f e r i n g with the h i e r a r c h y of r e g u l a t i o n and l e t h a l to the c e l l . i i i . DETERMINING PHENOTYPE. The phenotype of a l l mutant groups and c o n t r o l s are presented i n Table 3. Four mutant phenotypes were i d e n t i f i e d : no Hf produced ( g l , g2, g3, g4, and g l O ) , a low amount of abnormal or wildtype Hf produced (g5), Hf produced and shed i n t o the medium (g7, g8, and g9, F i g u r e 8), and Hf produced but abnormal i n adhesiveness e i t h e r due to the chemical nature of the Hf m a t e r i a l or due to a p h y s i c a l abnormality r e l a t e d to the s t a l k (g6). A l l Hf-mutants were colony-acetate-minus: mutant c o l o n i e s were unable to bind to c e l l u l o s e - a c e t a t e d i s c s . Mutant groups g7, g8, and g9 were colony-acetate-minus but H f - a c e t a t e - p l u s because of t h e i r Hf-shedding c h a r a c t e r (Table 3, F i g u r e 8). That i s , because the Hf m a t e r i a l d i d not remain attached to the s t a l k , the c e l l s d i d not remain on the c e l l u l o s e - a c e t a t e , and there was no p r o t e i n a c e o u s m a t e r i a l to be s t a i n e d with Coomassie blue . However, the Hf m a t e r i a l could be v i s u a l i z e d with the use of FITC-WGA (Table 3). Mutant groups g5 and g6 were a l s o colony-acetate-minus and H f - a c e t a t e - p l u s . For these mutant groups, the lower l e v e l s of WGA p o s i t i v i t y compared with w i l d t y p e CB2A (Table 3) suggested that the colony-adhesion assay was simply not s e n s i t i v e enough to 4 0 (A) <B) FIGURE 8. FLUORESCEIN-CONJUGATED LECTIN LABELLING OF THE CAULOBACTER HOLDFAST. Shown are combined f l u o r e s c e n c e and phase-c o n t r a s t microscopy images. C e l l s i n both images are l a b e l l e d with FITC-Wheat germ a g g l u t i n i n . (A) C.crescentus CB2A ( w i l d t y p e ) c e l l s show h o l d f a s t m a t e r i a l attached to s t a l k s of s i n g l e c e l l s or i n the c e n t r e of r o s e t t e s . (B) Mutant group g8 c e l l s ( h o l d f a s t - s h e d d i n g CB2A mutant) show h o l d f a s t m a t e r i a l t h a t i s detached from c e l l s . M a g n i f i c a t i o n i s approximately 1850X. Photographs are courtesy of Dr. J . Smit. 41 d e t e c t these lower l e v e l s of colony-adhesiveness to c e l l u l o s e -a c e tate . No Caulobacter c e l l s l a b e l l e d with three of the seven l e c t i n s : ConA, PNA, RCA1 (Table 2). A l l mutant groups t h a t s t i l l produced Hf (g5, g6, g7, g8, and g9) showed v a r i o u s degrees of p o s i t i v i t y f o r b i n d i n g of the remaining four l e c t i n s (WGA, DBA, SBA, UEA) to Hf, f o r the a b i l i t y of c e l l s to r o s e t t e , and f o r the a b i l i t y of Hf to bind to g l a s s and c e l l u l o s e - a c e t a t e c o v e r s l i p s ( Table 3). For example, although mutant groups g7, g8, and g9 were a l l c l a s s i f i e d as Hf-shedding mutants, they e x h i b i t e d somewhat d i f f e r i n g a b i l i t i e s to adhere to v a r i o u s s u b s t r a t a . A l l mutant groups except g6 showed t h a t the amount of Hf produced - which was assessed by the degree of p o s i t i v i t y i n b i n d i n g of FITC-WGA to Hf (WGA, Table 3) - was the same as the observed extent of Hf-adhesiveness to g l a s s or c e l l u l o s e - a c e t a t e ( H f /Gl and Hf/Ac, Table 3). T h e r e f o r e , mutant group g6 appeared to express more Hf m a t e r i a l than was i n v o l v e d i n adhesion to s u b s t r a t a . CLONING OF HOLDFAST-RELATED AND TN5-MUTATED DNA. On average, e l e c t r o p o r a t i o n of DH5-alpha c e l l s with the d i l u t e d l i g a t i o n mixtures produced two clones (those s e l e c t e d on Cm20/Km20) with the expected Tn5_-mutated i n s e r t per thousand e l e c t r o p o r a n t s (those s e l e c t e d on Cm20). The frequency of e l e c t r o p o r a t i o n was 10E-4 per i n i t i a l r e c i p i e n t . Host chromosomal DNA, enzymes used f o r c l o n i n g , plasmids e x t r a c t e d , and the approximate s i z e s of cloned fragments are shown i n Table 4. 