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UBC Theses and Dissertations

Role of the outer membrane of Pseudomonas aeruginosa in antibiotic resistance Nicas, Thalia Ioanna 1982

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ROLE OF THE OUTER MEMBRANE OF PSEUDOMONAS AERUGINOSA IN ANTIBIOTIC RESISTANCE by T h a l i a l o a n n a N i c a s B.Sc. U n i v e r s i t y o f C a l g a r y , 1975 M.Sc. U n i v e r s i t y o f A l b e r t a , 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (D e p a r t m e n t o f M i c r o b i o l o g y ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September 1982 (c) T h a l i a l o a n n a N i c a s , 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. T h a l i a I. Nicas Department of Microbiology The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 23. September 1982. Date DE-6 (3/81) ABSTRACT It was demonstrated that induction of a major outer protein, HI, was associated with increased resistance to chelators of divalent cations such as EDTA and to the cationic a n t i b i o t i c s polymyxins and aminoglycosides. Outer membrane protein HI was the major c e l l u l a r protein i n c e l l s grown in Mg - d e f i c i e n t medium (0.02 mM Mg ) and i n mutants selected for resistance to polymyxin. Increase i n protein HI was associated 2 + with decrease i n c e l l envelope Mg . Induction of protein HI 2 + was prevented by supplementation of Mg - d e f i c i e n t medium with 0.5 mM Mg 2 +, C a 2 + , Mn 2 + or S r 2 + , but not by Zn 2 + , Ba 2 +, or S n 2 + . C e l l s grown i n C a 2 + , Mn 2 + or Z n 2 + showed enhanced levels of these cations as main major c e l l envelope associated cation. Only c e l l s grown i n the presence of those cations which f a i l e d to prevent HI induction were resist a n t to chelators, polymyxin B and gentamicin. Protein HI overproducing c e l l s also demonstrated altered streptomycin uptake. It was further demonstrated that aminoglycosides could in t e r a c t with the outer membrane so as to make i t more permeable 2+ . to other substances. Mg inh i b i t e d aminoglycoside-mediated permeabilization. Both aminoglycosides and polymyxin B could be 2+ shown to displace a small amount of Mg from the c e l l envelope. A mutant severely deficient, in outer membrane protein F was isolated. Permeability of this s t r a i n was studied by measuring hydrolysis of a chromogenic beta-lactam by periplas-mic beta-lactamase. It was found that outer membrane permea-b i l i t y of P. aeruginosa was low compared to E. c o l i and that loss of protein F caused a further decrease. The results sug-gest that only a small proportion of protein F molecules form functional channels in wild type c e l l s so that the hydrophilic pathway of uptake across the outer membrane is r e l a t i v e l y i n e f f i c i e n t . Cationic a n t i b i o t i c s such as aminoglycosides and polymyxins may use an alternate pathway of "s e l f promoted" permeation. It is proposed that EDTA, polymyxin and aminogly-cosides act by attacking a c r i t i c a l divalent cation binding s i t e on the lipopolysaccharide. Protein HI i s proposed to act by replacing divalent cations at this s i t e , preventing the action of these agents. iv TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS iv L i s t of Tables v i i i L i s t of Figures ix L i s t of Abbreviations x ACKNOWLEDGEMENTS xi INTRODUCTION 1 1. Medical importance of Pseudomonas aeruginosa 1 2. A n t i b i o t i c resistance of Pseudomonas 2 aeruginosa 3. Mechanisms of a n t i b i o t i c resistance 4 4. The c e l l envelope and permeability 6 5. The gram-negative outer membrane 8 a. Lipopolysaccharide 9 b. Protein 10 6. The outer membrane of P. aeruginosa and i t s 12 role in a n t i b i o t i c permeability METHODS 1. Media and growth conditions 17 2. Bact e r i a l strains 17 3. Isolation of mutants de f i c i e n t in outer 21 membrane protein 4. Bacteriophage and bacteriocin studies 23 a. Bacteriophages and p a r t i a l 23 characterization of their receptors b. Aeruginocin studies 25 c. Isolation of protein s p e c i f i c phages 26 from nature d. Bacteriophage s e n s i t i v i t y testing 28 CONTENTS (cont'd) e. Isolation of phage-resistant mutants f. Phage characterization by adsorption to whole c e l l s 5. A n t i b i o t i c and chelator s u s c e p t i b i l i t y testing a. A n t i b i o t i c s b. Chelator and a n t i b i o t i c b a c t e r i o l y s i s and k i l l i n g c. Minimal in h i b i t o r y concentrations 6. S h i f t experiments 7. Membrane i s o l a t i o n and characterization of outer membrane, c e l l envelope, and whole c e l l proteins 8. Determination of c e l l envelope cation levels 9. Displacement of Mg2+ from the c e l l envelope by polymyxin B and aminoglycosides 10. Streptomycin uptake assays. 11. Enhancement of n i t r o c e f i n permeability by aminoglycosides and chelators 12. Measurement of outer membrane permeability by n i t r o c e f i n hydrolysis 13. Other assays RESULTS CHAPTER ONE S u s c e p t i b i l i t y to EDTA-Tris, Polymyxins, and Aminoglycosides 1. S u s c e p t i b i l i t y to polymyxin B and EDTA-Tris in Mg^ +-sufficient and - d e f i c i e n t media 2. S u s c e p t i b i l i t y of polymyxin B res i s t a n t mutants v i CONTENTS (cont'd) Page 3. Aminoglycoside resistance 42 4. Substitution of other cations for Mg 2 + 43 5. Minimal in h i b i t o r y concentrations of 45 polymyxin at various Mg2+ le v e l s 6. Streptomycin uptake and binding in 48 susceptible and resistant strains 7. Permeabilization of the outer membrane by 51 aminoglycosides 8. Other properties of polymyxin-resistant 52 mutants a. Resistance to chloramphenicol and 52 other a n t i b i o t i c s b. A b i l i t y to accept RPI 53 c. Loss of v i a b i l i t y in cold storage 53 9. Summary 54 CHAPTER TWO Outer Membrane Characterization 55 1. Outer membrane protein patterns 55 2. Effects of s h i f t from low to high Mg2+ 60 3. Divalent cation concentration of c e l l 62 envelopes and displacement of cations by aminoglycosides and polymyxin B 4. Comparison of EGTA and EDTA su s c e p t i b l i t y 63 of Ca2+ and Mg2+ grown c e l l s 5. Protein Hi induction in other Pseudomonas 67 strains 6. Summary 67 v i i CONTENTS (cont'd) Page CHAPTER THREE Isolation and Characterization of a Porin- 70 Deficient Mutant and Bacteriophage Studies 1. Isolation of outer membrane protein- 70 d e f i c i e n t strains by random heavy mutagenesis and i s o l a t i o n of phage s p e c i f i c for protein receptors 2. Characterization of a porin-deficient 76 isola t e 3. Bacteriophage s e n s i t i v i t y of outer 78 membrane protein mutants, mucoid iso l a t e s and serotype strains 4. Summary 7 2 CHAPTER FOUR Measurement of Outer Membrane Permeability 84 Summary 87 DISCUSSION LITERATURE CITED 90 109 v i i i LIST OF TABLES Page Table I B a c t e r i a l s t r a i n s 18 I I B a c t e r i o p h a g e s o u r c e s and r e c e p t o r s 24 I I I R e s i s t a n c e t o k i l l i n g by EDTA-Tris and p o l y m y x i n 41 B o f H103 and i t s p o l y m y x i n B r e s i s t a n t mutant H181. E f f e c t of M g 2 + c o n c e n t r a t i o n i n the growth medium IV R e s i s t a n c e of H103, i t s p o l y m y x i n d e r i v a t i v e s 44 H181 and H185 and a r e v e r t a n t H207 t o v a r i o u s a n t i b i o t i c s V E f f e c t of growth i n v a r i o u s d i v a l e n t c a t i o n s on 46 i n d u c t i o n o f o u t e r membrane p r o t e i n HI, l y s i s and k i l l i n g by EDTA-Tris, and k i l l i n g by p o l y m y x i n B and g e n t a m i c i n VI L e v e l s of o u t e r membrane p r o t e i n HI, c e l l 47 en v e l o p e M g 2 + c o n c e n t r a t i o n , and r e s i s t a n c e t o p o l y m y x i n s of H103 and i t s p o l y m y x i n B r e s i s t a n t d e r i v a t i v e s H181 and H185 and a r e v e r t a n t , H207: e f f e c t s of v a r y i n g M g 2 + c o n c e n t r a t i o n s i n the medium V I I D i v a l e n t c a t i o n s of c e l l e n velopes a f t e r growth 64 i n the presence of d i f f e r e n t c a t i o n s V I I I E f f e c t s of EGTA-Tris and EDTA-Tris on c e l l s grown 66 i n M g 2 + and C a 2 + IX R e s u l t s o f enrichment p r o c e d u r e f o r i s o l a t i o n of 75 o u t e r membrane p r o t e i n r e c e p t o r - s p e c i f i c phage X B a c t e r i o p h a g e s e n s i t i v i t y of o u t e r membrane 81 p r o t e i n mutants, s e r o t y p e s t r a i n s and mucoid i s o l a t e s X I B a c t e r i o p h a g e s u s c e p t i b i l i t y of s e r o t y p e s t r a i n s 82 of J P . a e r u g i n o s a performed u s i n g phages prop a g a t e d and c h a r a c t e r i z e d on P_. a e r u g i n o s a PA01 s t r a i n s X I I Rate of n i t r o c e f i n h y d r o l y s i s by i n t a c t c e l l s and 88 o u t e r membrane p e r m e a b i l i t y c o e f f i c i e n t C o f P. a e r u g i n o s a H103 ( R P l ) , i t s d e r i v a t i v e c a r r y i n g p l a s m i a R P l , Z61 (RPl) and E. c o l i UB1636 ( R P l ) i x LIST OF FIGURES Page F i g u r e 1. E f f e c t of a d a p t a t i o n on M g 2 + - d e f i c i e n t medium, 40 and o f m u t a t i o n t o po l y m y x i n B r e s i s t a n c e , on s u s c e p t i b i l i t y t o l y s i s by p o l y m y x i n B and EDTA-Tris 2. Uptake of [3JH] s t r e p t o m y c i n a t two 49 c o n c e n t r a t i o n s by the w i l d - t y p e s t r a i n H103 and the o u t e r membrane p r o d u c i n g s t r a i n H181 3. Time r e q u i r e d f o r i n i t i a t i o n of r a p i d uptake of 50 s t r e p t o m y c i n (EDP-II) i n the w i l d - t y p e s t r a i n H103 and the o u t e r membrane p r o t e i n HI o v e r p r o d u c i n g s t r a i n 4. E f f e c t of a d a p t a t i o n on M g 2 + - d e f i c i e n t medium 56 and o f m u t a t i o n t o p o l y m y x i n B r e s i s t a n c e on l e v e l s of p r o t e i n H i 5. E f f e c t o f growth i n d i f f e r e n t d i v a l e n t c a t i o n s 59 on i n d u c t i o n o f p r o t e i n HI 6. E f f e c t o f s h i f t from low to h i g h M g 2 + on l e v e l s 61 o f p r o t e i n HI and s u s c e p t i b i l i t y t o EDTA-Tris and p o l y m y x i n B 7. I n d u c t i o n of p r o t e i n HI i n Pseudomonas s t r a i n s 68 grown i n low M g 2 + 8. Membranes of h e a v i l y mutagenized s t r a i n s w i t h 72 a p p a r e n t p r o t e i n a l t e r n a t i o n s 9. C e l l e n velopes of w i l d - t y p e s t r a i n H103, i t s 77 p o r i n d e f i c i e n t mutant H283 and a r e v e r t a n t , H284 10. Model i l l u s t r a t i n g the proposed mechanism of 94 r e s i s t a n c e t o a m i n o g l y c o s i d e s , p o l y m y x i n B, and EDTA-Tris i n P. a e r u g i n o s a w i t h h i g h l e v e l s of p r o t e i n Hi X L I S T OF ABBREVIATIONS 600 Absorbance a t 600 nm. BM2 B a s a l medium number 2, a phosphate b u f f e r e d m i n i m a l medium ( G i l l e l a n d e t a l , 1974). EDTA E t h y l e n e d i a m i n e t e t r a a c e t e t a t e . EGTA E t h y l e n e g l y c o l - b i s ( b e t a - e t h y l e t h e r ) N , N ' t e t r a a c e t a t e . LPS L i p o p o l y s a c c h a r i d e . MIC M i n i m a l i n h i b i t o r y c o n c e n t r a t i o n . NTG N - m e t h y l - N ' - n i t r o - n i t r o s o g u a n i d i n e . PP2 P r o t e o s e peptone number 2 medium. SDS Sodium d o d e c y l s u l p h a t e . x i ACKNOWLEDGEMENTS I wish to thank R.E.W. Hancock for his warm fri e n d -ship and consistent encouragement. I am grateful for the support of a Frank Wesbrook scholarship during most of my studies. 1 INTRODUCTION Pseudomonas aeruginosa i s a gram-negative bacterium found widely distributed in the environment. During the l a s t 20-25 years P. aeruginosa has acquired notoriety as a major opportunistic pathogen. One of the primary reasons for i t s emergence in this role is i t s resistance to commonly used a n t i b i o t i c s . 1. Med i c a l importance'of Pseudomonas aerug inosa . The intro-duction of e f f i c i e n t anti-staphylococcal agents has played a major role in a l t e r i n g patterns of hospital-acquired infec-tions. Consequently, gram-negative bacteria have replaced staphylococci as the major source of nosocomial infecti o n s . P_. aeruginosa in p a r t i c u l a r is well known for i t s resistance to a n t i s e p t i c s , and is often d i f f i c u l t to eradicate. Extensive use of broad spectrum a n t i b i o t i c s has also favoured the selection of P. aeruginosa, so that i t is now a major potential hazard in the hospital environment. P_. aeruginosa does not cause disease in healthy, uninjured i n d i v i d u a l s . However, in individuals with impaired defense mechanisms, i t is one of the most frequently isolated pathogens. As modern medicine improves the survival rate for patients with severe burns, neoplastic disease and c y s t i c f i b r o s i s , an increasing incidence of Pseudomonas infections has been noted (Levine e_t a l . , 1974; Pennington e_t a l . 1975; Reynolds et a l . , 1 9 7 5 ) . Treatment with immunosuppressive and 2 cytotoxic drugs and c o r t i c o s t e r o i d s , as well as many hospital techniques (e.g., catheterization) which can res u l t in introduction of organisms into susceptible tissues have contributed to the growing prevalance of infections by gram-negative bacteria, e s p e c i a l l y J?. aeruginosa. P. aeruginosa is a predominant cause of burn-wound i n f e c t i o n s . For example, some studies have found that i t colonizes up to 6 0 % of patients by 5 days after injury (Pruitt e_t a l . , 1 9 7 9 ) . The frequency of P. aeruginosa is also extremely high among cancer patients, e s p e c i a l l y in patients with acute leukemia or other diseases re s u l t i n g in neutropenia (Rodriguez and Bodey, 1 9 7 9 ) . F a t a l i t y rates of such infections are reported to be as high as 5 0 - 8 0 % (Rodriguez and Bodey, 1 9 7 9 ) . P. aeruginosa is a major pathogen in patients with c y s t i c f i b r o s i s , where i t is the predominant bacterium associated with terminal pulmonary i l l n e s s . It i s also an important cause of hospital-acquired pneumonias, hospi-tal-acquired urinary tract i n f e c t i o n s , and one of the major causes of infect i o n of patients undergoing invasive procedures. 2. A n t i b i o t i c resistance of Pseudomonas aeruginosa. P. aeruginosa has been c h a r a c t e r i s t i c a l l y regarded as resistant to antimicrobial agents, although introduction of new antimi-c r o b i a l drugs s p e c i f i c a l l y developed for anti-pseudomonal ac-t i v i t y has somewhat changed this s i t u a t i o n (Bryan, 1 9 7 9 ) . The small number of a n t i b i o t i c s which show high i_n v i t r o a c t i v i t y against P. aeruginosa include some aminoglycoside a n t i b i o t i c s 3 such as gentamicin, tobramycin, amikacin and t h e i r recently developed analogues such as n e t i l m i c i n and sisomycin, polymyxin a n t i b i o t i c s such as polymyxin B and c o l i s t i n , and a variety of semi-synthetic beta-lactam a n t i b i o t i c s , including c a r b e n i c i l -l i n , i t s t h i e n y l analogue t i c a r c i l l i n , and some new broad spectrum cephalosporins such as cefotaxime and moxalactam. For a l l anti-pseudomonal a n t i b i o t i c s currently i n use, effectiveness i s limited by the narrow margin between safe and e f f e c t i v e doses (Young, 1979; Bryan, 1979). This i s par-t i c u l a r l y true for aminoglycosides, which are ototoxic and can cause renal damage, (Wersall ^t_ a_l. , 1969; Falco ejt a_l. , 1969) and for polymyxins, which are generally considered to be too toxic for parenteral use (Bryan, 1979). Furthermore, there has generally been a poor c o r r e l a t i o n between the i n v i t r o s u s c e p t i b i l i t y to antimicrobial agents and i n vivo therapeutic e f f i c a c y (Davis, 1974; F l i c k and Cuff, 1976). One possible cause for t h i s i s the occurrence of adaptive (non-mutational) resistance i n vivo, as has been demonstrated for a variety of a n t i b i o t i c s i n v i t r o (Brown and Melling, 1969; G i l l e l a n d et  a l . , 1974; Pechey and James, 1974). One of the properties of P. aeruginosa which i s becoming clear from recent studies i s that the c e l l envelope of P_. aeruginosa i s highly variable depending on the growth conditions to which the organism i s subjected (Brown and Melling, 1969; Costerton _et jal. , 1979; Hancock and Carey, 1981; Hancock et a l . , 1982). In agreement with t h i s , i t now appears that the outer membranes of c e l l s grown i n vivo are somewhat d i f f e r e n t from those grown i n v i t r o 4 (P.A. Sokol, D.E. Woods, CD. Cox, and B.H. Iglewski, manu-sc r i p t in preparation; K. Poole and R.E.W. Hancock, unpublished data). Some of these changes may well a f f e c t a n t i b i o t i c e f f i c a c y (Costerton et a l . , 1979). An additional cause of the poor correlation between in vivo and iri v i t r o effectiveness of a n t i b i o t i c s i s the antag-2+ 2+ onism of a n t i b i o t i c a c t i v i t y by Ca and Mg , which occurs with polymyxin and aminoglycoside a n t i b i o t i c s (Newton, 1954; Zimelis and Jackson, 1973). In the case of aminoglyclosides, divalent cation antagonism of a n t i b i o t i c action is of much greater significance in P_. aeruginosa than in other gram-negative bacteria (Madeiros e_t a_l., 1971). Differences of up to 3 2-fold have been observed in measurements of minimal inhib-i t o r y concentrations of gentamicin in Mueller Hinton medium with varying lev e l s of C a 2 + and Mg 2 + (Reller et a l . , 1974). It 2 + has been suggested by several workers that the s i t e of Mg and Ca^ antagonism is the c e l l envelope (Newton, 1954; Zimelis and Jackson, 1973; Brown, 1975). The study reported here offers evidence that the s i t e of competition is the outer membrane. 3. Mechanisms of a n t i b i o t i c resistance. Resistance of P_. aeruginosa to antimicrobial agents takes two general forms (Bryan, 1979). One of these is resistance to agents e f f e c t i v e against most other gram-negative bacteria, generally termed " i n t r i n s i c " resistance. The second form is emerging resistance to the more recently introduced anti-pseudomonal agents such as gentamicin and c a r b e n i c i l l i n . In many cases the l a t t e r form of 5 resistance may be attributed to inactivating enzymes usually sp e c i f i e d by R factors (Bryan, 1979). I n t r i n s i c resistance does not, however, require the presence of R factors, and i s present in nearly a l l strains of P. aeruginosa. A n t i b i o t i c inactivating enzymes appear to have only a minor role in i n t r i n s i c resistance. For example chroma-somally-specified, inducible cephalosporinase is reported to be present in a l l strains of P_. aeruginosa (Bryan, 1979; Sabath e_t a l . , 1965), which may in part account for resistance to some beta-lactam a n t i b i o t i c s . Other chromosomally-specified in a c t i v a t i n g enzymes have not been found (Bryan e_t a l . , 1975). Resistance at the l e v e l of the targets of a n t i b i o t i c a c t i v i t y also does not appear to account for i n t r i n s i c resistance. For example, Bryan et a l . (1975) have shown that protein synthesis by c e l l - f r e e systems derived from P. aeruginosa is f u l l y sensi-tiv e to i n h i b i t i o n by streptomycin, whereas whole c e l l s are quite r e s i s t a n t . S i m i l a r l y , Mirleman and Nuchamowitz (1979) have shown that the enzymes which synthesize peptidoglycan in P. aeruginosa are at least as sensitive to b e n z y l p e n i c i l l i n as those of E_. c o l i , whereas whole c e l l s are much more r e s i s t a n t . As neither a n t i b i o t i c i n a c t i v a t i o n nor altered targets of a n t i b i o t i c action would seem to account for the high a n t i b i o t i c resistance of P. aeruginosa, a p o s s i b i l i t y to be considered i s that a n t i b i o t i c s are unable to reach their s i t e of a c t i v i t y . Bryan and colleagues have shown that defective uptake of amino-glycosides (Bryan, 1979) and tetracycline (Tseng and Bryan, 6 1974) correlates with resistance to these drugs. In addition, enhanced permeability of the outer membrane has been shown to correlate with increased s u s c e p t i b l i t y to 27 di f f e r e n t agents in the a n t i b i o t i c supersusceptible P_. aeruginosa mutant Z61 (Angus et. a l . , 1982). Thus perhaps the most l i k e l y explanation of i n t r i n s i c resistance is that P. aeruginosa is r e l a t i v e l y impermeable to a n t i b i o t i c s (Brown, 1975; Bryan, 1979). 