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Phosphate transport across the outer membrane of Pseudomonas aeruginosa Poole, Raymond Keith 1986

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PHOSPHATE TRANSPORT ACROSS THE OUTER MEMBRANE OF PSEUDOMONAS AERUGINOSA by RAYMOND KEITH POOLE B.Sc, The University of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFULLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA ^ A p r i l 1986 (c) Raymond Keith Poole, 1986 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) ABSTRACT When wild-type c e l l s of Pseudomonas aeruginosa were grown in a phosphate-limiting medium (0.2 mM orthophosphate) they were derepressed for the production of an outer membrane protein, designated protein P. This protein was p u r i f i e d to homogeneity and demonstrated to form channels in planar l i p i d bilayer membranes. In agreement with previous data, the channels formed by protein P were anion-specific (due to the presence of a binding s i t e for anions in the channel) and exhibited a marked s e l e c t i v i t y for phosphate 2-(HP04 ) over other anions (e.g chloride). These properties were not altered in protein P preparations p u r i f i e d free of lipopolysaccharide. Protein P was coinducible with the enzymes alkaline phosphatase and phospholipase C, and with a periplasmic protein of 34,000 molecular weight. Mutants of P.  aeruginosa, constitutive or non-inducible for these constituents, were isolated. This suggested that the genes encoding these products were part of a phosphate regulon. Alkaline phosphatase and phospholipase C were demonstrated to be secreted into the external medium upon induction, although this e x t r a c e l l u l a r release was s p e c i f i c and did not involve an increase in outer membrane permeability. The 34K periplasmic protein was p u r i f i e d and demonstrated to bind phosphate in v i t r o (Kd=0.34 uM). S p e c i f i c i t y studies revealed that inorganic phosphate polymers (up to P15) and i i arsenate could i n h i b i t the binding of orthophosphate to the binding protein, although organic phosphates (e.g. glucose-6-phosphate) could not. The a b i l i t y of the phosphate-binding protein and protein P to associate was demonstrated in v i t r o , with implications concerning the means by which phosphate crosses the outer membrane. Two major inorganic phosphate transport systems were identifed, of low (Km=l9.3 uM phosphate) and h i g h - a f f i n i t y (Km=0.39 uM phosphate), respectively. Mutants de f i c i e n t in the phosphate-binding protein were isolated and shown to lack the h i g h - a f f i n i t y phosphate uptake system, confirming the role of the binding protein in h i g h - a f f i n i t y phosphate transport in P. aeruginosa. In addition, a role for protein P in h i g h - a f f i n i t y phosphate transport was confirmed by the i s o l a t i o n of a Tn501 insertion mutant lacking porin protein P. This mutant exhibited a ten-fold increase in Km for h i g h - a f f i n i t y phosphate transport. The loss of these proteins in the respective mutants was correlated with a growth defect in a phosphate-deficient medium. Protein P, l i k e most porins, was isolated as an oligomer (trimer) in i t s native (functional) state, dissociating to non-functional monomers at high temperatures. A polyclonal antiserum s p e c i f i c for protein P trimers was raised and shown to cross-react with other phosphate-starvation-inducible outer membrane proteins of the families Pseudomonadaceae and Enterobactereaceae. This c r o s s - r e a c t i v i t y was observed only with the native, oligomeric forms of these proteins. No cr o s s - r e a c t i v i t y was seen with the constitutive porins produced by these strains, indicating that the cr o s s - r e a c t i v i t y of phosphate-limitation-inducible oligomeric outer membrane proteins was not due to any homologies r e l a t i n g to porin structure in general. Using a polyclonal antiserum s p e c i f i c for protein P monomers, no r e a c t i v i t y was observed with either the oligomeric or monomeric forms of any of the phosphate-limitation-inducible outer membrane proteins (except for protein P monomers). These data suggested that the common antigenic determinants present in these proteins were conserved in the native functional proteins only. Examination of some of the physical properties of the phosphate-starvation-inducible outer membrane proteins (e.g. molecular weight, peptidoglycan association, detergent s o l u b i l i t y ) revealed that these proteins could be grouped into two classes, represented by protein P of P. aeruginosa and protein PhoE of Escherichia c o l i . Those proteins resembling protein P were i d e n t i f i e d in members of the fluorescent Pseudomonads, including P. putida, P.  fluorescens, P. aureofaciens and P. chlororaphis. The pu r i f i e d proteins formed small, anion/phosphate-selective channels in planar l i p i d bilayers which were quite similar to protein P channels. iv TABLE OF CONTENTS PAGE Abstract i i Table of Contents v L i s t of Figures . x L i s t of Tables x i i i L i s t of Abbreviations xv Acknowledgements xvi I n t r o d u c t i o n 1 1 . The Gram-negative c e l l envelope • . . 1 a. The cytoplasmic membrane 1 b. The peptidoglycan 2 c. The outer membrane 3 2. The role of the outer membrane in transport . . . 6 a. The LamB protein 8 b. The PhoE protein 11 c. Iron-regulated outer membrane proteins . . .13 d. Others 14 3. Bacterial phosphate transport - with s p e c i f i c reference to E. c o l i 15 4. The pho regulon of E. c o l i 18 Methods 22 1. Media and growth conditions 22 2. Bacterial strains 23 v 3. C e l l fractionation and sodium dodecyl sulphate-polyacrylamide gel electrophoresis 23 4. P u r i f i c a t i o n of protein P 28 5. Acetylation of protein P 29 6. Immunological methods . 30 7. Preparation of protein P-phosphatidyl choline vesicles 30 8. Preparation of protein a f f i n i t y columns 31 a. Protein F-Sepharose 31 b. Protein P-Affigel-10 31 c. Phosphate-binding protein-Sepharose . . . .32 9. Preparation of protein P-specific antisera . . .32 a. Trimer-specific 32 b. Monomer-specific 34 c. Antiserum to protein P in phosphatidyl choline vesicles 35 10. Isolation of a protein P-deficient mutant . . . .35 a. Tn501 insertion mutagenesis 35 b. Selection of a protein P-deficient mutant using a protein P trimer-specific antiserum 36 11. Phosphate transport assays 37 12. Enzyme assays 38 13. Nitrocefin permeability assay 39 14. Osmotic shock and p u r i f i c a t i o n of the phosphate-binding protein 40 15. F i l t e r assay of phosphate binding 41 16. Equilibrium d i a l y s i s 42 17. Isolation of mutants lacking the phosphate-binding protein 42 vi 18. Construction of a rabbit anti-protein P immunoadsorbant column 44 19. Electrophoretic elution of protein P from SDS-polyacrylamide gels 44 20. P u r i f i c a t i o n of phosphate-starvation-inducible outer membrane proteins of the fluorescent Pseudomonads .45 21. Black l i p i d bilayer experiments 47 22. Modified ELISA procedure for demonstrating an association between protein P and the phosphate-binding protein 48 a. Preparation of protein P 48 b. Modified ELISA procedure 49 23. A f f i n i t y chromatography method for determining an association between protein P and the phosphate-binding protein 50 a. Phosphate-binding protein-Sepharose 4B a f f i n i t y column .50 b. Protein P-Affigel-10 a f f i n i t y column . . . .50 24. Isolation of regulatory mutants of alkaline phosphatase and phospholipase C 51 25. Other assays 52 Chapter One Outer membrane protein P: involvement in hi g h - a f f i n i t y phosphate transport in Pseudomonas aeruginosa S3 1. Induction of protein P by phosphate l i m i t a t i o n .53 2. Co-regulation with alkaline phosphatase, phospholipase C and a 34K periplasmic protein . .60 3. Outer membrane permeability 63 4. LPS-free protein P forms channels in planar l i p i d bilayer membranes 69 5. Isolation of a protein P-deficient mutant . . . .73 a. Preparation of a protein P trimer-specific antiserum 73 vi i b. Tn501 mutagenesis of P. aeruginosa 75 c. Isolation of a Tn501-induced protein P-deficient mutant 80 6. Phosphate transport 82 7. Growth in low phosphate medium 83 8. Summary 85 Chapter Two Role of a per.iplasmic phosphate-binding protein in phosphate transport in Pseudomonas aeruginosa 89 1. P u r i f i c a t i o n and properties of the periplasmic phosphate-binding protein 89 2. Isolation of mutants lacking the phosphate-binding protein 95 3. Phosphate transport 97 4. Kinetics of phosphate transport . . . . . . . . 100 5. Growth in phosphate-deficient medium . . .- . . 103 6. Physical association between outer membrane protein P and the periplasmic phosphate-binding protein 103 7. Summary 109 Chapter Three Immunological cr o s s - r e a c t i v i t y of phosphate-starvation-inducible outer membrane proteins of the families Enterobacteriaceae and Pseudomonadaceae 111 1. Phosphate starvation-induction of membrane proteins of the Pseudomonadaceae and the Enterobacter iaceae . . 111 2. Immunological c r o s s - r e a c t i v i t y of phosphate-starvation-inducible outer membrane proteins . 118 a. Cross-reactivity of protein oligomers in phosphate-limited c e l l envelopes 118 b. I d e n t i f i c a t i o n of the cross-reactive proteins 124 v i i i c. Cross-reactivity of phosphate-starvation-induced monomers 129 3. Summary 130 Chapter Four Characterization of protein P-like porins from the fluorescent Pseudomonadaceae 132 1. P u r i f i c a t i o n of the phosphate-starvation-inducible outer membrane proteins of the fluorescent Pseudomonads 132 2. Single channel experiments 135 3. Ion-selectivity .142 4. Phosphate i n h i b i t i o n of macroscopic conductance 145 5. Summary 152 D i s c u s s i o n 154 .1. . A phosphate regulon in Pseudomonas aeruginosa . 154 2. Properties of outer membrane protein P . . . . 158 3. The outer membrane of Pseudomonas aeruginosa as a permeability barrier to phosphate under l i m i t i n g conditions 162 4. Protein P and PhoE as members of two d i s t i n c t classes of phosphate-regulated porins 167 5. Conserved antigenic determinants in phosphate-starvation-inducible outer membrane (porin) proteins 173 Literature Cited 176 ix LIST OF FIGURES Growth of P. aeruginosa in a phosphate-deficient medium Growth y i e l d of P. aeruginosa as a function of the concentration of phosphate in a defined minimal medium SDS-polyacrylamide gel electrophoretogram of p u r i f i e d protein P and of outer membanes and shock f l u i d s of phosphate-deficient c e l l s of P. aeruginosa Induction by phosphate l i m i t a t i o n and l o c a l i z a t i o n of alkaline phosphatase and phospholipase C of P. aeruginosa Hi 03 SDS-polyacrylamide gel electrophoretogram of whole c e l l protein extracts and c e l l envelope and soluble (non-membrane) fractions of alkaline phosphatase regulatory mutants Outer membrane permeability during growth on phosphate-deficient medium SDS-polyacrylamide gel electrophoretogram of LPS associated with protein P Immunoblots of electrophoretically separated P. aeruginosa H103 c e l l envelopes and p u r i f i e d protein P, and whole c e l l s SDS-polyacrylamide gel electrophoretogram of outer membranes prepared from a protein P-def i c i e n t mutant of P. aeruginosa and i t s wild-type parent Induction of the 34K periplasmic protein by phosphate li m i t a t i o n 11 SDS-polyacrylamide gel electrophoretogram of p u r i f i e d phosphate-binding protein and whole c e l l protein extracts of alkaline phosphatase constitutive mutants of P. aeruginosa H242 91 12 Scatchard plot of phosphate-binding a c t i v i t y 93 13 Phosphate uptake in P, aeruginosa 99 14 Kinetics of phosphate uptake in P. aeruginosa 102 15 Growth of a phosphate-binding protein-deficient mutant in a phosphate-limited medium 105 16 SDS-polyacrylamide gel electrophoretogram of c e l l envelopes prepared from phosphate-deficient and phosphate-sufficient grown strains of the families Pseudomonadaceae and Enterobactereaceae 116 17 Interaction of protein P trimer-specific or monomer-specific antiserum with Western blots of p u r i f i e d protein P and P. aeruginosa PA01 str a i n H103 c e l l envelopes 120 18 Interaction of protein P trimer-specific antiserum with Western blots of c e l l envelope preparations of d i f f e r e n t bacteria grown under phosphate-deficient or s u f f i c i e n t conditions 122 19 Two-dimensional (unheated x heated) SDS-polyacrylamide gel electrophoretogram of p u r i f i e d protein P and c e l l envelopes prepared from phosphate-limited strains of the Pseudomonadaceae and the Enterobacteriaceae 126 20 SDS-polyacrylamide gel electrophoretogram of p u r i f i e d phosphate-starvation-inducible outer membrane proteins of the fluorescent Pseudomonadaceae 134 xi 21 S t r i p chart recordings of stepwise increases in the conductance of an oxidized cholesterol membrane caused by the phosphate-starvation-inducible outer membrane protein of P. putida 138 22 Histogram of the conductance fluctuations observed with membranes of oxidized cholesterol in the presence of the phosphate-starvation-inducible outer membrane protein of P. putida 140 23 Average single channel conductance of the phospate-starvation-inducible porin protein of P. aureofaciens as a function of the KC1 concentration in the aqueous solution bathing an oxidized cholesterol membrane 147 24 Phosphate i n h i b i t i o n of chloride flux through protein P channels 151 x i i LIST OF TABLES PAGE Bacterial strains 24 II Measurements of LPS associated with conventionally p u r i f i e d and electroeluted protein P 71 III Functional properties of conventionally p u r i f i e d and electroeluted protein P in planar l i p i d bilayer membranes 72 IV Plasmids tested for u t i l i t y in transposon insertion mutagenesis of P. aeruginosa 77 V Kinetics of h i g h - a f f i n i t y phosphate transport in a protein P-deficient mutant st r a i n and i t s wild-type parent 84 VI Growth of a protein P-deficient mutant and strains wild-type for protein P in a phosphate-limited medium 86 VII Substrate s p e c i f i c i t y of the phosphate-binding protein 94 32 VIII P-orthophosphate binding by periplasmic extracts of wild-type and mutant strains of P. aeruginosa 96 IX In v i t r o association of the phosphate-binding protein and outer membrane protein P 107 X Properties of the phosphate-starvation-inducible membrane proteins of the Enterobacteriaceae and the Pseudomonadaceae 113 XI Channel-forming properties of a f f i n i t y - p u r i f i e d and electroeluted phosphate-starvation-inducible outer membrane oligomers of the fluorescent Pseudomonads 141 xi i i XII Single channel conductance of phosphate-starvation-inducible porin proteins of the fluorescent Pseudomonads in salt s of varying anion and cation size 144 XIII Binding a f f i n i t i e s of phosphate-starvation-inducible porin proteins of the fluorescent Pseudomonads for chloride and orthophosphate 148 xiv LIST OF ABBREVIATIONS A405 / A600 Absorbance at 405/600 nm Cb C a r b e n i c i l l i n EDTA Ethylenediaminetetraacetate FCS Fetal calf serum Hepes N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate Kd dissociation constant KDO 2-keto-3-deoxy octulosonic acid Km Michaelis constant Kn Kanamycin LPS Lipopolysaccharide NPPC para-Nitrophenyl phosphorylcholine P1 5 phosphate polymer comprising 15 phosphate.units PBS Phosphate buffered saline pNPP para-Nitrophenyl phosphate SDS Sodium dodecyl sulphate Tc Tetracycline Tp Trimethoprim Tr i s Tris(hydroxymethyl)aminomethane XP 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine X V ACKNOWLEDGEMENTS F i r s t l y , I thank Bob Hancock, who should already know that he has my warmest appreciation for the guidance, encouragement and friendship he has shown me. As well, my labmates, past and present, have my enduring thanks for their camaraderie and fellowship, without which this experience would have been sorely lacking. To J e r r i , my kindred s p i r i t , whom I f i r s t met at the outset of thi s -long journey, I am grateful, for helping me maintain a semblance of sanity in the insane world that i s graduate school. Without her understanding, patience and unf a i l i n g confidence in me I could not have come so far. Lastly, I thank my family, especially my mom and dad, who have always encouraged but never pushed me, and who have always shown an interest in my work, even when i t was a l l 'Greek' to them. xvi INTRODUCTION Nutrient acquisition by prokaryotic organisms necessarily involves transmembrane translocation of solute molecules. In the case of gram-negative organisms there are two membranes which must be traversed during the unidirectional movement of nutrient molecules from the environment to the c e l l i n t e r i o r . The mechanisms by which thi s unidirectional transport occur are s p e c i f i c to the membranes being traversed. 