42 Probing f o r Tn5_ ( F i g u r e 6) showed the s i m i l a r i t y i n s i z e between the cloned fragments and the genomic DNA fragments of mutant groups that contained l i n k e d s i t e s of Tn5_-insert i o n . As w e l l , the d i g e s t i o n of plasmids pG3 and pG9 with one of the two r e s t r i c t i o n enzymes used f o r c l o n i n g proved t h a t the i n s e r t s of these plasmids were d i r e c t i o n a l l y cloned. Probing of mutant group genomic DNA with the cloned Tn5-c o n t a i n i n g DNA fragments ( F i g u r e 9) proved t h a t chromosomal DNA common to each mutant group t h a t contained l i n k e d s i t e s of Tn5-i n s e r t i o n was cloned from the four H f - r e l a t e d genomic r e g i o n s . Unlinked Tn5-mutated chromosomal DNA samples ( g l and glO) showed two bands when probed with a non-linked cloned i n s e r t (pG3 and pG9, r e s p e c t i v e l y ) : one band t h a t probed f o r the Tn5 element and the other t h a t probed f o r the f l a n k i n g chromosomal DNA cloned. The CB2A c o n t r o l probed only f o r the f l a n k i n g chromosomal DNA cloned and t h i s fragment was approximately the s i z e of the i n s e r t cloned minus the s i z e of the Tn5_ element. Probing of r e s t r i c t i o n enzyme d i g e s t e d plasmids pGl, pG5, and pG9 r e p r o d u c i b l y i d e n t i f i e d one or more s m a l l e r s i z e fragments ( F i g u r e s 6 and 9). Presumably those bands that appeared on b l o t s probed f o r both Tn5 and cloned i n s e r t s contained Tn5 and those t h a t o n l y appeared on b l o t s probed with the cloned i n s e r t s contained chromosomal DNA. That these s m a l l e r fragments appeared d e s p i t e g e l - p u r i f i c a t i o n of the e n t i r e cloned i n s e r t used f o r p r o b i n g ( F i g u r e 9) suggested t h a t they were a degraded form of the i n t a c t cloned i n s e r t . PRODUCTION OF DOUBLE HOLDFAST-MUTANT. 4 3 E N Z Y M E : E S / E S C/K P R O B E : G5 G9 G1 G3 6 . 0 - m 5 . 0 -2.5 -FIGURE 9. SOUTHERN BLOT COMPARISON OF CLONED HOLDFAST-RELATED DNA FRAGMENTS WITH CHROMOSOMAL DNA DIGESTS OF HOLDFAST-DEFECTIVE MUTANT GROUPS THAT CONTAIN LINKED SITES OF TN5-INSERTI0N: PROBING WITH CLONED DNA FRAGMENTS. A l l probes (10 to 15kb) c o n t a i n both Tn5_ and f l a n k i n g chromosomal DNA. C = C l a l , E = EcoRI • K = Kp_n I, S = Ss_LI, CB = C. crescentus CB2A ( w i l d t y p e ) , GX = the cloned plasmid pGX t h a t c o n t a i n s H f - r e l a t e d and Tn5_-mutated chromosomal DNA cloned from mutant group gX (where X = 1 to 10). Negative c o n t r o l s : CB2A chromosomal DNA cut with S/E (CB2A(S/E)) and glO(S/E) are both probed with pG9(S/E) and gl(C/K) i s probed with pG3(C/K). 44 To show t h a t a Tn5_-induced Hf c h a r a c t e r c o u l d be t r a n s f e r r e d to a d i f f e r e n t C aulobacter s t r a i n , one of the T n 5 - c o n t a i n i n g c l o n e s was e l e c t r o p o r a t e d i n t o a p r e v i o u s l y prepared u l t r a v i o l e t l i g h t - i n d u c e d Hf-shedding Caulobacter mutant, CB2A-B9. S e l e c t i o n f o r CB2A-B9::Tn5 double ( u l t r a v i o l e t l i g h t - and Tn5_-induced ) mutants on Km50/Sm50 p l a t e s occured at a frequency of three per 10E+6 v i a b l e c e l l s . Of the i s o l a t e s s e l e c t e d , approximately 10% (19/176) were Cm2-sensitive. The high frequency of Cm-resistance was not a t t r i b u t e d to maintenance by the r e c i p i e n t c e l l s of the v e c t o r plasmid pG5 s i n c e no plasmid was d e t e c t e d i n the few Cm2-r e s i s t a n t i s o l a t e s t e s t e d . Rather, these r e s u l t s suggested t h a t s i n g l e c r o s s o v e r mediated i n t e g r a t i o n of the v e c t o r i n t o CB2A-B9 occured f a r more f r e q u e n t l y than d i d e i t h e r double c r o s s o v e r mediated gene replacement or t r a n s p o s i t i o n . Of the Cm2-sensitive i s o l