4. T h e c e l l envelope and permeability. The c e l l envelope of P. aeruginosa, l i k e that of other gram-negative bacteria has been shown by electron microscopy to consist of three layers. These are the inner, or cytoplasmic membrane, the peptidogly-can, and the outer membrane. In some instances, especially i n -fections of children with c y s t i c f i b r o s i s , a capsule of mucoid material may also be present. The space between the inner and outer membrane, the periplasm, is the location of a variety of degradative enzymes, including those which inactivate a n t i b i o t -ics (Benveniste and Davies, 1973; Bryan, 1979). The inner membrane is the s i t e of s p e c i f i c , energy-requiring, transport systems, as well as components involved in energy generation and most of the enzyme systems involved in synthesis of the external wall layers, peptidoglycan and the outer membrane. The inner membrane constitutes a highly selective permeability bar r i e r (Costerton et a l . , 1974). Both the inner and outer face of the cytoplasmic membrane are thought to be hydrophobic (Machtiger and Fox, 1973), and i t has been demonstrated that 7 this membrane does not constitute an e f f e c t i v e barrier against hydrophobic substances (Teuber and M i l l e r , 1977). In contrast, the inner membrane is r e l a t i v e l y impermeable to hydrophilic substances. Such substances cross the inner membrane by means of substrate s p e c i f i c transport systems. Thus hydrophilic a n t i b i o t i c s are unable to enter the cytoplasm unless they are able to mimic a natural substrate and use i t s transport system, or disrupt the membrane s u f f i c i e n t l y to allow permeation. The peptidoglycan of P. aeruginosa does not appear to d i f f e r chemically from that of enteric organisms (Meadow, 1975), although i t does not appear to be covalently linked to an outer membrane protein analogous to the major lipoprotein of E_. c o l i (Hancock et a l . , 1981). However, i t seems unlikely that the peptidoglycan makes a major contribution to the low a n t i b i o t i c permeability of P. aeruginosa. As the targets of a n t i b i o t i c a c t i v i t y are either within the cytoplasm (e.g., the ribosomes) or within the c e l l envelope i t s e l f (e.g., the p e n i c i l l i n binding proteins exposed on the outer surface of the inner membrane), the common barrier which must be traversed is the outer membrane. There is a growing body of evidence that much of the i n t r i n s i c resistance of J?. aeruginosa may be accounted for on the basis of the per-meability properties of i t s outer membrane. The outer membrane of gram-negative bacteria has a major role as a permeability b a r r i e r . Nikaido (Nikaido and Nakai, 1979) has described two general pathways for d i f f u s i o n of small molecules across the 8 outer membrane, one for hydrophobic compounds and one for hydrophilic compounds. The hydrophobic pathway i s apparently unimportant i n the outer membrane of organisms such as E. c o l i , Salmonella and P_. aeruginosa, which synthesize complete lipopolysaccharides although i t s existence was demonstrated i n deep rough organisms. The hydrophilic pathway i s mediated by sp e c i f i c i n t e g r a l membrane proteins which form trans membrane channels or pores. Such molecules are generally termed "porins" (Nikaido and Nakai, 1979). 5. Properties of the outer membrane of gram-negative bacteria. The outer membrane of enteric bacteria has been extensively studied (for reviews see Nikaido and Nakae, 1979; DiRienzo et  a l . , 1980; Osborn and Wu, 1980) and considerable insight has been achieved with regard to both outer membrane structure and the r e l a t i o n s h i p of structure to the function of the outer mem-brane as a permeability b a r r i e r . The major components of the outer membrane are protein, phospholipid and lipopolysaccharide (LPS). Like other membranes, the outer membrane appears as a bi l a y e r i n the electron microscope. Studies of the outer mem-brane of E. c o l i and Salmonella (reviewed by Nikaido and Nakae, 1979) have shown that the outer membrane i s unusual, however, i n the extreme assymetry of d i s t r i b u t i o n of membrane compon-ents: v i r t u a l l y a l l the phospholipids are located on the inner face (except, possibly i n certain mutants) while v i r t u a l l y a l l the LPS i s on the outer surface. In contrast, the proteins of 9 o u t e r m e m b r a n e a r e p r e s e n t i n b o t h l a y e r s , a n d a r e , i n some c a s e s , m e m b r a n e s p a n n i n g ( E n d e r m a n e t a l . , 1 9 7 8 ) . a . L i p o p o l y s a c c h a r i d e . The g e n e r a l s t r u c t u r e o f L P S i s s i m i l a r i n a l l g r a m - n e g a t i v e b a c t e r i a a n d h a s b e e n r e v i e w e d b y 0rskov ^ t a ^ . , 1 9 7 7 . L P S i s a n a m p h i p a t h i c m o l e c u l e w i t h a h y d r o p h o b i c p o r t i o n , l i p i d A , b e l i e v e d t o b e e m b e d d e d i n t h e membrane a n d a h y d r o p h i l i c p o l y s a c c h a r i d e p o r t i o n , w h i c h e x t e n d s o u t f r o m t h e c e l l s u r f a c e . The d i s t a l p o r t i o n o f t h e p o l y s a c c h a r i d e , t h e 0 a n t i g e n , o f t e n c o n s i s t s o f o l i g o s a c -c h a r i d e r e p e a t i n g u n i t s , a n d s h o w s w i d e v a r i a b i l i t y e v e n w i t h i n a s i n g l e s p e c i e s . T h i s p r o p e r t y , w h i c h c a n b e s i m p l y s c r e e n e d b y u s i n g a n t i s e r a , a l l o w s f i n e s e r o l o g i c a l t y p i n g o f s t r a i n s . The p r o x i m a l p o r t i o n , t h e R - c o r e , s h o w s l e s s v a r i a b i l i t y w i t h g i v e n s p e c i e s , a n d o f t e n c o n t a i n s a u n i q u e e i g h t c a r b o n s u g a r ( 2 - k e t o - 3 - d e o x y o c t o n a t e ) , a n d a h e p t o s e . P h o s p h a t e a n d e t h a n -o l a m i n e p h o s p h a t e a r e a l s o p r e s e n t i n t h i s r e g i o n , a n d s t u d i e s w i t h f l u o r e s c e n t p r o b e s h a v e i n d i c a t e d b i n d i n g s i t e s f o r d i v a l e n t c a t i o n s i n t h i s p o r t i o n o f t h e L P S ( S c h i n d l e r e t a l . , 1 9 7 8 ) . The p r o p e r t i e s o f t h e L P S o f e n t e r i c b a c t e r i a a n d P_. a e r u g i n o s a r e s u l t i n a m e m b r a n e w h i c h i s h i g h l y i m p e r m e a b l e t o h y d r o p h o b i c c o m p o u n d s , i n c o n t r a s t t o t h e p e r m e a b i l i t y o f p h o s -p h o l i p i d b i l a y e r s t o s u c h c o m p o u n d s ( N i k a i d o a n d N a k a e , 1 9 7 9 ) . T h i s w o u l d a c c o u n t f o r t h e r e s i s t a n c e o f t h e s e o r g a n i s m s t o h y d r o p h o b i c a n t i b i o t i c s s u c h a s a c t i n o m y c i n D, e r y t h r o m y c i n a n d r i f a m p i c i n . I t s h o u l d b e n o t e d t h a t t h e h i g h l e v e l o f r e s i s t -a n c e o f P_. a e r u g i n o s a t o h y d r o p h o b i c a n t i b i o t i c s ( B r y a n , 1 9 7 9 ) 10 s t r o n g l y s u g g e s t s t h a t n o s i g n i f i c a n t h y d r o p h o b i c u p t a k e p a t h w a y e x i s t s i n t h i s o r g a n i s m e i t h e r . b . P r o t e i n . The p r o t e i n s o f o u t e r m e m b r a n e s a r e l a r g e l y r e s p o n s i b l e f o r t h e p e r m e a b i l i t y o f t h e o u t e r membrane t o h y d r o p h i l i c c o m p o u n d s . A n u m b e r o f o u t e r m e m b r a n e p r o t e i n s a p p e a r t o f u n c t i o n i n t h e t r a n s p o r t o f s p e c i f i c c o m p o u n d s a c r o s s t h e o u t e r m e m b r a n e . T h e s e i n c l u d e p r o t e i n s r e q u i r e d f o r t h e u p t a k e o f i r o n c h e l a t e s ( H a n c o c k e t a l . , 1 9 7 6 ) , n u c l e o s i d e s ( H a n k e , 1 9 7 6 ) , v i t a m i n B 1 2 ( D i m a s i et_ a _ l . , 1 9 7 3 ) , a n d m a l t o d e x -t r a n s a n d m a l t o s e ( S z m e l e m a n a n d H u f n u n g , 1 9 7 6 ) . S u c h p r o t e i n s a r e e s p e c i a l l y i m p o r t a n t i n t h e u p t a k e o f s u b s t r a t e s p r e s e n t a t v e r y l o w c o n c e n t r a t i o n s ( N i k a i d o a n d N a k a e , 1 9 7 9 ) . The m a j o r c o n t r i b u t i o n t o o u t e r m e m b r a n e p e r m e a b i l i t y , h o w e v e r , c o m e s f r o m p r o t e i n s c a l l e d p o r i n s , w h i c h f o r m g e n e r a l t r a n s m e m b r a n e d i f f u s i o n c h a n n e l s . S u c h p r o t e i n s , w h i c h g e n e r a l l y h a v e a n a p p a r e n t m o l e c u l a r w e i g h t o f 3 2 , 0 0 0 - 4 2 , 0 0 0 , a r e p r e s e n t i n a l l g r a m - n e g a t i v e b a c t e r i a s o f a r e x a m i n e d . T h e y a r e p r e s e n t i n h i g h c o p y n u m b e r , m e m b r a n e s p a n n i n g , a n d a r e g e n e r a l l y c l o s e l y a s s o c i a t e d w i t h p e p t i d o g l y c a n ( D i R i e n z o e t a l . , 1 9 7 8 ) . The p o r e - f o r m i n g f u n c t i o n o f t h e s e p r o t e i n s h a s b e e n e s t a b l i s h e d i n r e c o n s t i t u t i o n s t u d i e s w h e r e p o r i n i s i n c o r p o r a t e d i n t o p h o s -p h o l i p i d o r p h o s p h o l i p i d - L P S v e s i c l e s ( N a k a e , 1 9 7 6 a a n d 1 9 7 6 b ) o r i n t o b l a c k l i p i d b i l a y e r s ( B e n z e t a l . , 1 9 7 8 ) . E x p e r i m e n t s b y N i k a i d o a n d c o w o r k e r s ( N i k a i d o a n d N a k a e , 1 9 7 9 ) h a v e e s t a b -l i s h e d t h a t p o r i n c h a n n e l s a p p e a r t o h a v e a f a i r l y c o n s t a n t d i a m e t e r , t h u s l i m i t i n g t h e s i z e o f m o l e c u l e s a b l e t o c r o s s t h e 11 outer membrane, which consequently acts as a molecular sieve. Exclusion l i m i t s of E_. c o l i and Salmonella porins have been measured as 550-650 daltons (Decad and Nikaido, 1976) while that of P. aeruginosa is larger, 3000-9000 daltons (Hancock and Nikaido, 1978). The function of porins in c o n t r o l l i n g permeability has been confirmed in studies of mutants de f i c i e n t in these proteins (von Meyenburg and Nikaido, 1977; Lutkenhaus, 1977; Bavoil et a l . , 1977). More than one porin species has been found in several bacteria (Osborn and Wu, 1980), and alternate porins which can be induced by s p e c i f i c growth conditions (von Meyenburg and Nikaido, 1977; Tommassen and Lugtenberg, 1970; Hancock and Carey, 1980; Hancock e_t a l . , 1982) or prophage infect i o n (Schnaitman, 1974) are also known. It i s , however, unclear whether such porins are t r u l y capable of mediating generalized permeability in the same way that the "major" porins do. The contribution of proteins other than porins and s p e c i f i c transport proteins i s not known, but i t would appear l i k e l y that their role in determining permeability is for the most part i n d i r e c t . Porin alterations in E_. c o l i have been shown to a f f e c t the uptake of some beta-lactams, chloramphenicol, and tetracycline (Nikaido et a_l. , 1977; Van Alphen et. _al., 1978; Chopra and Eccles, 1978; Foulds, 1976) and i t would appear that these proteins are largely responsible for the permeability of hydrophilic a n t i b i o t i c s to the outer membrane. Only a single isolated example of an a n t i b i o t i c which uses a s p e c i f i c outer 12 m e m b r a n e t r a n s p o r t p r o t e i n t o c r o s s t h e o u t e r m e m b r a n e h a s b e e n f o u n d : a l b o m y c i n , w h i c h u s e s t h e T o n A f e r r i c h r o m e t r a n s p o r t -i n g p r o t e i n s ( B r a u n e t a l . , 1 9 7 6 ) — a l t h o u g h i t i s i m p o r t a n t t o n o t e t h a t a l b o m y c i n i s a f e r r i c h r o m e a n a l o g u e . 6 . The o u t e r m e m b r a n e o f P . a e r u g i n o s a a n d i t s r o l e i n  a n t i b i o t i c p e r m e a b i l i t y . A l t h o u g h t h e o u t e r membrane o f P . a e r u g i n o s a w o u l d a p p e a r t o b e s i m i l a r t o t h a t o f E. c o l i a n d S a l m o n e l l a i n i t s o v e r a l l d e s i g n , t h e r e a p p e a r t o b e s i g n i f i -c a n t d i f f e r e n c e s w h i c h may w e l l c o n t r i b u t e t o t h e d i f f e r e n t a n t i b i o t i c s u s c e p t i b i l i t y o f P_. a e r u g i n o s a . The p r o t e i n s o f t h e o u t e r m e m b r a n e o f P_. a e r u g i n o s a h a v e b e e n e x a m i n e d b y s e v e r a l l a b o r a t o r i e s ( S t i n n e t t a n d E a g o n , 1 9 7 3 ; M i z u n o a n d K a g e y a m a , 1 9 7 8 ; H a n c o c k a n d N i k a i d o , 1 9 7 8 ; H a n c o c k a n d C a r e y , 1 9 7 9 ) . S i x t o e i g h t m a j o r o u t e r membrane p r o t e i n s h a v e b e e n f o u n d . P o r i n a c t i v i t y h a s b e e n d e m o n s t r a t e d f o r p r o t e i n F ( H a n c o c k e t a l . , 1 9 8 0 ) . T h i s m o l e c u l e d i f f e r s f r o m p o r i n s o f e n t e r i c b a c t e r i a i n t h a t i t i s u n u s u a l l y u n s t a b l e t o s o d i u m d o d e c y l s u l p h a t e , a n d h a s t w o i n t r a - c h a i n d i s u l f i d e b r i d g e s ( H a n c o c k a n d C a r e y , 1 9 7 9 ) Two i n d u c i b l e p o r i n s h a v e a l s o b e e n d e m o n s t r a t e d . T h e s e a r e p r o t e i n D l , a p r o t e i n i n d u c e d d u r i n g g r o w t h o n g l u c o s e a s s o l e c a r b o n s o u r c e ( H a n c o c k a n d C a r e y , 1 9 8 0 ) , a n d p r o t e i n P , a n a n i o n - s e l e c t i v e p o r e w i t h a r e l a t i v e l y l o w e x c l u s i o n l i m i t , w h i c h a p p e a r s d u r i n g g r o w t h o n p h o s p h a t e - d e f i c i e n t m e d i u m ( H a n c o c k e t a l . , 1 9 8 1 ) . A l i p o p r o t e i n , p r o t e i n I , a n a l o g o u s t o t h e B r a u n 13 l i p o p r o t e i n o f E. c o l i (Braun, 1975), has been demonstrated i n P. a e r u g i n o s a , and, a l t h o u g h t h i s p r o t e i n i s p e p t i d o g l y c a n a s s o c i a t e d , i t may w e l l d i f f e r from E. c o l i p r o t e i n i n t h a t 30% o f the E_. c o l i p r o t e i n i s c o v a l e n t l y a t t a c h e d the the p e p t i d o -g l y c a n whereas the c o v a l e n t a s s o c i a t i o n of the P. a e r u g i n o s a p r o t e i n I i s d i s p u t e d (Mizuno and Kageyama, 1979; Hancock ejt a l . , 1981). In any c a s e , the amino a c i d c o m p o s i t i o n s of t h e s e two l i p o p r o t e i n s d i f f e r s u b s t a n t i a l l y . A second l i p o p r o t e i n has a l s o been demonstrated (Mizuno, 1979). In sodium d o d e c y l s u l p h a t e g e l e l e c t r o p h o r e s i s , t h i s p r o t e i n (H2) i s s e p a r a t e d from a p r o t e i n o f s i m i l a r m o l e c u l a r w e ight (HI) o n l y under c o n d i t i o n s d e s c r i b e d by Hancock and Carey (1979). The f u n c t i o n o f H2, and o f the o t h e r major o u t e r p r o t e i n s D2, E, and G, i s s t i l l unknown. A l t h o u g h i t has been e s t a b l i s h e d t h a t the s i z e of i n d i v i d u a l p o r i n c h a n n e l s i s l a r g e r i n P. a e r u g i n o s a than i n e n t e r i c b a c t e r i a (Benz and Hancock, 1981; Hancock and N i k a i d o , 1978) , t h e r e i s e v i d e n c e a c c u m u l a t i n g t h a t the number of f u n c t i o n a l pores and c o n s e q u e n t l y the t o t a l a r ea of pore a v a i l a b l e f o r d i f f u s i o n i s much l o w e r , so t h a t t o t a l p e r m e a b i l i t y v i a the h y d r o p h i l i c pathway i s r e l a t i v e l y low. Low o u t e r membrane p e r m e a b i l i t y has been shown i n in v i v o s t u d i e s of w i l d type P. a e r u g i n o s a (Angus e_t a l . , 1982). These workers showed t h a t an a n t i b i o t i c super s u s c e p t i b l e mutant of P. a e r u g i n o s a , Z61, s e l e c t e d as a n t i b i o t i c s u s c e p t i b l e (Zimmerman and R o s s e l e t , 1979) showed g r e a t l y enhanced o u t e r membrane p e r m e a b i l i t y , thus 14 providing evidence that low outer membrane permeability i s indeed a major determinant in i n t r i n s i c resistance to a n t i b i o t -i c s . In Z61, increased permeability and a n t i b i o t i c supersus-c e p t i b i l i t y were associated with an LPS a l t e r a t i o n (Kropinski et a l . , 1982). Low jLn vivo porin a c t i v i t y of wild type c e l l s correlates well with the low pore forming a c t i v i t y of protein F which has been found in in v i t r o studies (Benz and Hancock, 1981). These observations, however, did raise the question as to whether the actual porin was protein F, or some minor contaminant copurified with protein F. This study reports the i s o l a t i o n of a mutant severely d e f i c i e n t in protein F. Results of permeability studies with this mutant confirm the pore-forming function of protein F and indicate that less than 1% of protein F molecules form functional channels across the outer membrane. Another unusual property of the LPS of P_. aeruginosa is i t s unusually high phosphate content (Drewry et, a l . , 1971). This phosphate is associated with the core region, and i s , in part, as triphosphate (Wilkinson, 1981). The c e l l envelope of P_. aeruginosa also has very high levels of divalent cations (Brown and Wood, 1972), which may well be associated with these phosphate groups since phosphate carries a net negative charge at neutral pH. While P. aeruginosa is unusually resistant to a n t i b i o t i c s , i t is highly susceptible to chelators of divalent cations such as EDTA (Cox and Eagon, 1968), and to polymyxin a n t i b i o t i c s . For example, although treatment of E. c o l i with 15 EDTA i s i n s u f f i c i e n t to allow osmotic l y s i s unless lysozyme i s present (Leive, 1965), EDTA treatment of P. aeruginosa results in osmotically f r a g i l e c e l l s . Tris(hydroxymethylJaminomethane (Tris) maximizes this EDTA ef f e c t (Eagon and As b e l l , 1966). Treatment with EDTA results in the release of LPS-protein complexes with low (less than 10%) phospholipid content (Rogers et a l . , 1969; Stinnett and Eagon, 1975). These complexes can be v i s u a l i z e d by electron microscopy of freeze fractured c e l l s and appear to be d i s t i n c t aggregates in the plane of the membrane (Stinnett and Eagon, 1975). It has been concluded from these and other studies (Roberts ejt a l . , 1970; Kenward e_t a l . , 1979; Boggis et a l . , 1979) that divalent cations play a c r i t i c a l role in maintaining the s t a b i l i t y of the outer membrane. Polymyxins are amphipathic molecules consisting of a highly cat ionic peptide head and a hydrophobic t a i l (Storm e_t a l . , 1977). Like EDTA, polymyxins appear to act d i r e c t l y on the outer membrane, and are known to bind with high a f f i n i t y to LPS (Cooperstock, 1974; Schindler and Osborn, 1979). The action of polymyxins is inhibited by the presence of divalent cations (Newton, 1954). These observations have led to the suggestion that polymyxins and EDTA act at a common s i t e on the outer membrane, a divalent cation binding s i t e on the LPS which is required for outer membrane s t a b i l i t y (Brown, 1975). The study reported here provides experimental e v i -dence supporting this suggestion. Furthermore, evidence i s 16 presented that aminoglycoside a n t i b i o t i c s may also be active at this same s i t e in P. aeruginosa, and that disruption of this s i t e by aminoglycosides and similar c a t i o n i c substances may provide an alternate pathway across the outer membrane. This study also provides an explanation for the long-standing observation that the s u s c e p t i b i l i t y of P. aeruginosa to EDTA 2+ and polymyxin may be reversed by growth in Mg -limited medium (Brown and Melling, 1969). It is demonstrated that this i n -crease in resistance is associated with induction of a major outer membrane protein HI. It i s suggested that Hi acts to replace divalent cations at a c r i t i c a l divalent cation binding s i t e on the LPS, protecting this s i t e from attack by EDTA and cationic a n t i b i o t i c s . 17 METHODS 1. Media and growth c o n d i t i o n s . P r o t e o s e peptone no. 2 ( D i f c o , PP2) was used as a r i c h medium. The m i n i m a l medium used was B a s a l Medium No. 2 (BM2) d e s c r i b e d by G i l l e l a n d e_t a l . (1974), c o n t a i n i n g 10 mM FeS04, a n d e i t h e r 20 mM p o t a s s i u m s u c c i n a t e (BM2 s u c c i n a t e ) o r 0.4% ( w t / v o l ) g l u c o s e (BM2 g l u -c o s e ) . The u s u a l l e v e l of MgS04 added was 0.5 mM (Mg s u f f i c i e n t m e d i a ) . Mg" d e f i c i e n t media c o n t a i n e d 0.02 mM MgS04. Other c a t i o n s were added as c h l o r i d e s a l t s i n the amounts s p e c i f i e d i n the t e x t . L i q u i d c u l t u r e s were grown w i t h v i g o r o u s a e r a t i o n a t 37°C except where s t a t e d o t h e r w i s e . A l l g l a s s w a r e used w i t h d e f i n e d media was c l e a n e d by a u t o c l a v i n g w i t h d i s t i l l e d w a t e r . N u t r i e n t b r o t h ( D i f c o ) was used to grow c e l l s f o r s t r e p t o m y c i n uptake a s s a y s . 2. B a c t e r i a l s t r a i n s . Sources and p r o p e r t i e s of the p r i n c i p a l b a c t e r i a l s t r a i n s used i n t h i s s tudy are l i s t e d i n Table I . Pseudomonas a e r u g i n o s a PA01 s t r a i n H103 was used as the w i l d type and r e f e r e n c e s t r a i n t h r o u g h o u t . The o u t e r mem-brane of t h i s s t r a i n has p r e v i o u s l y been w e l l c h a r a c t e r i z e d (Hancock and Carey, 1979; Hancock e t a l . , 1981). S t r a i n s H185 and H181 were i n d e p e n d e n t l y i s o l a t e d from H103 by d i e t h y l s u l f a t e mutagenesis f o l l o w e d by s e l e c t i o n on 18 TABLE 1. B a c t e r i a l s t r a i n s . S t r a i n P r o p e r t i e s Source HI 03 H181 H185 H207 Z61 P. aeruginosa PAO 1 w i l d type Polymyxin B r e s i s t a n t mutants of HI03 Revertant of HI81 A n t i b i o t i c super-s u s c e p t i b l e P. aeruginosa A. K r o p i n s k i (Queen's U n i v e r s i t y Kingston, Ont.) This study This study W. Zimmerman (Ciba-Geigy, Basel Switzerland H251 H283 Revertant of Z61 P r o t e i n F - d e f i c i e n t mutant of H103 B.L. Angus, U.B.C. (Angus et a l 1982) This study H284 H321 H324 Revertants of H283 This study AK43 AK1160 AK1012 L P S - d e f i c i e n t mutants of H103 A. K r o p i n s k i AK1213 AK1114 UB1636(RP1) N o n - p i l i a t e d mutant of H103 N o n - p i l i t e d , non-f l a g e l l a t e d mutant of H103 E. c o l i K12 t r p h i s  s t r A l a c / ampr t e t r neo r k a n r A. K r o p i n s k i A. K r o p i n s k i P.M. Bennet ( U n i v e r s i t y of B r i s t o l , B r i s t o l U.K.) 19 BM2 s u c c i n a t e a g a r c o n t a i n i n g 50 u g / m l o f p o l y m y x i n B s u l p h a t e . M u t a g e n e s i s was c a r r i e d o u t b y s u s p e n d i n g 0 . 1 m l o f o v e r n i g h t c e l l s i n 5 m l s a t u r a t e d s o l u t i o n o f e t h y l s u l p h a t e i n 0 . 0 6 M p o t a s s i u m p h o s p h a t e b u f f e r pH 6 . 0 f o r 30 m i n . a t 2 5 ° C . C e l l s w e r e t h e n d i l u t e d 1 i n 50 i n P P 2 , a l l o w e d t o g r o w o v e r n i g h t , a n d p l a t e d o n BM2 s u c c i n a t e w i t h 50 u g / m l p o l y m y x i n B . C l o n e s w e r e s u b c u l t u r e d o n n o n - s e l e c t i v e m e d i a , t h e n r e t e s t e d o n p o l y -m y x i n . The r e v e r t a n t H207 a n d f i v e s i m i l a r r e v e r t a n t s o f H181 a n d H185 w e r e i s o l a t e d b y s c r e e n i n g c u l t u r e s w h i c h h a d b e e n h e l d a t 4°C f o r s e v e r a l w e e k s f o r l o s s o f p o l y m y x i n B r e s i s t -a n c e . The l e v e l s o f r e s i s t a n c e t o p o l y m y x i n B o n BM2 s u c c i n a t e a g a r p l a t e s w e r e 0 . 