1. The gram-negative c e l l envelope. Electron microscopic studies have confirmed that the c e l l envelope of gram-negative bacteria, including Psedomonas aeruginosa, consists of three layers, the cytoplasmic or "inner" membrane, the peptidoglycan or murein layer and the outer membrane (Lugtenberg and van Alphen, 1983). In some instances, and in s p e c i f i c strains, a capsule and/or additional (A)-layer i s also present external to the t r i p a r t i t e c e l l envelope (Glauert and Thornley, 1969; Schleytr, 1978; Troy, 1979). a. The cytoplasmic membrane. The membrane bounding the cytoplasm i s comprised of approximately equimolar amounts of phospholipid and protein in a t y p i c a l l i p i d bilayer (Lugtenberg and van Alphen, 1983). The hydrophobicity of this membrane (Machtiger and Fox, 1973) makes i t a barrier to hydrophilic molecules although hydrophobic molecules diffuse r e l a t i v e l y freely across i t 1 (Teuber et a l • , 1977). As such, the inner membrane functions as a highly specfic permeability barrier to hydrophilic molecules, with solute translocation dependent upon the presence of s p e c i f i c energy-requiring transport systems in the membrane (Wilson, 1978). In certain transport systems, water-soluble binding proteins present in the space between the inner and outer membrane (the periplasm (Mitchell, 1961)) function in concert with inner membrane transport proteins in the uptake of solute molecules (Oxender, 1972; Wilson and Smith, 1978; Hoshino and Nishio, 1982; Eisenberg and Phibbs, 1982). The energy for nutrient uptake i s derived from the electrochemical gradient of protons across the inner membrane (the proton motive force) generated from the primary active transport of H+ ions during respiration or ATP hydrolysis and/or from phosphate-bond energy in the form of ATP or related metabolites (Berger and Heppel, 1974; Harold, 1977; Hong et a l . , 1979). b. The peptidoqlycan. The peptidoglycan exists as a network of linear amino sugars (N-acetyl glucosamine and N-acetyl muramic acid) covalently linked via cross-bridges between tetrapeptides attached to N-acetyl muramic acid (Schleifer and Kandler, 1972). O r i g i n a l l y described as a r i g i d monolayer present in the periplasmic space (Braun et a l . , 1973) the peptidoglycan of enteric organisms has recently been suggested to exist as a hydrated 'gel' which probably occupies the entire space between the outer and cytoplasmic membranes (Hobot et a l . , 1984). In addition, 2 the peptidoglycan is covalently cross-linked to the outer membrane via a protein analogous to the major lipoprotein of Escherichia c o l i (Braun, 1975; Lugtenberg et a l . , 1977). Although the peptidoglycan of P. aeruginosa is not chemically di f f e r e n t from that of enteric organisms (Meadow, 1975), i t i s probably not covalently linked to the outer membrane (Hancock et a l . , 1981a). There is no evidence to suggest that the peptidoglycan provides a barrier to solute molecules during transport, although, together with membrane-derived oligosaccharides (MDOs) (van Golde et a l . , 1973) i t provides fixed l o c a l i z e d charges within the periplasm which gives r i s e to a Donnan potential across the outer membrane (inside negative)(Stock et a l . , 1977). This potential may play a role in the movement of appropriately charged substrate molecules across the outer membrane (Costerton, 1970). c. The outer membrane. The outer membrane i s comprised of phospholipids, protein and lipopolysaccharide (LPS) a l i p i d i c molecule unique to gram-negative bacteria (Lugtenberg and van Alphen, 1983). LPS i s an amphipathic molecule possessing a hydrophobic portion, l i p i d A, embedded in the membrane, and a hydrophilic polysaccharide portion extending out from the c e l l surface (Luderitz et a l . , 1982). The d i s t a l portion of the polysaccharide, the 0 antigen, usually consists of repeating oligosaccharide units which exhibit wide v a r i a b i l i t y even within a single species (Luderitz e t a l . , 1982). Like the inner membrane, the outer 3 membrane appears as a bilayer in the electron microscope (Glauert and Thornley, 1969). However, the outer membrane is unusual in that the phospholipid i s present exclusively (except, perhaps, in certain mutants) in the inner l e a f l e t of the bilayer, while es s e n t i a l l y a l l of the LPS occurs in the outer l e a f l e t (Muhlradt and Golecki, 1975; Funahara and Nikaido, 1980). The proteins of the outer membrane occur in both l e a f l e t s and in some cases actually span the entire membrane (Lugtenberg and van Alphen, 1983). Unlike the inner membrane, the outer membrane lacks a hydrophobic uptake pathway (Nikaido, 1976), probably due to the presence of negatively charged LPS molecules on i t s outer surface, and fuctions as a non-specific permeability barrier to hydrophilic molecules (Nikaido, 1979). In the case of enteric bacteria this serves to protect the organisms from the detergent-like actions of b i l e s a l t s , fatty acids and glycerides, as well as from proteolytic and l i p o l y t i c enzymes and glycosidases present in the gut (Lugtenberg and van Alphen, 1983). The P. aeruginosa outer membrane, which has been implicated in the high i n t r i n s i c resistance of this organism to a n t i b i o t i c s (Angus et a l . , 1982; Yoshimura and Nikaido, 1982; Nicas and Hancock, 1983), may well serve a similar role in nature since P. aeruginosa is commonly found in the s o i l where a n t i b i o t i c producing organisms are also found. 4 Hydrophilic solute molecules below a defined molecular weight cutoff (the exclusion l i m i t ) are capable of permeating the outer membrane via a passive d i f f u s i o n process (Nikaido, 1979) mediated by a class of proteins of molecular weights 35,000-45,000, ca l l e d porins. These proteins form w a t e r - f i l l e d channels through the hydrophobic core of the outer membrane (Hancock et a l . , 1979; Nikaido, 1979). Porins exist in the outer membranes as trimers (Tokunaga et a l . , 1979; Angus et a l . , 1983; Maezawa et a l . , 1983), are non-covalently attached to the peptidoglycan (Lugtenberg et a l . , 1977; Hancock et a l . , 1981a) and are usually isolated in association with LPS (Furukawa et a l . , 1979; Schindler and Rosenbusch, 1978). LPS association is not, however, essential for porin function in v i t r o (Parr et. a l . , in press) although i t has been proposed to be involved in modulating in vivo porin a c t i v i t y (Kropinski et a l . , 1982). The exclusion l i m i t of the P. aeruginosa outer membrane is s i g n i f i c a n t l y larger than that of E. c o l i or S.  typhimurium outer membranes (Mr 3,000-9,000 compared with 600-700) ( Nakae and Nikaido, 1975; Nakae, 1976; Hancock and Nikaido, 1978), an observation consistent with the formation in model systems of larger channels by the major porin protein F of P. aeruginosa (2.2 nm dia.) than by proteins OmpF (1.1 nm) and OmpC (1.0 nm), the major E. c o l i porins (Benz et a l . , 1985). Interestingly, however, the outer membrane of P. aeruginosa is less permeable than that of 5 E. c o l i (Angus et a l . , 1982; Yoshimura and Nikaido, 1982; Nicas and Hancock, 1983), which has lead to the suggestion that only a small percentage of protein F molecules form fuctional pores in vivo. 2. The role of the outer membrane in transport. The permeability of the outer membrane, mediated by porins, provides a pathway for the entry of nutrient molecules (e.g. sugars, amino acids, nucleosides, ions). Because porins in general are non-specific, exhibiting only weak ion-s e l e c t i v i t y in reconstituted planar bilayer membranes (Benz et a l . , 1985), nutrient molecules cross the outer membrane down their respective concentration gradients. The rate of dif f u s i o n (V) of solute molecules across the outer membrane can be described by the equation V = P x A x [C - C^3 (Fick's f i r s t law of diffusion) where P i s the permeability c o - e f f i c i e n t , A i s the area of the membrane and C and C o 1 represent the concentration of substrate outside (in the external medium) and inside (in the periplasm) the c e l l , respectively (Yoshimura and Nikaido, 1982). The presence of hi g h - a f f i n i t y periplasmic binding proteins, and of cytoplasmic membrane transport systems of low Km, functions to maintain low periplasmic concentrations of these solutes so that, in the presence of s u f f i c i e n t e x t r a c e l l u l a r concentrations of these nutrients, this gradient i s usually s u f f i c i e n t to transport levels of nutrients which are not li m i t i n g for transport or growth. However, s u f f i c i e n t l y low 6 external concentrations of nutrient molecules or a decrease in outer membrane permeability 'resulting from, for example, the porin-deficiency of mutant strains (Von Meyenburg, 1971; Lutkenhaus, 1977; Nicas and Hancock, 1983), have been demonstrated to decrease the rate of solute movement across the outer membrane such that the overall rates of transport (Lutkenhaus, 1977) and growth (Von Meyenburg, 1971) are limited by diffusion across the outer membrane. In the case of permeability mutants, reduction of outer membrane permeability increases the Km of the overall transport process despite the fact that upon d i f f u s i o n across the outer membrane a substrate i s transported across the cytoplasmic membrane via a sp e c i f i c system with a very low Km. Nonetheless, the effect of porin-deficiency on transport and growth is seen only at lower concentrations of substrate since i t i s th e o r e t i c a l l y ("from Pick's f i r s t law) and p r a c t i c a l l y (Von Meyenburg, 1971; Lutkenhaus, 1977) possible to restore a non-limiting rate of d i f f u s i o n across the outer membrane of porin-deficient mutants simply by increasing the external substrate concentration. Pseudorevertants of porin-deficient mutants which express novel porin proteins (Henning et a l . , 1977; Von Meyenburg and Nikaido, 1977; Van Alphen et a l . , 1978; Pugsley and Schnaitman, 1978; Chai and Foulds, 1979) restore the c e l l ' s a b i l i t y to transport non-limiting concentrations of solute across the outer membrane, in the presence of s u f f i c i e n t external concentrations of nutrient molecule. 7 Similarly, the ra t e - l i m i t i n g d i f f u s i o n of nutrient molecules across the outer membrane resulting from low extr a c e l l u l a r • nutrient concentrations i s compensated for in some bacteria by the synthesis of inducible outer membrane proteins which function in the f a c i l i t a t e d uptake of the l i m i t i n g nutrient. Examples include the phosphate-starvation-inducible PhoE proteins of the Enterobacteriaceae (Overbeeke and Lugtenberg, 1980; Sterkenburg et a l . , 1984; Bauer et a l . , 1985) and the iron regulated outer membrane proteins found in many b a c t e r i a l species (Ernst et a l . , 1978; Braun and Hantke, 1982; Sciortino and Finkl e s t e i n , 1983; Williams et a l . , 1984; Brown et a l . , 1984). In addition, novel membrane proteins are sometimes produced in cases where the nutrient molecule permeates the outer membrane poorly via the major porin proteins. Such proteins include the maltose/maltodextrin LamB (Ferenci and Boos, 1980) and the Tsx nucleoside transport (Hantke, 1976) proteins of E. c o l i . a. The LamB protein. Although inducible in E.  c o l i strains grown in non-limiting concentrations of maltose (Braun and Krieger-Bauer, 1977), the LamB protein, which functions as the phage lambda receptor, appears to be an essential component of maltose transport at low (< 10 uM) -but not at high (> 1 mM) concentrations of maltose (Szmelcman and Hofnung, 1975; Szmelcman et a l . , 1976). Studies involving the reconstitution of the p u r i f i e d protein into liposomes have demonstrated that the LamB protein indeed exhibits a marked preference for maltose over other 8 disaccharides, f a c i l i t a t i n g the d i f f u s i o n of maltose into lipososmes 40 times faster than, for example, sucrose (Luckey and Nikaido, 1980a). The LamB protein also serves as an e f f i c i e n t channel for the uptake of maltodextrins (up to maltoheptaose) which exceed the exclusion l i m i t s of the major E. c o l i porins (Luckey and Nikaido, 1980a; Nakae and I s h i i , 1980). Mutants deficient in LamB protein exhibit a 1000-fold increase in the Km for maltose transport (from 1.0 uM to 1 mM) (Szmelcman et a l , 1976) and are severely defective in transporting maltotriose (Szmelcman et a l , 1976), while maltotetraose and higher molecular weight maltodextrins are not accumulated at a l l (Ferenci, 1980). The a f f i n i t y of the channel for maltose and maltodextrins increases with increasing chain length of the dextrin (Km for maltose=14 mM; Km for maltodecanose=75 uM) (Ferenci e_t a l . , 1980) and has been attributed to binding s i t e s in/near the channel (Ferenci et a l . , 1980; Luckey and Nikaido, 1980b). The LamB protein is a component of a maltose operon in E. c o l i which includes, in addition to inner membrane transport proteins and cytoplasmic catabolic enzymes, a periplasmic h i g h - a f f i n i t y maltose binding protein (Dietzel et a l . , 1978). The binding protein was demonstrated to bind maltose and maltodextrins in the micromolar range (Wandersmann et a l . , 1979) in agreement with the observed kinetics of transport (Ks = 1.0 uM) (Szmelcman et a l . , 1976). Furthermore, a physical association between the 9 binding protein and the LamB porin was observed in v i t r o (Bavoil and Nikaido, 1981) confirming electron microscopic data which showed that the maltose-binding protein associated with the periplasmic face of LamB-containing outer membranes (Boos and Staehlin, 1981). Such an association was suggested to be necessary for the e f f i c i e n t transport of maltose and maltodextrins across the outer membrane in vivo (Wandersman et a l . , 1979; Luckey and Nikaido, 1983). In addition to i t s role in maltose and maltodextrin uptake, the LamB channel functions as a general d i f f u s i o n channel as well. In v i t r o studies have confirmed the a b i l i t y of a number of amino acids and unrelated sugars (Nakae, 1979; Luckey and Nikaido, 1980a) as well as ions (Boehler-Kohler et a l . , 1979) to permeate the channel. LamB may also be capable of replacing the major porins in vivo in revertants of porin-deficient mutants (Von Meyenburg and Nikaido, 1977). An analogous protein, designated protein D1, has been i d e n t i f i e d in the outer membrane of P. aeruginosa c e l l s growing in glucose-containing media (Hancock and Carey, 1979). Co-regulated with a binding protein-dependent high-a f f i n i t y glucose transport system (Midley and Dawes, 1973; Stinson et a l . , 1977), th i s 46,000 molecular weight protein forms channels in liposomes which are s e l e c t i v e l y permeable to glucose (Hancock and Carey, 1980). 10 b. The PhoE protein. Inducible in E. c o l i under conditions of phosphate-limitation (Overbeeke and Lugtenberg, 1980), porin protein PhoE was f i r s t i d e n t i f i e d in revertants of porin-deficient mutants (Henning et a l . , 1977; Van Alphen et a l . , 1978; Pugsley and Schnaitman, 1978; Chai and Foulds, 1979) . The p u r i f i e d protein has been demonstrated to form large (1 nm dia) (Benz et a l . , 1985), weakly anion-selective (Benz et a l . , 1984) channels in reconstituted bilayer membranes. Examination of the transport properties of mutants expressing PhoE (previously protein e) as the sole porin revealed that a wide range of nutrients (sugars, amino acids, nucleosides,ions) could permeate the channel in vivo (Lugtenberg et a l . , 1978; Van Alphen et a l . , 1978), consistent with the formation of a general d i f f u s i o n channel. The protein i s immunologically cross-reactive with the major porin proteins OmpF and OmpC (Overbeeke et a l . , 1980) , exhibiting 70 % amino acid homology with OmpF (Tommassen et a l . , 1982) and 61 % amino acid homology with OmpC (Mizuno et a l . , 1983). In addition, the cloned phoE and ompF genes have been demonstrated to hybridize in regions along their entire lengths (Tommassen et a l . , 1982). Despite these s i m i l a r i t i e s with the major porins, the PhoE channel exhibits properties consistent with a presumed role in phosphate acq u i s i t i o n . A component of the phosphate (pho) regulon in E. c o l i (Tommassen and Lugtenberg, 1982) (see section 4) the protein forms a channel which is more e f f i c i e n t in the uptake of anionic and phosphorylated 1 1 compounds than either OmpF or OmpC (Overbeeke and Lugtenberg, 1982). Overbeeke and Lugtenberg (1982) also demonstrated that a mutant deficient in PhoE grew more slowly than wild type in the presence of polyphosphate (P15) as the sole phosphate source. Furthermore, Korteland et a l (1982) have demonstrated that a PhoE-deficient mutant exhibited a 10-fold increase in the Km for phosphate transport compared with strains expressing a PhoE channel. Unfortunately, this result was obtained in a porin-deficient background, rather than a background wild type for the major porins. Therefore, i t was not possible to conclude whether the increase in Km for phosphate in the PhoE-deficient strain reflected a s p e c i f i c role for protein PhoE in phosphate transport, or whether any porin would have reversed the poor phosphate transport of the porin-deficient s t r a i n . PhoE proteins have been i d e n t i f i e d in other Enterobacteriaceae, including Salmonella typhimurium (Bauer et a l . , 1985) and Enterobacter cloacae (Verhoef et a l . , 1984). These proteins form anion-selective channels consistent with their presumed roles in phosphate acquisition. A 36 kDa outer membrane protein inducible by phosphate-limitation has also been i d e n t i f i e d in K l e b s i e l l a  aerogenes (Sterkenburg et a l . , 1984) although i t was not assayed for porin function. c. Iron-regulated outer membrane proteins. Although iron is an abundant metal in nature i t occurs under aerobic conditions (at pH 7) as f e r r i c hydroxide with low 12 