a t e s , approximately 50% (9/19) c l e a r l y expressed both group g5 (ROS ++, WGA ++, and Hf/Ac ++, Table 5) and CB2A-B9 (Hf-shedding, F i g u r e 8) phenotypic c h a r a c t e r i s t i c s . These i s o l a t e s were r e f e r r e d to as B9(G5) double Hf-mutants ( a l to a6, Table 5) and such a c l a s s i f i c a t i o n was confirmed as c o r r e c t by r e s u l t s from Southern b l o t a n a l y s i s of chromosomal DNA d i g e s t e d with EamHI ( F i g u r e 3). These r e s u l t s showed t h a t a l l B9(G5) i s o l a t e s had Tn5 i n s e r t e d i n a p o s i t i o n w i t h i n the chromosome t h a t was equal to t h a t found i n mutant group g5 ( F i g u r e s 3 and 4 ) . As expected, i s o l a t e s of non-g5 c h a r a c t e r ( b l to b6, Table 5, F i g u r e 3) d i d not have Tn5 i n s e r t e d i n the g5 chromosomal l o c a t i o n but r a t h e r appeared to t have been mutated by Tn5 t r a n s p o s i t i o n . Thus, i t appeared that among the 45 TABLE 5: PHENOTYPE OF DOUBLE MUTANTS STRAIN ROSETTE FORMATION WGA BINDING TO HOLDFAST HOLDFAST ADHESION TO cAc CB2A CB2A-B9 g5 +++ + ++ +++ ++++ ++ +++ +++ ++ CB2A-B9: :Tn5_: a l ++ a2 ++ a3 ++ a4 ++ a5 ++ a6 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ b l b2 b3 b4 b5 b6 ++++ ++++ ++++ +++ +++ +++ +++ ++++ ++++ ++++ ++++ ++++ CONTROLS: CB2A = C.crescentus u l t r a v i o l e t l i g h t - i n d u c e d ho h o l d f a s t - d e f e c t i v e mutant l i g h t - and Tn5-induced doubl mutant i s o l a t e s (B9(G5)) h o l d f a s t ) and mutant group g5 = double mutants t h a t expre (where X = 1 to 6). Plu s (+) value s are re Caulobac t e r CB2A) of r o s e t t e f l u o r e s c e n c e produced by ho l d f a s t : WGA = Wheat germ ( c e l l u l o s e - a c e t a t e c o v e r s l i p ) h o l d f a s t m a t e r i a l with WGA. s t r a i n ( w i l d t y p e ) , CB2A-B9 = ld f a s t - s h e d d i n g mutant, and g5 = group. CB2A-B9: :Tn5_ = u l t r a v i o l e t e mutants: aX = double h o l d f a s t -t h a t express both CB2A-B9 (shed ( l e s s h o l d f a s t ) c h a r a c t e r i s t i c s , bX ss CB2A or CB2A-B9 c h a r a c t e r i s t i c s l a t i v e amount (compared to wi l d t y p e formation or r e l a t i v e amount of FITC-conjugated l e c t i n bound to a g g l u t i n i n . H o l d f a s t adhesion to cAc i s v i s u a l i z e d by l a b e l l i n g of bound 46 Cm2-sensitive i s o l a t e s t h a t gene replacement and t r a n s p o s i t i o n occured with n e a r l y equal f r e q u e n c i e s . Since three Cm2-resistant i s o l a t e s ( c l to c3, F i g u r e 3) a l s o showed Tn5_ to be i n s e r t e d i n the g5 chromosomal l o c a t i o n and s i n c e only 3% (9/300) of Cm2-selected c o l o n i e s from two of these i s o l a t e s ( c l and c2) probed p o s i t i v e with pBR322, i t was concluded that the s c r e e n i n g of i s o l a t e s was not s t r i n g e n t enough to a c c u r a t e l y d i f f e r e n t i a t e between those with from those without chromosomally i n t e g r a t e d v e c t o r DNA. In a d d i t i o n , i t i s known tha t the s i z e of the DNA fragments f l a n k i n g both s i d e s of the transposon g r e a t l y a f f e c t s the p r o b a b i l i t y , and hence the observed frequency, of double r e c i p r o c a l recombination between the cloned DNA and the t a r g e t DNA (5, 42). T h e r e f o r e , the f r e q u e n c i e s of v e c t o r i n t e g r a t i o n , t r a n s p o s i t i o n , and gene replacement noted above were regarded only as t e n t a t i v e e stimates. 