8 , 7 5 , 7 5 , a n d 0 . 8 u g / m l f o r H 1 0 3 , H 1 8 1 , H185 a n d H 2 0 7 , r e s p e c t i v e l y . S t r a i n Z 6 1 , a m u t a n t o f PAO w h i c h h a s b e e n s h o w n t o b e h i g h l y s u s c e p t i b l e t o a w i d e r a n g e o f a n t i b i o t i c s ( Z i m m e r m a n , 1 9 7 9 ) , a n d H 2 5 1 , a f u l l r e v e r t a n t o f Z 6 1 , h a v e b e e n d e s c r i b e d b y A n g u s e t a l . ( 1 9 8 2 ) . S t r a i n H 2 8 3 i s a m u t a n t o f H 1 0 3 s e v e r e l y d e f i c i e n t i n o u t e r m e m b r a n e p r o t e i n F ( p o r i n ) . I s o l a t i o n o f t h i s s t r a i n a n d t h r e e r e v e r t a n t s o f H 2 8 3 w i t h w i l d t y p e l e v e l s o f p o r i n ( H 2 8 4 , H 3 2 1 a n d H 3 2 4 ) , i s d e s c r i b e d b e l o w . S t r a i n s A K 4 3 , A K 1 1 6 0 a n d A K 1 0 1 2 a r e L P S - a l t e r e d s t r a i n s o b t a i n e d f r o m A . K r o p i n s k i ( Q u e e n s U n i v e r s i t y , K i n g s t o n , O n t a r i o ) . A K 1 2 1 3 , a n o n - p i l i a t e d s t r a i n , a n d A K 1 1 1 4 , a n o n - p i l i a t e d , n o n - f l a g e l l a t e d s t r a i n , w e r e a l s o o b t a i n e d f r o m t h i s s o u r c e . 20 RPl was introduced into P. aeruginosa by conjugation with Escherichia c o l i UB1636 (RPl), kindly provided by P.M. Bennett, University of B r i s t o l , B r i s t o l , U.K. RPl carries re-sistance to a m p i c i l l i n (and c a r b e n i c i l l i n ) , neomycin, kanamy-ci n , and t e t r a c y c l i n e . Resistance to beta-lactam a n t i b i o t i c s i s mediated by a TEM-2 type beta-lactamase (Sykes and Mathews, 1976). In the case of Z61, selection was for resistance to 100 ug/ml neomycin. For a l l other strains selection was on 500 ug/ml c a r b e n i c i l l i n . Z61 (RPl) was maintained on 200 ug/ml neomycin while a l l other P. aeruginosa (RPl) strains were maintained on 200 ug/ml t e t r a c y c l i n e . A set of 17 serotype-specific strains were a kind g i f t from Dr. P. Liu, University of L o u i s v i l l e , L o u i s v i l l e , Kentucky. These strains were representatives of the Interna-t i o n a l Antigenic Typing Scheme (IATS) (commercially marketed by Difco Ltd., Detroit, Michigan) which contains as subsets the type strains from a l l other commonly-used P. aeruginosa sero-typing systems. They were named as follows: type 1 (ATCC 33348), type 2 (ATCC 33349), type 3 (ATCC 33350), type 4 (ATCC 33351), type 5 (ATCC 33352), type 6 (ATCC 33354), type 7 (ATCC 33353), type 8 (ATCC 33355), type 9 (ATCC 33356), type 10 (ATCC 33357), type 11 (ATCC 33358), type 12 (ATCC 33359), type 13 (ATCC 33360), type 14 (ATCC 33361), type 15 (ATCC 33362), type 16 (ATCC 33363), type 17 (ATCC 33364). Pseudomonas putida type s t r a i n (ATCC 12633) was obtained from the American Type Culture Co l l e c t i o n (ATCC), B r o c k v i l l e , Maryland. 21 S t r a i n s H325 a n d H329 w e r e i n d e p e n d e n t l y d e r i v e d m u c o i d d e r i v a t i v e s o f H103 s e l e c t e d f o r r e s i s t a n c e t o p h a g e 7 . T h e s e s t r a i n s w e r e n o t r e s i s t a n t t o t h i s p h a g e and h a v e r e m a i n e d m u c o i d a f t e r r e p e a t e d s u b c u l t u r e on P P 2 . 3 . I s o l a t i o n o f M u t a n t s D e f i c i e n t i n O u t e r M e m b r a n e P r o t e i n s . I s o l a t i o n o f m u t a n t s was a t t e m p t e d u s i n g r a n d o m h e a v y m u t a g e n e s i s . T h i s p r o c e d u r e was b a s e d on t h a t o f S u z u k i et. a l . ( 1 9 7 8 ) , who u s e d i t w i t h E s h e r c h i a c o l i t o o b t a i n c l a s s e s o f m u t a n t s f o r w h i c h no s e l e c t i o n p r o c e d u r e i s r e a d i l y a v a i l a b l e . The m u t a g e n e s i s p r o t o c o l u s e d was b a s e d on t h a t o f A d e l b e r g ejt a l . ( 1 9 6 5 ) . A m i d - l o g a r i t h m i c p h a s e c u l t u r e o f H103 w a s c o l l e c t e d b y c e n t r i f u g a t i o n , w a s h e d o n c e i n 50 mM s o d i u m p h o s p h a t e b u f f e r (pH 6 . 0 ) , a n d c o n c e n t r a t e d t w e n t y - f o l d i n t h e same b u f f e r . Washed c e l l s ( 0 . 5 m l ) w e r e a d d e d t o 1 m l o f 1 mg/ml N - m e t h y l - N 1 - n i t r o - n i t r o s o g u a n i d i n e ( N T G ) , a n d h e l d a t 37°C f o r 30 m i n . C e l l s w e r e t h e n c o l l e c t e d by c e n t r i f u g a -t i o n , r e s u s p e n d e d i n b u f f e r , d i l u t e d a n d p l a t e d o n P P 2 a g a r . P l a t e s w e r e e x a m i n e d a f t e r 7 2 h g r o w t h a t 3 0 ° C . P l a t e c o u n t s w e r e a l s o c a r r i e d o u t w i t h t h e u n t r e a t e d c e l l s u s p e n s i o n . The s u r v i v a l r a t e a f t e r t h i s m u t a g e n e s i s p r o c e d u r e was a b o u t 0 . 0 0 4 % . F i v e h u n d r e d c o l o n i e s w e r e p i c k e d f o r s c r e e n i n g . T h e s e w e r e t r a n s f e r r e d t o P P 2 p l a t e s , t h e n s u b c u l t u r e d f r o m s i n g l e c o l o n y i s o l a t e s t o P P 2 p l a t e s and P P 2 b r o t h . A f t e r g r o w t h a t 3 0 ° C , p l a t e s w e r e s t o r e d a t 4°C and b r o t h c u l t u r e s w e r e s u p p l e m e n t e d w i t h d i m e t h y l s u l f o x i d e t o 8% a n d s t o r e d a t 22 - 7 0 ° C . I n i t i a l s c r e e n i n g was d o n e u s i n g i n o c u l a f r o m P P 2 p l a t e s , and f r o z e n s t o c k s w e r e u s e d f o r s u b s e q u e n t s t u d i e s . The 5 0 0 s t r a i n s w e r e s c r e e n e d f o r o u t e r membrane p r o -t e i n d e f i c i e n c i e s u s i n g a s i m p l i f i e d m e t h o d f o r c e l l e n v e l o p e p r e p a r a t i o n . C u l t u r e s w e r e g r o w n i n 30 mL BM2 g l u c o s e ( e x c e p t f o r a u x o t r o p h s w h i c h w e r e g r o w n i n P P 2 ) a t 30°C f o r 18 t o 42 h , a n d c o l l e c t e d b y c e n t r i f u g a t i o n . C e l l e n v e l o p e s w e r e p r e p a r e d 2+ i n 30 mM T r i s HC1 (pH 7 . 4 ) w i t h Mg a s d e s c r i b e d b e l o w . T h i s m e t h o d y i e l d e d e n v e l o p e s w h i c h t e n d e d t o be e n r i c h e d i n o u t e r m e m b r a n e . The e n v e l o p e s w e r e r e s u s p e n d e d i n w a t e r t o a p p r o x i -m a t e l y 10 mg p r o t e i n / m l a n d r u n o n s o d i u m d o d e c y l s u l p h a t e (SDS) p o l y a c r y l a m i d e g e l s ( a s d e s c r i b e d b e l o w ) t o d e t e r m i n e t h e o u t e r m e m b r a n e p r o t e i n c o m p o s i t i o n . D e f i c i e n c i e s i n m a j o r p r o t e i n s w e r e e a s i l y d i s t i n g u i s h e d b y t h i s p r o c e d u r e . O u t e r m e m b r a n e s w e r e p r e p a r e d f r o m s t r a i n s w h i c h a p p e a r e d t o h a v e d e f i c i e n c i e s i n m a j o r o u t e r membrane p r o t e i n u s i n g t h e o n e - s t e p p r o c e d u r e r e f e r r e d t o b e l o w , a n d t h e s e o u t e r m e m b r a n e s w e r e e x a m i n e d b y SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e -s i s t o c o n f i r m t h e a l t e r a t i o n . 23 4 . Bacteriophage and bacteriocin studies. (a) Bacteriophages"and p a r t i a l characterization of t h e i r  receptors. A l l methods used in the handling of bacteriophages were described previously by Hancock and Reeves (1976). Phages were characterized using a p i l u s - d e f i c i e n t derivative of P. aeruginosa PA01, AK1144, and two lipopolysaccharide (LPS)-altered (rough) s t r a i n s , AK43 and AK1160, obtained from A. Kropinski (Queen's University, Kingston, Ontario). Phage sources and putative receptors are summarized in Table I I . Phages were obtained from the following sources: 2, 7, 21, 44, 68, 73, 109, 352, 1214, C21, F7, F8, F10, 119X, and M6 from T.L. P i t t (Public Health Laboratory, London, U.K.); G101, F116, D3c + 1 +, and D3c~ 1 + from T. Iijima (Institute for Fermentation, Osaka, Japan); PLS27 and E79 from A. Kropinski; PB1 and B39 from D.E. Bradley (Memorial University, St. John's, Newfoundland); SI from R. Warren (University of B r i t i s h Columbia); and 176p from J.D. Piguet (Institute of Hygiene, Geneva, Switzerland). These phages were p u r i f i e d from single plaques using H103 as a host s t r a i n except for PLS27, for which AK1160 was used as host. A l l other phages were isolated in the laboratory of R.E.W. Hancock, University of B r i t i s h Columbia, as host range mutants of phages which plated poorly on p i l u s -or LPS-deficient s t r a i n s . The phages which were selected for a b i l i t y to form plaques on AK1144 (p i l u s - d e f i c i e n t ) were B6B, B6C, and B6D (independent isolates derived from 352), B9F (from M6), B5A (from 119x), C7B (from 176p), B1A (from F7), C3A (from 24 TABLE II. Bacteriophage sources and receptors Phage Lab name Source Putative Receptor 3 73 A9A P i t t 1 3 p i l u s 119x B5 P i t t p i l u s M6 B9 P i t t p i l u s B39 C9 Bradley 0 p i l u s 44 A7 P i t t smooth LPS 109 B4 P i t t smooth LPS F8 B2 P i t t smooth LPS 1214 B7 P i t t smooth LPS 352 B6 P i t t smooth LPS PBl C8 Bradley smooth LPS SI D2 Warrend smooth LPS C3A C3A Hancock (C27) e smooth LPS E79 Dl Kropinskif smooth LPS PLS27 D8 Kropinski rough LPS 7 A2 P i t t protein 21 A4 P i t t p r o t e i n 68 A8 P i t t protein F10 B3 P i t t protein C21 C3 P i t t protein F116 D3 I i jimag protein G101 D4 Iijima protein D3c+1+ D6 I i j i m a protein A8A A8A Hancock (68) protein BIB BIB Hancock (F7) protein B6B B6B Hancock (352) protein B6C B6C Hancock (352) protein B9F B9F Hancock (M6) protein or LPS B9E B9E Hancock (M6) protein or LPS 2 Al P i t t p r o tein or LPS D3c~l + D5 Iijima protein or LPS B5A B5A Hancock (119x) pro t e i n or LPS C7B C7B Hancock (B39) protein or LPS A9B A9B Hancock (73) protein or LPS B7A B7A Hancock (1214) protein or LPS V1-V28 V1-V28 This study protein a See Nicas and Hancock, 1981. b T.L. P i t t , Public Health Laboratory, London, U.K. c D.E. Bradley, Memorial University, St. John's, Nfld. d R.A.J. Warren, U. of B r i t i s h Columbia. e R.E.W. Hancock, u. of B r i t i s h Columbia (parent s t r a i n in brackets) f A. Kropinski, Queen's University, Kingston, Ont. 9 T. Iijima, I n s t i t u t e for Fermentation, Osaka, Japan. 25 C21), and A8A (from 68). B7A was derived from 1214, selected on AK43 (an LPS-altered rough strain) . A9A was a contaminant or derivative of 73 unable to form plaques on AK1144 or AK43. Phage stocks were maintained at 4°C. Stocks were checked regularly for t i t r e and phenotype, and were re p u r i f i e d every 18 months . Phages M6, B39, 73, and 119x were characterized as p i l u s - s p e c i f i c by their i n a b i l i t y to form plaques on pilus-de-f i c i e n t s t r a i n s . Phages 44, 109, F8, E79, 1214, PB1, SI, 352, and C3A were characterized as PAO smooth LPS-specific since they f a i l e d to form plaques on AK43 and other LPS-altered strains and could be shown to adsorb to p u r i f i e d LPS. Phage PLS27 has been characterized by J a r r e l l and Kropinski as spe-c i f i c f or PAO rough core, and does not form plaques on smooth strains ( J a r r e l l and Kropinski, 1981). Phages 7, 21, 68, F10, C21, F116, G101, B6B, B6C, D3c~ 1 +, B1A, and A8A formed plaques well on LPS altered and p i l u s - d e f i c i e n t strains and f a i l e d to adsorb to LPS and thus appear to have protein receptors. Phages 2, D3C +1 +, B5A B7A, B9E, B9F, and C7B formed plaques well on p i l u s - d e f i c i e n t and wild-type s t r a i n s , but poorly on LPS-altered strains but f a i l e d to adsorb to LPS, and thus may have LPS or LPS-associated protein receptors. (b) Aeruginocin"studies. The aeruginocins used were a P. aeruginosa typing set obtained from A. Kropinski, in addition to the aeruginocins from strains H41, received from J . Govan, University of Edinburgh, Scotland, and PAF41 and PAH108 from B. 26 Holloway, Monash University, Clayton, A u s t r a l i a . The receptors of these aeruginocins are as yet uncharacterized, but they plate equally well on H103 and AK1144 although some plate only on AK43 (but not H103). Aeruginocins were prepared using the method of Kageyama (1964). Aeruginocin producing strains were grown at 30°C to an A600 o f 0.6-0.8, then treated with 1 ug/ml mitomycin C (Sigma Chemical Co., MO.) to induce aeruginocin production. Culture supernatants were pre-c i p i t a t e d with 10% polyethylene gl y c o l (PEG) then d i a l i s e d to remove the PEG. Aeruginocin s u s c e p t i b i l i t y was tested by spot-ting t h i s preparation on b a c t e r i a l lawns. Aeruginocin prepara-tions were very unstable and tended to lose a c t i v i t y rapidly when stored at 4°C. (c) Isolation of p r o t e i n - s p e c i f i c phages"from nature. Isolation of phages s p e c i f i c for outer membrane proteins was attempted using the enrichment method of Verhoef e_t a l . (1977 ). The basis of t h i s method is the use of a s t r a i n lacking a s p e c i f i c receptor to adsorb out the majority of phages, leaving those phage which have the missing protein as their receptor. The source of phages was influent sewage from the Iona Island sewage treatment plant, Vancouver, B.C. Eight c o l -l e c t i o n s were made, each on days following three rain-free days in the spring of 1980. Samples were centrifuged to remove debris and treated with chloroform to reduce the p o s s i b i l i t y of encountering pathogens. Undiluted chloroform-treated samples (3 ml) were mixed in equal volumes with 3.2% agar and 2% PP2 27 and 0.1 ml mid-logarithmic culture of H103 and spread onto PP2 plates to obtain estimates of the number of phages present. Most samples contained 10-100 plaque forming units/ml. Seven strains with apparent outer membrane protein d e f i c i e n c i e s (isolated by heavy mutagenesis as described below) were used in these studies as the adsorbing s t r a i n s . AK1012, a LPS-deficient s t r a i n , and AK1213, a p i l u s -def i c i e n t s t r a i n , were used as hosts for phage propagation in order to reduce the p o s s i b i l i t y of i s o l a t i n g phages with LPS or p i l u s receptors. For each sewage sample, 20 ml of sample was mixed with 20 ml double strength PP2 and 1 ml overnight culture of AK1012. After overnight growth, the c e l l s were spun out and the supernatant treated with 0.5 ml chloroform. The phage t i t r e s of these preparations on H103 were 5 x 10^ to 4 x 10 5 plaque forming units per ml. The phage preparations were then pooled, and the pooled preparations distributed in seven 8-ml aliquo t s . To each aliquot, c e l l s from 15 ml of a mid-logarith-mic phase culture of one s t r a i n with outer membrane protein d e f i c i e n c i e s were added. After 10 min incubation at 37°, the c e l l s were removed by centrifugation, and this adsorption pro-cedure then repeated using the same s t r a i n . The supernatant was then diluted one to one in PP2, inoculated with AK1213, and grown overnight at 30° for a second cycle of phage propagation. Two further cycles of adsorption and propagation were then carried out for each of the 7 preparations. Phage preparations 28 were diluted to about 10^ to 10^ plaque-forming units per ml before adsorption. AK1012 was used for the third propaga-tion and AK1213 for the fourth. The f i n a l phage preparations were then diluted and plated with H103 on 1.6% agar overlays to obtain single plaques. Plaques were picked out of the agar using a Pasteur pipette and suspended in 1 ml PP2 broth. These phage prepara-tions were then tested against H103, AK1012, AK1213, and the s t r a i n which had been used in the adsorption procedure. Sixty to 150 single plaque isolates were tested for each s t r a i n . Phage which plated on wild type P. aeruginosa but not on outer membrane protein d e f i c i e n t strains were kept for further test-ing. Each df these phage was re-i s o l a t e d from a single plaque. Single plaques were obtained by streaking the phage preparation onto PP2 with a loop, then slowly pouring 3 ml of PP2 with 1.6% agar onto the plate from a point near the centre of the streak. This method resulted in single plaques without carrying out di l u t i o n s of the stock. Five to 10 is o l a t e s from each phage were retested, and those with the desired host range were kept. (d) Bacteriophage s e n s i t i v i t y t e s t i n g . The method of bacteriophage s e n s i t i v i t y testing used was based on that of Hancock and Reeves (1976). Bacterial lawns were prepared from overnight or mid-logarithmic phase cultures, diluted 1 in 10 and spread on PP2 agar, either in PP2 agar overlays (0.1 ml c e l l s in 3 ml 1.6% agar) or by swabbing. Plates were allowed 29 to dry for 5 to 15 minutes at 25°C, and phage suspensions con-taining 107-10*10 plaque forming units/ml were spotted onto the plate, either with a multiple syringe inoculator as des-cribed by Hancock and Reeves (1976), or with a micropipettor set to d e l i v e r 7.5-10 ul/spot. Plates were read after incuba-tion for 18-24 h at 37°C or 40-48 h at 30°C. (e) Isolation"of'phage-resistant~ mutants. Phage-resistant mutants were isolated by co-plating 0.1 ml mid-logarithmic phase culture with l O ^ - i o ^ plaque forming units of phage on PP2 plates i n PP2 agar overlays. After 24 h growth, colonies were picked, then taken through three series of streaking and single colony i s o l a t i o n . The third i s o l a t e s were retested for phage s e n s i t i v i t y . (f) Phage characterization by adsorption to whole c e l l s . Approximately 10^ plaque-forming units of phage were mixed with 0.2 ml of overnight b a c t e r i a l culture or PP2 and held at 37° for 10 min. These preparations were then centrifuged to remove c e l l s , and the supernatants were diluted and plated to deter-mine phage t i t r e s . 30 5. A n t i b i o t i c and"chelator s u s c e p t i b i l i t y t e s t i n g . (a) A n t i b i o t i c s . Gentamicin sulphate and tobramycin were g i f t s from Schering Co. (Pte. C l a i r e , Quebec) and E l i L i l l y Co. (Indianapolis, Indiana). C a r b e n i c i l l i n was purchased from Ayerst Laboratories (Montreal, Quebec). Streptomycin sulphate, neomycin sulphate, kanamycin, tetracycline hydrochloride, chloramphenicol, benzyl p e n i c i l l i n , rifampicin, polymyxin B sulphate (8000 U/mg) and c o l i s t i n methane sulfonate (polymyxin E, 12,470 U/mg) were purchased from Sigma Chemical Co. Cefsulodin was kindly provided by Ciba Geigy A.G. (Basel, Switzerland). N i t r o c e f i n was a generous g i f t from Dr. C. O'Callaghan (Glaxo Group Research Ltd., Middlesex, U.K.). ( D) Chelator and a n t i b i o t i c bacteriolysis"and k i l l i n g . Testing of l y s i s by EDTA-Tris, ethyleneglycol-bis (2-amino-ethyl ether)N,N'-tetra acetate (EGTA)-Tris and polymyxin B was carried out on c e l l s in mid-logarithmic phase growth (absorb-ance at 600 nm (Agog) °f 0.30-0.60), which were collected centrifugation at 25°C and resuspended i n 10 mM EDTA or EGTA and 10 mM Tris-HCl buffer (pH 8.5) at 25°C or 75 ug/mL of poly-myxin B in 30 mM sodium phosphate buffer (pH 7.4) at 37°C. The Aggo was read at timed i n t e r v a l s . To test k i l l i n g by EDTA-Tris, EGTA-Tris, and polymyx-i n , mid-log c e l l s were centrifuged and resuspended at 100-fold 31 d i l u t i o n in either 30 mM sodium phosphate buffer pH 7.0, with 75 ug/ml polymyxin B or either 10 mM EDTA or 10 mM EGTA in 10 mM Tris-hydrochloride, pH 8.5. After 5 min incubation at 25°C, c e l l s were diluted and plated for viable counts on PP2 agar in PP2 agar (0.6% agar) overlays. To test k i l l i n g by gentamicin, the procedure was modified s l i g h t l y because gentamicin is active only on respiring c e l l s (Hancock, 1981). Centrifuged c e l l s prepared as above were resuspended in BM2 growth medium containing 5 ug/ml gentamicin in addition to succinate and iron at normal leve l s but with no other cations added, and the c e l l s incubated at 37°C with vigorous aeration for 5 min at which time viable counts were carried out as above. (c) Determination of minimal i n h i b i t o r y concentrations  (MIC). For determinations in defined l i q u i d media, a n t i b i o t i c resistance was measured in 1 ml volumes of BM2 succinate with the stated Mg l e v e l s . The inoculum used was approximately 10^ c e l l s of an overnight culture grown in medium ide n t i c a l to the test medium. The l e v e l of resistance was taken as the highest a n t i b i o t i c concentration showing v i s i b l e t u r b i d i t y a f t e r 24 h at 37°C. Other measurements of MIC were done on PP2 agar using the method described by Angus et a l (1982). A multisyringe 32 applicator was used to delive r to each plate 24 drops of approximately 2 ul which contained an estimated 1000 c e l l s from dil u t e d 18 h cultures. Plates were read a f t e r 18 and 48 h incubation at 37°C. 6. Shift"experiments. Overnight cultures (1 ml) grown in BM2-2+ succinate with 0.02 mM Mg were transferred to 200 ml of the same medium and grown to an Agoo of 0.15 to 0.20, at 2+ which point Mg was added to a f i n a l concentration of 0.5 mM. Twenty ml of culture were removed at this point, and at 15 min i n t e r v a l s , and these samples used to test polymyxin B and EDTA-Tris s e n s i t i v i t y , and for preparation of c e l l envelopes as described above. 7. Membrane~Isolation and Characterization of Outer~Membrane,  C e l l Envelope ~, and "Whole C e l l Proteins. For whole c e l l prepar-ations, overnight or logarithmic phase cultures were centrifug-ed and the c e l l s resuspended i n 2% SDS, 20 mM Tris-HCl pH 8.0. After treatment at 100°C for 10 min, residual c e l l s were remov-ed by centrifugation at 27,000 x g for 20 min. The resulting supernatant was sonicated (1 min, setting 5, Biosonik sonicator (Bronwill S c i e n t i f i c , N.Y.)) to shear DNA and reduce v i s c o s i t y , and the sample applied d i r e c t l y to the g e l . 