water s o l u b i l i t y (equilibrium concentration of 10 uM) (Braun and Hantke, 1982). C e l l s of E. c o l i , for example, require an iron concentration of approximately 0.1 uM for growth and to gain s u f f i c i e n t iron they must produce iron chelators (eg. ferrichrome; enterochelin) concomittant with transport systems for these chelates (Hantke and Braun, 1975; Wayne and Neilands, 1975). A number of high molecular weight outer membrane proteins have.been i d e n t i f i e d which are co-regulated with these chelators under i r o n - l i m i t i n g conditions (Braun et a l . , 1976; Hancock and Braun, 1976; Mcintosh and Earhart, 1977). Two of these, the products of the fhuA (tonA) (Hantke and Braun, 1975) and fepA (feuB, cbr) (Pugsley and Reeves, 1976; Mcintosh and Earhart, 1977; Wookey and Rosenberg, 1978) genes, function in the uptake of ferric-ferrichrome and ferric-'enterochelin, respectively. This was supported by data which demonstrated d i r e c t l y the a b i l i t y of the respective iron chelates to bind to their receptor proteins in the outer membrane (Braun and Hantke, 1977; Ichihara and Mizushima, 1978) and by the inactivation of s p e c i f i c transport systems in mutants deficient in the corresponding outer membrane proteins (Pugsley and Reeves, 1976; Wookey and Rosenberg, 1978). Similar proteins have been i d e n t i f i e d in other gram-negative bacteria, including Salmonella typhimurium (Ernst et a l . , 1978), Neisseria gonorrheae (Norqvist et a l . , 1978), Vibrio  cholerae (Sciortino and Finke l s t e i n , 1983), K l e b s i e l l a  aerogenes (Williams et a l . , 1984) and Pseudomonas aeruginosa 1 3 (Brown et a l . , 1984). d. Others. The transport of vitamin B12 (cyanocobalamin) by E. c o l i i s a biphasic process involving an energy-independent rapid binding phase followed by a energy-dependent phase (DiGirolamo and Bradbeer, 1971). The i n i t i a l vitamin B12 binding s i t e s are firmly embedded in the outer membrane (DiGirolamo et a l . , 1971; White et a l . , 1973) and have been i d e n t i f i e d as the protein products of the btuB (bfe) gene (White et a l . , 1973; DiMasi et a l . , 1973; Kadner and Liggins, 1973). A minor outer membrane protein, the btuB gene product has not been shown to exhibit any porin function, although , together with LPS and the OmpF protein, i t has been i d e n t i f i e d as a constituent of the c o l i c i n A receptor (Chai et a l . , 1982). The proximity of the vitamin B12 receptor and major porin protein OmpP in vivo may be si g n i f i c a n t in terms of • the mechanism by which vitamin B12 actually crosses the outer membrane. The transport of nucleosides by E. c o l i c e l l s reportedly involves an outer membrane protein, the tsx gene product, which forms an especially e f f i c i e n t channel for nucleosides (Hantke, 1976). Although nucleosides are capable of permeating the outer membrane via the OmpF and PhoE channels (van Alphen et a l , 1978), their d i f f u s i o n through these channels i s expected to be slow due to the large size of nucleosides. Furthermore, the exceptionally high V m a x of the nucleoside active transport system (Koch, 1971) probably necessitates a s p e c i f i c channel in the outer membrane. 14 3. Bacterial phosphate transport - with s p e c i f i c  reference to E. c o l i . inorganic phosphate transport has been characterized in a number of bacterial systems, including Staphylococcus aureus (Mitchell, 1954), Bac i l l u s  cereus (Rosenberg et a l , 1969), Micrococcus lysodeikticus (Friedberg, 1977), Streptococcus faecalis (Harold and Baarda, 1966), E. c o l i (Medveczky and Rosenberg, 1971) and P. aeruginosa (LaCoste et a l . , 1981). In each case, the transport i s concentrative, energy-dependent and inhibitable to varying degrees by arsenate, a phosphate analogue. In addition, the rate of and capacity for phosphate uptake appears, in many cases, to increase at low concentrations of phosphate suggesting that the transport systems involved are inducible. The transport of inorganic phosphate by E. c o l i has been characterized in d e t a i l and two major systems of' uptake, the PST and PIT systems, have been i d e n t i f i e d (Willsky et a l . , 1973). The PST system, which operates at approximately 20 % of capacity in the presence of high levels of phosphate (Rosenberg et a l . , 1977), i s completely derepressed under phosphate-limiting (< 1.0 mM) conditions (Rosenberg et a l . , 1977). A h i g h - a f f i n i t y system (Km = 0.16 to 0.43 uM) (Rosenber et a l . , 1977; Willsky and Malamy, 1980) the PST phosphate transport system i s responsible for the bulk of phosphate transport under l i m i t i n g conditions. The PIT system, which operates c o n s t i t u t i v e l y , i s of low a f f i n i t y (Km = 25 to 38 uM) (Rosenberg et a l . , 1977; Willsky 15 and Malamy, 1980), and i s the major transport system for phosphate under phosphate-sufficient conditions. The PST system is comprised of the products'of at least 5 genes (pst, phoU, phoV, phoS, phoT) (Levitz et a l , 1984), one of which, the product of the phoS gene, functions as a periplasmic phosphate-binding protein (Gerdes and Rosenberg, 1974). The remaining gene products have not been i d e n t i f i e d , although they are probably l o c a l i z e d in the cytoplasmic membrane (Tommassen and Lugtenberg, 1982). The phosphate-binding protein is inducible under conditions of phosphate-li m i t a t i o n (Yagil et a l . , 1976) and binds phosphate with h i g h - a f f i n i t y (Kd=0.8 uM) (Medveczky and Rosenberg, 1970), accounting for the i n d u c i b i l i t y and low Km of the PST transport system. Osmotic shock and spheroplast formation, both of which result in the release of the binding protein from whole c e l l s , have been demonstrated to inactivate the PST system (Gerdes et a l . , 1977). Typical of binding protein-dependent transport systems in general, phosphate uptake via the PST system u t i l i z e s phosphate bond energy, in the form of ATP or a related metabolite, as the energy source (Rosenberg et a l . , 1979). The recent demonstration that the ornithine-arginine binding protein of E. c o l i is phosphorylated - dephosphorylated during substrate transport (Celis, 1984) may be a clue as to the role ATP (or a related metabolite) plays in binding protein-dependent transport. Although arsenate was capable of i n h i b i t i n g phosphate uptake via the PST system (Ki = 0.39 uM) (Willsky and Malamy, 1980) 16 i t was not transported by th i s system and c e l l s expressing only the PST phosphate transport system were capable of growth in the presence of arsenate. The low-affinity PIT phosphate transport system involves the product of a single known gene, p i t (Bennet and Malamy, 1970; Willsky et a l . , 1973; Sprague et a l . , 1975), which i s probably an inner membrane protein. Inorganic phosphate uptake via this system i s not sensitive to spheroplast formation (Rosenberg et a l . , 1977), consistent with the absence of a binding protein associated with i t . Characteristic of shock-resistant transport systems, phosphate transport via the PIT system is coupled to the proton motive force (Rosenberg et a l . , 1979). PIT-mediated phosphate transport is inhibited by arsenate which appears to be transported equally well by t h i s system (Willsky and Malamy, 1980). Cel l s expressing only the PIT system cannot grow .in an arsenate-containing medium in which they suffer an almost t o t a l depletion of i n t r a c e l l u l a r ATP levels (Willsky and Malamy, 1980). In addition to the two major inorganic phosphate uptake systems described above, three organophosphate transport systems have also been i d e n t i f i e d in E. c o l i with roles in hexose phosphate (the uhp operon) (Romberg and Smith, 1969) and glcerol-3-phosphate (the qlpT (Lin, 1976) and ugp (Schweizer et a l . , 1982) operons) transport. Two of these, involving the constituents of the glucose-6-phosphate-inducible uhp operon and the glycerol-3-phosphate-inducible 17 glpT operon appear to be pathways for the uptake of inorganic phosphate as well (Willsky and Malamy, 1974). The other involves the products of the ugpA and uqpB genes (Schweizer et a l . , 1982), which encode an inner membrane permease and a periplasmic glycerol-3-phosphate-binding protein, respectively (Tommassen and Lugtenberg, 1982). This system is derepressed upon phosphate-limitation (Argast and Boos, 1980) and forms part of a phosphate or pho regulon in E. c o l i (Schweizer et a l . , 1982; Tommassen and Lugtenberg, 1982) (see below). Interestingly, P. aeruginosa also appears to possess two major transport systems for inorganic phosphate, of low and h i g h - a f f i n i t y , respectively (LaCoste et a l . , 1981), as well as an uptake system for glycerol-3-phosphate (Siegel and Phibbs, 1979). The transport of inorganic phosphate by P. aeruginosa is somewhat sensitive to osmotic shock, consistent with the involvement of a periplasmic binding protein. In addition, the two uptake systems appear to exhibit different energy requirements similar to the situation in E. c o l i . 4. The pho regulon of E. c o l i . Under conditions of phosphate-limitation, wild type c e l l s of E. c o l i are derepressed for the synthesis of numerous proteins (Tommassen and Lugtenberg, 1982), and at least 18 phosphate-starvation-inducible genes have been described (Wanner et a l . , 1981). The roles of these gene products in the 18 scavenging of phosphate and phosphate-containing molecules from a d i l u t e environment has, in many cases, been addressed (Tommassen and Lugtenberg, 1982). The phosphate-starvation-inducible proteins which have been i d e n t i f i e d include periplasmic binding proteins for phosphate ( the phoS gene product) (Gerdes and Rosenberg, 1974) and glycerol-3-phosphate (the uqpB gene product) ( Argast and- Boos, 1980), a periplasmic alkaline phosphatase (the phoA gene product) (Torriani, 1960; Brickman and Beckwith, 1975), cytoplasmic membrane permeases for phosphate ( the pst gene product) (Rosenberg et a l . , 1977) and glycerol-3-phosphate (the ugpA gene product) (Argast and Boos, 1980), an outer membrane pore-forming protein (the phoE gene product) (Overbeeke and Lugtenberg, 1980) and a cytoplasmic polyphosphatase (Yagil, 1975). Several presumably cytoplasmic regulatory molecules, including the products of the phoB (Makino et a l . , 1982; Tommassen and Lugtenberg, 1982), phoM (Ludtke et a l . , 1984) and phoR (Tommassen et a l . , 1982) genes have also been i d e n t i f i e d . Most i f not a l l of these proteins are part of a single regulon, designated the pho regulon, which exhibits some s i m i l a r i t y to the maltose regulon of E. c o l i which is also inducible for an outer membrane protein, a periplasmic binding protein, cytoplasmic membrane c a r r i e r proteins and cytoplasmic catabolic enzymes (Hengge and Boos, 1983). Phosphate-starvation-inducible proteins i d e n t i f i e d in P. aeruginosa include an ex t r a c e l l u l a r phospholipase C (Stinson and Hayden, 1979) and an alkaline phosphatase which occurs 19 in both the periplasm and the ex t r a c e l l u l a r medium (Hou e_t a l . , 1966; Cheng et a l . , 1979). A number of additional phosphate-repressible proteins have been i d e n t i f i e d in phospholipase C regulatory mutants of Pseudomonas aeruginosa (Gray et a l . , 1982) although their functional a c t i v i t i e s have not been elucidated. The components of the E. c o l i pho regulon are under the control of a complex network of regulatory proteins which includes the products of three known genes - phoB, phoM and phoR (Tommassen and Lugtenberg, 1982). As a model for the regulation of pho regulon constutuents, production of the phoA gene product (alkaline phosphatase) has been studied in d e t a i l (Echols and Garen, 1961; Brickman and Beckwith, 1975; Bracha and Yagil, 1973; Wanner and L a t t e r e l l , 1980 ). Mutant studies and studies involving the cloned genes have indicated that the phoB gene product functions'as a transcriptional activator of phoA (Bracha and Yagil, 1973; Brickman and Beckwith, 1975), while the phoR gene product acts as a repressor (high phosphate) (Echols et a l . , 1961) and activator (low phosphate) (Wanner and L a t t e r e l l , 1980), the l a t t e r function being at least p a r t i a l l y replaceable by the phoM gene product (Wanner and L a t t e r e l l , 1980). Based on results of mutant studies i t was also concluded that the phoB gene product was i t s e l f co-regulated with alkaline phosphatase and that phoB transcription was probably under the control of the phoR and phoM gene products (Tommassen et a l . , 1982). It remains to be seen whether phosphate acts 20 d i r e c t l y (as co-repressor) or i n d i r e c t l y in regulating this process. 21 METHODS 1. Media and growth conditions. The minimal medium used in this study contained 0.1 M N-2-hydroxy-ethyl piperazine-N'-2-ethanesulfonate (Hepes) (pH 7.0), 0.5 mMMgS04, 7 mM (NH 4) 2S0 4, 20 mM potassium succinate or 0.4 % (wt/vol) glucose as the carbon source, 0.1 % (wt/vol) trace ion solution (as described by Hancock et a l . , 1981b) and either 0.2 mM potassium phosphate buffer (pH 7.0) for phosphate-deficient medium (with exceptions, see below) or 0.6-1.0 mM potassium phosphate buffer (pH 7.0) for phosphate s u f f i c i e n t medium. Amino acids were included, as required, at a f i n a l concentration of 1 mM. When the culture organism was Pseudomonas cepacia, Pseudomonas pseudomallei or Pseudomonas  acidovorans the phosphate-deficient medium contained 0.1 mM phosphate. When- the culture organism was K l e b s i e l l a  pneumoniae, Enterobacter aerogenes or Serratia marce'sens the phosphate-deficient medium contained 0.15 mM phosphate. Xanthamonas maltophilia (previously Pseudomonas maltophilia) cultures were supplemented with 1 mM methionine. L-broth [1 % (wt/vol) tryptone/ 0.5 % (wt/vol) yeast extract/ 0.05 % (wt/vol) NaCl] and proteose peptone No. 2 [1 % (wt/vol)] were used as the rich media throughout. Liquid cultures were grown with vigorous aeration at 37°C unless otherwise indicated. Strains were routinely maintained on L-broth agar (L-agar) or phosphate-sufficient medium agar plates prepared by including 2 % (wt/vol) 22 Bactoagar (Difco) in L-broth and phosphate-sufficient minimal medium respectively. A n t i b i o t i c s were used in selective media at the following concentrations: tetracycline (Tc), 200 ug/ml; kanamycin (Kn), 300 ug/ml; c a r b e n i c i l l i n (Cb), 1 mg/ml; mercuric chloride (HgC^), 15 ug/ml and trimethoprim (Tp), 1 mg/ml. 2. Bacterial strains. The ba c t e r i a l strains and plasmids used in t h i s study are l i s t e d in Table I. Plasmid pMTlOOO was introduced into strain H103 by conjugation with PA01594(pMT1000) on L-agar plates. Equal volumes of mid-log phase donor and recipient c e l l s (grown in L-broth) were mixed and pelleted by centrifugation. The supernatant was decanted and the pelle t resuspended gently in 0.05 ml of L-broth. The c e l l s were spread over approximately one-third of the surface of an L-agar plate and incubated for 2 h at . 