47 DISCUSSION The three main g o a l s of t h i s t h e s i s were to i d e n t i f y and c l o n e r e g i o n s of the Caulobacter CB2 H f - r e l a t e d genomic DNA, to assess the f u n c t i o n of these r e g i o n s , and to use these clones i n c o n j u n c t i o n with whatever f u n c t i o n a l i n f o r m a t i o n was determined to support f u r t h e r i n v e s t i g a t i o n i n t o the molecular and chemical b a s i s of Hf-mediated adhesion. In v a r y i n g degrees, a l l of these g o a l s were accomplished. The methods employed here c l e a r l y i d e n t i f i e d four d i s t i n c t r e g i o n s i n the CB2 genome t h a t have an e f f e c t on Hf f u n c t i o n (Table 4 and F i g u r e s 6, 7, and 9). The r e s u l t s from a l l r e s t r i c t i o n enzyme d i g e s t s of chromosomal DNA o u t s i d e of the Tn5_ element suggest t h a t these f o u r regions do not o v e r l a p and there was no evidence t h a t any two of them were contiguous. There remains the p o s s i b i l i t y t h a t any two of the four l a r g e Tn5_-c o n t a i n i n g A s e l fragments may be l i n k e d (Table 4, F i g u r e 7). I f the H f - r e l a t e d l o c i are l o c a t e d at the a p p r o p r i a t e ends of these fragments f o r contiguousness, then t h i s c ould be r e s o l v e d by a repeat of the PFGE-analysis with another enzyme that cuts the CB2 genome i n f r e q u e n t l y . Each of the four H f - r e l a t e d r e g i o n s , with the exception of t h a t cloned from mutant group g5, represented a c l u s t e r of m u t a t i o n a l s i t e s : gl/g2, g3/g4/g6/gl0, and g7/g8/g9. Double r e s t r i c t i o n enzyme a n a l y s i s of chromosomal DNA from these ten mutant groups would determine the s p a t i a l r e l a t i o n s h i p between the s i t e s of Tn5_-insertion w i t h i n each c l u s t e r , thereby p r o v i d i n g some i n d i c a t i o n as to the number of genes i n v o l v e d . As w e l l , such mapping could r e s o l v e whether the s i t e s are unique and c l u s t e r e d , 48 as i n an operon, or whether the mutations are the same s i t e rearranged by i l l e g i t i m a t e recombination induced d u r i n g the t r a n s p o s i t i o n event. The t r a n s p o s i t i o n of Tn5_ i n the CB2 genome was c l e a r l y not completely random as i n d i c a t e d by the hotspot of t r a n s p o s i t i o n observed i n c e r t a i n mutants of the T n 5 - l i b r a r y ( F i g u r e s 2 and 3), the high frequency of Tn5_-insertion observed i n c e r t a i n Hf-r e l a t e d l o c i (g2, g3: Table 3), the absence of s m a l l d i f f e r e n c e s between s i t e s of T n 5 - i n s e r t i o n w i t h i n each of the ten mutant groups, and the absence of p l e i o t r o p y (with the p o s s i b l e exception of g6 mutants) f o r the three p o l a r - r e l a t e d f u n c t i o n s t e s t e d . T h e r e f o r e , there may be other H f - r e l a t e d r e g i o n s of DNA i n the CB2 genome that were not i d e n t i f i e d . One approach to i d e n t i f y other H f - r e l a t e d r e g i o n s u s i n g the e x i s t i n g Tn5_-library, would be to complement u l t r a v i o l e t l i g h t - or c h e m i c a l l y - i n d u c e d Hf-mutants by t r a n s d u c t i o n with the t r a n s d u c i n g C.crescentus phage-CR30. S e l e c t e d Tn5_-mutants could then be p o s i t i v e l y screened f o r colony-adhesiveness to c e l l u l o s e - a c e t a t e and would be expected to have the Tn5_ element l o c a t e d next to H f - r e l a t e d DNA. Except f o r mutant groups g5 and g6, groups that contained l i n k e d s i t e s of Tn5_-insert ion were found to have the same or s i m i l a r phenotype: gl/g2 (no H f ) , g3/g4/gl0 (no H f ) , g7/g8/g9 (shed H f ) . Mutations i n genomic DNA that a f f e c t Hf f u n c t i o n might be expected to y i e l d s t r u c t u r a l Hf-mutants ( d e f e c t i v e or no adhesion (45, 57)), r e g u l a t o r y Hf-mutants (under- and over-p r o d u c t i o n of Hf m a t e r i a l ( J . Smit, unpublished r e s u l t s ) ) , mutants p l e i o t r o p i c f o r p o l a r - r e l a t e d f u n c t i o n s (45, 57), and 49 s t r u c t u r a l mutants i n other c e l l u l a r components that have an a n c i l l a r y e f f e c t upon Hf f u n c t i o n (shedding mutant ( 4 4 ) ) . Examples from most of these c a t e g o r i e s of mutants were i d e n t i f i e d among the ten H f - d e f e c t i v e mutant groups. The most c