33 To prepare c e l l envelopes, c e l l s from overnight or logarithmic phase cultures were collected by centrifugation, resuspended in 10 mM sodium phosphate buffer (pH 7.4) or 30 mM 2+ Tris-HCl, pH 7.4 containing 2 mM Mg and 10 ug/ml pancreatic deoxyribonuclease I (Sigma Chemical Co.) and broken in a French Press at 14,000 p s i . Whole c e l l s were removed by centrifuga-tion (1000 x g, 10 min) and the res u l t i n g supernatant diluted in the same buffer and centrifuged at 160,000 x g for 2 hr. The c e l l envelope p e l l e t was resuspended in deionized water. Outer membranes were prepared using the two methods described by Hancock and Carey (1979). One, o r i g i n a l l y des-cribed by Hancock and Nikaido (1978), employs a single step and a four-step sucrose gradient and yields two outer membrane fractions with indistinguishable protein composition. The second i s a more rapid method using only one sucrose gradient which yie l d s a single outer membrane band (Hancock and Carey, 1979) . Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis was performed using the 14% acrylamide system containing 0.07 M NaCl in the running gel previously described by Hancock and Carey (1979). Only in the presence of 0.07 M NaCl or with high acrylamide monomer concentrations are outer membrane proteins Hi and H2 separated (Hancock and Carey, 1979). S o l u b i l i z a t i o n conditions were 88°C for 10 min in reduction mix containing 2-mercaptoethanol. Ratios of protein Hi to H2 were calculated from dens-34 i t o m e t e r t r a c i n g s made o n a H e l e n a Q u i c k S c a n J r . D e n s i t o m e t e r ( H e l e n a L a b o r a t o r i e s , B e a u m o n t , T e x a s ) o f C o o m a s i e b r i l l i a n t b l u e R 5 0 - s t a i n e d g e l s . O u t e r m e m b r a n e p r o t e i n H2 w a s u s e d a s a r e f e r e n c e s i n c e i t was o n e o f t h e m a j o r p r o t e i n s o f t h e c e l l a n d i t s l e v e l v a r i e d w i t h l i t t l e g r o w t h c o n d i t i o n s a s j u d g e d b y SDS g e l e l e c t r o p h o r e s i s o f w h o l e c e l l p r o t e i n s . 8 . D e t e r m i n a t i o n o f c e l l e n v e l o p e c a t i o n l e v e l s . C e l l e n v e l -o p e s p r e p a r e d a s d e s c r i b e d a b o v e w e r e e x t r a c t e d b y t h e m e t h o d o f K e n w a r d e t a l . ( 1 9 7 8 ) a n d c a t i o n l e v e l s d e t e r m i n e d b y a t o m i c a b s o r p t i o n s p e c t r o s c o p y c a r r i e d o u t b y S u s a n J a s p a r a n d S u s a n L i p t a k ( D e p a r t m e n t o f C i v i l E n g i n e e r i n g , U n i v e r s i t y o f B r i t i s h C o l u m b i a ) , C a n a d i a n M i c r o a n a l y t i c a l C o r p . , V a n c o u v e r , B . C . , o r D r . S . Ma a n d D r . D . H . C o p p ( D e p a r t m e n t o f P h y s i o l o g y , U n i v e r s i t y o f B r i t i s h C o l u m b i a ) . 9 . D i s p l a c e m e n t o f Mg f r o m t h e c e l l e n v e l o p e b y p o l y m y x i n B  a n d a m i n o g l y c o s i d e s . C e l l s g r o w n t o a n AgQO ° f 0 * 6 0 w e r e i n c u b a t e d w i t h 1 mM KCN ( t o p r e v e n t a m i n o g l y c o s i d e u p t a k e ) f o r 15 m i n a t 37° w i t h a e r a t i o n . S t r e p t o m y c i n , g e n t a m i c i n o r p o l y -m y x i n w a s t h e n a d d e d a n d t h e c e l l s i n c u b a t e d a f u r t h e r 10 m i n . C e l l e n v e l o p e s w e r e t h e n p r e p a r e d a s d e s c r i b e d a b o v e , a n d a n a l y s i s o f d i v a l e n t c a t i o n s was c a r r i e d o u t , a s d e s c r i b e d a b o v e , o n l y o p h i l i z e d e n v e l o p e s . 35 10. Streptomycin uptake'assays. [ H]-dihydrostreptomycin (1.2 Ci/mmol) (Amersham Searle, Oakville, Ontario) was diluted by the addition of nonradioactive streptomycin to a s p e c i f i c a c t i v i t y of 50 uCi/mg streptomycin. C e l l s were grown by shak-ing at 37°C to an Agoo o f 0.5-0.6 i n unmodified nutrient broth, and the uptake assay started by the addition, to the growing c e l l s , of streptomycin to the desired f i n a l concentra-tions. At defined times, 1.0 ml samples were removed and the c e l l s collected by f i l t r a t i o n onto n i t r o c e l l u l o s e f i l t e r s (0.45 um, M i l l i p o r e , Bedford, Mass.) which had been presoaked in 0.1 M L i C l p r i o r to use. The f i l t e r e d c e l l s were then washed twice with 3 ml of 0.1 M L i C l , dried at 60°C for one hour, then assayed for r a d i o a c t i v i t y in a toluene-based s c i n t i l l a n t . This method was based on those of Holtje (1978) and Bryan and c o l -leagues (1976) for streptomycin uptake assays. T r i a l experi-ments demonstrated that the f i l t e r preparation and washing techniques were c r i t i c a l , as previously suggested (Holtje, 1978; Bryan, 1976) but that the methods of either of the above authors were s a t i s f a c t o r y . Nutrient broth was chosen as the medium for uptake assays because i t has r e l a t i v e l y low Mg l e v e l s (Nicas and Bryan, 1978), and has previously been shown suitable (Bryan et: a l . , 1976). BM2, the phosphate buffered minimal medium used elsewhere in this study was judged unsuitable as high phosphate was found to i n h i b i t aminoglycoside uptake, necessitating the use of large amounts of l a b e l . 36 11. Enhancement of n i t r o c e f i n permeability by aminoglycosides  and chelators. The assay used was modified from that of O'Callaghan et a l . (1972). H103 growing in the presence of 0.2 mg/ml benzyl p e n i c i l l i n or H103 (RPl) growing in the presence of 20 ug/ml tetracycline was grown to an Agon of 0.50 to 0.60, harvested by centrifugation at 25°C and resuspended in 20 mM sodium phosphate buffer pH 7.0 at an AgQO o f 1.50 to 2.0. For studies with chelators, the c e l l s were then dil u t e d 1 in 10 in EDTA-Tris or EGTA-Tris at a f i n a l concentra-tion of 10 mM EDTA or EGTA and 10 mM Tris-HCl pH 8.5. After 2 min at 25°C, 0.1 ml of c e l l suspension was quickly mixed with 0.65 ml of n i t r o c e f i n (12.5 ug/ml, in phosphate buffer) and the hydrolysis of n i t r o c e f i n monitored spectrophotometrically by measurement of the increase in absorbance at 540 nm. Rates of hydrolysis for untreated c e l l s were also measured, and hydrol-ysis rates were expressed as the r a t i o of hydrolysis rates in treated c e l l s to rates of untreated c e l l s . In studies with gentamicin, 1 ml of gentamicin was added to 0.1 ml of c e l l suspension to give a f i n a l concentation of 10-100 ug/ml. After 2 min at 25°C, 0.6 ml of n i t r o c e f i n (250 ug/ml) was added and hydrolysis of the n i t r o c e f i n monitored as above. 12. Measurement of outer'membrane permeability by n i t r o c e f i n  hydrolysis. A method based on the technique of Zimmermann and Rosselet (1980) as modified by Angus et a^ L. (1982) was develop-ed, since neither the o r i g i n a l technique nor the modification 37 enabled a measurement of outer membrane permeability for s t r a i n H283. In p a r t i c u l a r , a r e l a t i v e l y substantial release of p e r i -plasmic beta-lactamase was found during the resuspension of c e l l s after centrifugation. Therefore, the technique was further modified as follows: aeruginosa or E_. c o l i strains containing the RPl plasmid were grown overnight in PP2 broth at 37°C in the presence of 200 ug/ml tetracycline or in the case of Z61 (RPl) with 20 ug/ml tetracycline to ensure retention of the plasmid. (Retention of the plasmid under these conditions was confirmed by comparing plate counts on PP2 and PP2 with 200 ug/ml c a r b e n i c i l l i n or 200 ug/ml neomycin.) The overnight c u l -tures were diluted 1 in 20 into fresh PP2 broth and grown to an A600 of 0.6 to 0.8. A 0.1 ml sample of c e l l s was placed in the sample cuvette of a Perkin-Elmer (Oak Brook, 111.) Lambda 3 dual beam spectrophotometer. Another 1.5 ml sample was taken at the same time and centrifuged for 1 min at 9000 x g in an Eppendorf microcentrifuge model 5412 (Brinkman Instruments, Westbury, N.Y.). The c e l l - f r e e supernatant was decanted and 0.1 ml added to the reference cuvette of a Perkin-Elmer Lambda 3 spectrophotometer. To both reference and sample cuvettes, 0.8 ml of a 0.1 mg/ml solution of the chromogenic beta-lactam n i t r o c e f i n (O'Callaghan et. a l . , 1972) was added and the d i f f e r -e n t i a l rate of conversion of n i t r o c e f i n to n i t r o c e f o i c acid followed over time at an absorbance of 540 nm using a coupled Perkin-Elmer model 581 s t r i p chart recorder. Since both sample and reference cuvettes contained supernatants, the d i f f e r e n t i a l 38 rate of hydrolysis was a measure of whole c e l l hydrolysis of n i t r o c e f i n . Control experiments showed that the rate of hydrolysis did not increase over time, indicating that c e l l breakage was not occurring. In a l l experiments with the outer membrane protein mutants H283 (RPl) and H181 (RPl), the culture used was checked for protein F deficiency or HI overproduction by examining whole c e l l protein p r o f i l e s on SDS polyacrylamide gels a f t e r each experiment. 13. Other assays. The protein assay used was that of Schacterle and Pollack (1973). Levels of 2-keto-3-deoxyocton-ate were estimated by the method of Osborn et. a l . (1963). 39 CHAPTER"ONE  SUSCEPTIBILITY TO EDTA-TRIS,  POLYMYXINS AND AMINOGLYCOSIDES 7 + 1. S u s c e p t i b i l i t y to-polymyxin B~and EDTA-Tris in Mg^  s u f f i c i e n t and d e f i c i e n t media. Wild type J?. aeruginosa PA01 7 + 7 + s t r a i n H103, grown on Mg - s u f f i c i e n t medium (0.5 mM Mg was sensitive to polymyxin B and EDTA k i l l i n g (Table III) and 7 + l y s i s (Fig. 1). In contrast, H103 grown under Mg -d e f i c i e n t 7 + (0.02 mM Mg ) conditions was 70 to 700 fo l d more resistant to these agents (Fig. 1; Tables III and V),in agreement with pre-viously published results (Brown and Melling, 1969; Gi l l e l a n d et a l . , 1974). This resistance could be reversed by culturing 7 + H103 on Mg^ - s u f f i c i e n t medium for a few generations. A l l l y s i s and k i l l i n g experiments were done on mid-logarithmic phase c e l l s after control experiments showed that s u s c e p t i b i l i t y to l y s i s by EDTA-Tris and by polymyxin B varied with the growth phase of the culture. When 18 h (late s t ation-2+ ary phase) cultures grown in Mg s u f f i c i e n t medium were transferred to fresh medium, s u s c e p t i b i l i t y to l y s i s increased throughout early logarithmic phase, and reached a maximum in middle and late logarithmic phase. As c e l l s entered stationary phase, s u s c e p t i b i l i t y decreased, so that 18 h cultures were highly r e s i s t a n t to l y s i s , exhibiting only 5 to 20% of the 2+ l e v e l of l y s i s seen in mid-log c e l l s . C e l l s grown in Mg d e f i c i e n t medium followed a similar pattern, except that de-40 A . E D T A - T r i s B . P o l y m y x i n B A T i m e ( m i n ) T i m e ( m i n ) F i g u r e " 1 . E f f e c t o f a d a p t a t i o n on Mg^ - d e f i c i e n t m e d i u m , a n d o f m u t a t i o n t o p o l y m y x i n B r e s i s t a n c e , on s u s c e p t i b i l i t y o f c e l l s t o l y s i s b y p o l y m y x i n B and E D T A - T r i s . A , l y s i s b y E D T A - T r i s ; B, l y s i s b y p o l y m y x i n B. S y m b o l s : W i l d t y p e s t r a i n H103 g r o w n i n Mg - d e f i c i e n t (0.02) mM m e d i u m ( A ) ; s t r a i n H103, (A), p o l y m y x i n B r e s i s t a n t m u t a n t s H181, (•) and H185, (fP) and r e v e r t a n t H207, (O) g r o w n i n M g 2 + - s u f f i c i e n t (0.5 mM) m e d i u m . TABLE III. Resistance to k i l l i n g by EDTA-Tris and polymyxin B of H103 and its polymyxin B resistant mutant H181. Effect of Mg2+ concentration in the growth medium. Strain 04. Mg concentration during growth (mM) Survivors (%) a EDTA-Tris Polymyxin B H103 0.02 0.5 65 0.9 15 O.02 H181 0.02 77 42 0.5 76 22 a Cells were treated for 5 minutes in lOmM EDTA in lOmM- Tris-HCl (pH8.5) or 75 ug/ml polymyxin B in phosphate buffer (pH 7.4). 42 c r e a s e i n s u s c e p t i b i l i t y began somewhat e a r l i e r , i n l a t e l o g a r -i t h m i c phase growth, and t h e s e c e l l s were more r e s i s t a n t than 9 + c e l l s i n Mg s u f f i c i e n t medium d u r i n g a l l phases of growth. 2+ 2+ The growth r a t e s of c e l l s growing i n Mg - s u f f i c i e n t and Mg d e f i c i e n t medium were i d e n t i c a l i n l o g phase, a l t h o u g h lower 2 + l e v e l s of Mg (below 0.01 mM) r e s u l t e d i n decreased growth 2 + r a t e . For c u l t u r e s grown i n 0.015-0.05 mM mg , the growth 2 + y i e l d was p r o p o r t i o n a l t o the amount' of Mg added. 2. S u s c e p t i b i l i t y o f p o l y m y x i n r e s i s t a n t mutants. Two p o l y -myxin B r e s i s t a n t mutants of H103, s t r a i n s H181 and H185, were r e s i s t a n t t o EDTA-Tris and p o l y m y x i n B k i l l i n g ( T a b le I I I ) and 2 + l y s i s ( F i g . 1 ) , i r r e s p e c t i v e of the medium Mg c o n c e n t r a t i o n . The mutant phenotype was s t a b l e f o r up t o 12 c o n s e c u t i v e s i n g l e 2 + c o l o n y i s o l a t i o n s on Mg - s u f f i c i e n t medium. S i x spontaneous r e v e r t a n t s o f H181 and H185 ( e . g . , s t r a i n H207) had r e g a i n e d a l l o f the w i l d type p r o p e r t i e s of s t r a i n H103 (see F i g . 1 and b e l o w ) . These d a t a suggest t h a t H181 and H185 each have a s i n g l e m u t a t i o n r e s u l t i n g i n p h e n o t y p i c a l t e r a t i o n s m i m i c k i n g 9+ t h o s e of the Mg - l i m i t e d , a d a p t i v e l y r e s i s t a n t s t r a i n H103. 3. A m i n o g l y c o s i d e " r e s i s t a n c e . S i n c e Mg" has been p r e v i o u s l y shown t o a n t a g o n i z e the a c t i o n of a m i n o g l y c o s i d e s , as w e l l as p o l y m y x i n s ( Z i m e l i s and J a c k s o n , 1973), the mutants were t e s t e d f o r enhanced r e s i s t a n c e t o t h r e e r e p r e s e n t a t i v e a m i n o g l y c o s i d e a n t i b i o t i c s , g e n t a m i c i n , s t r e p t o m y c i n and t o b r a m y c i n . S t r a i n s 43 H181 and H185 were consistently 4-fold more resistant to genta-micin and streptomycin and 2-fold more resistant to tobramycin (Table IV). In contrast they were equally susceptible to c a r b e n i c i l l i n and tetracycline when compared to s t r a i n H103. Strain H103 grown in Mg - d e f i c i e n t medium was also shown to be much more resist a n t to gentamicin than H103 grown 2+ in Mg - s u f f i c i e n t medium (Table V) when resistance was measured in a common assay medium. Studies reported elsewhere (Hancock, Raffle and Nicas, 1981) have shown that H103 grown in 2+ Mg - d e f i c i e n t medium shows a marked decrease in s e n s i t i v i t y to k i l l i n g by gentamicin over a broad range of a n t i b i o t i c concen-t r a t i o n s . The le v e l of resistance achieved was shown to be similar to that of the polymyxin resis t a n t mutants H181 and H185 . 4. Substitution"of other'cations for'Mg^ . Results of sup-2 + plementation of Mg d e f i c i e n t medium with other cations are shown in Table V. These results confirmed and extended the findings of Boggis et a l . (1979) with respect to the effects of di f f e r e n t metal cations on s u s c e p t i b i l i t y to polymyxin and EDTA-Tris. Ce l l s grown with Mg 2 +, C a 2 + , Mn 2 + or S r 2 + were at leas t 1000 times more sensitive to polymyxin B, 100 times more sensitive to EDTA-Tris and 10 times more sensitive to gentami-2+ c i n than were c e l l s grown in low Mg ; s u s c e p t i b i l i t y to l y s i s in EDTA-Tris was also much enhanced in such c e l l s . In contrast, 2+ 2+ 2+ c e l l s grown with the several other cations (Ba , Zn , Sn , 44 TABLE IV. Resistance of H103, its polymyxin B resistant derivatives H181 and H185 and a revertant H207 to various antibiotics. 3. b Resistance (ug/ml) ' Strain Gentamicin Streptomycin Tobramycin Carbenicillin Tetracycline H103 1 8 0.5 16 8 H181 4 32 1 16 8 H185 4 32 1 16 8 H207 1 8 0.5 16 8 a Resistance levels were determined as described in Methods, using liquid BM2 medium with 0.5 mM Mg2+. k The differences observed, while not large, were reproducibly obtained in 6-10 individual experiments. 45 3+ + A l , Na ) , w e r e i n d i s t i n g u i s h a b l e f r o m c e l l s g r o w n i n l o w Mg i n t h e i r r e l a t i v e l y l o w s u s c e p t i b i l i t y t o p o l y m y x i n B , E D T A -T r i s a n d g e n t a m i c i n , p r o v i d i n g t h a t a common a s s a y m e d i u m was u s e d . 5 . M i n i m a l i n h i b i t o r y c o n c e n t r a t i o n s o f p o l y m y x i n a t v a r i o u s Mg^"1" l e v e l s . A l t h o u g h g r o w t h o f P_. a e r u g i n o s a i n m e d i a w i t h 2 + l o w M g " i n c r e a s e s r e s i s t a n c e t o p o l y m y x i n when s u s c e p t i b i l i t y i s t e s t e d i n a common a s s a y m e d i u m , t h e p r e s e n c e o f a d d e d e x -2 + t e r n a l Mg c a n p r o t e c t b o t h r e s i s t a n t a n d s u s c e p t i b l e c e l l s f r o m t h e p o l y m y x i n a c t i o n ( N e w t o n , 1 9 6 4 ; K l e m p e r e r e t a l . , 1 9 7 9 ) . T h e MIC t o p o l y m y x i n B a n d c o l i s t i n ( p o l y m y x i n E ) s h o w n 2 + m T a b l e V I r e f l e c t t h e s e Mg e f f e c t s . T h u s , a l t h o u g h c e l l s g r o w n i n 5 mM M g 2 + a r e c l e a r l y h i g h l y s u s c e p t i b l e t o k i l l i n g b y 2 + p o l y m y x i n B i n t h e a b s e n c e o f Mg ( T a b l e I I I ) , t h e y a p p e a r t o b e r e l a t i v e l y r e s i s t a n t i n MIC m e a s u r e m e n t s d o n e i n t h e p r e s -2+ 2+ e n c e o f Mg ( T a b l e V I ) . S i n c e a t t h i s Mg c o n c e n t r a t i o n 2 + t h e r e was a l a r g e m o l a r e x c e s s o f Mg o v e r p o l y m y x i n B ( e . g . , 6 0 - f o l d a t 1 0 0 u g / m l p o l y m y x i n B , 1 0 0 0 - f o l d a t 4 u g / m l p o l y m y x -i n B ) , t h e r e s u l t s may b e e x p l a i n e d b y c o m p e t i t i o n f o r a s i t e 2 + o n t h e L P S n o r m a l l y o c c u p i e d b y Mg ( s e e F i g . 10 a n d D i s c u s -s i o n ) . C o m p e t i t i o n b e t w e e n Mg a n d t h e p o l y m y x i n s c o u l d a l s o e x p l a i n t h e 2 . 5 - f o l d d i f f e r e n c e i n t h e r e s i s t a n c e s o f H 1 8 1 a n d H185 2+ g r o w n a n d t e s t e d i n 0 . 5 mM Mg r e l a t i v e t o H 1 0 3 g r o w n i n 0 . 0 2 2 + mM Mg , ( T a b l e V I ) w h e r e a s t h e r e s i s t a n c e s o f t h e s t r a i n s 2+ g r o w n a t t h e s e Mg c o n c e n t r a t i o n s w e r e s i m i l a r when t e s t e d i n TABLE V. Effect of growth in various divalent cations on induction of outer membrane protein HI, lysis and k i l l i n g by EDTA-Tris, and k i l l i n g by polymyxin B and gentamicin. Mg2+ during growth (mM) Other cations during growth (mM) Induction of protein H l a % Lysis by EDTA-Tris" Polymyxin B % Survivors 0 EDTA-Tris Gentamicin 0.02 - + 11/5 15.0 65.0 11.0 0. 02 Ba2+ (0.5) + 6. 0 20.3 14.6 21.5 0. 02 Sn2+ (0.5) + 16.0 20.5 11.7 10.5 0.02 Zn2+ (0.5) + 14.0 20. 2 13.0 19. 5 0.02 Ca2+ (0.5) - 51. 5 O.01 O.l 1.56 0.02 Mn2+ (0.5) - 53.5 <0.01 O.l 1.65 0.02 Sr2+ (0.5) - 51.7 *0.01 O.l 0.68 0.5 - - 52.3 SO. 01 <0.1 .2.01 Induction of Hi as judged from sodium dodecyl sulphate polyacrylamide gels of c e l l envelope proteins, see Fig. 3. Lysis was measured as decrease in A ^ Q Q after 1 5 min in 1 0 mM EDTA,10 mM Tris-HCl pH 8 . 5 . Cells were treated for 5 min with 7 5 ug/ml polymyxin B in phosphate buffer, 1 0 mM EDTA in 1 0 mM Tris-hydrochloride, or 5 ug/ml gentamicin in growth medium without added divalent cations. TABLE VI. Levels of outer membrane protein Hi, c e l l envelope Mg2+ concentration and resistance to polymyxins of H103 and i t s polymyxin B resist a n t derivatives H181 and H185 and a revertant H207: e f f e c t of varying Mg2+ concentrations i n the growth medium. Strain Mg 2 + concentration Outer Membrane Cations in Resistance (ug/ml) d during growth Proteins:Ratio c e l l envelope Polymyxin B C o l i s t i n (mM) Hl:H2 a (ug/mg p r o t e i n ) b Mg 2 + C a 2 + H103 H181 HI 8 5 0.02 0. 5 5.0 0.02 0. 5 5.0 0.02 0.5 5.0 4.7 0.7 0. 2 6.3 4. 7 3.4 6.4 -c 2.9 4.8 17.1 20. 5 3.8 8.9 11. 6 2.9 8.8 12.6 1. 2 0. 9 0.8 1.4 1.1 1.3 1.0 10 1 4 100 25 100 100 25 100 50 4 25 200 100 200 200 100 200 H207 0. 5 0.8 a C e l l envelopes were iso l a t e d and subjected to SDS polyacrylamide gel electrophoresis. The ratios of proteins Hi to H2 were calculated from densitometer tracing of stained gels loaded with a standard amount of protein. Outer membrane protein H2 was used as a reference since i t was one of the major proteins of the c e l l and i t s l e v e l s varied very l i t t l e with growth conditions. b Levels obtained by atomic adsorption spectroscopy. c "-" means not done. d Resistance was determined in l i q u i d BM2 succinate using the given Mg2+ concentrations. 48 the absence of Mg^ (Table I I I ) . The relationship of the resistance shown in Table VI and properties of the outer membrane is described below (Chapter Two). 6. Streptomycin uptake and binding in'susceptible and  resis t a n t s t r a i n s . The pattern of streptomycin uptake in both strains H103 and H181 ( i l l u s t r a t e d by a t y p i c a l experiment F i g . 2) followed three phase ki n e t i c s as described by Bryan and colleagues for other strains (Bryan and Van Elzen, 1976): an instantaneous binding phase, an early slow uptake phase (EDP-I) and a l a t e r rapid uptake phase (EDP-II). An extensive series of experiments was performed in an attempt to demonstrate differences in the apparent amount of streptomycin binding to cyanide-treated [ i . e . , non-streptomycin transporting (Bryan and Van Den Elzen, 1977)] or -untreated wild type s t r a i n H103 or mutant H181 c e l l s . For fiv e separate experiments done at eight d i f f e r e n t concentrations of streptomycin (data not shown), s t a t i s t i c a l analysis of the data suggested that there was no s i g n i f i c a n t d i f f e r e n t (P >0.5) in aminoglycoside binding to the two s t r a i n s . Thus, any apparent differences in streptomycin binding to the two strains (e.g., as seen at 10 ug/ml in F i g . 