30°C. The mating mixture was then resuspended in 1 ml of phosphate-sufficient medium, centrifuged and washed several times in the same medium. Transconjugants were selected at 30°C on phosphate-sufficient minimal medium containing 100 ug/ml Tc. 3. C e l l fractionation and sodium dodecyl sulfate- polyacrylamide gel electrophoresis. Whole c e l l protein extracts were obtained as described by Nicas and Hancock (1980). B r i e f l y , overnight cultures were centrifuged and 23 Table I. Bacterial strains Strain Description Source/ Reference P. aeruginosa PAO HI 03 Hl03(pMT1000) H242 H287 H553 H556 H576 H585 H586 H587 PA01594 PA01 wild type contains plasmid pMTlOO PA01 thr-102 ATCC #19305 Tn501 insertion mutant of H103 non-derepressible for alkaline phosphatase, phospholipase C, phosphate-binding protein and protein Tn501 insertion mutant of H103 requiring arginine Tn501 insertion mutant of H103 non-derepressible for protein P Phosphate-binding protein deficient mutant of H242 selected as constitutive for alkaline phosphatase Alkaline phosphatase constitutive mutant of H242 producing a defective phosphate-binding protein Mutant stra i n of H242 constitutive for alkaline phosphatase, phospholipase C, phosphate-binding protein and protein P. met-28 ilv-202 rmo-53 str-1 Hancock & Carey, 1979 This study S. Benson This study This study This study This study This study This study M. Tsuda, Tokyo 24 Table I. - continued Strain Description Source/ Reference P. aeruginosa PAO PAO1594(pMT1000) Pseudomonadaceae contains plasmid pMTlOOO Tsuda et a l , 1984 P. putida ATCC # 12633 T p. fluorescens ATCC # 949 p. chlororaphis ATCC # 9446 T p. aureofaciens ATCC # 13985T p. cepac ia ATCC # 25609 T p. pseudomallei ATCC # 23343 T p. ac idovorans strain 29 p. st u t z e r i ATCC # 17588T p. syringae ATCC # 19310T p. solanacearum ATCC # 1 1696T p. maltophilia ATCC # 13637T Warren, 1 979 Enterobacteriaceae Escherichia c o l i K12 strain HMS174 OmpF+ OmpC+ Escherichia c o l i K12 strain JF700 OmpF" OmpC+ Escherichia c o l i K12 strain JF694 OmpF" OmpC" PhoE c R.A.J. Warren, U.B.C. Foulds and Chai, 1978 Foulds and Chai, 1979 25 Table I. - continued Strain Description Source/ Reference Salmonella typhimurium strain SL1906 Stocker et a l , 1979 OmpC OmpD OmpF Kl e b s i e l l a pneumoniae ATCC # 13883 T Enterobacter aeroqenes ATCC # 13048 T Serratia marcesens ATCC # 13880 T a ATCC, American Type Culture Co l l e c t i o n ; only the relevant phenotypes are indicated; T, type s t r a i n ; PhoE c, constitutive for PhoE 26 the c e l l s resuspended in 2 % (wt/vol) sodium dodecyl sulfate (SDS)/ 20 mM Tris-HCl (pH 8.0). After treatment at 100°C for 10 min, residual c e l l s were removed by centrifugation at 27,000 x g for 10 min and the resulting supernatant sonicated (1 min, setting 5, Biosonik sonicator, Bronwill S c i e n t i f i c , Rochester,NY) to shear DNA and reduce v i s c o s i t y . The preparation of c e l l envelopes was based on the method of Nicas and Hancock (1980). C e l l s from overnight or logarithmic phase cultures were centrifuged, resuspended in 15 mM Tris-HCl (pH 8.0) containing 10 ug/ml pancreatic deoxyribonuclease I (Sigma Chemical Co., St. Louis, MO) and broken in a French pressure c e l l (Aminco) at 11,500-13,000 p s i . Unbroken c e l l s were removed by centrifugation (1,000 x g for 10 min) and the resulting supernatant centrifuged at 160,000 x g for 1 h. The c e l l envelope p e l l e t was resuspended in deionized water. Outer membranes were prepared using the two-step gradient method described by Hancock and Carey (1979). C e l l envelopes prepared in 15 mM Tris-HCl (pH 8.0)/ 20 % (wt/vol) > -sucrose were layered onto a sucrose step gradient of 60 % (wt/vol) (top layer) and 70 % (wt/vol) (bottom layer) and centrifuged overnight at 183,000 x g in a Beckman SW41 Ti rotor (Beckman Instruments Inc., Palo Alto, CA). A single outer membrane band was obtained at the interface of the two sucrose solutions. Triton X-100-Tris, Triton X-100-Tris-EDTA and Triton X-100-Tris-lysozyme extraction of outer membranes was exactly 27 as described by Hancock et a l . (1981a). SDS-polyacrylamide gel electrophoresis was performed as described by Hancock and Carey (1979) using a 12 % (wt/vol) acrylamide running gel. Two-dimensional (unheated x heated) SDS-polyacrylamide gel electrophoresis was based on a method described previously (Russel, 1976). Samples s o l u b i l i z e d at room temperature were electrophoresed on an SDS-polyacrylamide slab gel and the lanes excised (1st dimension). The gel s t r i p s were then placed in screw capped tubes conta-ining 2 % (wt/vol) SDS/20 mM Tris-HCl (pH 6.8) and heated at 88°C for 10 min. The heated gel s t r i p s were l a i d horizontally across the top of a second SDS-polyacrylamide slab gel, sealed in place with 0.8 % agarose (Biorad, Richmond, CA) and electrophoresed again (2nd dimension). Where indicated, urea was included in the second dimension slab gels at a f i n a l concentration of 6M. 4. P u r i f i c a t i o n of protein P. The s o l u b i l i z a t i o n in Triton X-100-EDTA of outer membranes from phosphate-deficient c e l l s and chromatography on a DEAE-Sephacel column was exactly as described previously for protein D1 p u r i f i c a t i o n (Hancock and Carey, 1980). Protein P-containing fractions (exhibiting some contamination with protein F) were pooled and concentrated 5-fold by d i a l y s i s against 20 % (wt/vol) polyethyleneglycol 20,000 (Sigma Chemical Co.). To th i s pooled concentrate a 4-fold excess of SDS over Triton X-100 28 ( i . e . 2 % (wt/vol) SDS) was added and the solution was made 3 mM for sodium azide. This solution was added to a Sepharose 4B column (46 x 2 cm) pre-equilibrated with 0.1 % (wt/vol) SDS/ 5 mM Tris-HCl (pH 8.0)/ 3 mM sodium azide (column buffer) and eluted with column buffer. Three-m i l l i l i t e r fractions were collected at 12 ml/h and tested for absorbance at 280 nm and for protein composition on SDS-polyacrylamide gels. Protein P, s l i g h t l y contaminated with protein F, eluted just after t h e v o i d volume, whereas the Triton X-100 eluted in subsequent f r a c t i o n s . The protein P-containing fractions were again pooled, concentrated and reapplied to the Sepharose 4B column described above. The resultant protein P peak was homogeneous as determined by SDS-polyacrylamide gel electrophoresis. 5. Acetylation of protein P. Protein P was acetylated using acetic anhydride as described by Tokunaga et a l (1981). Protein P (500 ug in 1.2 ml of 0.1 % (wt/vol) SDS/ 10 mM Tris-HCl (pH 8.0)/ 3 mM sodium azide) was diluted in 50 mM sodium phosphate buffer (pH 6.8)/ 0.1 % (wt/vol) SDS to a f i n a l volume of 2 ml. The reaction was started by the addition of 2 ul of acetic anhydride, which was subsequently added at 10 min intervals over a period of 1 h. The pH of the solution was monitored with a microprobe and maintained at approximately 7 with aliquots of 5 M NaOH. The solution was allowed to s i t for an additional hour before i t was dialysed against one l i t e r of 35 mM sodium phosphate buffer 29 (pH 6.8)/ 0.1 % (wt/vol) SDS for 4 h. The d i a l y s i s buffer was changed and d i a l y s i s continued overnight in the same buffer. 6. Immunological methods. Antigen s p e c i f i c i t y and t i t r e of the various antisera was determined by the enzyme-linked immunosorbent assay (ELISA) as described by Mutharia and .Hancock (1983) using 20 ug/ml f i n a l concentration of antigen in the wells. The Western immunoblot procedure, involving the electrophoretic transfer of SDS-polyacrylamide gel electrophoretograms to n i t r o c e l l u l o s e and subsequent immunostaining, has been described previously (Mutharia and Hancock, 1983). In cases where a peroxidase conjugate was used as the second antibody, azide was omitted from a l l buffers and the blots developed using the peroxidase substrate descibed below. 7. Preparation of protein P-phosphatidyl choline v e s i c l e s . Phosphatidyl choline.(0.5 umol in CHCl^) was dried under N 2 and dessicated for 30 min at room temperature. Protein P (200 ug) was added to the dried l i p i d and vortexed for 30 sec. Deionized water was added to the p r o t e i n - l i p i d solution to make a f i n a l volume of 1 ml. The l i p i d was scraped from the sides of the tube with a spatula and the solution vortexed a further 30 sec. Following sonication (4 pulses of 15 sec each at setting 50, Biosonik sonicator, Bronwill) the vesicle solution was cooled on ice and stored 30 at -20°C. 8. Preparation of protein a f f i n i t y columns. a. Protein F-sepharose. Protein F was p a r t i a l l y p u r i f i e d according to the procedure of Yoshimura et a l . (1983), omitting the column chromatography step. The resultant preparation, which had only minor contamination with protein H2, was approximately 90 % pure as judged by SDS-polyacrylamide gel electrophoresis. The p a r t i a l l y p u r i f i e d protein was passed across a Biogel P-10 (Biorad) column (15x1.5 cm) equilibrated with 0.1 M NaHC03 (pH 8.3)/ 0.5 M NaCl/ 0.1 % (wt/vol) SDS. Peak fractions (measured at an absorbance of 280 nm) were pooled and the protein (approximately 3.5 mg) was cross-linked to CNBr-activated Sepharose 4B beads (approximately 0.5 g dry weight) as recommended by the manufacturer (Pharmacia, Upsalla, Sweden). The column was stored at 4°C in phosphate-buffered saline (pH 7.4) (PBS) (Mutharia and Hancock 1983) containing 0.1 % (vol/vol) Triton X-100. The f i n a l column volume was approximately 1 ml. Prior to use, the column was washed exhaustively with PBS to remove excess detergent. b. Protein P - a f f i - g e l 10. P u r i f i e d protein P was passed across a Biogel P-10 (Biorad) column equilibrated with 0.1 M acetic acid/sodium acetate buffer, pH 5.0/ 0.1 % (wt/vol) SDS. Peak fractions, measured at an absorbance of 280 nm, were pooled and the protein (1.5 mg) cross-linked to approximately 0.65 ml of A f f i - g e l 10 (Biorad) for 2 h at 31 room temperature as recommended by the manufacturer. The column was stored at 4°C in PBS containing 0.1 % (wt/vol) Triton X-100. Prior to use, the column was washed exhaustively with PBS to remove excess detergent. c. Phosphate-binding protein-Sepharose. P u r i f i e d phosphate-binding protein was passed across a Biogel P-10 (Biorad) column (at 20 % of column volume) equilibrated with 0.1 M NaHC03/ 0.5 M NaCl (pH 8.0) (coupling buffer) and the protein-containing fractions were pooled ( f i n a l volume of 5 ml at an absorbance at 280 nm of 0.38) and coupled to CNBr-activated Sepharose 4B (Pharmacia) exactly as described for protein F. The f i n a l column volume was approximately 1 ml. The column was stored in 20 mM Tris-HCl (pH 8.0) at 4°C. 9. Preparation of protein P-specific antisera. a. Trimer-specif i c . • Antibodies to protein P trimers were raised in New Zealand White rabbits using the following immunization schedule. Protein P (50 ug) was injected subcutaneously at weekly intervals over a three week period. Following t h i s , the rabbits were rested, without injection, for three weeks. This cycle of three weeks of weekly immunization followed by three weeks without injection was repeated twice more, before a f i n a l subcutaneous injection of protein P (50 ug) was given. For the f i r s t two injections, protein P (diluted in PBS) was mixed 1:1 with Freund's Incomplete Adjuvant (Difco, Detroit, MI, USA), otherwise i t was injected in PBS alone. Two weeks after the 32 f i n a l i n j e c t i o n , blood was collected and the serum obtained after centrifugation of clotted blood. The resultant antiserum contained antibodies to lipopolysaccharide (LPS) and porin protein F as well as to protein P. Thus in order to generate a protein P trimer-s p e c i f i c antiserum i t was necessary to remove these contaminating a c t i v i t i e s . The antiserum was f i r s t adsorbed against whole c e l l s of P. aeruginosa PA01 strain H103 as follows. C e l l s from a 10 ml overnight culture in L-broth were harvested by centrifugation in a table top centrifuge and washed twice with Hank's Balanced Saline Solution (Gibco, Burlington, Ont, Can). The c e l l p e l l e t was resuspended d i r e c t l y into 1.0 ml of the antiserum, placed in a 1.5 ml polypropylene centrifuge tube (Evergreen S c i e n t i f i c , Los Angeles, CA, USA) and incubated.for 45 min at room temperature in an end-over-end shaker. The c e l l s were then pelleted and the antiserum-containing supernatant adsorbed a second time against a fresh batch of washed c e l l s . Whole c e l l adsorbtion e f f e c t i v e l y removed a l l antibodies directed against smooth LPS as measured by ELISA and confirmed by Western immunoblots. There was, however, no decrease in antibody t i t r e to protein F (or protein P). The'adsorbed antiserum (600 ul) was then incubated for 45 min at room temperature on a protein F-Sepharose a f f i n i t y column (2.5x0.7 cm). At the completion of the incubation period, the column was washed with 4 ml of PBS and the unbound antibodies collected in 400 ul fractions. Fractions 33 containing antibodies to protein P, as determined by ELISA, were pooled to y i e l d a protein F-adsorbed antiserum. Adsorption of the antiserum on the protein F-Sepharose column f a c i l i t a t e d removal of 99 % of the antibody a c t i v i t y to protein F, with no decrease in antibody t i t r e to protein P. The protein F-adsorbed antiserum, however, could only poorly distinguish between phosphate-limited c e l l s producing protein P (eg. strain H103) and phosphate-limited c e l l s defective in protein P production (eg. strain H553). Therefore, the protein F-adsorbed antiserum was subsequently adsorbed twice against phosphate-limited P. aeruginosa strain H553 c e l l s as described above for strain H103. The resultant antiserum was protein P trimer-specific (see Chapter One). b. Monomer s p e c i f i c . Antibodies to protein P monomers were raised as follows. P u r i f i e d protein P was heated at 88°C for 15 min to promote the heat denaturation of trimers to form monomers (see Chapter One). After cooling, the protein (20 ug) , suspended in 0.1 ml PBS (Mutharia and Hancock, 1983), was injected interperitoneally into Balb/c mice (Department of Microbiology breeding colony, University of B r i t i s h Columbia, Vancouver, Canada). The injection was repeated.on days 14, 28, 35, 42, 45 and 50. The blood was" collected 7 days after the f i n a l injection and the serum obtained after centrifugation of clot t e d blood. Antibodies to LPS, as measured by ELISA using p u r i f i e d LPS as the antigen, were removed by adsorbtion against whole c e l l s of 34 P. aeruginosa PA01 stra i n HI 03 (see above). c. Antiserum to protein P in phosphatidyl choline  v e s i c l e s . Antiserum to protein P vesicles was raised in Balb/c mice as follows. Protein P vesicles (20 ug protein P) were preincubated at 37°C in the presence of monoclonal antibodies MA5-8 (LPS core-specific) (Hancock et a l . , 1983a) and MA1-8 (LPS 0 antigen-specific) (Hancock et a l . , 1983a) for 30 min prior to subcutaneous i n j e c t i o n . The monoclonals were previously t i t r a t e d against protein P vesicl e s in the ELISA to determine the amounts of the 2 antisera required to block a l l LPS molecules present in a given amount of protein P-containing v e s i c l e s . The injections were repeated on days 14,21,28,35,42,56,70,84, and 98. One week after the f i n a l injection, the blood was collected and the antiserum obtained after centrifugation of clot t e d blood. 10. Isolation of a protein P-deficient mutant. a. Tn50l insertion mutagenesis. P. aeruginosa Hl03(pMT1000) was cultured overnight in L-broth at 30°C in the presence of HgCl 2. Dilutions were plated onto L-agar plates containing HgCl 2 and incubated at 42°C. Colonies growing up after 24 h (insertion mutants) were picked onto grids on fresh L-agar plates containing HgCl 2 and incubated once again at 42°C. After 24 h, these plates were then re p l i c a plated onto L-agar plates containing HgCl 2 and onto phosphate-deficient minimal medium plates, followed by 35 incubation at 42°C. The replicas on r i c h medium were retained as a master set from which desired mutants, once i d e n t i f i e d , could be rescued. The replicas grown on the phosphate-deficient minimal medium plates were screened for protein P-deficient mutants. b. Selection of a protein P-deficient mutant using a  protein P trimer-specific antiserum. Bacterial clones resistant to HgCl 2 at 42°C and r e p l i c a plated onto phosphate-deficient minimal medium plates were transferred by contact onto n i t r o c e l l u l o s e f i l t e r discs (Schleicher and Schuell Inc., Keene, NH, USA, type BA85, 82 mm). The n i t r o c e l l u l o s e replicas were subsequently screened, by a modification of the procedure of Helfman et a l (1983), for the absence of protein P using the above-described protein P trimer-specific antiserum. Blotted f i l t e r s were placed in individual Petri dishes in 10 ml of 50 mM Tris-HCl (pH 7.4)/ t50 mM NaCl/ 5 mM MgCl 2 containing 3 % (wt/vol) bovine serum albumin and shaken gently for 1 h at 37°C. The f i l t e r s were then washed twice at room temperature for 10 min in 10 ml of Tris-buffered saline (0.9 % NaCl/ 10 mM Tris-HCl (pH 7.4); Towbin et a l 1979) with shaking, followed by three 5 min washes in 10 ml PBS. Protein P trimer-specific antiserum (with an ELISA t i t r e of 2000, indicating that antibodies to protein P were detectable at a 1/2000 d i l u t i o n of the antiserum) was diluted 1:249 in PBS containing 3 % (wt/vol) bovine serum albumin (10 ml) and then incubated on the f i l t e r s overnight 36 at room temperature with shaking. The f i l t e r s were subsequently washed three times for 10 min at room temperature in 10 ml PBS. A f f i n i t y p u r i f i e d goat a n t i -rabbit IgG (H+L)-peroxidase conjugated antibody (Cappel Laboratories, West Chester, PA, USA) diluted 1:999 in 10 ml PBS containing 3 % (wt/vol) bovine serum albumin was then incubated on the f i l t e r s at 37°C, again with shaking. After 2 h of incubation, the f i l t e r s were washed twice for 10 min each in 10 ml of PBS at room temperature, followed by three washes of 10 min each in 10 ml of Tris-buffered saline. Peroxidase substrate (30 mg chloro-4-naphthol (Sigma) in 10 ml of methanol/ 50 ml of Tris-buffered s a l i n e / 0.02 ml H 20 2 (30 % (vol/vol)) was then added (10 m l / f i l t e r ) and the f i l t e r s incubated at 37°C u n t i l colour developed. Those colonies f a i l i n g to develop colour were i d e n t i f i e d and picked from the master plates and screened for the absence of protein P in SDS-polyacrylamide gels of phosphate-limited c e l l envelopes. 11. Phosphate transport assays. Overnight cultures of P.  aeruginosa grown in phosphate-deficient medium were harvested by f i l t r a t i o n and washed with three volumes of minimal Hepes-buffered medium without phosphate. Washed c e l l s were resuspended by vortexing in the same phosphate-less medium at a f i n a l absorbance at 600 nm of 0.2-0.3 and stored on ice u n t i l needed. C e l l s could be stored on ice for up to 3 h without any change in c e l l density or any 37 signs of c e l l damage (measured as release of periplasmic alkaline phosphatase into the medium). Pr«ior to assaying phosphate accumulation, c e l l s were shaken at 37°C for 5-25 min. To assay phosphate uptake, 1 ml samples of prewarmed c e l l s were added to 10 ml culture tubes containing radioactively labelled orthophosphate. The c e l l s were vortexed to ensure adequate aeration and 200 u l aliquots were removed at various times and f i l t e r e d on n i t r o c e l l u l o s e membrane f i l t e r cups (0.45 urn dia, Amicon Corp) in an Amicon vacuum manifold. F i l t e r e d c e l l s were washed twice with 1.5 ml of minimal Hepes-buffered medium containing 1 mM unlabelled phosphate. The f i l t e r s were then removed and counted in 3 ml of PCS s c i n t i l l a t i o n c o c k t a i l (Amersham). In some cases i t was necessary to dil u t e c e l l s in prewarmed phosphate-less minimal Hepes-buffered medium at the time of assay (1 ml f i n a l volume) due to excessively rapid transport rates of undiluted c e l l cultures. 