l e a r example of mutations i n H f - r e l a t e d s t r u c t u r a l genes were the Hf-minus mutants (gl/g2 and g3/g4/gl0). Presumably these mutants were e i t h e r aberrant i n the b i o s y n t h e s i s of the Hf m a t e r i a l or i n the t r a n s p o r t of the Hf m a t e r i a l to the c e l l s u r f a c e . In the case of the shedding mutants (g7/g8/g9), the Hf m a t e r i a l was produced, t r a n s p o r t e d to the c e l l s u r f a c e , and adhesive. Moreover, shed Hf i s capable of mediating r o s e t t e formation with Hf-minus mutant c e l l s (44). That i s , to the extent t h a t the Hf m a t e r i a l can be t e s t e d f u n c t i o n a l l y , shed Hf appears to be e q u i v a l e n t to wildtype Hf except i n i t s attachment to the pol e of the c e l l . T h e r e f o r e , although the mutation i n shedding mutants was H f - r e l a t e d , i t i s probably not r e l a t e d to the s t r u c t u r a l Hf genes. The extent to which the Hf m a t e r i a l and H f - r e l a t e d DNA was i n v e s t i g a t e d here d i d not pr o v i d e a means with which to unambiguously d i s t i n g u i s h between r e g u l a t o r y and s t r u c t u r a l mutants. Nonetheless, t h a t mutant groups g5 and g6 appeared to produce l e s s (WGA ++, Table 3) than wi l d t y p e l e v e l s (WGA +++) of the Hf m a t e r i a l suggested t h a t these mutants were aberrant i n the r e g u l a t i o n of Hf ex p r e s s i o n . A l t e r n a t e l y , d e s p i t e the f a c t t h a t mutant groups g5 and g6 appeared to express e q u i v a l e n t l e v e l s of the Hf m a t e r i a l , g6 mutants'did not produce any r o s e t t e s and were shown to bind l e s s w e l l than g5 mutants to g l a s s and c e l l u l o s e -50 a c e t a t e . I t may be that mutant group g6 has c h e m i c a l l y a l t e r e d Hf m a t e r i a l . The bent s t a l k c h a r a c t e r i s t i c of group g6 f u r t h e r complicated i t s c h a r a c t e r i z a t i o n . That i s , i t may have been t h a t adhesion to other Hf and s u b s t r a t a by g6 mutants was p h y s i c a l l y i n h i b i t e d by t h e i r bent s t a l k . Moreover, the c o i n c i d e n c e of Hf and s t a l k a b e r r a t i o n s i n a s i n g l e mutant suggested t h a t group g6 mutants might be p l e i o t r o p i c . Whether or not i t i s i n v o l v e d i n the p r o d u c t i o n of Hf, r e g u l a t i o n of Hf, or r e g u l a t i o n of p o l a r f u n c t i o n s , the g6 locus was c l e a r l y l i n k e d to H f - r e l a t e d l o c i <g3/g4/gl0: Table 4 and F i g u r e s 4, 5, 6, and 9). In a d d i t i o n to t h e i r u s e f u l n e s s f o r the f u t u r e i s o l a t i o n of w i l d t y p e H f - r e l a t e d chromosomal DNA, the Tn5-mutated clones were a l s o u s e f u l f o r i n v i v o s i t e - d i r e c t e d mutagenesis as was shown by the r e s u l t s of the gene replacement experiment. Such mixing of Tn5_-induced mutation with u l t r a v i o l e t l i g h t - or c h e m i c a l l y -induced mutation f a c i l i t a t e s the i s o l a t i o n of Hf m a t e r i a l f o r chemical a n a l y s i s . As shown here, a Tn5_-derived Hf-mutant c h a r a c t e r could be t r a n s f e r r e d to a Hf-shedding r e c i p i e n t c e l l . Presumably, any Tn5_-derived Hf-mutant could be analyzed i n t h i s way. Conversely, i t should be p o s s i b l e to t r a n s f e r the Tn5_-d e r i v e d shedding phenotype i n t o any u l t r a v i o l e t l i g h t - or c h e m i c a l l y - i n d u c e d Hf-mutant. T h i s l a s t approach has been s u c c e s s f u l l y employed with the use of the plasmid pG9 and a Hf-overproducing CB2A mutant to produce a Hf-overproducing and Hf-shedding double mutant ( J . Smit, unpublished r e s u l t s ) . 51 REFERENCES 1. Agabian,N. and L.Shapiro. 1970. S t a l k e d b a c t e r i a : p r o p e r t i e s of d e o x y r i b o n u c l e i c a c i d b a c t e r i a pahge CBK. J. V i r o l . 5 _ : 795-800. 