2) were shown by more careful analysis to be not s i g n i f i c a n t . Scatchard analysis of the data from one experiment suggested in 7 the order of 2 - 5 x 10 potential binding s i t e s for strepto-mycin per c e l l . The large number of non-specific binding s i t e s on the c e l l (since there are only about 2 - 4 x 10° molecules 49 - 8 30 TIME (min) Figure 2. Uptake of [3H] streptomycin at two concentrations by the wild type s t r a i n H103 and the outer embrane protein HI overproducing s t r a i n H181. Symbols: O H103, 2 ug/ml streptomycin; • H103, 10 ug/ml streptomycin; ^ H181, 2 ug/ml streptomycin; 4L H181, 10 ug/ml streptomycin. 50 3 io ~io ioi Streptomycin added (pg/ml) Figure'3» Time r e q u i r e d f o r i n i t i a t i o n of r a p i d uptake of streptomycin (EDP-II) i n the w i l d type s t r a i n s H103 ( © ) and the outer membrane p r o t e i n Hi overproducing s t r a i n H181 ( £ ) . the p o i n t s r e p r e s e n t the means of three experiments; the given l i n e s were drawn by l i n e a r r e g r e s s i o n a n a l y s i s of the p o i n t s with c o r -r e l a t i o n c o e f f i c i e n t ( r 2 ) of 0.98 f o r H103 and 0.97 f o r H181. 51 of LPS per P. aeruginosa c e l l ) , and the high background f i l t e r adsorption of streptomycin even under the stringent washing procedures used may well have acted together to obscure expect-ed differences in aminoglycoside binding. The major a l t e r a t i o n in the ki n e t i c s of streptomycin uptake seen in the resistant s t r a i n , H181, was that at a l l con-centrations of a n t i b i o t i c used, t r a n s i t i o n from the early slow phase of uptake EDP-I to the l a t e r rapid phase EDP-II was delayed in the resist a n t s t r a i n (Fig. 3). This difference was consistently observed in seven separate experiments, each using several le v e l s of a n t i b i o t i c . 7. Permeabilization of'the outer membrane by aminoglycosides. The a b i l i t y of EDTA and polymyxin to interact with and disrupt the outer membrane of gram-negative bacteria is well known (Leive, 1965; Cooperstock, 1974; G i l l e l a n d and Murray, 1976; Michael and Eagon, 1966; Rosenthal and Strom, 1977). The a b i l i t y of aminoglycosides to interact with the outer membrane was investigated by examining their a b i l i t y to enhance outer membrane permeability to other agents. It has been reported elsewhere (Hancock, Raffle and Nicas, 1981) that treatment with gentamicin made P. aeruginosa susceptible to l y s i s by lysozyme, an.enzyme normally unable to penetrate the outer membrane to reach i t s s i t e of a c t i v i t y , peptidoglycan. Conditions known to block aminoglycoside transport and k i l l i n g did not af f e c t gentamicin-lysozyme l y s i s . These conditions 52 i n c l u d e d t r e a t m e n t w i t h K C N , t h e u n c o u p l e r s d i n i t r o p h e n o l a n d s o d i u m a z i d e , i n h i b i t o r s o f p r o t e i n s y n t h e s i s ( c h l o r a m p h e n i c o l a n d t e t r a c y c l i n e ) a n d t h e s t r A m u t a t i o n . G e n t a m i c i n - p r o m o t e d l y s o z y m e l y s i s was h o w e v e r c o m p l e t e l y i n h i b i t e d b y 1 mM M g 2 + ( H a n c o c k , N i c a s a n d R a f f l e , 1 9 8 1 ) . P e r m e a b i l i z a t i o n o f t h e o u t e r m e m b r a n e b y g e n t a m i c i n i n s t r a i n H 1 0 3 w a s a l s o e x a m i n e d b y m e a s u r i n g t h e h y d r o l y s i s o f a c h r o m o g e n i c b e t a - l a c t a m , n i t r o c e f i n . A n i n c r e a s e d r a t e o f h y d r o l y s i s i n i n t a c t n o n - g r o w i n g c e l l s w o u l d i n d i c a t e i n c r e a s e d p e r m e a t i o n o f t h e b e t a - l a c t a m t h r o u g h t h e o u t e r m e m b r a n e t o t h e p e r i p l a s m i c b e t a - l a c t a m a s e . The r a t e o f h y d r o l y s i s c o u l d b e i n c r e a s e d 3 . 5 f o l d o v e r t h a t o f u n t r e a t e d s t r a i n H 1 0 3 c e l l s b y p r e t r e a t m e n t w i t h 1 0 0 u g / m l g e n t a m i c i n a n d 1 . 7 - f o l d w i t h 10 u g / m l g e n t a m i c i n [ t h e a c t u a l c o n c e n t r a t i o n s o f g e n t a m i c i n p r e s e n t d u r i n g t h e a s s a y w e r e i n f a c t 15 u g / m l a n d 1 . 5 u g / m l , r e s p e c t i v e l y ] . T r e a t m e n t w i t h 50 mM EDTA i n c r e a s e d t h e r a t e o f h y d r o l y s i s 1 0 - f o l d . A s i n t h e c a s e o f g e n t a m i c i n - m e d i a t e d l y s o z y m e a c t i v i t y , p e r m e a b i l i z a t i o n t o n i t r o c e f i n c o u l d b e t o t a l l y i n h i b i t e d b y 1 mM M g 2 + . 8 . O t h e r p r o p e r t i e s o f p o l y m y x i n r e s i s t a n t s t r a i n s (a ) R e s i s t a n c e t o c h l o r a m p h e n i c o l a n d o t h e r a n t i b i o t i c s . The r e f e r e n c e s t r a i n u s e d , P . a e r u g i n o s a P A 0 1 s t r a i n H 1 0 3 , was p r e v i o u s l y m u t a t e d t o h i g h 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 (MIC o f 2 0 0 - 4 0 0 u g / m l c o m p a r e d t o 1 0 - 2 0 f o r m o s t i s o l a t e s ; B . H o l l o w a y , p e r s o n a l c o m m u n i c a t i o n ) . The p o l y m y x i n r e s i s t a n t 53 mutants H181 and H185 were found to have l o s t this high l e v e l of chloramphenicol resistance, and had MIC of 20 ug/ml. The revertants of H181 and H185 regained the same high levels of chloramphenicol resistance as the parent. Strains H181 and H185 did not d i f f e r from H103 with regard to their s u s c e p t i b i l i t y to rifampicin, cefsulodin, t i c a r c i l l i n , C u 2 + and A g 2 + , as well as c a r b e n i c i l l i n and tetracycline as shown in Table IV. (b) A b i l i t y to accept RPl. The plasmid RPl could be con-jug a l l y transferred e a s i l y into H103 from either E. c o l i or other P. aeruginosa strains with a transfer frequency of 1.0-2.4 x 10 -^ transconjugants per r e c i p i e n t . In contrast, the polymyxin resistant mutant H181 accepted the plasmid at 10 3 to 4 — f t —7 10 - f o l d lower frequency, 2.0 x 10 to 6.7 x 10 . Frequen-cies of conjugation did not d i f f e r according to the a n t i b i o t i c used for s e l e c t i o n . (c) Loss of v i a b i l i t y in cold storage. It was found that cultures of H181 and H185 which had been stored at 4°C for sev-era l weeks gave r i s e to a high proportion of revertant clones when subcultured and tested for polymyxin resistance. In an attempt to explain this phenomenon, the loss of v i a b i l i t y of wild type and polymyxin resistant mutants after storage was compared. It was found that after one week in l i q u i d growth medium at 4°C, viable counts of H103 showed 15-45% su r v i v a l , in contrast to 1.2-4.6% sur v i v a l of H181. Of the surviving H181 54 clones, only 0.3-1% remained polymyxin r e s i s t a n t . Cultures stored for 1-2 weeks at -70°C in 10% dimethyl sulfoxide showed a si m i l a r difference in v i a b i l i t y between H103 and H181. 2+ 9. Summary. Ce l l s grown in Mg - d e f i c i e n t medium (0.02 mM 2+ Mg ) were more resistant to the action of EDTA, polymyxin 2 + B, and aminoglycosides than were c e l l s grown in Mg - s u f f i c i e n t 2+ 2+ 2+ 2+ medium. Ca , Sr , or Mn could substitute for Mg in reversing resistance while several other cations could not. Mutants selected for polymyxin resistance resembled c e l l s grown 2 + in low Mg in their resistance to cationic a n t i b i o t i c s and EDTA. These mutants also showed altered uptake of aminoglyco-sides. It was shown that aminoglycosides could interact with the outer membrane in wild type c e l l s so as to make i t more permeable to other substances. Aminoglycoside-mediated pj. permeabilization could be inhibited with Mg . 55 CHAPTER TWO OUTER MEMBRANE C H A R A C T E R I Z A T I O N 1 . O u t e r membrane p r o t e i n p a t t e r n s . O u t e r membrane p r o t e i n H I w a s s h o w n t o b e i n c r e a s e d up t o 2 4 - f o l d i n Mg - l i m i t e d H 1 0 3 c e l l s ( F i g . 4 , c o m p a r e g e l E w i t h F ; T a b l e V I ) , w h i l e t h e p r o -t e i n G l e v e l w a s d e p r e s s e d 3 - f o l d . U n d e r t h e s e c o n d i t i o n s , HI was b y f a r t h e m a j o r c e l l u l a r p r o t e i n a s j u d g e d b y SDS p o l y -a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f w h o l e c e l l p r o t e i n s . The p o l y m y x i n B r e s i s t a n t m u t a n t s H 1 8 1 a n d H 1 8 5 h a d c o n s t i t u t i v e l y h i g h l e v e l s o f p r o t e i n H I ( F i g . 4 , g e l s B a n d C) w h i c h v a r i e d 2 + o n l y 2 - f o l d w i t h c h a n g i n g Mg c o n c e n t r a t i o n s i n t h e m e d i u m ( T a b l e V I ) . T h e l e v e l o f p r o t e i n G w a s a l s o d e p r e s s e d i n b o t h H 1 8 1 a n d H 1 8 5 . S i n c e o t h e r s t r a i n s w i t h g r e a t l y d e p r e s s e d l e v -e l s o f p r o t e i n G ( e . g . , H a n c o c k a n d C a r e y , 1 9 7 9 ) h a d n o a l t e r a -t i o n i n p o l y m y x i n B o r EDTA r e s i s t a n c e , o r i n l e v e l s o f p r o t e i n H I , i t w a s c o n c l u d e d t h a t t h e d e c r e a s e i n t h i s p r o t e i n i s u n r e l a t e d t o t h e r e s i s t a n c e o b s e r v e d i n t h e a d a p t e d o r m u t a n t s t r a i n s . S i m i l a r l y , a v a r i e t y o f g r o w t h c o n d i t i o n s ( e . g . , p y r u v a t e a s a c a r b o n s o u r c e , l i m i t i n g N H 4 + ) c o u l d d e p r e s s l e v -e l s o f p r o t e i n G w i t h o u t e f f e c t o n p r o t e i n H I l e v e l s . L e v e l s o f p r o t e i n D2 a l s o a p p e a r e d t o b e s l i g h t l y l o w e r . The l e v e l o f p o r i n ( p r o t e i n F ) a p p e a r e d t o b e s o m e w h a t r e d u c e d i n m e m b r a n e s w i t h h i g h e r l e v e l s o f p r o t e i n H i . H o w e v e r , p o r i n f u n c t i o n a p p e a r e d t o b e u n a l t e r e d i n t h e s e s t r a i n s ( s e e C h a p t e r F o u r ) . 56 A B C D E F Figure 4. Effect of adaptation on Mg2+-deficient medium and mutation to polymyxin B resistance on le v e l s of protein H i . Gels A and B - whole c e l l preparations of strains H103 and H185, respectively, grown with 5 mM Mg 2 +; Gels C and D - outer mem-brane preparations of strains H181 and H103, respectively, grown with 0.5 mM Mg2+; Gels E and F - c e l l envelopes of H103 grown with 0.02 mM Mg2+ and 5 mM Mg 2 +, respectively. In the protein patterns of whole c e l l s , c e l l envelopes or outer membranes of H103 grown on low (0.02 mM) Mg 2 + or the polymyxin B resistant mutants H181 and H185 grown on high (5 mM) Mg^+, a large increase in outer membrane protein Hi was observed, while pro-tein G was somewhat decreased compared to H103 grown on high Mg2+. The high molecular weight protein seen in gel E but not in gel F c e l l envelopes is an inner membrane protein of unknown function induced in either H103 or H181 grown in low Mg 2 +. 57 A n i n n e r m e m b r a n e p r o t e i n o f 7 5 , 0 0 0 d a l t o n s w a s a l s o o b s e r v e d i n a l l s t r a i n s ( e . g . , F i g . 4 , G e l E ) g r o w n on Mg - d e f i c i e n t 2+ m e d i u m b u t n e v e r i n s t r a i n s g r o w n on Mg - s u f f i c i e n t m e d i u m . T h u s , i t w a s c o n c l u d e d t h a t t h i s p r o t e i n a l s o i s u n r e l a t e d t o t h e p h e n o m e n a r e p o r t e d h e r e . A l l s i x r e v e r t a n t s h a d r e g a i n e d 2+ w i l d t y p e membrane p r o t e i n p a t t e r n s a t a l l Mg c o n c e n t r a t i o n s ( e . g . , H 2 0 7 , T a b l e V I ) . A l t h o u g h p i l i n h a s a s i m i l a r m o l e c u l a r w e i g h t t o p r o t e i n H I ( P a r y n c h y c h , 1 9 7 9 ) , t h e y a r e d i s t i n c t p r o t e i n s a s d e t e r m i n e d u s i n g a p i l i n s a m p l e k i n d l y p r o v i d e d b y W. P a r y n c h y c h ( U n i v e r s i t y o f A l b e r t a , E d m o n t o n , A l b e r t a ) . I t w a s d e t e r m i n e d t h a t t h e a m o u n t o f p r o t e i n p e r mg ( d r y w e i g h t ) o f c e l l e n v e l o p e ( 5 4 + 16%) a n d t h e a m o u n t o f 2 - k e t o - 3 - d e o x y o c t o n a t e p e r mg o f p r o t e i n ( 8 7 + 7 ug/mg) d i d n o t v a r y s i g n i f i c a n t l y b e t w e e n w i l d t y p e c e l l s g r o w n i n Mg -s u f f i c i e n t m e d i u m a n d e i t h e r p o l y m y x i n r e s i s t a n t m u t a n t s o r 2+ c e l l s g r o w n i n Mg d e f i c i e n t m e d i u m (P >0 .5 b y S t u d e n t ' s t t e s t ) . T h e c h a n g e i n t h e l e v e l s o f p r o t e i n H I i n t h e o u t e r m e m b r a n e w e r e r e f l e c t e d i n p a r t b y t h e d e g r e e o f r e s i s t a n c e t o 2 + t w o p o l y m y x i n a n t i b i o t i c s a n d b y t h e Mg c o n c e n t r a t i o n o f t h e c e l l e n v e l o p e s o f s t r a i n s H103, H181, H185 a n d H207 g r o w n a t v a r i o u s m e d i u m Mg c o n c e n t r a t i o n s ( T a b l e V I ) . T h u s , a 7 - f o l d i n c r e a s e i n H I l e v e l s b e t w e e n s t r a i n s H 1 0 3 a n d H 1 8 1 , g r o w n i n BM2 2 + m i n i m a l s u c c i n a t e m e d i u m c o n t a i n i n g 0 . 5 mM Mg was a s s o c i a t e d w i t h a 2 5 - f o l d i n c r e a s e i n p o l y m y x i n B a n d c o l i s t i n r e s i s t a n c e . 2 + S i m i l a r l y , s t r a i n H 1 0 3 g r o w n o n Mg - d e f i c i e n t - m e d i u m h a d 7 58 fold higher le v e l s of protein HI than the same st r a i n grown on 2 + Mg - s u f f i c i e n t medium, and was 10-fold more resistant to poly 2+ myxins. It is well established that high Mg concentrations in the medium i n h i b i t the action of polymyxins (Newton, 1954). In the experiments described in Table VI, an increase in poly-2+ myxin resistance was observed in the presence of 5 mM Mg despite l e v e l s of protein HI similar to that of c e l l s grown in 0.5 mM Mg 2 +. As described above (Chapter One, Section 5), t h i 2+ e f f e c t could be accounted for by competition between Mg and 2+ polymyxin B for binding s i t e s . The large molar excess of Mg over polymyxin at 5 mM polymyxin could account for the higher MIC in high Mg 2 +. This could also explain why the MIC of H181 2 + and H185 in 0.5 mM Mg indicate higher resistance than H103 2 + grown in 0.02 mM Mg despite the high degree of s i m i l a r i t y between these c e l l s in their l e v e l of HI (Table VI) and r e s i s t 2 + ance to polymyxin k i l l i n g in the absence of Mg (Table I I I ) . 2 + Results of supplementation of Mg -d e f i c i e n t medium with other divalent cations are shown in F i g . 5. Induction of 2 + protein Hi was prevented in Mg - d e f i c i e n t media supplemented with 0.5 mM C a 2 + , Mn 2 +, or S r 2 + , as well as 0.5 mM Mg 2 +, but not with 0.5 mM Zn 2, S n 2 + , B a 2 + (Fig. 5), A l 3 + , or 1 mM Na + (data not shown). As shown in Table V, c e l l s grow in cations which prevent HI induction are highly susceptible to k i l l i n g by polymyxin, EDTA-Tris and gentamicin, whereas c e l l s grown in cations which f a i l e d to prevent induction of HI were 2+ • indistinguishable from c e l l s grown in low Mg^ - in their r e l a t i v e l y low s u s c e p t i b i l i t y to these agents. 59 A B C D E F G H Figure 5. Effect of growth in di f f e r e n t divalent cations on induction of protein HI. Sodium dodecyl sulphate polyacrylamide gel of c e l l envelopes from c e l l s grown in the presence of d i f -ferent divalent cations. Lane A, 0.02 mM Mg 2 +; Lane B, 0.5 mM Mg2+; Lanes C-H, 0.0 2 mM Mg 2 + plus 0.5 mM C a 2 + (C), 0.5 mM Mn 2 + (D), 0.5 mM S r 2 + (E) , 0.5 mM B a 2 + (F), 0.5 mM S n 2 + (G) or 0.5 mM Z n 2 + (H). In order to ensure that a l l the protein Hi ran at the head modified position, s o l u b i l i z a t i o n was carried out at 100°C for 10 min (as described by Hancock and Carey, 1979). This results in p a r t i a l heat modification of protein F to run with a lower r e l a t i v e mobility at the position F* (Hancock & Carey, 1979). 60 2. E f f e c t of shift'from low to high Mg" . To further confirm the rela t i o n s h i p between the presence of outer membrane protein Hi and resistance, s h i f t experiments were carried out. 2 + C e l l s growing in 0.02 mM Mg in early logarithmic phase growth 2 + ( A600 ° f 0.15-0.2) were supplemented with Mg to 0.5 mM, and lev e l s of Hi in the c e l l envelope and s u s c e p t i b i l i t y to poly-myxin B and EDTA-Tris were followed over time. As described 2+ above, 0.02 mM Mg allows the same growth rate as 0.5 mM up to 2+ an Agoo of 0.6, so these c e l l s could not be considered Mg limited for growth. Control experiments demonstrated that 2 + c e l l s grown in 0.02 mM Mg showed increasing levels of protein 2+ Hi as the medium became depleted in Mg . However, the s h i f t 2 + to high Mg was performed on early logarithmic phase c e l l s , which showed only moderately higher l e v e l s of protein Hi (2- to 2+ 3-fold) in order to avoid any non-specific effects of Mg starvation [e.g., stringent response (St. John and Goldberg, 1980), ribosome effects (e.g., Gestland, 1966; Schlessinger et a l . , 1967)]. Addition of Mg 2 + did not a l t e r the growth rate (about 42 min generation time), and the c e l l s remained in logarithmic growth phase throughout the sampling period. The r a t i o of outer membrane protein HI to protein H2, calculated from densitometer tracings of Coomassie blue stained gels was used to estimate r e l a t i v e levels of protein Hi in c e l l enve-lopes. The time required for the r e l a t i v e l e v e l of protein HI 61 Min. after addition of Mg Figure 6. Effect of s h i f t from low to high Mg2 + on levels of protein HI and s u s c e p t i b i l i t y to EDTA-Tris and polymyxin B. Cel l s growing in 0.02 mM Mg2+ received Mg 2 + to a f i n a l concentration of 0.5 mM at tine 0. Samples of cultures were subsequently removed at intervals and assayed for protein HI levels and s e n s i t i v i t y . A. Decrease in r a t i o of protein H1 :H2 measured from stained gels of c e l l envelopes (levels of protein H2 are constant under the conditions used). B. Increase in s e n s i t i v i t y to b a c t e r i c i d a l action of EDTA-Tris (0) or polymyxin B ( f ) . Ce l l s sampled at the given times were treated for 5 min with either 10 mM EDTA in 10 mM Tris-HCl pH 8.5 or 75 ug/ml polymyxin B in phosphate buffer, then plated for viable counts. C. Increase in s e n s i t i v i t y to b a c t e r i o l y t i c action of EDTA-Tris. Lysis was measured as the decrease in Agoo after 15 min treatment with 10 mM EDTA-Tris in 10 mM Tris-HCl pH 8.5 62 to decrease by one-half was estimated as about 38 min, close to the time for one c e l l d i v i s i o n . After 45 to 60 min, the pro-t e i n HI l e v e l s t a b i l i z e d at the levels previously seen in c e l l s 2+ grown in Mg s u f f i c i e n t medium. Increase in s u s c e p t i b i l i t y to k i l l i n g by polymyxin B and EDTA-Tris and l y s i s by EDTA-Tris followed a very similar time course (Fig. 6). There was a short lag before the c e l l s increased in s e n s i t i v i t y to the ba c t e r i c i d a l action of EDTA. However, s e n s i t i v i t y to l y s i s by EDTA more cl o s e l y p a r a l l e l e d decrease in protein HI. 3. Divalent cation concentration of c e l l envelopes and d i s -placement of cations by aminoglycosides and polymyxin B. As indicated in Table VI, decrease in levels of pro-te i n HI was accompanied by an increase in c e l l envelope Mg content. In experiments with wild type c e l l s and three lev e l s 2+ 2+ of Mg , there was a reciprocal relationship between Mg levels in the c e l l envelope and protein HI levels (correlation c o e f f i c i e n t of 0.99 by lin e a r regression). For the protein Hi 2+ overproducing mutants grown in Mg - s u f f i c i e n t medium, protein HI le v e l s were about 7-fold greater than wild type l e v e l s , 2 + while envelope Mg was reduced about 2-fold. The relationship between decrease in protein HI lev-els and increase in c e l l envelope Mg 2 + suggested that HI may act by replacing Mg 2 + at a s i t e susceptible to chelator and a n t i b i o t i c s . This was tested by examining whether polymyxin or 63 7+ aminoglycoside could act to displace Mg in whole c e l l s . C e l l s were pre-treated with KCN to prevent inner membrane up-7 + take of aminoglycosides. When c e l l s grown in 0.5 mM Mg were 2+ treated with 50 ug/ml of polymyxin B, c e l l envelope Mg content was reduced by about 10%. Treatment with 25 ug/ml 2+ gentamicin or 50 ug/ml streptomycin reduced Mg level s by about 3.5 - 5%. This suggests that these agents can act to 2+ displace Mg , but that a r e l a t i v e l y small number of sit e s are involved. 2+ Table VII shows that when c e l l s were grown with Ca , 2+ 2+ Mn , or Zn as the major divalent cation, high levels of the major divalent cation were incorporated into the c e l l envelope. 2+ The lev e l s of Zn were s i g n i f i c a n t l y lower than the levels of C a 2 + or Mg 2 + (P <0.05 by Student's unpaired t-test) in c e l l s 7+ 7+ grown with 0.5 mM Ca and Mg , respectively. 4. Comparison of -EGTA ~and'EDTA s u s c e p t i b i l i t y ' o f ~Ca 2 + and 2+ Mg grown c e l l s . Since c e l l s were always provided with 2+ some Mg as a growth factor, and c e l l envelopes a l l contained 7 + s i g n i f i c a n t l e v e l s of Mg"6, (Table V) , i t was attempted to 2+ determine whether EDTA was exerting i t s eff e c t on Ca grown 2+ c e l l s by removal of the small amounts of Mg or by removal of 2+ 2+ the major cation Ca (Table I I I ) . Ce l l s grown in Ca were equally susceptible to l y s i s by EGTA (which can be regarded as 2+ 2+ a Ca - s p e c i f i c chelator) and EDTA (which chelates both Ca TABLE VII. Divalent cations of c e l l envelopes after growth in the presence of different cations. 2+ Mg present during growth (mM) Other cations present during growth (mM) Divalent 2+ Mg cations in (nmol/mg „ 2 + Ca c e l l envelope dry wt) 2+ 2+ Mn Zn Total b 0. 02 - 54.7 <18 <0.4 <1 54.7 0. 02 „ 2+ Ba (0.5) 48. 5 <18 0. 02 r, 2 + Sn (0.5) 53. 0 <18 0.02 „ 2 + Zn (0.5) 19.5 <L8 95 114. 5 0.02 o 2+ Ca (0.5) 26.3 147 173.3 0. 02 2+ Mn (0.5) 25.0 <18 111 136 0. 02 Sr (0.5) 26. 0 <L8 0. 5 - 122.5 <18 <0.4 <1 122. 5 0.5 2+ Ca (0.5) 76. 5 115.5 192 Determined by atomic absorption spectroscopy; means of up to 5 separate determinations on 2 to 3 separate samples. i 2+ Mg plus other cation present during growth. 