12. Enzyme assays. Alkaline phosphatase was measured using para-nitrophenyl phosphate (pNPP) as the chromogenic substrate at a f i n a l concentration of 1 mg/ml in 0.1 M T r i s -HCl (pH 8.5). The assay was read at an absorbance of 410 nm. Beta-lactamase was measured using, as the substrate, the chromogenic beta-lactam n i t r o c e f i n at a f i n a l concentration of 0.06 mg/ml in 50 mM sodium phosphate buffer (pH 7). The assay was read at an absorbance of 540 nm. Phospholipase C a c t i v i t y was measured using para-nitrophenyl phosphorylcholine 38 (NPPC) as the chromogenic substrate at a f i n a l concentration of 120 mg/ml in 60 % (vol/vol) g l y c e r o l / 0.25 M Tris-HCl (pH 7.4). The assay was read at an absorbance of 410 nm. 13. Nitrocefin permeability assay. Outer membrane permeability was determined using a modification of the method of Angus et a l . (1982). Intact c e l l beta-lactamase a c t i v i t y was measured on growing cultures by f i r s t taking aliquots of c e l l s and dividing them in two. One fraction was taken up into a syringe and slowly squeezed through a mi l l i p o r e f i l t e r of 0.22 urn pore size to obtain a culture supernatant while the other fraction was l e f t u n f i l t e r e d . Equal volumes of each fraction were transferred to separate cuvettes. The cuvette containing the culture supernatant was placed in the reference beam of a Perkin-Elmer (Norwalk, CT, USA) Lambda 3 dual beam spectrophotometer. The other cuvette, containing intact c e l l s and supernatant, was placed in the sample beam. Nitrocefin, a chromogenic beta-lactam, was added to each cuvette ( f i n a l concentration of 0.06 mg/ml) 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 nitr o c e f o i c acid was recorded at an absorbance of 540 nm using a coupled Perkin-Elmer model 561 chart recorder. The recorded beta-lactamase a c t i v i t y was a direct measure of intact c e l l a c t i v i t y . Because beta-lactamase has been shown to be periplasmic, the a c t i v i t y of intact c e l l s at a given concentration i s limited by the d i f f u s i o n of the beta-lactam, in this case n i t r o c e f i n , across the outer membrane 39 rather than by the amount of enzyme. From theory (Zimmermann and Rosselet, 1977), the steady state rate of hydrolysis of beta-lactam in intact c e l l s ( v i n t ) equals the rate of beta-lactam d i f f u s i o n across the outer membrane (Vp) and hence provides a measure of outer membrane permeability. Permeability parameters (C) were calculated using the formula v i n t = V D = c ^ S o u t ~ S i n ^ a c C 0 E " d i n g to Zimmermann and Rosselet (1977), where C = permeability parameter; S Q u t= concentration of substrate (nitrocefin) outside the c e l l and S. = concentration of substrate inside the c e l l ( i . e . in the in periplasm) (which i s << s t , and thus n e g l i g i b l e ) . 14. Osmotic shock and p u r i f i c a t i o n of the phosphate-binding  protein. P. aeruginosa PA01 str a i n H103 was grown in phosphate-deficient medium to induce the synthesis of the periplasmic 34K protein. Induced c e l l s were harvested by centrifugation (10,000 x g for 10 min) and subjected to two rounds of the Tris-HCl/ MgCl 2/ cold shock procedure described by Hoshino and Kageyama (1980). The shocked c e l l s were removed by centrifugation (10,000 x g for 10 min) and the supernatant concentrated approximately 50-fold via Amicon pressure f i l t r a t i o n using a PM10 m i c r o f i l t e r (Amicon Corp, Danvers, MA, USA). Remaining whole c e l l s and debris were removed by centrifugation in a c l i n i c a l table top centrifuge. The concentrated shock f l u i d s were then desalted by passage over a Biogel P-10 (Biorad) column equilibrated with 20 mM Tris-HCl (pH 7.4). The eluted 40 protein peak was applied to a DEAE-Sephacel (Pharmacia) column also equilibrated with 20 mM Tris-HCl (pH 7.4). The binding protein did not bind to DEAE-Sephacel at t h i s pH and was c o l l e c t e d in the flow through fraction. Binding protein containing fractions were pooled and applied to a CM-Sepharose (Pharmacia) column equilibrated with 20 mM sodium acetate-acetic acid buffer (pH 5.0). At this pH the binding protein bound to the column and was eluted with a NaCl gradient of 0.1 to 0.4 M. The binding protein eluted at between 0.1 and 0.2 M NaCl as a single peak of homogeneous protein as determined by SDS-polyacrylamide gel 32 electrophoresis. P-orthophosphate-bindmg a c t i v i t y was monitored at a l l stages of the p u r i f i c a t i o n . 15. F i l t e r assay of phosphate binding. Periplasmic extracts (shock fluids).and p u r i f i e d phosphate-binding protein were screened for their a b i l i t y to bind phosphate u t i l i z i n g a n i t r o c e l l u l o s e f i l t e r binding assay based on the methodology described by Lever (1972) for the h i s t i d i n e -binding protein. B r i e f l y , protein extracts were added to Eppendorf tubes in a f i n a l volume of 250 ul containing 1 uM 32 P-orthophosphate (specific a c t i v i t y = 1 mCi/umol phosphate, Amersham) and 10 mM Tris-HCl (pH 8.0). After 5 min at 23°C 100 ul aliquots were removed and f i l t e r e d on n i t r o c e l l u l o s e membrane f i l t e r s (Millipore Corp., Bedford, MA, USA, type HA, 0.45 urn pore s i z e ) . After washing once with 600 ul of 10 mM L i C l , the f i l t e r s were removed and 41 counted in 5 ml of PCS aqueous s c i n t i l l a t i o n c o c k t a i l (Amersham). To determine the specicity of the phosphate binding, various i n h i b i t o r s , as indicated, were included in the reaction mixture. 16. Equilibrium d i a l y s i s . To determine the Kd for orthophosphate binding to the phosphate-binding protein, the equilibrium d i a l y s i s technique was employed. D i a l y s i s bags (Spectrapor, 6.4 mm dia, Spectrum Medical Industries Inc., Los Angeles, CA, USA) were f i l l e d with 7 ug of p u r i f i e d phosphate-binding protein in a f i n a l volume of 300 u l . The binding protein solutions were dialysed against 40 ml of radioactively labelled orthophosphate in 50 ml conical tubes. The concentration of phosphate ranged from 0.1 uM to 5.0 uM. After d i a l y s i s for 24 h at 4°C, 25 ul aliquots (in duplicate) were removed from the d i a l y s i s bags and from the solutions surrounding the bags and counted separately in 3 ml of PCS aqueous s c i n t i l l a t i o n c o c k t a i l (Amersham). 17. Isolation of mutants lacking the phosphate-binding  protein. Diethyl sulfate mutagenesis of P. aeruginosa PA01 strain H242 was carried out as described by T. Nicas (Ph.D. Thesis, U.B.C., Vancouver, Can, 1983) with modifications. An overnight culture (0.1 ml) was resuspended in 5 ml of a saturated solution of diethyl sulfate in 50 mM sodium Hepes buffer (pH 7.0) for 30 min at 25°C. C e l l s were then diluted 1:49 into proteose peptone No. 2 broth and allowed to grow 42 overnight at 37°C. After overnight growth, mutagenized c e l l s were harvested by centrifugation (10,000 x g for 10 min) and washed three times in phosphate-less minimal Hepes-buffered medium. The washed c e l l s were resuspended in the same medium and d i l u t i o n s were plated onto phosphate-s u f f i c i e n t medium agar plates (1.0 mM phosphate) containing 20 ug/ml 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine (XP) (Bachem). It was necessary to dissolve the XP in dimethylsulfoxide prior to i t s addition to plates but the f i n a l concentration of dimethylsulfoxide was < 1 % (vol/vol). This medium i d e n t i f i e d alkaline phosphatase constitutive mutants as blue-green colonies, the colour resulting from the hydrolysis of XP by alkaline phosphatase. Wild-type c e l l s , which were repressed for alkaline phosphatase production in t h i s medium, were non-pigmented. (Alkaline phosphatase c o n s t i t u t i v i t y i s a phenotype of phosphate-binding protein-deficient mutants in E. c o l i (Brickman and Beckwith, 1975)). After overnight growth at 37°C blue-green pigmented colonies were picked and cultured overnight in phosphate-deficient medium (to induce the phosphate-binding protein). Shock f l u i d s and whole c e l l extracts were obtained and screened using SDS-polyacrylamide gel electrophoresis for the absence of the phosphate-binding protein under inducing conditions. 43 18. Construction of a rabbit anti-protein P immunoadsorbant  column. Protein P trimer-specific antibodies were p u r i f i e d free of serum proteins by incubating the protein P trimer-s p e c i f i c rabbit antiserum (200 ul) on the above protein P-a f f i - g e l a f f i n i t y column (1.8x0.7 cm). After incubation at room temperature for 45 min the column was washed with 3 ml PBS followed by 3 ml PBS + 0.25 M NaCl, to wash off unbound and non-specifically bound material, respectively. Bound antibodies were eluted with 3 ml 0.1 M glycine-HCl (pH 2.5) and fractions (300 ul) c o l l e c t e d in tubes containing enough s o l i d T r i s base (Schwartz-Mann, Cambridge, MA) to increase the pH of the fractions to between 7 and 9. The fractions containing antibody to protein P, as measured by ELISA (Mutharia and Hancock, 1983), were pooled and dialysed against one l i t r e of 0.1 M borate buffer (pH 9.0) (0.1 M boric acid (pH 9.0)/0.1 M KC1) for 24 h at 4°C, with one buffer change. The dialysed antibodies (2.3 ml at an absorbance at 280 nm of 0.22) were then cross-linked to 0.5 gm CNBr-activated Sepharose 4B (Pharmacia) in 0.1 M borate buffer (pH 9.0) for 16 h at 4°C as recommended by the manufacturer. The f i n a l column volume was approximately 1.0 ml. The column was stored at 4°C in PBS. 19. Electrophoretic elution of protein P from SDS- polyacrylamide gels. Phosphate-limited outer membranes of P. aeruginosa PA01 strain H103 were s o l u b i l i z e d in Triton X-100-Tris-EDTA (pH 8.0) to release protein P. Protein P-44 containing fractions, s o l u b i l i z e d at 23°C in a • s o l u b i l i z a t i o n mix without 2-mercaptoethanol (Hancock and Carey, 1979)", were loaded onto SDS-polyacrylamide gels (1.5 mm thickness) and electrophoresed to separate protein components. A portion of the gel was stained with Coomassie blue to locate the appropriate trimeric band of protein P and the corresponding region of the gel was cut from the unstained-unfixed portion of the gel. The porin-containing gel was crushed with a glass rod to increase i t s surface area and then placed in d i a l y s i s bags in the presence of PBS (Mutharia and Hancock, 1983) and 0.1 % (wt/vol) SDS. The proteins in the preparations were electroeluted at 50V for 2 h at 4°C in a Biorad transblotting c e l l . The buffer used for electroelution was the same as that used for Western immunoblots. 20. P u r i f i c a t i o n of phosphate starvation-induced outer  membrane proteins of the fluorescent Pseudomonads. Two methods were used for the p u r i f i c a t i o n of phosphate starvation-induced outer membrane proteins. The f i r s t was an a f f i n i t y chromatography method u t i l i z i n g an immunoadsorbant column. Triton X-100 insoluble c e l l envelopes prepared from 100 ml stationary phase cultures of phosphate-limited c e l l s were s o l u b i l i z e d in 1 ml 2 % (wt/vol) Triton X-100/20 mM Tris-HCl, pH 8.0/10 mM EDTA containing 1 mg/ml lysozyme at 37 C for 30 min. The Triton X-100-EDTA-lysozyme soluble fractions (300 ul) were 45 subsequently incubated on the anti-protein P immunoadsorbant column at room temperature. After 45 min incubation, the column was washed successively with 3 ml of 2 % TX-100/20 mM Tris-HCl, pH 8.0 and 3 ml of 2 % (wt/vol) Triton X-100/20 mM Tris-HCl, pH 8.0/0.5 M NaCl, to remove unbound and non-s p e c i f i c a l l y bound material respectively. Material bound s p e c i f i c a l l y to the column was eluted with 3 ml of 1 % (wt/vol) Triton X-100/0.1 M glycine-HCl (pH 2.5), and fractions collected in tubes containing s o l i d T r i s base as described above. Peak fractions, as determined by SDS-polyacrylamide gel electrophoresis, were pooled. The second method u t i l i z e d electroelution from SDS-polyacrylamide gels. Phosphate-limited c e l l envelopes were so l u b i l i z e d in Triton X-100/Tris-HCl (pH 8.0)/EDTA after lysozyme treatment to release phosphate-starvation-inducible proteins. Solubilized proteins (300 ul) were incubated at 23°C in a s o l u b i l i z a t i o n mix without 2-mercaptoethanol, loaded onto individual SDS-polyacrylamide gels (1.5 mm thickness) and electrophoresed to separate protein components. The phosphate-starvation-induced protein oligomers were then excised from the gels and elecroeluted as described above. The eluted proteins (5 ml f i n a l volume) were concentrated 10-fold against s o l i d polyethylene glycol (20,000 molecular weight, Sigma Chemical Co., St. Louis, MO, USA) before being dialysed at room temperature for 16 h against one l i t r e of 0.1 % (wt/vol) Triton X-100/20 mM Tris-HCl (pH 8.0) with one buffer change. 46 21. Black l i p i d bilayer experiments. The methods used for black l i p i d bilayer experiments have been described in d e t a i l (Benz et a l . , 1978; Benz and Hancock, 1981). The apparatus consisted of a Teflon chamber with two compartments separated by a small hole (0.1 mm for single channel experiments; 2 mm for macroscopic conductance experiments). A membrane was formed across the hole by painting on a solution of 1.5 % (wt/vol) oxidized cholesterol in n-decane. Bilayer formation was indicated by the membrane turning o p t i c a l l y black to incident l i g h t . In single channel conductance experiments, conductance through the pores was measured after application of a given voltage, using a pair of Ag/AgCl electrodes inserted into the aqueous solutions on both sides of the membrane. The current through the pores was boosted by a preamplifier, monitored by a storage oscilloscope and recorded on a s t r i p chart recorder. The procedure for macroscopic conductance i n h i b i t i o n experiments has been described (Hancock and Benz, submitted). B r i e f l y , the Ag/AgCl electrodes were replaced with Calomel electrodes and the current through the pores was monitored with a Keithley 610B electrometer. After addition of p u r i f i e d protein P to the solution bathing the l i p i d bilayer membrane the increase in conductance (measured as current increase) was followed for 15-25 min or u n t i l the rate of increase had slowed considerably. At this time membrane conductance had increased 2-4 orders of magnitude. 47 The bathing solutions in both compartments were s t i r r e d gently (approximately 60 revolutions per minute) with a magnetic s t i r bar and aliquots of phosphate added to both compartments. Su f f i c i e n t time (30-90 sec) was allowed for the new current level to be established before addition of subsequent aliquots. 22. Modified ELISA procedure for demonstrating an  association between protein P and the phosphate-binding  protein. a. Preparation of protein P. A solution of p u r i f i e d protein P ( approximately 1 mg in 0.5 % (wt/vol) SDS) was made 2 % (vol/vol) for Triton X-100 ( f i n a l volume of 500 ul) and passed across a Sepharose 6B (Pharmacia) column (20x1.5 cm) equilibrated with 0.1 % (vol/vol) Triton X-100/ 10 mM Tris-HCl (pH 7.4)/ 3 mM azide (column buffer). The column was eluted with column buffer and 600 ul fractions collected at 10 ml/h. Protein-containing fractions (as determined by absorbance at 280 nm) were pooled and concentrated 5-fold against 20 % (wt/vol) polyethylene glycol 20,000 (Sigma) ( f i n a l volume 1 ml). The concentrated protein was passed a second time across the Sepharose 6B column described above and protein-containing fractions (600 ul) again pooled and concentrated to y i e l d a p u r i f i e d protein P solution in Triton X-100. In some experiments, protein P was used as an enriched (but not pure) preparation obtained from the NaCl gradient-eluted, protein P-containing fractions from the 48 DEAE column described in section 3. These preparations were dialysed against 10 mM Tris-HCl (pH 7.4) for 6-8 h prior to being used in the ELISA. b. Modified ELISA procedure. Phosphate-binding protein, p u r i f i e d as described above and diluted in carbonate/bicarbonate buffer (Ruitenberg et a l . , 1974), was used to coat the bottom of the wells of polyvinyl chloride microtitre plates (Falcon 3912 Microtest III, Becton Dickinson Labware, Oxnard, CA) exactly as described for conventional ELISA (Mutharia and Hancock, 1983). After blocking unbound si t e s in the wells with f e t a l c a l f serum (FCS) [1 % (vol/vol) in 10 mM Tris-HCl (pH 7.4)] for 45 min, protein P, was added to the phosphate-binding protein-containing wells diluted in a solution of 10 mM Tris-HCl (pH 7.4) containing 0.5-1 % (vol/vol) Triton X-100 and 1 % (vol/vol) FCS. After incubation for 2 h at 37°C, the wells were washed 4 times with 1 % Triton X-100/ 10 mM Tris-HCl (pH 7.4) and the protein P trimer-specific antiserum (diluted 1:999 in Triton-Tris (pH 7.4) + 1 % FCS) was incubated on the wells for 2 h at 37°C. The plates were again washed 4 times with T r i t o n - T r i s (pH 7.4) and subsequently incubated for 2 h at 37°C in the presence of a goat-anti-rabbit IgG (H+L)-alkaline phosphatase conjugated antibody (Cappell) diluted 1:999 in Triton-Tris (pH 7.4)/ 1 % FCS. After washing 4 times with Tr i t o n - T r i s (pH 7.4), paranitrophenyl phosphate (pNPP) was added to the wells at 1 mg/ml f i n a l concentration in 10 % (vol/vol) 49 diethanolamine buffer (pH 9.8) (Ruitenberg et a l . , 1974) and incubated at 3'7°C or 23°C u n t i l colour developed. The colour reaction was assayed by measuring absorbance at 405 nm using a Titretek Multiscan ELISA reader (Flow Labs, McLean, VA). A l l incubations were at 37°C. 23. A f f i n i t y chromatography method for determining an  association between protein P and phosphate-binding protein. a. Phosphate-binding protein-Sepharose 4B a f f i n i t y  column. Triton-Tris-EDTA soluble phosphate-deficient outer membrane (enriched for protein P) (300 ul) was incubated on the phosphate-binding protein Sepharose 4B column at 4°C, 23°C or 37°C. After 1 h incubation, the column was washed with 3 ml of 0.1 % (vol/vol) Triton X-100/ 20 mM Tris-HCl (pH8.0)/ 10 mM EDTA (pH 8.0), followed by 3 ml of the same solution containing '1 M NaCl. The eluted fractions (400 ul) were collected, dialysed overnight in 20 mM Tris-HCl (pH 8.0) and concentrated 5-fold against 20 % (wt/vol) polyethylene glycol 20,000 before being run on SDS-polyacrylamide gels. b. . Protein P-Affigel-10 a f f i n i t y column. Phosphate-binding protein-containing crude shockates, obtained by Tris-MgCl2~cold shock treatment of phosphate-limited c e l l s , were desalted by passage across a Biogel P-10 (Biorad) column equilibrated with 20 mM Tris-HCl (pH 8.0) and incubated (300 ul) on a protein P-Affigel-10 column equilibrated with 0.1 % (vol/vol) Triton X-100 at 4°C, 23°C 50 or 37°C. After 1 h, the column was washed with 3 ml 20 mM Tris-HCl (pH 8.0) + Triton X-100, followed by 3 ml 20 mM Tris-HCl .