2. B a r r e t t , J . T . , R.H.Croft, D.M.Ferber, C.J.Gerardot, P . V. Schoenlein, and B.Ely. 1982. Genetic mapping with Tn5_-derived auxotrophs of Caulobacter c r e s c e n t u s . J . B a c t e r i o l . 15_i(2) : 888-898. 3. Berg,D.E., A.Weiss, and L . C r o s s l a n d . 1980. P o l a r i t y of Tn5_ i n s e r t i o n mutations i n E s c h e r i c h i a c o l i . J . M i c r o b i o l .14,2.(2): 439-446. 4. Brent,R. and M.Ptashne. 1980. The lexA gene product r e p r e s s e s i t s own promoter. Proc.Natl.Acad.Sci.USA 77(4): 1932-1936. 5. de B r u i j n , F . J . 1987. Transposon Tn5 mutagenesis to map genes. Meth.Enzymol.151: 175-196. 6. de B r u i j n , F . J . and J.R.Lupski. 1984. The use of transposon Tn5_ mutagenesis i n the r a p i d g e n e r a t i o n of c o r r e l a t e d p h y s i c a l and g e n e t i c maps of DNA segments cloned i n t o multicopy plasmids -a review. Gene 22.: 131-149. 7. B r u n o v s k i s , I . and W.C.Summers. 1972. The process of i n f e c t i o n with c o l i p h a g e T7. V i r o l o g y 5J1: 322-327. 8. Calvin,N.M. and P.C.Hanawalt. 1988. High e f f i c i e n c y t r a n s f o r m a t i o n of b a c t e r i a l c e l l s by e l e c t r o p o r a t i o n . J . B a c t e r i o 1.120.(6): 2796-2801. 9. Chen,H., J.X.Gray, M.Nayudu, M.A.Djordjevic, M.Batley, J.W.Redmond, and B.G.Rolfe. 1988. F i v e g e n e t i c l o c i i n v o l v e d i n the s y n t h e s i s of a c i d i c e x o p o l y s a c c h a r i d e s are c l o s e l y l i n k e d i n the genome of Rhizobium spp. s t r a i n NGR234. Mo1.Gen.Genet.212:310-316. 10. Costerton,J.W., K.-J.Cheng, G.G.Geesey, T.I.Ladd, J . C . N i c k e l , M.Dasgupta, and T . J . M a r r i e . 1987. B a c t e r i a l f i l m s i n nature and d i s e a s e . Ann.Rev.Microbiol.41: 435-464. 11. Costerton,J.W., R . T . I r v i n , and K.-J.Cheng. 1981. The b a c t e r i a l g l y c o c a l y x i n nature and d i s e a s e . Ann . Rev. M i c r o b i o l . 3_5_: 299-324. 12. Darzins,A., S.K.Wang, R.I.Vanags, and A.M.Chakrabarty. 1985. C l u s t e r i n g of mutations a f f e c t i n g a l g i n i c a c i d b i o s y n t h e s i s i n mucoid Pseudomonas aeruginosa. J . M i c r o b i o l . 1 M ( 2 ) : 516-524. 13. Dolph,P.J., D.R.Majerczak, and D.L.Coplin. 1988. C h a r a c t e r i z a t i o n of a gene c l u s t e r f o r exopolysaccharide b i o s y n t h e s i s and v i r u l e n c e i n E r w i n i a s t e w a r d i i . J . M i c r o b i o l .120.(2) : 865-871. 52 14. Dower,W.J., J . F . M i l l e r , and C.W.Ragsdale. 1989. High e f f i c i e n c y t r a n s f o r m a t i o n of E s c h e r i c h i a c o l i by high v o l t a g e e l e c t r o p o r a t i o n . N u c l . A c i d Res. (In p r e s s ) . 15. Dry,P.J. 1988. A qu i c k and easy method f o r the p u r i f i c a t i o n of DNA from c h o r i o n i c v i l l u s samples. N u c l . A c i d Res. 16.(5): 7730. 16. Dworkin,M. 1985. Cauloba c t e r , p.50-67. In: Developmental b i o l o g y of the b a c t e r i a , Benjamin/Cumming P u b l i s h i n g , C a l i f o r n i a . 17. E l y , B . and R.Croft. 1982. Transposon mutagenesis i n CanInhacter c r e s c e n t u s . J . B a c t e r i d .I4JL(2) : 620-625. 18. E l y , B . and C.Gerardot. 1988. Use of p u l s e d - f i e l d - g r a d i e n t g e l e l e c t r o p h o r e s i s to c o n s t r u c t a p h y s i c a l map of the Caulobacter crescentus genome. Gene 68: 323-333. 19. F l e t c h e r , M . 1987. How do b a c t e r i a a t t a c h to s o l i d s u r f a c e s ? M i c r o b i o l . S c i . 1 ( 5 ) : 133-136. 20. F o s t e r , T . J . 1984. A n a l y s i s of plasmids w i t h transposons. Meth.Microbiol.1Z: 197-226. 21. Fukuda,A., K.Miyakawa, H.Iida, and Y.Okada. 1976. Regula t i o n of p o l a r s u r f a c e s t r u c t u r e s on Caulobacter c r e s c e n t u s : p l e i o t r o p i c mutations a f f e c t the c o o r d i n a t e morphogenesis of f l a g e l l a , p i l i , and phage r e c e p t o r s . Molec.Gen.Genet.149: 167-173. 