65 and Mg^ "1- at high e f f i c i e n c y ) . EGTA had no measurable effect on 2+ c e l l s grown on 0.5 mM Mg as the sole divalent cation. EGTA-2+ T r i s had some b a c t e r i c i d a l a c t i v i t y on Ca grown c e l l s , but none 2+ on Mg grown c e l l s . However, the b a c t e r i c i d a l a c t i v i t y of EGTA was several orders of magnitude lower than that of EDTA. Permeabilization of the outer membrane as a result of chelator treatment was also measured, by examining hydrolysis of a chromogenic beta-lactam n i t r o c e f i n . An increase in the rate of hydrolysis indicates increased permeation of the beta-lactam through the outer membrane to the periplasmic beta-lactamase (Angus et a l . , 1982), and thus provides a sensitive and s p e c i f i c technique for demonstrating disruption of the outer membrane permeability b a r r i e r . EGTA- and EDTA-Tris 2+ treatment were of similar e f f i c i e n c y in permeabilizing Ca grown c e l l s to n i t r o c e f i n , producing hydrolysis rates about 30 2+ times higher than those seen in unbroken c e l l s . Mg grown c e l l s were s i m i l a r l y affected by EDTA, but were not affected by 2+ EGTA. Furthermore, i f Ca grown c e l l s which had been treated with EGTA were subsequently treated with EDTA, only a small increase (5-10%) in the rate of hydrolysis was observed. A similar increase was seen in EDTA treated Ca grown c e l l s subsequently treated with EGTA. A l l of the above experiments were repeated using an RPl plasmid-encoded beta-lactamase, with e s s e n t i a l l y i d e n t i c a l r e s u l t s . TABLE VIII. Effects of EGTA-Tris and EDTA-Tris on cells grown in Mg and Ca . Cations during growth (mM) % Lysis % k i l l i n g EGTA EDTA EGTA EDTA Increase in' nitrocefin hydrolysis^ EGTA ix-Tj EDTA 2+ Mg 0.5' 51 >99.99 32 2+ 2+ Mg 0.02; Ca 0.5 40 36 37 >99.99 30 29 See Table V. Ratio of rate of nitrocefin hydrolysis in cells treated for 2 min with 10 mM EGTA or EDTA in 10 mM Tris-HCl pH 8.5 to rate of hydrolysis in untreated cells. 67 5. Protein HI induction in other Pseudomonas s t r a i n s . The 17 strains in a c o l l e c t i o n representing the 17 P_. aeruginosa sero-types of the International Antigenic Typing Scheme (IATS) have been shown to have outer membrane protein patterns extremely si m i l a r to that of P. aeruginosa PAO H103 (Mutharia, Nicas and Hancock, 1982). A l l these strains have a protein equivalent to protein HI with respect to heat m o d i f i a b i l i t y and molecular weight. When these strains and a type s t r a i n of P. putida were 2+ grown in Mg d e f i c i e n t BM2 succinate a l l but one, the IATS 6 s t r a i n , showed large increases in levels of protein Hi similar to that seen in H103 (Fig. 7). 6. Summary. Outer membrane protein HI was present as the major c e l l u l a r protein both in c e l l s which acquired resistance 2 + to polymyxin B, aminoglycosides and EDTA by growth in Mg -d e f i c i e n t medium and in polymyxin-resistant mutants. Those 2+ cations which could substitute for Mg in reversing resistance to these agents also prevented induction of protein HI. In s h i f t experiments the time course of loss of protein HI corre-lated with that of increase in s u s c e p t i b i l i t y to cationic a n t i -b i o t i c s and chelators. Increase in protein HI was associated 2+ 2+ with a decrease in c e l l envelope Mg . C e l l s grown in Ca , 2+ 2+ Mn , or Zn had levels of those cations in their c e l l envelopes similar to the levels of Mg^ seen in c e l l s grown in 2+ 2+ Mg - s u f f i c i e n t medium. In c e l l s grown in Ca , but not c e l l s 6 8 A B 1 2 3 4 5 6 7 8 9 101112 13 141516 Figure 7. Induction of protein HI i n Pseudomonas strains grown i n low Mg 2 +. SDS polyacrylamide gels of c e l l envelopes of c e l l s grown in BM2 succinate with 0.02 mM Mg 2 +. A. H181; B. Pseudomonas putida type s t r a i n ; 1-16 IATS serotype str a i n s of P. aeruginosa types 1-16. A l l strains were inducible for protein HI except for the type 6 s t r a i n . The type 17 s t r a i n i s not shown. 69 9 + grown in Mg , the calcium s p e c i f i c chelator EGTA had effects s i m i l a r to those of EDTA. Both aminoglycosides and polymyxin B 2+ could be shown to displace Mg from the c e l l envelope. 70 CHAPTER THREE ISOLATION AND CHARACTERIZATION OF A PORIN  DEFICIENT-MUTANT"AND"BACTERIOPHAGE - STUDIES 1. Isolation of outer membrane'protein d e f i c i e n t ~ s t r a i n s ~ b y  random heavy'mutagenesis and i s o l a t i o n of phage s p e c i f i c  for protein'receptors. Isolation of mutants was carried out by random heavy mutagenesis followed by screening of c e l l envelopes for protein a l t e r a t i o n s . This method has been used in E. c o l i to obtain classes of mutants for which no selection i s readily available such as mutants in p e n i c i l l i n binding pro-teins, l i p o p r o t e i n and c a r d i o l i p i n synthetase (Suzuki e_t a l . , 1978). This approach was adopted after preliminary studies showed that selection procedures for outer membranes protein mutants in other organisms were often inapplicable to P. aeruginosa and were generally slow, laborious and i n e f f i c i e n t . The mutagenesis procedure was designed to generate multiple mutations in the survivors. It was anticipated that any outer membrane protein d e f i c i e n t mutants isolated by this method could then be used to i d e n t i f y p o t e n t i a l l y useful selective agents such as bacterio-phage and bacteriocins (see below). NTG was chosen as the mutagen after preliminary experiments showed that mutagenesis with diet h y l sulphate to similar survival levels yielded fewer auxotrophic mutants. In order to avoid selecting mainly strains r e s i s t a n t to the mutagen, the treatment time chosen was 71 within the range where treatment time was s t i l l l i n e a r l y related to s u r v i v a l . Preliminary experiments showed a linear r e l a t i o n s h i p for up to 1 h treatment. Under the conditions chosen (30 min treatment with 1 mg/ml NTG) the survival was 0.004%, suggesting a high mutation frequency. High frequency of mutation among the 500 survivors was also indicated by preliminary screening which showed 19% unable to grow on minimal medium, 15% unable to grow at 42°C, 3% unable to serve as host for p i l u s s p e c i f i c phages (M6 and B39), 23% unable to serve as host for LPS s p e c i f i c phage (Phage 44), 6% c a r b e n i c i l l i n r e s i s t a n t , and 0.4% with the r e l a t i v e l y rare mutation of resistance to high levels (0.1 mg/ml) of streptomycin. Screening of the 500 strains for membrane protein alterations yielded mutants severely d e f i c i e n t in 4 of the 7 major outer membrane proteins, F, G, Hi and H2 (Fig. 8). Two were found which appeared to have protein F with a s l i g h t l y altered molecular weight present in somewhat reduced amounts. The outer membrane protein alterations were confirmed on SDS polyacrylamide gels of p u r i f i e d outer membranes. No strains d e f i c i e n t in proteins I, Dl, or D2 were found. A number of strains appeared to have moderately high molecular weight (50,000-80,000) proteins present in large amounts (e.g., T133 Fig. 8, Gel D). It was not established whether these were inner or outer membrane proteins. 72 A B C D E F G H I J K L M Figure 8. Membranes of heavily mutagenized strains with apparent protein a l t e r a t i o n s . Arrows indicate a l t e r a t i o n s . Gels A, H, M - H103 (wild type) outer membrane (OM); Gel B -T316 c e l l envelope (CE) (Hi d e f i c i e n t ) ; Gel C - T509 CE (G d e f i c i e n t ) ; Gel D - T133 CE (extra protein); Gel E - T513 CE (H2 d e f i c i e n t ) ; Gel F - H283 CE (F d e f i c i e n t ) ; Gel G - T129 (Hi d e f i c i e n t ) ; Gels I and K - T817 OM (F altered); Gels J and L T941 OM ( F altered); Gels K, L and M were s o l u b i l i z e d without 2-mercaptoethanol so that F runs in the F* p o s i t i o n . 7 3 Bacteriocins and bacteriophages have outer membrane receptors. Strains resistant to these agents are usually de-f i c i e n t in those receptors, and bacteriophage and bacteriocins which use s p e c i f i c outer membrane components can often be used as a means of selecting outer membrane protein d e f i c i e n t mutants (for examples see reviews by Osborne and Wu, 1 9 8 0 ; and DiRienzo e_t al.., 1 9 7 8 ) . An attempt was made to use 7 strains with altered outer membrane proteins isolated by random muta-genesis to i d e n t i f y agents which could then be used to select for s p e c i f i c outer membrane protein mutants. The 7 strains were tested for s e n s i t i v i t y to a set of 2 2 aeruginicins and 3 2 phages. P a r t i a l characterization of the receptors of these phages had shown that 1 9 of them probably had outer membrane protein receptors (Table I I ; Nicas and Hancock, 1 9 8 0 ) . How-ever, no bacteriophage with obvious potential u t i l i t y was found in the c o l l e c t i o n , and none of protein d e f i c i e n t mutants was resista n t to any aeruginocin. A second, more d i r e c t approach attempted was the iso-l a t i o n of phage of the desired s p e c i f i c i t y from nature using the enrichment technique of Verhoef et a l . ( 1 9 7 7 ) . This method i s based on the use of bacteria lacking a s p e c i f i c receptor to adsorb out the majority of phages. Adsorption is followed by propagation, and the process repeated for several cycles. Each of the 7 outer membrane protein-altered strains isolated was used as the adsorbing s t r a i n in separate experi-ments . 74 Results of these enrichments are summarized in Table IX. The procedure was successful in i s o l a t i n g phage unable to plate on 3 of the 7 protein-deficient or protein-altered strains used. No attempt was made to establish whether the 3 groups of phages unable to form plaques on s p e c i f i c outer membrane-altered mutants each represented a set of multiple isolates of the same phage or several d i f f e r e n t phages with the same host range. Four phage isolates were/ found which did not form plaques on the G d e f i c i e n t s t r a i n T509. These isolates did form plaques on the second G d e f i c i e n t s t r a i n , T817. Nine mutants re s i s t a n t to this group of phages were is o l a t e d . None had any membrane protein alterations apparent on SDS polyacryl-amide gels. Nine phage isolates (V20-V28) were found which did not form plaques on T316, a s t r a i n d e f i c i e n t in both protein HI and LPS. These phages were able to form plaques on other LPS-altered s t r a i n s . However, 19 of 20 mutants resistant to these phages had altered LPS as judged by i n a b i l i t y to form plaques on LPS receptor-specific phages. Of the 14 resis t a n t strains examined on SDS polyacrylamide gels, 3 had apparently reduced le v e l s of Hi (data not shown). These were named H317, H318 (isolated against phage V28) and H319 (isolated against V27) . A spontaneous revertant of H318, named H318b, was also found. A number of phage isolates which did not form plaques on the porin d e f i c i e n t s t r a i n H283 were also obtained (phages TABLE IX. R e s u l t s o f enrichment p r o c e d u r e f o r i s o l a t i o n o f o u t e r membrane p r o t e i n r e c e p t o r - s p e c i f i c phages. Adsorbing Outer Membrane # I s o l a t e s T e s t e d # I s o l a t e s Not S t r a i n A l t e r a t i o n 3 A f t e r Enrichment P l a t i n g on D e f i c i e n t S t r a i n H283 F-deficient 161 15 (V1-V15) T817 F-altered; G-deficient 60 9 (V16-V19) T941 F-altered 120 0 T509 G-deficient 60 0 T129 Hl-deficient 60 0 T316 Hl-deficient; LPS altered 120 9 (V20-V28) T513 H2-deficient 120 0 See F i g . 8. I s o l a t e d e s i g n a t i o n s . 76 V 1 - V 1 5 ) . H o w e v e r , r e v e r t a n t s o f H 2 8 3 w h i c h a p p e a r t o h a v e n o r m a l l e v e l s o f p o r i n w e r e a l s o r e s i s t a n t t o t h e s e p h a g e s , s t r o n g l y i n d i c a t i n g t h a t t h e i r r e c e p t o r i s s o m e t h i n g o t h e r t h a n p o r i n ( s e e b e l o w ) . 2 . C h a r a c t e r i z a t i o n o f a p o r i n d e f i c i e n t i s o l a t e . A s t h e p r o t e i n F d e f i c i e n t s t r a i n H 2 8 3 w a s i s o l a t e d a f t e r h e a v y m u t a g e n e s i s , m o r e t h a n o n e m u t a t i o n m i g h t b e e x p e c t e d i n t h i s s t r a i n . To c i r c u m v e n t t h i s p r o b l e m , 3 i n d e p e n d e n t s p o n t a n e o u s r e v e r t a n t s w e r e i s o l a t e d . Two o f t h e s e , H 2 8 4 a n d H 3 2 4 w e r e i s o l a t e d f o r t u i t o u s l y a f t e r g r o w t h o n BM2 g l u c o s e . The t h i r d , H 3 2 1 , was s e l e c t e d f o r b y e n r i c h i n g f o r s t r a i n s w i t h h i g h e r g r o w t h r a t e s b y r e p e a t e d s u b c u l t u r e o f c e l l s g r o w i n g i n l i q u i d m e d i u m . The i s o g e n i c i t y o f t h e r e v e r t a n t s a n d H 2 8 3 w a s c o n f i n -e d b y t h e i r p h a g e s e n s i t i v i t y p a t t e r n s ( s e e b e l o w ) . T r a n s f e r o f t h e s p e c i f i c m u t a n t g e n e r e s p o n s i b l e f o r p o r i n d e f i c i e n c y w a s n o t a t t e m p t e d s i n c e t h e o n l y a v a i l a b l e m e t h o d o f s c r e e n i n g was SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f c e l l e n v e l o p e s , a n d f r e q u e n c i e s o f g e n e t r a n s f e r i n P_. a e r u g i n o s a t e n d t o b e q u i t e l o w . P r o t e i n F h a s b e e n s h o w n t o b e p r e s e n t i n a b o u t 1 - 3 x 1 0 5 c o p i e s / c e l l o f H 1 0 3 . The r e v e r t a n t s H 2 8 4 ( F i g . 9 , G e l C ) , H 3 2 1 , a n d H324 ( d a t a n o t s h o w n ) c o n t a i n e d a p p a r e n t l y n o r m a l l e v e l s o f p r o t e i n F . I n c o n t r a s t , t h e o u t e r m e m b r a n e s o f t h e m u t a n t s t r a i n H 2 8 3 d i d n o t c o n t a i n o b s e r v a b l e l e v e l s o f p r o t e i n F ( F i g . 9 , G e l s B a n d E ) . The p a t t e r n o f o t h e r o u t e r m e m b r a n e p r o t e i n s was t h e same i n t h e m u t a n t H283 a n d i t s r e v e r t a n t s . 77 [ •1 s Figure 9. C e l l envelopes of wild type s t r a i n H103, i t s porin d e f i c i e n t mutant H283, and a revertant, H284. Gel A, D. - H103; Gel B, E. - H283; Gel C. - H284. Cells envelopes for gels A, B, and C were s o l u b i l i z e d i n normal reduction mix before applica-t i o n to the ge l . For gels E and D, 2-mercaptoethanol was omit-ted from the s o l u b i l i z a t i o n mix, so that protein F runs in the F* p o s i t i o n . The amount of protein F detectable i n H283 i s less than 1% that of H103 or H284. 78 When t h e e l e c t r o p h o r e t i c m o b i l i t y o f p r o t e i n F f r o m H 1 0 3 a n d t h e r e v e r t a n t s was i n c r e a s e d b y o m i s s i o n o f 2 - m e r c a p -t o e t h a n o l f r o m t h e s o l u b i l i z a t i o n b u f f e r ( H a n c o c k a n d C a r e y , 1 9 7 9 ) , no o u t e r membrane p o l y p e p t i d e f r o m s t r a i n H 2 8 3 w a s s i m i -l a r l y 2 - m e r c a p t o e t h a n o l m o d i f i e d ( c . f . F i g . 9 , G e l s D a n d E ) . A l l o t h e r m a j o r o u t e r membrane p r o t e i n s a p p e a r e d t o b e p r e s e n t i n H 2 8 3 i n q u a n t i t i e s c o m p a r a b l e t o t h o s e o f t h e r e v e r t a n t , a l t h o u g h t h e a b s e n c e o f t h e l a r g e p r o t e i n F b a n d on SDS p o l y a c r y l a m i d e g e l s r e s u l t e d i n a p p a r e n t e n h a n c e m e n t o f m i n o r b a n d s . S t r a i n s H 2 8 3 a n d i t s r e v e r t a n t s w e r e s i m i l a r t o t h e p a r e n t s t r a i n H 1 0 3 i n t h e i r a b i l i t y t o p r o d u c e t w o i n d u c i b l e o u t e r membrane p r o t e i n s : D l , a g l u c o s e - i n d u c i b l e p o r i n ( H a n c o c k a n d C a r e y , 1 9 8 0 ) , a n d p r o t e i n H I . 3 . B a c t e r i o p h a g e s e n s i t i v i t y o f o u t e r membrane p r o t e i n m u t a n t s , m u c o i d i s o l a t e s , a n d s e r o t y p e s t r a i n s . A s b a c t e r -i o p h a g e s a n d b a c t e r i o c i n s h a v e o u t e r m e m b r a n e r e c e p t o r s , t e s t -i n g t h e s u s c e p t i b i l i t y o f s t r a i n s t o t h e s e a g e n t s i s a u s e f u l m e t h o d o f r e v e a l i n g o u t e r m e m b r a n e a l t e r a t i o n s . No d i f f e r e n c e s w e r e f o u n d i n t h e s u s c e p t i b i l i t i e s o f t h e p o l y m y x i n B - r e s i s t a n t m u t a n t s H 1 8 1 a n d H 1 8 5 t o 2 4 p h a g e s a n d 22 a e r u g i n o c i n s w h e n c o m p a r e d t o t h e w i l d t y p e s t r a i n , H103 ( T a b l e X ) . The p o r i n d e f i c i e n t s t r a i n H 2 8 3 a l s o s h o w e d n o d i f f e r e n c e s i n s e n s i t i v i t y when t e s t e d a g a i n s t t h e a e r u g i n o c i n s a n d 3 3 p h a g e s . S i n c e some o f t h e p h a g e s w e r e s m o o t h L P S o r p i l u s r e c e p t o r s p e c i f i c , i t c a n b e c o n c l u d e d t h a t H 1 8 1 , H185 a n d H 2 8 3 h a d s m o o t h L P S a n d w e r e p i l i a t e d . The 3 r e v e r t a n t s o f H 2 8 3 w e r e a l s o s e n s i t i v e t o 79 a l l t h e s e p h a g e s , e x c e p t f o r H284 w h i c h was r e s i s t a n t t o p h a g e s w i t h p i l u s r e c e p t o r s . T h i s s t r a i n was a l s o n o n - m o t i l e , s u g g e s t i n g i t was d e f e c t i v e i n f l a g e l l a r f u n c t i o n , a s w e l l a s p i l i . A s e t o f 15 p h a g e s ( V 1 - V 1 5 ) a b l e t o p l a t e on t h e w i l d -t y p e H 1 0 3 , a p i l u s - d e f i c i e n t s t r a i n , A K 1 2 1 3 , a n d a n L P S -d e f i c i e n t s t r a i n , A K 1 0 1 2 , b u t u n a b l e t o f o r m p l a q u e s on H283 was i s o l a t e d f r o m n a t u r e ( s e e a b o v e ) . R e v e r t a n t s o f H283 w e r e a l s o r e s i s t a n t t o t h e s e p h a g e s . Two o f t h e s e p h a g e s , V4 a n d V 7 , c o u l d be s h o w n t o a d s o r b e f f i c i e n t l y t o w h o l e c e l l s o f t h e w i l d t y p e H103 ( 9 5 - 1 0 0 % r e d u c t i o n i n p h a g e t i t r e ) , b u t r e l a t i v e l y p o o r l y t o t h e p o r i n d e f i c i e n t m u t a n t H283 o r i t s r e v e r t a n t H284 ( 2 - 1 0 % r e d u c t i o n i n p h a g e t i t r e ) . T h i s s u g g e s t s t h a t H283 a n d i t s d e r i v a t i v e s l a c k a membrane r e c e p t o r f o r t h e s e p h a g e s w h i c h i s p r e s e n t i n w i l d - t y p e c e l l s . The s p e c i e s P . a e r u g i n o s a c a n be d i v i d e d i n t o 17 s e r o t y p e s b a s e d on a n t i g e n i c d i f f e r e n c e s i n t h e i r L P S ( B r o k o p p a n d F a r m e r , 1 9 7 9 ; L a n y i et. a l . , 1 9 7 9 ) . T h e s e d i f f e r e n c e s r e f l e c t c h e m i c a l v a r i a t i o n s i n t h e L P S 0 s i d e - c h a i n ( K o v a l a n d M e a d o w , 1 9 7 5 ) . P i l i a l s o v a r y f r o m s t r a i n t o s t r a i n ( B r i n t o n , 1 9 8 1 ) . A s b a c t e r i o p h a g e s a r e t h o u g h t t o i n t e r a c t w i t h c h e m i -c a l l y u n i q u e s i t e s o n s p e c i f i c c e l l s u r f a c e c o m p o n e n t s ( e . g . , B r a u n a n d K e i g e r - B a u e r , 1 9 7 5 ) , t h e a b i l i t y o f a g i v e n p h a g e t o f o r m p l a q u e s o n s e v e r a l s t r a i n s i n d i c a t e s a r e c e p t o r common t o t h e s e s t r a i n s . I t s h o u l d h o w e v e r be n o t e d t h a t l a c k o f s e n s i -t i v i t y t o a b a c t e r i o p h a g e d o e s n o t n e c e s s a r i l y i m p l y a b s e n c e o f t h e r e c e p t o r p r o t e i n s i n c e s i n g l e a m i n o a c i d s u b s t i t u t i o n s i n 80 such proteins has been shown to r e s u l t in resistance to phages which normally use this protein as a receptor (e.g., E. c o l i CR63; Braun and Keiger-Bauer, 1977). Receptors of the phages used had been p a r t i a l l y characterized allowing them to be separated into f i v e groups (see Table II, Methods). Results of s e n s i t i v i t y testing are l i s t e d in Table X and summarized in Table XI. The results with the 13 phages that had uncharacter-ized protein receptors indicated that these receptor s i t e s were quite well conserved. For example, phage 7 formed plaques on a l l 17 £. aeruginosa serotyping strains tested, while phages B9F and F116 plated on 16 and 11 s t r a i n s , respectively. None of the bacteria studied appeared rough as tested either with a bacteriophage which used rough but not smooth LPS as i t s receptor, or by examination of gross colony morphology. With the exception of IATS type 11 bacteria, a l l typing strains plated one or more smooth LPS s p e c i f i c phages (Tables X and XI). Indeed, 9 of the 17 serotypes plated 7 or more of the 9 smooth LPS-specific phages. This indicated that there is some homology ( i . e . , the receptor site) in the 0 antigens of d i f f e r e n t serotyping strains despite the prior observation of s i g n i f i c a n t chemical v a r i a t i o n s . It was further observed that only 3 of the 17 strains plated phages s p e c i f i c for PA01 p i l i in agreement with the finding that p i l i d i f f e r from s t r a i n to s t r a i n as demonstrated for P. aeruginosa and other bacteria. Taken o v e r a l l , the results suggested that the surfac-es of P_. aeruginosa c e l l s were moderately well conserved, since TAB LE X. Bacteriophage s e n s i t i v i t y of outer membrane protein mutants, serotyping s t r a i n s , and mucoid i s o l a t e s . Phage B a c t e r i a l S t r a i n s 3 A B C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 .15 16 17 D E F G H I J K 119x S s s s s s M6 S s s s s s s 339 S s s S' s s 44 S s s s s s s s s s E79 S s s s s s s s F8 S s s s s s s s 109 s s s s s s 1214 s s s s s s s s s PBl s s s s s s s C3A s s s s s s s SI s s s s s s s 352 s s s s s s s s s s PLS27 s 2 s s s s s s D3c +1 + s s s B5A s s s s s B7A s s s s s s s s s C7B s s s s s s s s s B9E s s s s s s s s B9F s s s s s s 7 s s s s s s s s s s s s 21 s s s s s 68 s s s s s s s s C21 s s s s s s s s s s F116 s s s s s s s s s s G101 s s s s s s s B1A s s s s s s s s A8A s s s s s s s s s B6B s s s s s s s s s s D3c~l + s s s s s s s s s s B6C F10 V1-V15 s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s 3 s . s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s - - s s s s s s s s - - s s s s s s - - s s s s s s r s s - - c s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s •s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s 's s s s s s s s s s s s s s s s s s s s s s s s s s s s s - - s s s s s s s - - s s s s s s s s s s s - - s s s s s s - - s s s s s s s s - - s s s s s A: H103 (wild type); B: AK1213 ( p i l u s d e f i c i e n t ) ; C: AK1012 (LPS-de f i c i e n t ) ; 1-17: IATS serotype s t r a i n s 1-17; D,E: pro t e i n HI overproducers, H181 and H185; F: H283 ( p o r i n - d e f i c i e n t ) : G, H, I: revertants of H283 (H284, H321, H324); J , K: mucoid s t r a i n s , H329 and H325. "S" means s e n s i t i v e ; "-" means not tested; no entry means r e s i s t a n t . TABLE XI. Bacteriophage susceptibility of serotype strains of P_. aeruginosa performed using phages propagated and characterized on P_. aeruginosa PA01 strains. Number of Phages to Which Strain is Susceptible Bacterial Smooth LPS Rough LPS Pilus Possible Possible Cumulative Strain specific specific specific LPS protein sus ceptib i l i t y phages (9) phage (1) phages (2 ) receptor receptor (%) phages (6) phages (13) H103 9 0 2 6 13 100% Pilus deficient 9 0 0 5 13 90% LPS altered 0 1 2 0 13 50% 0 Serotype 1 9 0 0 4 12 83% 2 9 0 0 4 12 83% 3 9 0 0 5 11 83% 4 9 0 0 5 12 87% 5 9 0 0 3 13 83% 6 7 0 2 2 7 60% 7 7 0 2 3 6 60% 8 3 0 0 0 4 23% 9 1 0 0 0 1 7% 10 1 0 0 3 4 27% 11 0 0 0 0 2 7% 12 1 0 0 0 5 20% 13 3 0 0 2 6 37% 14 . 