(pH 8.0)/ 1 M NaCl + Triton X-100. Eluted fractions (400 ul) were dialysed overnight in 20 mM Tris-HCl (pH 8.0), concentrated 5-fold against polethylene glycol as described above and run on SDS-polyacrylamide gels. 24. Isolation of regulatory mutants of alkaline phosphatase  and phospholipase C. P. aeruginosa was mutagenized using diethylsulphate or Tn501 insertion into the chromosome (see above) and mutagenized c e l l s were plated onto phosphate-s u f f i c i e n t minimal plates (for selection of constitutive mutants) or phosphate-deficient minimal plates (for selection of non-inducible mutants) containing XP (Bachem). As described above, th i s compound yields a blue-green colour when hydrolyzed by alkaline phosphatase. Alkaline phosphatase constitutive mutants were i d e n t i f i e d as blue-green colonies on phosphate-sufficient plates and mutants non-inducible for alkaline phosphatase were i d e n t i f i e d as non-pigmented colonies on phosphate-deficient plates. To distinguish regulatory mutants from mutants in the alkaline phosphatase gene, mutant colonies were tested for their phospholipase C phenotypes. Thus, mutagenized colonies were transferred by contact onto Whatman 3M paper previously soaked in NPPC (a chromogenic substrate for phospholipase C (see above)). Colonies demonstrating phospholipase C a c t i v i t y turned rapidly yellow on the f i l t e r paper. 51 Regulatory mutants were i d e n t i f i e d as those colonies constitutive or non-inducible for both enzyme a c t i v i t i e s . 25. Other assays. Protein determinations were made by either the method of Schacterle and Pollack (1973), using bovine serum albumin as the standard, or the method of Warburg et a l (1941), using absorbance at 260 and 280 nm. 2-keto-3-deoxy-octulosonic acid (KDO) was measured using the method of Osborn (1963). The s i l v e r staining procedures for protein (Wray et a l . , 1981) and LPS (Tsai and Frasch, 1982) have been described . 52 CHAPTER ONE Outer membrane protein P; involvement in h i g h - a f f i n i t y • phosphate transport in Pseudomonas aeruginosa 1. Induction of protein P by phosphate l i m i t a t i o n . Growth of P. aeruginosa PA01 strain H103 in a phosphate-deficient medium (0.2 mM phosphate) was characterized by an i n i t i a l logarithmic rate of growth indistinguishable from that observed for phosphate s u f f i c i e n t (0.6 mM phosphate) c e l l s (60 min doubling time) (Fig. 1). The onset of phosphate-li m i t a t i o n in the phosphate-deficient c e l l culture was detectable as a marked decline in growth rate (> 2.5 h doubling time) which contrasted with the phosphate-s u f f i c i e n t .ce l l culture which continued to grow with a doubling time of ca. 60 min (Fig. 1). At concentrations of phosphate below 0.2 mM the t o t a l growth y i e l d was dependent upon the concentration of phosphate in the medium (Fig. 2) indicating that phosphate was indeed l i m i t i n g for growth at these concentrations. Examination of the outer membranes prepared from P. aeruginosa c e l l s harvested 4 h after the onset of phosphate li m i t a t i o n revealed the presence of a novel protein (Fig. 3, lane 2), hereafter referred to as protein P, not present in c e l l s grown in a phosphate-sufficient medium (Fig. 3, lane 1). A major protein of ca. 22,000 molecular weight present in the outer membranes of c e l l s grown in phosphate-sufficient medium (Fig. 1, lane 1) was consistently absent from the outer 53 Figure 1. Growth of P. aeruginosa in a phosphate deficient medium. Overnight cultures of P. aeruginosa PA01 stra i n H103 grown in phosphate-sufficient medium were harvested, washed twice with phosphate-deficient medium and resuspended in phosphate-deficient (X—X) or phosphate-sufficient (0—0) medium at an absorbance at 600 nm of 0.20 (A500)• Growth was followed by the time dependent increase in A f i n n. 54 0 60 120 180 240 300 360 Time (m in ) 55 Figure 2. Growth y i e l d of Pseudomonas aeruginosa as a function of the concentration of phosphate in a defined minimal medium. An overnight culture of P. aeruginosa H103 grown in phosphate-sufficient medium was harvested, washed twice in phosphate-free Hepes-buffered minimal medium and resuspended in the or i g i n a l volume of the same phosphate-free medium. Aliquots (0.1 ml) were added to flasks containing Hepes-buffered minimal medium and varying concentrations of phosphate and allowed to grow overnight at 37 C. The growth y i e l d was determined from the culture density (measured as A, 0 Q) obtained after overnight incubation. 56 0.8H Phosphate Concentration ( J J M ) 57 1 2 3 A 5 6 7 Figure 3. SDS-polyacrylamide gel electrophoretogram of pu r i f i e d protein P and of outer membranes and shock f l u i d s of phosphate-sufficient and phosphate-deficient c e l l s of P. aeruginosa H103. Lane 1, outer membrane of phosphate-sufficient H103; lane 2, outer membrane of phosphate-deficient H103; lane 3, p u r i f i e d protein P so l u b i l i z e d at 75°C; lane 4, p u r i f i e d protein P so l u b i l i z e d at 55°C; lane 5, p u r i f i e d protein P so l u b i l i z e d at 25°C; lane 6, unconcentrated shock f l u i d of phosphate-sufficient H103; lane 7, unconcentrated shock f l u i d of phosphate-deficient H103. Because of the dil u t e nature of the samples in lanes 6 and 7 only major proteins were detected. Samples were s o l u b i l i z e d at 88°C prior to electrophoresis unless otherwise indicated. P, protein P monomer; P*f protein P oligomer (trimer; Angus et a l . , 1983); F, protein F. membranes of a l l phosphate-limited P. aeruginosa strains examined, including a protein P-deficient mutant (H576) (Fig. 9). Additional minor alterations in the outer membrane protein banding patterns of phosphate-sufficient and phosphate-deficient grown c e l l s were consistently observed, and may r e f l e c t differences in growth stage since phosphate-limited c e l l s were routinely harvested several hours after the onset of l i m i t a t i o n when the growth rate was substantially lower than that observed for phosphate-s u f f i c i e n t c e l l s (Fig. 1). By previously published c r i t e r i a (Hancock et a l . , 1981) protein P was not a peptidoglycan-associated protein although i t s i n a b i l i t y to be s o l u b i l i z e d in 2 % SDS/ 20 mM Tris-HCl (pH 8.0) suggested that i t was at least weakly associated with the peptidoglycan. Using the procedure outlined in Methods, a highly p u r i f i e d preparation of this protein was obtained (Fig. 3, lane 3)» The apparent molecular weight of the p u r i f i e d protein in SDS-polyacrylamide gels after s o l u b i l i z a t i o n in SDS at 75°C was 48,000 (48K), corresponding exactly to the apparent molecular weight in outer membranes. Upon s o l u b i l i z a t i o n at temperatures < 60°C, however, the protein ran at an apparently higher molecular weight (Fig. 3, lanes 4 and 5) suggesting that the native form of the protein was an oligomer. The oligomeric nature of protein P was confirmed by cross-linking data which indicated that the native protein was, in fact, a trimer (Angus et a l . 1983). 59 The formation by protein P of SDS-stable oligomers in polyacrylamide gels is» consistent with properties of known enteric porin proteins (Lugtenberg and van Alphen, 1983), but in contrast to the previously described P. aeruginosa porin proteins F (Hancock and Carey, 1979) and D1 (Hancock and Carey, 1980) which did not demonstrate oligomer formation in SDS-polyacrylamide gels. 2. Co-regulation with alkaline phosphatase, phospholipase C  and a 34K periplasmic protein. When phosphate became li m i t i n g for growth as indicated by a decline in growth rate (Fig. 4A) detectable levels of the enzymes alkaline phosphatase and phospholipase C were produced by wild-type P. aeruginosa c e l l s , and the levels increased with time (Fig. 4, panels B and C). A protein of molecular weight 34K was also observed as the major protein in the periplasm (releasable by Tris-MgCl 2~cold shock) of c e l l s grown in phosphate-deficient (Fig. 3, lane 7) but not phosphate-s u f f i c i e n t (Fig. 3, lane 6) media. In addition to their supernatant a c t i v i t i e s (Fig. 4B), alkaline phosphatase and phospholipase C exhibited cell-associated a c t i v i t y which was lo c a l i z e d to the periplasm (Fig. 4C). The above data indicated that protein P was co-regulated with the enzymes alkaline phosphatase and phospholipase C and the 34K periplasmic protein. To obtain support for this genetically, mutants non-inducible (H553) or constitutive (H587) for alkaline phosphatase were 60 Figure 4. Induction by phosphate-limitation and l o c a l i z a t i o n of alkaline phosphatase and phospholipase C of P. aeruginosa H103. A) Growth in phosphate-de f i c i e n t medium. B) Supernatant a c t i v i t i e s and C) Periplasmic a c t i v i t i e s of alkaline phosphatase (X—X) and phospholipase C ( • — • ) . Logarithmic-phase c e l l s in phosphate-sufficient medium (1 mM Pi) were washed and resuspended in phosphate-deficient medium (0.2 mM Pi) at time zero. Ce l l s were harvested at various times during growth. Supernatants were obtained after removal of c e l l s by centrifugation and periplasmic extracts were obtained using the Tris-MgCl 2 cold shock procedure of Hoshino and Kageyama (1980). Enzyme assays were carried out as described in Methods. The measurements in panels B and C were representative data of 5 separate experiments. 61 62 i s o l a t e d (see legend t o F i g . 5 ) . Mutan t s t r a i n H553 was s i m i l a r l y n o n - i n d u c i b l e f o r p h o s p h o l i p a s e C, and e x a m i n a t i o n of t he p r o t e i n complement o f t h i s mutant r e v e a l e d t h a t i t was a l s o n o n - i n d u c i b l e f o r p r o t e i n P ( F i g . 5, l a n e C; c f . w i l d - t y p e , l a n e D) and the 34K p e r i p l a s m i c p r o t e i n ( F i g . 5, l ane E, c f . w i l d - t y p e , l a n e F ) . L i k e w i s e , t he a l k a l i n e phosphatase c o n s t i t u t i v e mutan t s t r a i n H587 was a d d i t i o n a l l y c o n s t i t u t i v e f o r p h o s p h o l i p a s e C (measured by NPPC h y d r o l y s i s ) as w e l l as p r o t e i n P and the 34K p e r i p l a s m i c p r o t e i n ( F i g . 5, l ane A; c f . w i l d - t y p e , - l a n e B ) , a l b e i t a t l e v e l s below t h a t o b t a i n e d i n t h e f u l l y d e r e p r e s s e d w i l d -t y p e ( l a n e s D and F ) . These d a t a s u p p o r t t he e x i s t e n c e of a phosphate r e g u l o n i n P. a e r u g i n o s a ana logous t o t h e pho r e g u l o n i n E. c o l i (Tommassen and L u g t e n b e r g , 1 9 8 2 ) . 3. Outer membrane p e r m e a b i l i t y . A l k a l i n e phosphatase i s c h a r a c t e r i s t i c a l l y a p e r i p l a s m i c marker i n g r a m - n e g a t i v e b a c t e r i a and some a l k a l i n e phosphatase and p h o s p h o l i p a s e C a c t i v i t y was a lways d e t e c t a b l e i n t he p e r i p l a s m of p h o s p h a t e - l i m i t e d P. a e r u g i n o s a c e l l s ( F i g . 4C) . However, a p o r t i o n o f t h e a l k a l i n e phosphatase and the m a j o r i t y o f t h e p h o s p h o l i p a s e C a c t i v i t y of p h o s p h a t e - l i m i t e d c e l l s was r e l e a s e d i n t o the c u l t u r e s u p e r n a t a n t (see b e l o w ) . The e x t r a c e l l u l a r r e l e a s e of t hese enzymes by p h o s p h a t e - l i m i t e d c e l l s c o u l d be e x p l a i n e d by a breakdown i n t he o u t e r membrane p e r m e a b i l i t y b a r r i e r , r e l e a s i n g them f rom a p e r i p l a s m i c l o c a t i o n . C o n v e r s e l y , a mechanism of s p e c i f i c 63 A B C D E F Figure 5. SDS-polyacrylamide gel electrophoretogram of whole c e l l protein extracts and c e l l envelope and soluble (non-membrane) fractions of alkaline phosphatase regulatory mutants. Mutants of P. aeruginosa H103 constitutive (H587) and non-inducible (H585) for alkaline phosphatase were isolated following diethylsulphate mutagenesis as described in Methods. Lane A, whole c e l l protein extract of phosphate-s u f f i c i e n t H587; lane B, whole c e l l protein extract of phosphate-sufficient H103; lane C, c e l l envelope of phosphate-deficient H553; lane D, c e l l envelope of phosphate-deficient H103; lane E, soluble fracti o n of phosphate-deficient H553; lane F, soluble fracti o n of phosphate-deficient H103. A l l samples were s o l u b i l i z e d at 88°C for 10 min prior to electrophoresis. P, protein P; 34K, 34,000 molecular weight periplasmic protein. 64 secretion across the outer membrane may be responsible for their release, in the absence of any gross permeability changes. To test the s p e c i f i c i t y of alkaline phosphatase and phospholipase C release, the d i s t r i b u t i o n of two other proteins normally l o c a l i z e d within the periplasm, the constitutive RP1-encoded beta-lactamase and the 34K protein, were examined at the time of enzyme induction and secretion. The results in Fig. 6 demonstrated that beta-lactamase remained almost wholly periplasmic (as T r i s - M g C ^ T e l e a s a b l e enzyme) during growth on phosphate-deficient medium, with only 6 % of the t o t a l a c t i v i t y present in the supernatant (extracellular medium) 2 h after the onset of phosphate l i m i t a t i o n . The 34K protein, although present as the major protein in the periplasm upon induction (Fig. 3), was undetectable in the supernatant as determined by SDS-polyacrylamide gel electrophoresis of 50-fold' concentrated supernatants. In contrast, up to 58 % of the t o t a l alkaline phosphatase a c t i v i t y and 87 % of the t o t a l phospholipase C a c t i v i t y were found in the supernatant 2h post-induction (Fig. 4B). These results confirmed that the release of alkaline phosphatase and phospholipase C by whole phosphate-limited c e l l s was indeed s p e c i f i c and not explainable by a general increase in outer membrane leakiness. Furthermore, LPS (measured as KDO) or major outer membrane proteins were not detected in 50-fold concentrated supernatants, supporting the absence of membrane breakdown during release. 65 The periplasmic location of beta-lactamase (Hancock et a l . , 1981) and the demonstration by Angus et a l . (1982) that n i t r o c e f i n is taken up by the hydrophilic (porin) pathway, provided a means by which outer membrane permeability could be measured d i r e c t l y , as a function of n i t r o c e f i n uptake and hydrolysis. Furthermore, treatment of c e l l s with EDTA, an agent known to break down the outer membrane thus increasing permeability (Hague and Russel, 1974), is associated with a 10-fold increase in n i t r o c e f i n hydrolysis (Hancock et a l . , 1981). From the results of n i t r o c e f i n permeability assays, permeability c o e f f i c i e n t s (C) were calculated (see Methods) as a function of growth in phosphate-deficient medium. No increase in outer membrane permeability was detected concomittant with, enzyme release (Fig. 6B). In fact, the only a l t e r a t i o n in outer membrane permeability which was detected over the 2.5 h of the experiment was a general 2.8-fold decrease in permeability (Fig. 6). Given the low permeability of the P. aeruginosa outer membrane and the lack of an increase in permeability during phosphate-limited growth, i t seemed reasonable to hypothesize that protein P may function as a phosphate porin, mediating the uptake of phosphate from a di l u t e environment. 