22. G i l c h r i s t , A . , W.Bingle, and J.Smit. 1989. I n t r o d u c t i o n of plasmids i n t o freshwater and marine Ca u l o b a c t e r s by e l e c t r o p o r a t i o n . (In p r e p ) . 23. Hanahan,D. 1985. Techniques f o r t r a n s f o r m a t i o n of K.cjali, p.109-114. in.: D.M.Glover ( e d ) , DNA c l o n i n g , a p r a c t i c a l approach. Volume 1, IRL Press, Oxford. 24. Hanahan,D. 1983. S t u d i e s on t r a n s f o r m a t i o n of E s c h e r i c h i a c a i i with plasmids. J . Mol. B i o l . 16jo_: 557-580. 25. Isaac,D.H. 1985. B a c t e r i a l p o l y s a c c h a r i d e s , p. 141-184. In.: Atkins,E.D. ( e d ) , P o l y s a c c h a r i d e s , t o p i c s i n s t r u c t u r e and morphology. VCH P u b l i s h e r s , F l o r i d a . 26. Isberg,R.R. and M.Syvanen. 1985. Tn5_ transposes independently of c o i n t e g r a t e r e s o l u t i o n : evidence f o r an a l t e r n a t i v e model f o r t r a n s p o s i t i o n . J . M o l . B i o l . 182: 69-78. 27. Johnson,R. and B.Ely. 1979. A n a l y s i s of nonmotile mutants of the dimorphic bacterium Caulobacter c r e s c e n t u s . J . B a c t e r i o 1 . 1 3 2 ( 1 ) : 627-634. 28. Johnson,R. and B.Ely. 1977. I s o l a t i o n of spontaneously d e r i v e d mutants of Caulobacter c r e s c e n t u s . G e n e t i c s Q6: 25-32. 53 29. Johnson,R.C., J.C.Yin, and W.S.Reznikoff. 1982. C o n t r o l of the Tn5_ t r a n s p o s i t i o n i n E s c h e r i c h i a c o l j i s mediated by p r o t e i n from the r i g h t repeat. C e l l 2Q_: 873-882. 30. Jorgensen,R.A., S.J.Rothstein, and W.S.Reznikoff. 1979. A r e s t r i c t i o n enzyme cleavage map of Tn5_ and l o c a t i o n of a r e g i o n encoding neomycin r e s i s t a n c e . Mol .Gen .Genet .17J7_: 65-72. 31. Kado,C.I. and S.Liu. 1981. Rapid procedure f o r d e t e c t i o n and i s o l a t i o n of l a r g e and s m a l l plasmids. J . B a c t e r i o l . 1 4 5 ( 3 ) : 1365-1373. 32. Kleckner,N. 1977. T r a n s l o c a t a b l e elements i n p r o k a r y o t e s : review. C e l l 11: 11-23. 33. Krebs,M.P. and W.S.Reznikoff. 1986. T r a n s c r i p t i o n a l and t r a n s l a t i o n a l i n i t i a t i o n s i t e s of IS50: c o n t r o l of transposase and i n h i b i t o r e x p r e s s i o n . J.Mol.Biol.192: 781-791. 34. L a g e n a u r , C , S.Farmer, and N.Agabian. 1977. A d s o r p t i o n p r o p e r t i e s of s t a g e - s p e c i f i c Caulobacter phage-CBK. V i r o l o g y 7J7: 401-407. 35. Long,S., J.W.Reed, J.Himawan, and G.C.Walker. 1988. Genetic a n a l y s i s of a c l u s t e r of genes r e q u i r e d f o r s y n t h e s i s of the c a l c o f l u o r - b i n d i n g e x opolysaccharide of Rhizobium m e l i l o t i . J.Bacteriol.l7_£L(9): 4239-4248. 36. M a n i a t i s , T . , F r i t s c h , E . , Sambrook,J. 1982. Molecular c l o n i n g , a l a b o r a t o r y manual. Cold S p r i n g Harbor, New York. 37. Marshall,K.C. 1985. Mechanisms of b a c t e r i a l adhesion at s o l i d - w a t e r i n t e r f a c e s , p. 133-161. In.: Savage, D.C. and F l e t c h e r , M . ( e d s ) , B a c t e r i a l adhesion: mechanisms and p h y s i o l o g i c a l s i g n i f i c a n c e . Plenum Press, New York. 38. Meinkoth,J. and M.Wahl. 1987. Nick t r a n s l a t i o n . Meth.Enzymol. 3J5_2_: 91-94. 39. Merker,R.I. and J.Smit. 1988. C h a r a c t e r i z a t i o n of the adhesive h o l d f a s t of marine and freshwater C a u l o b a c t e r s . Appl .Environ .Microbiol-54.(8) : 2078-2085. 40. Murray,N.E., W.J.Brammar, and K.Murray. 1977. Lambdoid phages that s i m p l i f y the recovery of in. v i t r o recombinants. Mol. Gen. Genet. 150.: 53-61. 41. Newton,A. 1989. D i f f e r e n t i a t i o n i n C a u l o b a c t e r : f l a g e l l u m development, m o t i l i t y , and chemostaxis, p.199-220. In: Hopwood,D.A. and K.F.Chater ( e d s ) , G e n e t i c s of b a c t e r i a l d i v e r s i t y . Academic Press, Toronto. 42. Noti,J.D., M.N.Jagadish, and A.A.Szalay. 1987. S i t e - d i r e c t e d Tn5_ and transplacement mutagenesis: methods to i d e n t i f y s y m b iotic n i t r o g e n f i x a t i o n genes i n slow-growing Rhizobium. Meth.Enzymol.151: 197-217. 