7 0 0 1 8 53% 15 3 0 0 1 5 30% 16 8 0 2 2 6 60% 17 1 0 0 2 4 23% Nos. of phages reacting with 9/9 0/1 0/2 2/6 11/13 73% 45% of serotype strains 83 9 of the 17 strains plated 53% or more of the phages screened. The antigenic and physical s i m i l a r i t y of outer membrane pro-teins of P. aeruginosa has been further studied by Mutharia e_t a l . (1982). Two mucoid derivatives of H103 o r i g i n a l l y isolated by selection for resistance to phage 7 were also tested. One of these was i d e n t i c a l to the parent in i t s phage s e n s i t i v i t y , while the other was sensitive to over 60% of the phages, i n d i -cating that mucoidy does not necessarily impede phage s e n s i t i v -i t y , in agreement with previous results (Martin, 1973). 4. Summary. Random heavy mutagenesis with nitrosoguanidine was used to iso l a t e mutants d e f i c i e n t in outer membrane proteins F, G, HI, and H2. Three revertants of the protein F-de f i c i e n t s t r a i n were also i s o l a t e d . Attempts to isolate phage with outer membrane protein s p e c i f i c receptors yielded one group of phages which could be used to select for mutants d e f i c i e n t in protein Hi and a group of phages unable to form plaques on the protein F-deficient s t r a i n or i t s revertants. Revertants of the protein F-deficient s t r a i n had a protein band on SDS polyacrylamide gels which was indistinguishable from protein F in i t s electrophoretic mobility both in the presence and absence of 2 mercaptoethanol, while no such protein was detectable on gels of the protein F-deficient s t r a i n . Phage s e n s i t i v i t y of wild type, protein F-deficient and HI overpro-ducing mutants were e s s e n t i a l l y the same, indicating that the 84 mutants were p i l i a t e d , had smooth LPS, and lacked any gross surface a l t e r a t i o n s . Phage studies with a set of strains representing the 17 serotypes of P. aeruginosa indicated considerable conservation of phage receptors. i 85 CHAPTER FOUR  MEASUREMENT OF"OUTER MEMBRANE P E R M E A B I L I T Y O u t e r membrane p r o t e i n F h a s b e e n c h a r a c t e r i z e d a s a p o r i n o n t h e b a s i s o f i r i v i t r o s t u d i e s ( H a n c o c k e_t a l . , 1 9 7 9 ; B e n z a n d H a n c o c k , 1 9 8 1 ) . S u c h s t u d i e s h a v e i n d i c a t e d t h a t a l t h o u g h p r o t e i n F f o r m s s u b s t a n t i a l l y l a r g e r c h a n n e l s t h a n t h e p o r i n s o f e n t e r i c b a c t e r i a ( H a n c o c k e_t a l . , 1 9 7 9 ; N i k a i d o , 1 9 8 0 ) , t h e in v i t r o a c t i v i t y o f p r o t e i n F i s r e l a t i v e l y l o w i n t h a t o n l y a s m a l l p r o p o r t i o n o f t h e p o r i n p r o t e i n s f o r m f u n c -t i o n a l c h a n n e l s ( A n g u s e t a l . , 1 9 8 2 ; B e n z a n d H a n c o c k , 1 9 8 1 ) . Low o u t e r membrane p e r m e a b i l i t y o f w i l d - t y p e P_. a e r u g i n o s a c e l l s h a s a l s o b e e n s h o w n in v i v o ( A n g u s et. a l . , 1 9 8 2 ) . The l o w a c t i v i t y o f p r o t e i n F _in v i t r o a n d in v i v o r a i s e s t h e p o s s i b i l i t y t h a t t h e e f f e c t s o b s e r v e d c o u l d be d u e t o a m i n o r p r o t e i n c o - p u r i f y i n g w i t h p r o t e i n F r a t h e r t h a n p r o t e i n F i t -s e l f . T h i s h y p o t h e s i s was t e s t e d b y e x a m i n i n g t h e p e r m e a b i l i t y o f a s t r a i n s e v e r e l y d e f i c i e n t i n p r o t e i n F , H 2 8 3 . I s o l a t i o n o f t h i s s t r a i n a n d i t s r e v e r t a n t s ( w h i c h h a v e n o r m a l l e v e l s o f p r o t e i n F) i s d e s c r i b e d a b o v e . The t e c h n i q u e e m p l o y e d was b a s e d on t h a t o f Z immerman a n d R o s s e l e t ( 1 9 7 7 ) . T h i s t e c h n i q u e i s b a s e d on t h e c o n c e p t t h a t , p r o v i d i n g e n o u g h b e t a - l a c t a m a s e i s p r e s e n t i n t h e p e r i -p l a s m , t h e n t h e b e t a - l a c t a m a s e a c t i v i t y o f i n t a c t c e l l s w i l l be l i m i t e d b y t h e r a t e o f d i f f u s i o n o f b e t a - l a c t a m a c r o s s t h e 86 outer membrane to the periplasmic beta-lactamase. Thus, the equilibrium rate of hydrolysis of beta-lactam by intact c e l l s ( V i n t a c t ) i s equal to the rate of d i f f u s i o n (Vpjiff). This allows c a l c u l a t i o n of an outer membrane permeability c o e f f i c i e n t C using the following equation of Zimmermann and Rosselet: V i n t = V D i f f = C< Sout " Sin> where S Q U £ i s the beta-lactam concentration added and S^ n i s the periplasmic concentration of beta-lactam [which can be calculated from the Michaelis-Menten equation]. The beta-lac-tam used in these measurements was the chromogenic cephalospor-in n i t r o c e f i n (0'Callaghan, 1972). The beta-lactamase used was the periplasmic TEM-2 enzyme encoded by the plasmid RPl, which was introduced into each of the strains used by conjugation. Measurement of t o t a l beta-lactamase a c t i v i t y in c e l l s broken by passage through a French press was carried out to confirm that periplasmic a c t i v i t y was in excess. Beta-lactamase a c t i v i t y of broken c e l l s varied l i t t l e from s t r a i n to s t r a i n and was 7 to 500-fold higher than the a c t i v i t y of whole c e l l s . Other stud-ies (Angus et. a l . , 1982) have demonstrated that the temperature c o e f f i c i e n t of n i t r o c e f i n hydrolysis by intact c e l l s ( V D ^ f f ) is consistent with n i t r o c e f i n entering via a hydrophilic pathway. In addition, i t was established here that n i t r o c e f i n hydrolysis in intact c e l l s was d i r e c t l y proportional to the concentration of n i t r o c e f i n added (S o n i.) for strains H103 (RPl) and E. c o l i 87 UB1636 (RPl) over an 8-fold range of substrate concentrations (0.025-0.02 mg/ml), as predicted by the above d i f f u s i o n equation. The results shown in Table XII revealed that s t r a i n H283 was s i g n i f i c a n t l y less permeable than i t s parent s t r a i n H103 or the revertant H284. While the standard deviations of these r e s u l t s were high, the range of rates of hydrolysis of n i t r o c e f i n in intact c e l l s of H283 and H103 or H284 did not overlap and the means were c l e a r l y d i f f e r e n t as judged by the Student t - t e s t . As an additional control, previous results demonstrating that the a n t i b i o t i c super-susceptible mutant Z61 i s s i g n i f i c a n t l y more permeable than H103 or i t s f u l l revertant H251 (Angus et a l . , 1982) were confirmed using this modified assay. In contrast, there was no s i g n i f i c a n t a l t e r a t i o n in permeability of the aminoglycoside and polymyxin resi s t a n t mutant H181. An E_. c o l i K-12 s t r a i n UB1636 had much greater permeability than any of the P. aeruginosa strains studied here . Summary. Outer membrane permeability was measured using the rates of hydrolysis, in intact c e l l s , of a chromogenic beta-lactam, n i t r o c e f i n , by periplasmic beta-lactamase. It was shown that the protein F-deficient mutant H283 had reduced out-er membrane permeability r e l a t i v e to i t s parent or revertant, indicating that this protein is the major outer membrane porin. Protein HI overproducing strains were not altered in outer TABLE XII. Rate of n i t r o c e f i n hydrolysis by in t a c t c e l l s , and outer membrane permeability c o e f f i c i e n t s C, of P_. aeruginosa s t r a i n H103 (RPl) , i t s RPl plasmid-containing derivatives, Z61 (RPl) and E. c o l i s t r a i n C127 (RPl). S t r a i n Phenotype Nos. of Rate of n i t r o c e f i n hydro- Outer Membrane Significance, by Determinations l y s i s i n intact c e l l s permeability Student t - t e s t , of (pmol n i t r o c e f i n min -l c o e f f i c i e n t C difference from mg c e l l dry wt~l) a (sec~l mg c e l l H103 (RPl) dry wt" 1 x 10 4) H103 (RPl) Parent 12 60 ± 17 4.1 H181 (RPl) GM, PX, EDTA 9 62 ± 36 4. 2 p>0. 5 re s i s t a n t , protein Hi overproducing H251 (RPl) Revertant of Z61 7 58 ± 13 3.9 p>0.5 H284 (RPl) revertant of H283 7 59 ± 30 4.0 p>0. 5 H283 (RPl) protein F-deficient 5 9.8 ± 7.6 0. 7 p<0. 01 Z61 (RPl) a n t i b i o t i c super- 12 360 ±170 24.9 p<0. 001 susceptible UB1636 (RPl) E. c o l i 13 740 ±390 50.6 p<0.001 mean ± standard deviation 89 membrane permeability. An a n t i b i o t i c supersusceptible s t r a i n of J P . aeruginosa was found to be s i g n i f i c a n t l y more permeable than i t s revertant or the wild type. An E. c o l i s t r a i n had much greater permeability than any of the P_. aeruginosa strains used here. 90 DISCUSSION This study demonstrates that the outer membrane of Pseudomonas aeruginosa plays a major role in the a n t i b i o t i c resistance of this organism. The results of the permeability studies (Table XII) were consistent with the hypothesis that a n t i b i o t i c resistance can be explained on the basis of low permeability of the P. aeruginosa outer membrane due to the properties of protein F. Although in v i t r o experiments have indicated that the area of individual protein F channels is up to 3-fold larger than the area of E_. c o l i porin channels (Benz and Hancock, 1981), and that the number of porin molecules per c e l l i s about the same in E. c o l i and P. aeruginosa (Benz and Hancock, 1982; Rosenbusch, 1974), P. aeruginosa was shown to have an outer membrane permeability c o e f f i c i e n t (C) s i g n i f i -cantly lower than that of E. c o l i . This was especially s t r i k -ing when i t is considered that the size of n i t r o c e f i n (520 daltons) approaches the exclusion l i m i t of E. c o l i porins. It would thus be expected that d i f f u s i o n of n i t r o c e f i n would be slowed by f r i c t i o n a l and s t e r i c interactions with the walls of the channel as discussed by Nikaido and Rosenburg (1981), so that the d i f f u s i o n constant C would be decreased when compared to a larger channel such as protein F (Hancock and Nikaido, 1978). In order to correct for the apparent reduction in C due to smaller channels, the permeability to n i t r o c e f i n of single pores of P. aeruginosa porin compared to those of E. c o l i 91 porins may be corrected t h e o r e t i c a l l y (Nicas and Hancock, 1982) using Fick's law and the Renkin equations (Renkin, 1954; Lakshminarayaniah, 1969, p.325). This cal c u l a t i o n can be made assuming a hydrated radius of 0.44-0.53 nm for n i t r o c e f i n (similar to that of a disaccharide) and using previous estimates for the r a d i i of E_. c o l i porin lb and P. aeruginosa protein F (0.65 nm and 1.1 nm, respectively; Benz and Hancock, 1981; Nikaido and Rosenberg, 1981). Using such values one arrives at the conclusion that the s t e r i c e f f e c t of interaction of n i t r o c e f i n with E_. c o l i porin channel walls w i l l r e s u l t in an apparent 11-fold decrease in the C value of E. c o l i pores r e l a t i v e to the C value for single pores of P. aeruginosa. Taken together with the 12-fold difference in C value shown in this study, the calculated t o t a l difference in outer membrane pore area available for the d i f f u s i o n of n i t r o c e f i n in P. aeruginosa was 132-fold lower. Since the actual area of a P. aeruginosa channel i s three times that of E. c o l i , the t o t a l number of active and functional porin channels (per c e l l dry weight) would be less than 1/400 of that of E_. c o l i or about 100 per c e l l assuming 4 x 10 4 porin trimers per c e l l (Rosenbusch, 1974). This difference in permeability correlates well with the low porin a c t i v i t y of protein F which has been observed both iri v i t r o and jjri vivo (Angus e_t al^., 1982; Benz and Hancock, 1981) and also correlates with the known high i n t r i n s i c resistance of P. aeruginosa to hydrophilic a n t i b i o t i c s . 92 It has previously been demonstrated (Angus et a l . , 1982) that the apparent number of functional channels can be increased by LPS mutation in s t r a i n Z&l, but no explanation has emerged as to why more than 99% of protein F molecules do not form functional pores in wild type c e l l s . The evidence report-ed here from studies with protein F-deficient c e l l s argues in favour of the p o s s i b i l i t y that the actual porin is protein F rather than a minor contaminating protein. The protein F-d e f i c i e n t mutant had a s i g n i f i c a n t l y lower outer membrane permeable c o e f f i c i e n t C compared to i t s parent or to revertant s t r a i n s . Since s t r a i n H283 does have a measurable C value, i t may well not be t o t a l l y p o r i n - d e f i c i e n t , but rather porin pro-tein F-deficient, in that other porin proteins may be present at lower lev e l s in the outer membrane. Black l i p i d bilayer studies of fractions from porin d e f i c i e n t mutants of E. c o l i (Benz e_t a l . , 1978) have provided evidence that a more cation-selective channel i s responsible for residual porin a c t i v i t y in these s t r a i n s . In the case of P. aeruginosa, two other induc-i b l e porin proteins, P and Dl (Hancock and Carey, 1980; Hancock et a l . , 1982), are possible candidates for providing the r e s i d -ual porin a c t i v i t y of the protein F-deficient s t r a i n H283. The low le v e l of active porin in wild-type c e l l s sug-gests that the hydrophilic pathway is a r e l a t i v e l y i n e f f i c i e n t means of traversing the outer membrane of P. aeruginosa. It would thus be expected that hydrophilic a n t i b i o t i c s , including aminoglycosides, would be r e l a t i v e l y i n e f f e c t i v e against 93 P. aeruginosa. However, this study offers evidence that highly ca t i o n i c a n t i b i o t i c s e f f e c t i v e against P. aeruginosa, i . e . , aminoglycosides and polymyxins, may cross the membrane by means of an alternate "self-promoted" pathway. This study demonstrates that the outer membrane is a major determinant of the a c t i v i t y of polymyxins, aminoglyco-sides and EDTA. A close correlation between levels of outer membrane protein Hi and s u s c e p t i b i l i t y to chelators, polymyxin B, and gentamicin was shown under a variety of d i f f e r e n t growth conditions. In s h i f t experiments, the decrease in protein Hi brought about by increasing Mg2+ in the growth medium clos-ely p a r a l l e l e d increase in s u s c e p t i b i l i t y to EDTA-Tris and polymyxin B. When other divalent metal cations were substitut-ed for Mg2+, only c e l l s grown in those cations which prevented induction of protein HI were susceptible to EDTA, polymyxin B and gentamicin. It has been suggested that polymyxins, chelators (Brown, 1975; Nicas and Hancock, 1980), and aminoglycosides (Hancock, 1981; Hancock et. a l . , 1981) act at a common s i t e on the outer membrane. A model for the action of these agents on the outer membrane is i l l u s t r a t e d in Fig. 10. It is proposed that these agents act at a s i t e on the outer membrane, possibly a polyphosphate s i t e on the lipopolysaccaride and that divalent cations bound at this s i t e would be required for the s t a b i l i t y of the outer membrane. Thus EDTA would act to remove divalent cations by chelation, while the cationic a n t i b i o t i c s , polymyx-94 SENSITIVITY °69 QM. RESISTANCE QM. Figure'10. Model i l l u s t r a t i n g the proposed mechanism of resistance to aminoglycosides, polymyxin B, and EDTA-Tris in P_. aeruginosa with high lev e l s of protein HI. O.M. outer membrane; R core-the heptose, KDO, rough core region of the LPS; O Ag-the somatic antigen of P. aeruginosa LPS; (P) n-the polyphosphate portion of the P. aeruginosa LPS; Hl-major outer membrane pro-tein Hi, which appears in large amounts in s p e c i f i c mutants and in c e l l s grown in Mg2+ d e f i c i e n t medium. It is proposed that the crosslinking of the negatively charged polyphosphate regions of the LPS by Mg + i s important for outer membrane s t a b i l i t y in sensitive c e l l s . EDTA by removing Mg2+, and the highly cationic a n t i b i o t i c s aminoclycosides and polymyxin B by displacing Mg2+, lead to disruption of the outer membrane permeability b a r r i e r . It i s proposed that in c e l l s with high levels of protein HI, this protein replaces Mg2+ at s p e c i f i c s i t e s in the outer membrane. Protein Hi thus protects the polyphosphate s i t e on the LPS from attach by aminoglycosides, polymyxin B and EDTA and makes the c e l l r e l a t i v e l y r e s i s t a n t to these agents. 95 ins and aminoglycosides, would act by competing for the l i p o -polysaccharide binding s i t e . Protein HI i s proposed to act by replacing divalent cations at this s i t e on the lipopolysac-charide, thus protecting i t from attack by these agents. Both polymyxin and EDTA are known to interact with P. aeruginosa LPS (Cooperstock, 1974; Michaels and Eagon, 1966). An early consequence of polymyxin B and EDTA action i s permea-b i l i z a t i o n of the outer membrane (Brown and Melling, 1969; Iida and Koike, 1974; Laporte et a l . f 1977; Rosenthal and Strom, 1977) and polymyxin B has been shown to cause blebbing of the outer monolayer of the outer membrane, which is the sole loca-tion of c e l l u l a r LPS (Gil l e l a n d and Murray, 1976; Schindler and Teuber, 1975). Schindler and Osborn (1979) have demonstrated that the 2-keto-3-deoxyoctonate-lipid A region of Salmonella  typhimurium LPS has high a f f i n i t y binding s i t e s for polymyxin B (Kd = 0.3 to 0.5 uM - approximately 10-fold higher than for polymyxin-phospholipid interactions) and for Mg2+ and Ca2+. E a r l i e r studies by Newton (1954) demonstrated that Mg2+ and polymyxin B competed for a P. aeruginosa c e l l u l a r s i t e , which he postulated to be polyphosphate in nature. P_. aeruginosa LPS has been demonstrated to have an especially high phosphate concentration (Dewry et a l . , 1971), and may have up to 8 moles of phosphate per mole of LPS in the heptose-KDO region (A. Kropinski, personal communication). There is evidence that some of this phosphate is present as triphosphate (Wilkinson, 1981) . Since phosphoryl and phosphodiester groups 96 are negatively charged at neutral pH, this could contribute to the r e l a t i v e l y high Mg 2 + content of P. aeruginosa c e l l envelopes (Brown and Woods, 1 9 7 2 ) . Aminoglycosides are also highly c a t i o n i c , and have been shown to bind to P_. aeruginosa LPS (Day et a l . , 1 9 7 8 ) . The a b i l i t y of aminoglycosides to interact with the outer membrane and promote a s i g n i f i c a n t a l t e r a t i o n in i t s permeability was shown by both c e l l l y s i s in the presence of lysozyme and aminoglycosides (Hancock, Raffle and Nicas, 1 9 8 1 ) , and increased hydrolysis of n i t r o c e f i n in the presence of gentamicin. Lysozyme i s normally inactive on gram-negative bacteria as i t i s unable to penetrate the outer membrane to reach i t s s i t e of a c t i v i t y , the peptidoglycan. It was shown that gentamicin and streptomycin acted to overcome this penetration b a r r i e r , allowing lysozyme to attack the peptidoglycan and lyse the c e l l s . Lysozyme i t s e l f i s known to bind to outer membranes (Day e_t a l . , 1 9 7 8 ) , and this a b i l i t y may have contributed to the e f f i c i e n c y of i t s permeation in aminoglycoside-treated c e l l s . This is further suggested by the r e l a t i v e l y i n e f f i c i e n t gentamicin-mediated permeabilization of outer membranes to n i t r o c e f i n , a chromogenic beta-lactam. In fac t , this implies that aminoglycoside-mediated permeabiliza-tion has some s p e c i f i c i t y for cationic substrates such as lysozyme and aminoglycosides. The permeabilization observed cannot be attributed to aminoglycoside k i l l i n g as i t was shown that i t occurs under conditions in which aminoglycosides are known not to be transported or l e t h a l (Bryan and Van Den Elzen, 97 1 9 7 6 ; H u r w i t z a n d R o s a n o , 1 9 6 1 ) s u c h a s i n c h l o r a m p h e n i c o l - o r K C N - t r e a t e d c e l l s ( H a n c o c k e t a l 1 9 8 1 ) . A m i n o g l y c o s i d e s a r e c l e a r l y a b l e t o p r o m o t e t h e p a s s a g e o f o t h e r m o l e c u l e s t h r o u g h t h e o u t e r m e m b r a n e , a n d i t s e e m s l i k e l y t h a t t h e y a r e a l s o c a p a b l e o f p r o m o t i n g t h e i r own t r a n s p o r t . I t i s p r o p o s e d t h a t a m i n o g l y c o s i d e u p t a k e a n d k i l -l i n g i n P_. a e r u g i n o s a r e q u i r e s i