66 Figure 6. Outer membrane permeability during growth on phosphate-deficient medium. Panel A shows growth after transfer to phosphate-deficient medium at time zero as described in the legend to Figure 4. Panel B shows beta-lactamase a c t i v i t y in the supernatant (•—•) and periplasm (•—•) and the outer membrane permeability c o e f f i c i e n t C (X—X) (expressed in ml/min/mg whole c e l l protein) calculated as described in Methods. 67 60 120 180 Time (min ) 240 68 4. LPS-free protein P forms channels in planar l i p i d  bilayer membranes. During the course of this study, protein P was demonstrated to form small (0.6 nm diameter), water-f i l l e d channels in l i p i d bilayer membranes which were sp e c i f i c for anions (Hancock et a l . , 1982). The basis of this s p e c i f i c i t y was shown to be lysine residues in or near the channel (Hancock et a l . , 1983), which also formed a binding s i t e for phosphate (Hancock and Benz, submitted). Data in the l i t e r a t u r e suggests that the a b i l i t y of porins to form channels i s dependent upon an association with LPS (Schindler and Rosenbusch, 1978). Furthermore, i t has been proposed that LPS.may function to modulate porin a c t i v i t y (Kropinski et a l . , 1982). To determine i f any of the properties hitherto attributed to protein P were related to i t s association with LPS ( LPS i s invariably detected in p u r i f i e d preparations of protein P (see below)) the protein was p u r i f i e d free of LPS by electroeluting the protein trimer out of SDS-polyacrylamide gels as described in Methods. Protein P isolated by electroelution lacked detectable LPS (<2-3.8X10 mol/mol protein) as measured by ELISA using LPS-specific monoclonal antibodies (Table II) and as observed by s i l v e r staining for LPS in polyacrylamide gels (Fig. 7). In contrast, the conventionally p u r i f i e d protein contained s i g n i f i c a n t levels of LPS (1-1.7 mol/mol protein) (Table II; F i g . 7). LPS-free protein P was s t i l l capable of forming channels in l i p i d bilayer membranes with a mean single channel conductance in 1 M KC1 (234 pS) 69 1 2 Figure 7. SDS-polyacrylamide gel electrophoretogram of LPS associated with protein P. Conventionally p u r i f i e d (lane 1) and electroeluted (lane 2) protein P were electrophoresed on SDS-polyacryalimide gels after s o l u b i l i z a t i o n at 23°C for 10 min and stained for LPS using the pocedure of Tsai and Frasch (1982). The area of densest stain in lane 1 occurs at the tracking dye front. 70 Table II. Measurements of LPS associated with conventionally p u r i f i e d and electroeluted protein P Associated LPS (mol/mol protein) Assay Method Conventionally P u r i f i e d Electroeluted SDS-PAGE3 1.7 <2X10~2 ELISA b 1.1 <3.8X10~2 Conventionally p u r i f i e d and electroeluted protein P were electrophoresed on SDS-polyacrylamide gels and stained for LPS using the method of Tsai and Frasch (1982). Contaminating LPS was estimated by comparing s i l v e r stained electrophoretograms of d i l u t i o n s of the protein P preparations with s i l v e r stained electrophoretograms of d i l u t i o n s of pure LPS of known concentration. b Protein P preparations were s e r i a l l y d i luted and used to coat the bottoms of microtitre wells. Monoclonal antibodies s p e c i f i c for P. aeruginosa LPS were then used to detect the presence of LPS at each d i l u t i o n . Based on the detection l i m i t s of the antibodies employed (approximately 50 ng LPS), derived from ELISA analysis of s e r i a l l y diluted pure LPS preparations of known concentration, the LPS levels could be estimtated from the highest d i l u t i o n which s t i l l gave a positive LPS response. 71 Table I I I . Functional properties of conventionally p u r i f i e d and electroeluted protein P in planar l i p i d bilayer membranes Pu r i f i c a t i o n Average single Number of S e l e c t i v i t y Procedure channel conductance events (Pc/Pa) in 1 M KC1 (pS) Conventional 235 317 <0.0l b Electroeluted 234 224 0.005 a Permeability ratio of K + to Cl derived from the Goldman-Hodgkin-Katz equation as described by Hancock et a l . (1982) b Taken from Benz et a l . (1985) 72 almost indistinguishable from that obtained for the conventionally p u r i f i e d , LPS-contaminated protein (235 pS) (Table I I I ) . Furthermore, the LPS-free protein remained anion-specific (Pc/Pa = 0.005) and single channel conductance through LPS-free protein P channels was observed to saturate at high concentrations of KC1, consistent with a binding s i t e in the channel for anions. These properties were in agreement with the published properties of the conventionally p u r i f i e d protein (Hancock et a l . , 1982; Table I I I ) . LPS association was therefore not required for channel formation by protein P and was not responsible for the functional properties of this protein in v i t r o . 5. Isolation of a protein P-deficient mutant. a. Preparation of a protein P trimer-specific antiserum. A polyclonal rabbit antiserum raised against p u r i f i e d protein P trimers (see Methods) reacted s p e c i f i c a l l y with the native trimer form of the protein (Fig. 8A, lane 3), exhibiting no reaction with heat-dissociated monomers (Fig. 8A, lane 4). The smearing pattern evident in the reaction of the antibody with electrophoresed protein P trimers suggested some heterogeneity. This may be due to an association of the trimer form of the protein with LPS or due to aggregation of the trimers. Nevertheless, a l l of the material in the smear reacting with the antibody was protein P as confirmed by the a b i l i t y to convert this 73 1 2 3 A Figure 8. Immunoblots of electrophoretically separated P.aeruginosa H103 c e l l envelopes and p u r i f i e d protein P, and whole c e l l s . A) C e l l envelopes from phosphate-s u f f i c i e n t c e l l s (lane 1), phosphate-limited c e l l s (lane 2) and p u r i f i e d protein P (lanes 3 and 4) were separated on SDS-polyacrylamide gels after s o l u b i l i z a t i o n at 23°C (lanes 1-3) or 88°C (lane 4) for 10 min. After electrophoretic transfer to n i t r o c e l l u l o s e , the blots were interacted with the protein P-specific polyclonal antiserum and subsequently immunostained using a peroxidase-conjugated goat-anti-rabbit IgG antibody and a histochemical stain for peroxidase (see Methods). B) A colony immunoblot showing the interaction of the protein P-specific polyclonal antiserum with phosphate-limited Tn501 insertion mutants of P. aeruginosa PA01 str a i n H103. The protein P-deficient mutant, s t r a i n H576, i s indicated by the arrowhead. 74 material to protein P monomers by heating (see Chapter Three, Fig. 19A) . The s p e c i f i c i t y of the polyclonal antiserum to protein P was demonstrated by the a b i l i t y of the antiserum to react with a component present in envelopes from phosphate-starved c e l l s (Fig. 8A, lane 2) which was absent in envelopes from phosphate-sufficient c e l l s (Fig. 8A, lane 1). The reaction p r o f i l e was very similar to that seen with p u r i f i e d protein P trimers (Fig. 8A, lane 3). b. Tn50l mutagenesis of P. aeruginosa. In our search for a suitable vehicle for use in the transposon insertion mutagenesis of P. aeruginosa PA01 (HI 03), a number of vectors were tested (see Table IV). One class, which included plasmids pME9 and pME3l9, were temperature sensitive for maintainance due to mutation, so that selection for transposon-encoded resistance at the non-permissive temperature (42°C) resulted in insertions into the PAO chromosome. Recovery of the transposable element at the non-permissive temperature was usually (> 98 %) associated with recovery of a l l plasmid a n t i b i o t i c resistance markers as well, indicating that the entire plasmid had probably inserted. The second class of vectors tested included plasmids pUW942 (::Tn50l), pUW964 (::Tn5), pAS8Rep-1 (::Tn7) and pKPlOO (:;Tn5-132). These plasmids were hybrids comprising the broad host range transfer functions of the Inc P-1 75 Abbreviations: Cb r, c a r b e n i c i l l i n resistant; Kn r, kanamycin resistant; Tp r, trimethoprim resistant; Tc r, tetracycline resistant; Hg r, mercury resistant; t s , temperature sensitive. Genotype symbols are according to Bachmann (1983). Only the relevant phenotypes are indicated. b A.derivative of Tn5 carrying a T c r determinant in place of the Kn r determinant (Berg and Berg, 1983). c The temperature sensitive phenotype is due to a mutation in t r f A (Rella et a l , 1985). d A derivative of Tn5 carrying the Trf determinant of plasmid R751 (Rella et a l , 1985). 76 Table IV. Plasmids tested for u t i l i t y in transposon insertion mutagenesis of P. aeruginosa Plasmid Description' Source/ Reference pAS8Rep-1 RP4-ColE1 hybrid/rep(RP4)::Tn7 (Tra + Cb r Kn r T c s Tp r) pUW942 pAS8Rep-1:;Tn501 (Tra + Cb r Kn r T c s Tp r Hg r) pKP100 putative pAS8Rep-1:;Tn5-132b (Tra + Cb r Kn r Tc r Tp r) pRK2013 RK2-ColE1 hybrid T r a + Kn r pUW964 pRK20l3(Kan::Tn7)::Tn5 (Tra + Cb s Kn r T c s Tp r) PME319 RP1 t S [Rep A t S Rep B t S ] (carries TnJ_) (Tra + Cb r Kn r Tc r) pME305 RP1 t s with a 12 kb deletion c in Kn r, primase and IS21 (Tra + Cb r Kn s Tc r) pME9 pME305::Tn5-751 (Tra + Cb r Kn r Tc r Tp r) PMO190 a temperature-sensitive mutant of R68 (=RP4) pMTlOOO PMO190::Tn50l Sato et a l , 1981 Weiss and Falkow, 1983 Figurski & Helinski, 1979 Weiss et a l , 1983 Haas et a l , 1981 Rella et a l , 1985 Rella et a l , 1985 Tsuda et a l , 1984 Tsuda et a l , 1984 77 plasmids (eg. RP1) and the narrow host range r e p l i c a t i o n functions of the ColE1-like plasmids. Thus, these vectors could be transferred from E. c o l i to P. aeruginosa but were unable to replicate in this recipient (Bagdasarian et a l . , 1979). Selection for transposon-encoded resistances in P. aeruginosa PAO strains mated with E. c o l i strains harbouring these plasmids revealed colonies with insertions in the chromosome. Almost without exception, insertion events associated with these vectors involved insertion of the whole plasmid, as indicated by the recovery of a l l plasmid markers. The plasmid markers were stably maintained in P. aeruginosa and were not readily transferrable to a second host str a i n indicating that they were, indeed, present as inserts in the chromosome. Although curing of transposon-mediated whole plasmid inserts (co-integrates), to leave a single copy of the" transposon in the mutated gene, has been documented (Harayama et a l . , 1981; Tsuda et a l . , 1984), the consistent i s o l a t i o n of whole plasmid inserts made the vectors outlined above unsuitable for our needs. The i s o l a t i o n of a protein P-deficient mutant, which involved a negative selection, required the screening of thousands of potential mutants. It was thus desirable to have a system whereby mutants generated would represent resolved, single transposon inserts, without the requirement for additional manipulations to obtain the desired insertional mutation. 78 Plasmid pMTlOOO, recently described by Tsuda et a l . (1984), i s a temperature-sensitive R68 plasmid carrying a Tn501 element. Insertion mutants in P.aeruginosa can be readily selected on HgCl 2~containing plates at 42°C. After r a i s i n g the incubation temperature of P. aeruginosa PA01 st r a i n H103 (pMTlOOO) to the r e s t r i c t i v e temperature (42°C), colonies resistant to HgCl 2 were isolated at a frequency of > 1x10~ 3/viable c e l l . Of these, approximately 30 % (3X10~ 4/ viable c e l l ) apparently represented whole plasmid inserts in that they were resistant to Cb, Tc and Kn, as well as to HgCl 2 r and this proportion decreased to < 15 % after a single passage on HgCl 2~containing plates. The remainder of the mercury resistant colonies were sensitive to Cb, Tc and Kn. This, together with the high frequency of i s o l a t i o n suggested that they were Tn501 insertion mutants. Examination of colonies growing on HgCl 2~containing plates at 42°C revealed the existence of two colony morphologies which could be correlated to the type of insertion event which had occurred in these clones in the rescue of the Tn501 element. Colonies containing whole plasmid inserts (Hg r, Tc r, Kn r, Cb r at 42°C) were t y p i c a l l y f l a t , translucent and i r r e g u l a r l y shaped. Colonies containing a resolved Tn501 insertion (Hg r, T c s , Kn s, Cb s at 42°C) were opaque, dome-shaped and generally c i r c u l a r , t y p i c a l of wild type. The i s o l a t i o n , in the majority of cases, of resolved Tn501 inserts in P. aeruginosa PA01 strain H103 meant that 79 the Tn501 insertion mutagenesis was s i g n i f i c a n t l y simpler than published procedures (Harayama et a l . , 1981; Tsuda et a l . , 1984) requiring no curing of plasmid sequences. In addition, the mutagenic c a p a b i l i t y of plasmid pMTlOOO (Tsuda et a l . , 1984) was confirmed in P. aeruginosa PA01 strain H103 by the i s o l a t i o n of auxotrophs (frequency=2x10 /Hg — 4 r colony), mutants deficient in pigment production (6x10 /Hg colony), and a number of pho regulon mutants, including phosphate-binding protein-deficient mutants (2x10 4/Hg r colony), alkaline phosphatase constitutive mutants (1x10 / Hg r colony) and alkaline phophatase-deficient mutants (3.3x10~ 4/Hg r colony). c. Isolation of a Tn501-induced protein P- deficient mutant. In order to confirm a role for protein P in phosphate transport in P. aeruginosa, i t was necessary to isolate a mutant deficient in protein P. Plasmid pMTlOOO-mediated Tn501 insertion mutants, isolated as resistant to HgCl 2 at 42°C, were transferred from phosphate-deficient minimal medium plates to n i t r o c e l l u l o s e by contact and screened for the absence of protein P using a protein P-s p e c i f i c antiserum. Of 3,200 mercury resistant colonies screened, only one f a i l e d to react strongly with the protein P-specific antiserum in the colony blot assay (see F i g . 8B). SDS-polyacrylamide gel electrophoresis of c e l l envelopes of this mutant (designated strain H576), grown under phosphate-deficient conditions, confirmed the absence of detectable 80 1 2 3 4 5 Figure 9. SDS-polyacrylamide gel electrophoretogram of outer membranes prepared from a protein P-deficient mutant of P. aeruginosa and i t s wild type parent. Lane 1, p u r i f i e d protein P. The outer membranes were prepared from: lane 2, phosphate-sufficient H103 (wild type) c e l l s ; lane 3, phosphate-deficient H103 c e l l s ; lane 4, phosphate-sufficient H576 (mutant) c e l l s ; lane 5, phosphate-deficient H576 c e l l s . A l l preparations were s o l u b i l i z e d at 88°C prior to electrophoresis such that protein P (P) ran as the monomer. 81 protein P (see F i g . 9, lane 5). In contrast, the parent stra i n H103 grown under the same conditions produced large quantities of protein P (Fig. 9, lane 3). Western immunoblots of electrophoretically-separated c e l l envelope and whole c e l l proteins confirmed the absence of detectable protein P in phosphate-limited mutant c e l l s using both a protein P trimer-specific and monomer-specific antiserum. The mutant, l i k e i t s parent, was normally derepressible for alkaline phosphatase and phospholipase C under conditions of phosphate deficiency. In addition, the presence of the phosphate-binding protein in shock f l u i d s and whole c e l l extracts of the mutant was confirmed using SDS-polyacrylamide gel electrophoresis and Western immunoblotting with a phosphate-binding protein s p e c i f i c antiserum (data not shown). These results supported the s p e c i f i c loss of protein P in t h i s mutant, and distinguished t h i s s t r a i n from a class of mutants isolated previously which were p l e i o t r o p i c a l l y d e f i c i e n t in the phosphate-regulated components of P. aeruginosa, including protein P (see F ig. 5). 6. Phosphate transport. Phosphate transport in wild-type P. aeruginosa is characterized by the presence of two major systems of uptake, of low and h i g h - a f f i n i t y , respectively (LaCoste et a l . , 1981). When stationary phase, phosphate-starved c e l l s of P. aeruginosa were pre-incubated, with aeration, at 37°C for only 5 min prior to transport assays, 82 i t was possible to examine h i g h - a f f i n i t y phosphate transport alone, since i t was found necessary to incubate c e l l s for longer periods (15-25 min) at 37°C for the low-affinity uptake system to become operative. Thus i t was possible to precisely examine what role, i f any, protein P played in h i g h - a f f i n i t y phosphate transport by comparing phosphate uptake in the protein P-deficient mutant with that of i t s parent H103. Compared with the wild type parent s t r a i n , the protein P-deficient mutant was s i g n i f i c a n t l y defective in phosphate transport, exhibiting a Km for h i g h - a f f i n i t y transport roughly 10 times greater than that of the parent (Table V). No effect on the Vmax of the system was seen, however, as a result of the loss of protein P in the mutant (Table V). This confirmed the involvement of protein P in h i g h - a f f i n i t y phosphate transport in P. aeruginosa. It was not possible to accurately determine the kinetic parameters of low-affinity phosphate transport in the mutant owing to the simultaneous operation of a h i g h - a f f i n i t y uptake component (see Chapter Two, section 4 ) . However, the rates of phosphate transport measured in the mutant (H576) and wild-type (H103) were comparable at higher concentrations of phosphate (> 25 uM) suggesting that low-a f f i n i t y phosphate uptake was not affected by the protein P-deficiency of the mutant. 