54 43. O ' N e i l l , E . , G . K e i l y , and R.Bender. 1984. Transposon Tn5 encodes streptomycin r e s i s t a n c e i n n o n e n t e r i c b a c t e r i a . J.Bacteriol.l£9(l): 388-389. 44. Ong,C. and J.Smit. 1989. The adhesive h o l d f a s t of C a u l o b a c t e r s : a n a l y s i s of mutants d e f e c t i v e i n the attachment of the o r g a n e l l e to the c e l l . (In p r e p ) . 45. Poindexter,J.S. 1981. The C a u l o b a c t e r s : u b i q u i t o u s unusual b a c t e r i a . M i c r o b i o l . Rev. 4J1( 1) : 123-179. 46. Poindexter,J.S. 1964. B i o l o g i c a l p r o p e r t i e s and c l a s s i f i c a t i o n of the Caulobacter group. B a c t e r i o l . Rev.2Jl(3): 231-295. 47. Purucker,M., R.Bryan, K.Amemiya, B.Ely, and L.Shapiro. 1982. I s o l a t i o n of a Caulobacter gene c l u s t e r s p e c i f y i n g f l a g e l l u m p r o d u c t i o n by u s i n g nonmotile Tn5_ i n s e r t i o n mutants. Proc. N a t l . Acad. S c i . 79.: 6797-6801. 48. Quigley,N.B. and P.R.Reeves. 1987. Chloramphenicol r e s i s t a n c e c l o n i n g v e c t o r based on pUC9. Plasmid 12:54-57. 49. Reed,K.C. and Mann,D.A. 1985. Rapid t r a n s f e r of DNA from agarose g e l s to nylon membranes. Nuc . A c i d . Res . 13_( 20 ) : 7207-7221. 50. S i l h a v y , T . J . , M.L.Berman, and L.W.Enquist. 1984. Experiments with gene f u s i o n s . Cold S p r i n g Harbor Laboratory, 303p. 51. Simon,R., U . P r i e f e r , and A.Puhler. 1983. A broad host range m o b i l i z a t i o n system f o r i n v i v o g e n e t i c e n g i n e e r i n g : transposon mutagenesis i n gram negative b a c t e r i a . Bio/technology 1: 784-790. 52. Smit,J., D.A.Grano, R.M.Glaeser, and N.Agabian. 1981. P e r i o d i c s u r f a c e a r r a y i n Caulobacter c r e s c e n t u s : f i n e s t r u c t u r e and chemical a n a l y s i s . J . B a c t e r i o l . 1 4 6 : 1135-1150. 53. Southern,E.M. 1975. D e t e c t i o n of s p e c i f i c sequences among DNA fragments separated by g e l e l e c t r o p h o r e s i s . J . Mol. B i o l . 98_: 503-517. 54. Sriv a s t a v a , R . , V.B.Sinha, and B . S . S r i v a s t a v a . 1989. Chromosomal t r a n s f e r and i n v i v o c l o n i n g of genes i n V i b r i o  c h o l e r a e u s i n g RP4::mini-Mu. Gene 75: 253-259. 55. Sutherland,I.W. 1985. B i o s y n t h e s i s and composition of gram ne g a t i v e b a c t e r i a l e x t r a c e l l u l a r and c e l l w a l l p o l y s a c c h a r i d e s . Ann. Rev. M i c r o b i o l . 39_: 243-270. 56. Sutherland,I.W. 1983. M i c r o b i a l e x o p o l y s a c c h a r i d e s - t h e i r r o l e i n m i c r o b i a l adhesion i n aqueous systems. CRC C r i t . R e v . M i c r o b i o l . 1 0 ( 2 ) : 173-201. 55 57. Umbreit,T.H. and J.L.Pate. 1978. C h a r a c t e r i z a t i o n of the h o l d f a s t r e g i o n of wil d t y p e c e l l s and h o l d f a s t mutants of A s t i c c a c a u l i s biprosthecum. Arch. M i c r o b i o l . X18_: 157-168. 58. Verma,N.K., N.B.Quigley, and P.R.Reeves. 1988. O-Antigen v a r i a t i o n i n Salmonella spp.: r f b gene c l u s t e r s of three s t r a i n s . J . M i c r o b i o l . 1 2 0 ( 1 ) : 103-107. 59. Wood,N.B., A.V.Rake, and L.Shapiro. 1976. S t r u c t u r e of Caulobacter d e o x y r i b o n u c l e i c a c i d . J . B a c t e r i o l . 126_( 3 ): 1305-1315 . 60. Wu,A.M. 1985. A t a b l e of l e c t i n carbohydrate s p e c i f i c i t i e s . In: Bog-Hansen,T.C. and J.Breborowicz ( e d s ) , L e c t i n s : b i o l o g y , b i o c h e m i s t r y , c l i n i c a l b i o c h e m i s t r y . Vol.4: 629-636. 61. Y i n , J . C , M.Krebs, and W . S . Reznikof f . 1988. The e f f e c t of dam methyl a t i o n on Tn5 t r a n s p o s i t i o n . J.Mol.Biol.199: 35-46. 62. Y i n , J.C. and W . S . Reznikof f . 1988. p2 and I n h i b i t i o n of Tn5_ t r a n s p o s i t i o n . J . M i c r o b i o l . 1 2 H ( 7 ) : 3008-3015. 

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