n t e r a c t i o n w i t h a d i v a l e n t c a t i o n b i n d i n g s i t e a t t h e o u t e r m e m b r a n e . T h i s i n t e r a c t i o n p r o m o t e s u p t a k e o f t h e a n t i b i o t i c i n t o t h e p e r i p l a s m , p e r m i t -t i n g f u r t h e r t r a n s p o r t a t t h e l e v e l o f t h e c y t o p l a s m i c m e m b r a n e . I t w a s o b s e r v e d t h a t M g 2 + w a s a b l e t o i n h i b i t a m i n o g l y c o s i d e m e d i a t e d p e r m e a b i l i t y t o b o t h n i t r o c e f i n a n d t o l y s o z y m e . T h i s i n h i b i t i o n , c o u p l e d w i t h t h e r e s u l t s o f k i l l i n g e x p e r i m e n t s w i t h p r o t e i n H l - o v e r p r o d u c i n g s t r a i n s , s t r o n g l y s u g g e s t s t h a t t h e s i t e o f a m i n o g l y c o s i d e a c t i v i t y a t t h e o u t e r membrane i s a M g 2 + b i n d i n g s i t e . C o m p e t i t i o n a t t h e l e v e l o f t h e o u t e r m e m b r a n e may w e l l e x p l a i n t h e u n u s u a l l y h i g h a n t a g o n i s m o f a m i n o g l y c o s i d e s b y d i v a l e n t c a t i o n s ( M a d e i r o s e t a l . , 1 9 7 1 ; Z i m e l i s a n d J a c k s o n , 1 9 7 3 ) i n P . a e r u g i n o s a , a l t h o u g h i t i s p r o b a b l e t h a t o t h e r s i t e s o f c o m p e t i t i o n a l s o e x i s t . I t h a s b e e n s h o w n t h a t t h o s e a m i n o g l y c o s i d e s w h i c h a r e h i g h l y a c t i v e a g a i n s t P_. a e r u g i n o s a , s u c h a s g e n t a m i c i n , a r e much m o r e e f f i c i e n t a t i n d u c i n g p e r m e a b i l i z a t i o n t o l y s o z y m e t h a n a r e l e s s a c t i v e a m i n o g l y c o s i d e s s u c h a s s t r e p t o m y c i n ( H a n c o c k , N i c a s a n d R a f f l e , 1 9 8 1 ) . T h e s e r e s u l t s a l s o s u g g e s t t h a t t h e a b i l i t y o f a m i n o g l y c o s i d e s t o p r o m o t e p e r m e a b i l i z a t i o n o f t h e 98 o u t e r m e m b r a n e may i n f a c t b e a m a j o r d e t e r m i n a n t i n t h e i r a c t i v i t y . I n t h i s l i g h t , i t i s i n t e r e s t i n g t h a t some o f t h e new a n t i - P s e u d o m o n a l a m i n o g l y c o s i d e s d i f f e r f r o m t h e i r p a r e n t c o m p o u n d s l a r g e l y i n t h e i r e f f i c i e n c y o f t r a n s p o r t ( L e e e t a _ l . , 1 9 7 8 ) r a t h e r t h a n t h e i r i n h i b i t i o n o f r i b o s o m a l f u n c t i o n . I m p r o v e d a b i l i t y t o i n t e r a c t w i t h t h e o u t e r membrane c o u l d , i n p a r t , a c c o u n t f o r t h e i r i n c r e a s e d t r a n s p o r t . S t u d i e s o f a m i n o g l y c o s i d e t r a n s p o r t i n H I o v e r p r o d u c -i n g s t r a i n s a l s o p r o v i d e e v i d e n c e t h a t t h e o u t e r m e m b r a n e p l a y s a m a j o r r o l e i n a m i n o g l y c o s i d e a c t i v i t y . A m i n o g l y c o s i d e u p t a k e i n b o t h E . c o l i a n d JP. a e r u g i n o s a h a s b e e n s h o w n t o o c c u r i n t h r e e c o n s e c u t i v e p h a s e s ( B r y a n a n d V a n D e n E l z e n , 1 9 7 5 a n d 1 9 7 6 ) : a n i n i t i a l r a p i d e l e c t r o s t a t i c b i n d i n g , f o l l o w e d b y a n e a r l y s l o w u p t a k e p h a s e ( E D P - 1 ) , a n d a l a t e r r a p i d u p t a k e p h a s e ( E D P - I I ) . The b i n d i n g i s e n e r g y i n d e p e n d e n t , w h i l e t h e t w o l a t t e r p h a s e s a r e e n e r g y r e q u i r i n g a n d o c c u r o n l y i n p r e s e n c e o f a n e n e r g i z e d c y t o p l a s m i c m e m b r a n e a n d e l e c t r o n t r a n s p o r t ( B r y a n a n d V a n D e n E l z e n , 1 9 7 5 a n d 1 9 7 6 ) . E D P - I I may c o i n c i d e w i t h o r f o l l o w t h e o n s e t o f l o s s o f v i a b i l i t y s i n c e b o t h E D P - I I a n d l e t h a l i t y c a n b e i n h i b i t e d b y c h l o r a m p h e n i c o l ( B r y a n a n d V a n Den E l z e n , 1 9 7 5 ; H u r w i t z a n d R o s a n o , 1 9 6 1 ) , a n d d o n o t o c c u r r i b o s o m a l l y r e s i s t a n t ( s t r A ) s t r a i n s ( B r y a n a n d V a n D e n E l z e n , 197 5 ) . A l t e r a t i o n o f r i b o s o m a l a f f i n i t y i n o t h e r m u t a n t s a l s o a f f e c t s u p t a k e (Ahmad e t a l • , 1 9 8 0 ) . C o m p a r i s o n o f s t r e p t o m y c i n u p t a k e i n w i l d t y p e s t r a i n s w i t h u p t a k e i n r e s i s t a n t m u t a n t s w h i c h o v e r p r o d u c e p r o t e i n H I , s h o w e d t h a t i n 99 t h e r e s i s t a n t s t r a i n s t h e l a t e r a p i d p h a s e E D P - I I w a s a l w a y s d e l a y e d ( F i g . 2 , 3 ) . I t w o u l d t h u s a p p e a r t h a t t h e o u t e r mem-b r a n e h a s a c r i t i c a l i n f l u e n c e o n t h e e v e n t s r e q u i r e d t o i n i t i -a t e E D P - I I . S o m e w h a t s i m i l a r d e l a y s i n t h e o n s e t o f E D P - I I h a v e b e e n o b s e r v e d f o r c e r t a i n E . c o l i m u t a n t s w i t h a l t e r e d r i b o s o m a l a f f i n i t y t o a m i n o g l y c o s i d e s (Ahmad e t _al_. , 1 9 8 0 ) . H o w e v e r , s i n c e t h e m u t a n t w a s s e l e c t e d f o r r e s i s t a n c e t o p o l y m y x i n a n d E D T A , a n d s i n c e r i b o s o m a l e f f e c t s a r e u s u a l l y s p e c i f i c t o g i v e n a m i n o g l y c o s i d e s ( H a n c o c k , 1 9 8 1 ) , i t i s u n l i k e l y t h a t o u r m u t a n t s a l s o h a v e r i b o s o m a l a l t e r a t i o n s . F u r t h e r m o r e , i t was d e m o n s t r a t e d t h a t s i n g l e s t e p r e v e r t a n t s o f s t r a i n s H 1 8 1 a n d H185 t o p o l y m y x i n s e n s i t i v i t y h a d l o w l e v e l s o f p r o t e i n H I o n M g 2 + - s u f f i c i e n t m e d i u m a s w e l l a s w i l d t y p e a m i n o g l y c o s i d e a n d EDTA s u s c e p t i b i l i t i e s , s u g g e s t i n g t h a t t h e d i f f e r e n t p h e n o t y p i c a l t e r a t i o n s i n t h e m u t a n t s h a d a common b a s i s . The i n c r e a s e i n r e s i s t a n c e t o a m i n o g l y c o s i d e s c o n f e r -r e d b y p r o t e i n H I i s s m a l l e r t h a n t h a t s e e n f o r p o l y m y x i n . T h i s c o u l d b e d u e t o a l t e r n a t i v e b i n d i n g o r u p t a k e s i t e s f o r a m i n o g l y c o s i d e s , a r e q u i r e m e n t f o r f e w e r b i n d i n g s i t e s f o r a m i n o g l y c o s i d e a c t i v i t y , o r t o a l o w e r a f f i n i t y o f a m i n o g l y c o -s i d e s f o r t h e p r o p o s e d L P S b i n d i n g s i t e . S c h i n d l e r a n d O s b o r n e ( 1 9 7 9 ) h a v e d e m o n s t r a t e d t h a t p o l y m y x i n h a s a h i g h e r a f f i n i t y f o r S a l m o n e l l a t y p h i m u r i u m L P S t h a n Mg 2 " 1 " , w h i l e o t h e r s h a v e d e m o n s t r a t e d t h a t m o d e r a t e l y h i g h l e v e l s o f M g 2 + a r e n e c e s -s a r y t o i n h i b i t p o l y m y x i n a c t i v i t y ( D a v i s , 1 9 7 4 ) . I n t h e c a s e o f a m i n o g l y c o s i d e s , a n t a g o n i s m b y M g 2 + o c c u r s a t q u i t e l o w 100 Mg 2 + l e v e l s , and this competition tends to mask the protec-tiv e e f f e c t s of protein HI when the protein is induced by growth in low Mg 2 +. Thus, in MIC measurements of aminogly-coside s u s c e p t i b i l i t y , P. aeruginosa c e l l s in low Mg 2 + were apparently more susceptible than c e l l s in high Mg 2 +. When the d i f f e r e n t i a l competitive effects of Mg 2 + were eliminated by comparison of loss of v i a b i l i t y in a common assay medium (Tables III and V; Hancock, Raffle and Nicas, 1981), c e l l s grown in Mg 2 + - d e f i c i e n t medium were actually more resistant to aminoglycoside k i l l i n g than c e l l s grown in M g 2 + - s u f f i c i e n t medium. The increase in resistance seen in outer membrane protein Hi overproducing strains cannot be attributed to a general decrease in outer membrane permeability. The major pore-forming protein (protein F) was only s l i g h t l y reduced i n amount in protein HI overproducing s t r a i n s . Furthermore, s e n s i t i v i t y to both c a r b e n i c i l l i n and tetracycline [which use the so-called hydrophilic (porin-mediated) pathway in E_. c o l i (Nikaido and Nakae, 1974)] was not altered in these strains (Table IV) and their growth rate on Mg 2 +-deficient media was unaffected suggesting that the porin is functionally normal. The finding that the HI overproducing mutants were unchanged in the i r s e n s i t i v i t y to phages, including LPS recept-or s p e c i f i c phages, also suggested that there are no major surface a l t e r a t i o n s . Normal porin function was confirmed by the lack of s i g n i f i c a n t difference in permeation of n i t r o c e f i n 101 between wild type and protein HI overproducing strains (Table XII). This suggests that the hydrophilic pathway (Nikaido and Nakae, 1974) of passive permeation through the hydrophilic pores formed by porin (Hancock and Nikaido, 1978; Nikaido and Nakae, 1979) may not be the major route taken by aminoglycoside a n t i b i o t i c s in P. aeruginosa. Although the large pore size of P_. aeruginosa (Hancock and Nikaido, 1978) would not be expected to o f f e r any barrier to permeation of such a n t i b i o t i c s , there i s evidence that most of the pores at any given time are not in an active, open state (Benz and Hancock, 1981; and above). However, i t i s possible that in other organisms where a greater proportion of porins are in the active state (Benz e_t a l . , 1980) and there are less Mg2+ binding s i t e s on the c e l l surface (Brown and Melling, 1969), the hydrophilic pathway of a n t i b i o t i c uptake may offer an alte r n a t i v e , e f f i c i e n t means of streptomycin permeation. Evidence for this is provided by the finding of Foulds and Chai (1978), who demonstrated that a porin l a d e f i c i e n t mutant of E. c o l i defective in the hydro-p h i l i c uptake pathway was somewhat more resistant to kanamycin and gentamicin, and studies with E. c o l i omp B mutants (which are d e f i c i e n t in both major porins) have shown a 4-fold reduc-tion in aminoglycoside resistance (V. Raffle, E. Buenaventura, and R.E.W. Hancock, unpublished r e s u l t s ) . Furthermore, strep-tomycin action in other organisms such as E^ . c o l i should be, and i s , less affected by Mg2+ antagonism (Madeiros e_t a l . , 1971) as would be predicted. 102 An alternative explanation for some of the results presented here might be that protein HI i s a magnesium binding outer membrane protein which also binds gentamicin and strepto-mycin and thus s p e c i f i c a l l y l i m i t s access of aminoglycosides to porin. However, s i g n i f i c a n t differences in the binding of streptomycin to mutant st r a i n H181 with high protein HI l e v e l s , when compared to our wild type s t r a i n H103, could not be demon-strated. In addition, at a streptomycin concentration where we could demonstrate a large difference in k i l l i n g of strains H181 and H103 (Hancock, Raffle and Nicas, 1981) less than 0.5% of the added streptomycin become bound to cyanide-treated c e l l s , suggesting that binding did not s i g n i f i c a n t l y a l t e r the effec-ti v e concentration of streptomycin in the medium. Also, mutant strains had less Mg 2 + in their outer membranes, suggesting that protein Hi i s unlikely to be a s p e c i f i c Mg 2 + binding protein. F i n a l l y , the above alternative does not explain aminoglycoside-mediated permeabilization of outer membranes or the unsually high Mg 2 + antagonism of aminoglycosides in wild type P. aeruginosa (Madeiros et a l . , 1971). Thus, the above alternative model seems unlikely, although we cannot rigorously exclude that binding of aminoglycosides to protein HI contributes to the phenotype of the mutants. Resistance to polymyxin acquired during growth under conditions other than low divalent cations has been reported by other workers. G i l l e l a n d and colleagues (Gilleland and Murray, 1976; G i l l e l a n d and Lyle, 1979) and Brown and Watkins (1970) 103 studied P_. aeruginosa trained to grow on very high levels (750 ug/ml) of polymyxin. Such strains exhibit a large number of alterations in phospholipid content, readily extractable l i p i d , wall phosphorous, LPS and envelope protein (Brown and Watkins, 1970; G i l l e l a n d and Lyle, 1979). In addition, u l t r a s t r u c t u r a l alterations in these strains d i f f e r from those of c e l l s grown in Mg2+-deficient medium (Gilleland and Murray, 1976). Thus, this type of resistance cannot be compared to the r e s i s t -ance reported here. P. aeruginosa may also acquire resistance to polymyxin B by growth on branched-chain amino acids and branched-chain facyl derivatives as sole carbon source (Conrad et a l . , 1979). This resistance appears to be related to changes in fatty acid composition of readily extractable l i p i d s (Conrad et a l M 1981), and d i f f e r s from the resistance reported here in that i t does not a f f e c t aminoglycoside s u s c e p t i b i l i t y . Alterations in Mg 2 + content, readily extractable l i p i d and reduction in phospholipid have also been observed in polymyxin resist a n t c l i n i c a l isolates of P. aeruginosa and other gram-negative bacteria (Brown and Wood, 1972) . Yet another mechanism of acquired polymyxin B resistance has been shown in P. fluorescens, where growth under phosphate-limiting condi-tions reduces the amount of membrane phospholipids and brings about the synthesis of a novel cationic l i p i d , ornithine amide l i p i d . It is thus clear that there are a variety of mechanisms for a c q u i s i t i o n of polymyxin resistance, as would be expected for an a n t i b i o t i c which interacts with both outer and inner 104 membrane components to exert i t s l e t h a l effects (Storm ejt a l . , 1977). The common factor in most of these forms of resistance may be the reduction of the amount or a v a i l a b i l i t y of negativ-ely charged membrane components, either on phospholipid or on LPS, with which the a n t i b i o t i c may int e r a c t . The model presented here for protein HI mediated resistance shares this property. In agreement with t h i s , Vaara and co-workers (Vaara et al.., 1979; Vaara, 1981) have reported a class of LPS mutants in Salmonella typhimurium (pmrA) which are polymyxin re s i s t a n t and have LPS with reduced binding a f f i n i t y for polymyxin. No information is available on the aminoglycoside resistance of these s t r a i n s . Membrane changes similar to those reported in this study may also be responsible for the gentamicin r e s i s t -ance of c l i n i c a l isolates of P. aeruginosa with reduced amino-glycoside transport reported by Bryan e_t a l . (1976) and the adaptive resistance to gentamicin and EDTA reported by Pechey and James (1974 ) . C a 2 + , Mn 2 + and S r 2 + were able to substitute for Mg 2 + both in preventing HI induction and in allowing s u s c e p t i b i l i t y to these agents. These four cations showed very similar e f f e c t s , implying that they are equivalent in their a b i l i t y to regulate protein Hi. Regulation of protein HI production by cations could be mediated by a common receptor for these cations. Similar receptor s p e c i f i c i t y has been seen for the chemotaxis receptor for Mg 2 + in S_. typhimurium and E_. c o l i (Koshland, 1979). A l t e r n a t i v e l y , the expression of HI may be 105 sensitive to the t o t a l amounts of divalent cation in the outer membrane i t s e l f , or to divalent cations bound at s p e c i f i c s i t e s in the outer membrane. Alt e r a t i o n of outer membrane protein composition in response to changes in other outer membrane components (Van Alphen e_t a l . , 1976; DiRienzo and Inouye, 1979) i s known to occur in IS. c o l i . The a b i l i t y to respond to condi-tions at the outer membrane by a l t e r a t i o n of outer membrane protein is also seen in the modulation of levels of major outer proteins of E. c o l i in response to an osmotic pressure gradient across the outer membrane (Kawaji e_t a l . , 1979). As c e l l envelopes isolated from c e l l s grown with d i -valent cations other than Mg 2 + contained some Mg 2 +, we considered the p o s s i b i l i t y that the s i t e of a c t i v i t y of amino-glycosides, polymyxins and EDTA could involve this small amount of Mg 2 + rather than a s i t e occupied by other cations. The ef f e c t of EGTA on C a 2 + grown c e l l s , however, strongly suggested that C a 2 + i s replacing Mg 2 + at the target s i t e . EGTA chelates C a 2 + e f f i c i e n t l y but Mg 2 + poorly ( K e f f 10.4 vs. 4.7, Roberts et a l . , 1970). C a 2 + grown c e l l s appeared as sensitive to the outer membrane effects of EGTA as they are to those of EDTA, as shown both by the di r e c t measurement of l y s i s and by increase in the rate of hydrolysis of n i t r o c e f i n . EGTA, however, did not have the high b a c t e r i -c i d a l a c t i v i t y of EDTA, suggesting that additional s i t e s are involved in the b a c t e r i c i d a l action of EDTA. Other workers (Boggis et a l . , 1979) have noted that s e n s i t i v i t y to l y s i s by 106 EGTA i s d e p e n d e n t o n t h e a m o u n t o f C a 2 + p r e s e n t i n t h e g r o w t h m e d i u m , b u t t h a t s e n s i t i v i t y t o EGTA i s d e c r e a s e d i f M g 2 + a s w e l l a s C a 2 + i s p r e s e n t , s u g g e s t i n g t h a t e i t h e r c a t i o n may o c c u p y t h e s i t e a t t a c k e d b y t h e t w o c h e l a t o r s . T h e n u m b e r o f c a t i o n b i n d i n g s i t e s i n v o l v e d i n t h e a c t i o n o f E D T A , p o l y m y x i n a n d g e n t a m i c i n o n t h e o u t e r m e m b r a n e may w e l l r e p r e s e n t a s m a l l p r o p o r t i o n o f t h e t o t a l n u m b e r o f s i t e s . S c h i n d l e r a n d O s b o r n ( 1 9 7 9 ) h a v e d e m o n s t r a t e d a t l e a s t t w o c l a s s e s o f C a 2 + a n d M g 2 + b i n d i n g s i t e s o n t h e l i p o -p o l y s a c c h a r i d e o f S a l m o n e l l a t y p h i m u r i u m w i t h w i d e l y d i f f e r i n g a f f i n i t i e s , w h i l e o n l y h i g h a f f i n i t y b i n d i n g s i t e s f o r p o l y m y x -i n B w e r e f o u n d . The i n v o l v e m e n t o f a s m a l l n u m b e r o f s i t e s i s s u g g e s t e d b y t h e d i s p l a c e m e n t o f o n l y 3 . 5 t o 10% o f t h e c e l l e n v e l o p e M g 2 + f r o m c e l l s t r e a t e d w i t h a m i n o g l y c o s i d e s o r p o l y m y x i n B . A r e l a t i v e l y s m a l l p r o p o r t i o n o f c r i t i c a l t a r g e t s i t e s f o r p o l y m y x i n s , E D T A - T r i s , a n d g e n t a m i c i n , c o u l d a l s o e x p l a i n why c e l l s g r o w n i n Z n 2 + a r e n o t s e n s i t i v e d e s p i t e t h e h i g h l e v e l s o f Z n 2 + i n c e l l e n v e l o p e s a n d t h e v e r y h i g h e f f i c i e n c y o f Z n 2 + c h e l a t i o n b y EDTA . I t was o b s e r v e d t h a t Z n 2 + g r o w n c e l l s h a d h i g h l e v e l s o f p r o t e i n H I , a n d t h a t t h e i r l e v e l o f d i v a l e n t c a t i o n s w a s 1 5 - 3 5 % l o w e r t h a n t h e l e v e l f o u n d i n c e l l s g r o w n i n C a 2 + o r M n 2 + a n d 8% l o w e r t h a n c e l l s g r o w n i n 0 . 5 mM M g 2 + . I t may b e t h a t o n l y t h o s e d i v a l e n t c a t i o n b i n d i n g s i t e s w h i c h a r e s e n s i t i v e t o a t t a c k b y c a t i o n i c a n t i b i o t i c s a r e p r o t e c t e d b y p r o t e i n H I , a n d t h e r e m a i n i n g s i t e s w h i c h c a n b e o c c u p i e d b y Z n 2 + ( o r i n d e e d 107 any of the divalent cations studied) are not required for membrane s t a b i l i t y . This would suggest that these c r i t i c a l divalent cation binding s i t e s can only be occupied by s p e c i f i c divalent cations, i . e . , Mg 2 +, C a 2 + , S r 2 + , or Mn 2 +. Al t e r n a t i v e l y , protein HI could only displace divalent cations from these s i t e s when induced by l i m i t a t i o n of these divalent cations. Although there appears to be a linear re c i p r o c a l relationship between the decrease in Mg 2 + l e v e l s and increase in protein HI in the c e l l envelope of wild type c e l l s , i t would appear that resistance to chelators and catio n i c a n t i b i o t i c s correlates more closely with the presence and amounts of protein HI than with absolute "levels of divalent cations in the c e l l envelope. This i s supported by studies of the mutant strains r e s i s t a n t to these agents, which overproduce protein HI to about 7-fold wild type levels in 0.5 mM Mg 2 + but show a reduction of less than 2-fold in their envelope Mg 2 + l e v e l s . Protection of a r e l a t i v e l y small proportion of divalent cation binding s i t e s would thus appear to be s u f f i c i e n t to confer resistance. In summary, i t is concluded that the outer membrane of P. aeruginosa i s a major determinant of the a n t i b i o t i c resistance of this bacterium. Two separate l i n e s of evidence support t h i s conclusion: 1) Alteration of the outer membrane, for example, by growth in low Mg 2 + or by mutation to HI overproduction, a l t e r s resistance to cationic a n t i b i o t i c s and chelators. The data presented here suggest that these agents 108 are e f f e c t i v e against P_. aeruginosa because they are able to a c t i v e l y disrupt the outer membrane; and 2) The outer membrane of P. aeruginosa has been shown to be r e l a t i v e l y impermeable despite the greater channel size of i t s porins, possibly due to there being a r e l a t i v e l y small number of active, functional, channels. Previous studies with other bacteria have suggested two major pathways for a n t i b i o t i c uptake across the outer membrane. These are the hydrophilic or porin-mediated pathway and the hydrophobic pathway which is apparently r e s t r i c t e d to deep rough mutants of E. c o l i and Salmonella. 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