83 Table V. K i n e t i c s of h i g h - a f f i n i t y phosphate t r a n s p o r t i n a p r o t e i n P - d e f i c i e n t mutant s t r a i n and i t s w i l d -type p a r e n t S t r a i n Km (uM) Vmax (umol/min/mg c e l l p r o t e i n ) H1 03 0.39 + 0.07 5.34 + 0.59 H576 3.60 + 0.64 5.56 + 0.66 I n i t i a l r a t e s of phosphate t r a n s p o r t at v a r i o u s c o n c e n t r a t i o n s of phosphate were p l o t t e d as an E a d i e - H o f s t e e p l o t , from which k i n e t i c parameters were d e r i v e d by l e a s t squares a n a l y s i s . The r e s u l t s a re the mean v a l u e s + s t a n d a r d d e v i a t i o n s of f o u r e x p e r i m e n t s . 8 4 7. Growth in low phosphate medium. It was of interest to determine i f the transport differences attributable to a lack of protein P were s i g n i f i c a n t in terms of the growth c a p a b i l i t i e s of the c e l l under phosphate-limiting conditions (under which conditions protein P i s normally derepressed). Thus wild-type strain H103 and mutant stra i n H576 c e l l s were grown in phosphate-deficient medium for 14-16 hrs to deplete internal phosphate pools and thus make growth dependent on transported phosphate. These c e l l s were then placed in a Hepes-buffered minimal medium containing 50 uM phosphate. Typ i c a l l y , a lag period of 30-45 min was observed followed by logarithmic growth for 2-4 h at a very reduced rate, after which the c e l l s stopped growing. Determination of the rate of growth during this period revealed that the protein P-deficient mutant grew more slowly than i t s wild type parent strain H103 (Table VI). To eliminate possible growth differences attributable to the presence of a Tn501 element in the chromosome of the protein P-deficient mutant, an arginine auxotroph, str a i n H556, obtained by Tn501 insertion mutagenesis, was used as the protein P-derepressible control. Again, the mutant lacking protein P exhibited a slower rate of growth than the strain producing wild type levels of protein P (Table VI). The 18-35 % increase in doubling time characterized by the protein P mutant strain stresses the importance of protein P channels in the outer membrane of P. aeruginosa c e l l s growing in a phosphate-limited environment. 85 Table VI. Growth of a protein P-deficient mutant and strains wild type for protein P in a phosphate-limited medium Experiment 3 Strain* 5 Doubling time c (h) H103 12.82 +1.18 H576 15.87 + 0.60 H103 9.98 + 0.90 H576 15.43 + 2.57 H556 13.55 + 0.33 H576 19.60 + 1.62 H556 11.27 + 0.53 H576 13.67 + 0.33 Overnight cultures, grown in phosphate-deficient medium, were resuspended in t r i p l i c a t e in Hepes-buffered minimal medium containing 50 uM phosphate at A 6 Q 0 = 0.20 and growth measured by the increase in A^QQ. b H103, wild type PA01; H556, arginine requiring Tn50l insertion mutant; H576, Tn501 insertion mutant def i c i e n t in protein P. c Doubling times represent the reciprocal of growth rate, u, calculated from a plot of ln A 6 0 Q vs. time (min) using least squares analysis. Results are expressed as the mean doubling times + standard deviations for three cultures (see above). Variations from experiment to experiment in growth rates obtained for a given str a i n r e f l e c t technical d i f f i c u l t i e s in obtaining precisely the same degree of phosphate l i m i t a t i o n every time. For a given experiment, however, the degree of l i m i t a t i o n was the same for each s t r a i n . 86 8. Summary. When wild-type c e l l s of Pseudomonas aeruginosa PA01 were grown in a medium containing 0.2 mM or less inorganic phosphate (phosphate-deficient medium) a new major outer membrane protein, P, was induced. The protein was pu r i f i e d and demonstrated to form SDS-resistant oligomers in polyacrylamide gels, a property shared by most known porins. The enzymes alkaline phosphatase and phospholipase C, as well as a major periplasmic protein of 34K were co-induced in a phosphate-deficient medium at the onset of phosphate-li m i t a t i o n i d e n t i f i a b l e by a marked decrease in growth rate. Mutants constitutive or non-inducible for alkaline phosphatase and phospholipase C were isolated and demonstrated to be s i m i l a r l y constitutive and non-inducible, respectively, for protein P and the 34K periplasmic protein consistent with the existence of a phosphate regulon in P. aeruginosa. The phosphate-starvation-inducible enzymes alkaline phosphatase and phospholipase C were secreted into the growth medium upon induction although enzyme release was shown not to be associated with a breakdown in the outer membrane or an increase in outer membrane permeability. As such, an increase in outer membrane permeability, which could conceivably increase the rate of phosphate movement across the outer membrane, i s not the means by which P. aeruginosa adapts to a phosphate-limited environment. Protein P has been demonstrated to form small, anion-specific channels and the protein p u r i f i e d free of LPS exhibited unaltered channel-forming properties 87 in planar bilayer membranes. In order to demonstrate a role for protein P channels in phosphate transport in P. aeruginosa, a transposon insertion mutant de f i c i e n t in protein P was sought. A number of transposon delivery systems were tested which yielded, for the most part, whole plasmid inserts. Plasmid pMTlOOO (Tsuda et a l . , 1984), a temperature-sensitive R68 plasmid carrying the transposon Tn501, was successfully employed in the i s o l a t i o n of a Tn501 insertion mutant lacking protein P under normally inducing conditions. To identify the mutant deficient in protein P, a protein P-specific polyclonal antiserum was used. This mutant, strain H576, was d e f i c i e n t in h i g h - a f f i n i t y phosphate transport, exhibiting a Km for uptake (3.60 uM phosphate) almost ten times greater than that of the wild-type str a i n (0.39 uM phosphate). There was, however, no change in the Vmax for h i g h - a f f i n i t y transport as a result of the loss of protein. P in this mutant. The protein P-deficiency of the mutant correlated with a growth defect in a phosphate-limited medium, resulting in an 18-35 % decrease in growth rate compared with the wild-type. 88 CHAPTER TWO Role of a periplasmic phosphate-binding protein in  phosphate transport in Pseudomonas aeruginosa 1. P u r i f i c a t i o n and properties of the periplasmic  phosphate-binding protein. Phosphate l i m i t a t i o n of P. aeruginosa PA01 strain HI 03 c e l l s resulted in the induction of a major protein of molecular weight 34,000 (Fig. 10). Present as the major protein in the periplasm (releasable by cold osmotic shock) (Fig. 11, lane A) i t was readily p u r i f i e d , (Fig. 11, lane B), using the procedure outline in Methods. A Scatchard plot of the data obtained from equilibrium d i a l y s i s binding studies (Fig. 12). revealed that the p u r i f i e d protein bound one molecule of phosphate per molecule of protein (n = 0.91 from the Scatchard plot) with a Kd of 0.34 + 0.05 uM (mean of three Kd determinations + standard deviation). The s p e c i f i c i t y of the phosphate-binding protein was tested using a number of potential inh i b i t o r s of phosphate binding (Table VII). The organic phosphates glucose-6-phosphate, glycerol-3-phosphate and adenosine-5'-monophosphate did not compete with orthophosphate for binding, even at 1000-fold excess over orthophosphate (Table VII). In contast, polymers of phosphate from P2 (pyrophosphate) to P15, as well as arsenate, inhibited the binding of orthophosphate to the binding protein (Table VII). 89 Figure 10. Induction of the 34K periplasmic protein by phosphate l i m i t a t i o n . C e l l s grown under phosphate-s u f f i c i e n t conditions were harvested, washed in phosphate-free minimal Hepes-buffered medium and resuspended in phosphate-deficient minimal Hepes-buf f ered medium at an absorbance at 600 nm of 0.20. Ce l l s were shaken at 37 C, and at 15 min intervals c e l l samples were removed and whole c e l l protein extracted and run on SDS-polyacrylamide gels (lanes A-M). A l l samples were s o l u b i l i z e d at 88 C for 10 min prior to electrophoresis. P, protein P. 90 A B C D E F G Figure 11. SDS-polyacrylamide gel electrophoretogram of p u r i f i e d phosphate-binding protein and whole c e l l protein extracts of alkaline phosphatase constitutive mutants of P. aeruginosa H242. Lane A, 50-fold concentrated shock f l u i d of phosphate-limited s t r a i n H242 c e l l s ; lane B, p u r i f i e d phosphate-binding protein; lane C, whole c e l l protein extract of phosphate-limited st r a i n H585; lane D, whole c e l l protein extract of phosphate-limited str a i n H586; lane E, whole c e l l protein extract of phosphate-limited strain H587; lanes F and G, whole c e l l protein extracts of phosphate-s u f f i c i e n t and phosphate-deficient H242, respectively. A l l samples were s o l u b i l i z e d at 88°C prior to electrophoresis. P, protein P; PBP, phosphate-binding potein. 91 F i g u r e 12. S c a t c h a r d p l o t of p h o s p h a t e - b i n d i n g a c t i v i t y . E q u i l i b r i u m d i a l y s i s b i n d i n g a s s a y s were performed w i t h 7 ug of b i n d i n g p r o t e i n and v a r y i n g amounts of phosphate. V r e p r e s e n t s nmol phosphate bound per nmol p h o s p h a t e - b i n d i n g p r o t e i n . L r e p r e s e n t s the - c o n c e n t r a t i o n of phosphate. 92 93 Table VII. Substrate s p e c i f i c i t y of the phosphate-binding protein Inhibitor I n h i b i t i o n 3 (%) 0.1 mM 1 .0 mM Arsenate 17 61 Pyrophosphate (P2) 40 75 Tripolyphosphate (P3) 50 80 Trimetaphosphate (Cyclic P3) 65 93 Polyphosphate (P5) 62 N.D. Polyphosphate (P15) 41 N.D. Orthophosphate (P1) 95 >99 Glucose-6-phosphate 0 0 Glycerol-3-phosphate 0 N.D. Adenosine-5'-monophosphate 0 0 a Representative data from three determinations; N.D., not determined 94 2. Isolation of mutants lacking the phosphate-binding  protein. Mutants lacking the phosphate-binding protein of E. c o l i (designated phoS) are constitutive for alkaline phosphatase (Willsky et a l . , 1973). Therefore, to obtain mutants lacking the phosphate-binding protein in P. aeruginosa, alkaline phosphatase constitutive mutants were selected using the procedure of Brickman and Beckwith (1975). Of nine alkaline phosphatase constitutive mutants obtained, four lacked the phosphate-binding protein on SDS-polyacrylamide gels (e.g. strain H585: Fig . 11, lane C). The remainder were constitutive for a l l measured phosphate-regulated constituents in addition to alkaline phosphatase, including protein P, phospholipase C and the phosphate-binding protein, t y p i c a l of the regulatory mutants described in Chapter One (e.g. H587 Fig . 11, lane E). The absence of the phosphate-binding protein in periplasmic (and whole c e l l extracts) of phosphate-limited c e l l s of mutant strain H585 (Fig. 11, lane C) and in extracts of the uninduced ( i . e . phosphate-sufficient) wild-type parent strain H242 (Fig. 11, lane F) was correlated 32 with an i n a b i l i t y of these extracts to bind P-orthophosphate (Table VIII). In contrast, periplasmic extracts of the induced (phosphate-limited) parent strain (H242) (Fig. 11, lane G) and the alkaline phosphatase constitutive mutants retaining the phosphate-binding protein (H587) (Fig. 11, lane E) demonstrated excellent binding of 32 P-orthophosphate (Table VIII). Interestingly, extracts of 95 T a b l e V I I I 32 a P - o r t h o p h o s p h a t e b i n d i n g b y p e r i p l a s m i c e x t r a c t s o f w i l d t y p e a n d m u t a n t s t r a i n s o f P . a e r u g i n o s a S t r a i n P h o s p h a t e - b i n d i n g p r o t e i n 32 c P - o r t h o p h o s p h a t e " b o u n d ( c p m ) H 5 8 5 H 5 8 6 H 5 8 7 H 2 4 2 ( P h o s p h a t e -s u f f i c i e n t ) H 2 4 2 ( P h o s p h a t e -d e f i c i e n t ) 2 , 3 7 0 3 , 5 0 0 1 8 , 1 0 0 2 , 4 0 4 1 7 , 1 0 0 T e n m l c u l t u r e s w e r e g r o w n o v e r n i g h t u n d e r p h o s p h a t e -d e f i c i e n t c o n d i t i o n s ( e x c e p t a s i n d i c a t e d ) a n d s h o c k f l u i d s o b t a i n e d a s d e s c r i b e d i n M e t h o d s . A l i q u o t s ( 2 5 u l ) w e r e i n c u b a t e d i n t h e p r e s e n c e o f 0 . 5 uM o r t h o p h o s p h a t e ( s p e c i f i c a c t i v i t y = 1 m C i / m l ) i n a f i n a l v o l u m e o f 2 5 0 u l a n d p h o s p h a t e - b i n d i n g m e a s u r e d u s i n g t h e f i l t e r - b i n d i n g a s s a y o f L e v e r ( 1 9 7 2 ) a s d e s c r i b e d i n M e t h o d s . b T h e p r e s e n c e ( + ) o r a b s e n c e ( - ) o f t h e p h o s p h a t e - b i n d i n g p r o t e i n i n p e r i p l a s m i c e x t r a c t s w a s d e t e r m i n e d b y S D S -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 ( s e e F i g . 5 ) . c R e p r e s e n t a t i v e d a t a f r o m t w o d e t e r m i n a t i o n s 96 alkaline phosphatase-constitutive strain H586 apparently contain the phosphate-binding protein (Fig. 11, lane D) yet 32 f a i l to bind P-orthophosphate (Table VIII). This mutant may well represent a structural gene mutation which does not al t e r expression of the binding protein but does aff e c t a c t i v i t y . 3. Phosphate transport. The involvement of the periplasmic phosphate-binding protein in phosphate uptake in vivo was examined using a wild-type strain containing the phosphate-binding protein (strain H242) and a mutant lacking the binding protein (strain H585). The loss of the binding protein in H585 resulted in a marked deficiency in phosphate transport compared with the parental st r a i n (H242) (Fig. 13). The rapid plateauing observed for the uptake curve of the parental st r a i n indicated that the available phosphate was being depleted, precluding the determination of an accurate rate of transport at the concentration of phosphate used in the experiment depicted in Fig . 13. Indeed, i t was necessary to d i l u t e the wild--type parental c e l l s 1:4 compared with mutant c e l l s in order to obtain a comparable rate of phosphate uptake (Fig. 13). At lower concentrations of phosphate i t was often necessary to d i l u t e wild-type c e l l s 1:19 in order to obtain linear rates of transport. 97 F i g u r e 13. P h o s p h a t e u p t a k e i n P. a e r u g i n o s a . The p r o c e d u r e f o r t r a n s p o r t a s s a y s i s d e s c r i b e d i n Methods. The c o n c e n t r a t i o n of p h o s p h a t e was 2.5 uM. A l l c e l l s were a s s a y e d a t an a b s o r b a n c e a t 600 nm of 0.30 e x c e p t as i n d i c a t e d . S t r a i n H242 (• • ) ; s t r a i n H585 (A A); s t r a i n H242 d i l u t e d 1:4 ( f i n a l k^nn = 0.06) ( 0 — 0 ) . 6 0 0 98 0.6 i i i r 10 20 30 AO Time (seconds) 99 4. Kinetics of phosphate transport. In wild-type c e l l s of P. aeruginosa, two major components of phosphate uptake were observable (Fig. 14B), confirming preliminary results (LaCoste et a l . , 1981). The h i g h - a f f i n i t y component of uptake was characterized by an apparent Km of 0.46 + 0.10 uM phosphate and a Vmax of 5.4 + 0.2 nmol phosphate taken up/min/mg c e l l protein while the low-affinity component was characterized by an apparent Km of 12.0 + 1.6 uM phosphate. The extrapolated Vmax value for the 'low-affinity' curve (16.0 + 1.5 nmol/min/mg c e l l protein) actually represented the sum of both the high and low-affinity parameters. Given that the extrapolated Vmax of the h i g h - a f f i n i t y system in the wild-type was 5.4 nmol/min/mg c e l l protein, the actual Vmax of the low-affinity system could be estimated as approximately 11 nmol/min/mg c e l l protein. This was in good agreement with the value derived from the phosphate-binding protein-deficient mutant'strain H585 containing only the low-affinity transport system. In the mutant H585, only a single phosphate uptake component with a Km of 19.3 + 1.4 uM phosphate and a Vmax of 12.1 +0.5 nmol/min/mg c e l l protein was observable (Fig. 14A). (Kinetic constants represent the mean of at least three determinations + standard deviation). Thus, the loss of the phosphate-binding protein by mutation in H585 correlated with the loss of high-a f f i n i t y phosphate uptake. 100 Figure 14. Kinetics of phosphate uptake in P. aeruginosa. Data from uptake assays performed at various concentrations of phosphate was plotted as an Eadie-Hofstee plot with kinetic constants derived by least squares analysis. A) phosphate-binding protein-deficient mutant stra i n H585 containing only a single phosphate uptake system. B) wild-type strain H242 containing two phosphate uptake systems. 101 102 5. Growth in phosphate-deficient medium. In order to test whether the defect in h i g h - a f f i n i t y phosphate transport resulting from the phosphate binding protein-deficiency in H585 could be correlated with a growth defect, the growth of wild-type and mutant c e l l s in phosphate-deficient medium was followed by measuring the time dependent increase in absorbance at 600 nm. Upon resuspension of phosphate-s u f f i c i e n t c e l l s in phosphate-deficient medium (under which conditions the phosphate-binding protein would be induced in the wild-type), both strains were seen to grow logarithmically (Fig. 15) after a short lag (not shown). However, the phosphate-binding protein-deficient mutant H585 grew at a markedly slower rate (doubling time of 124 min) compared with the wild-type H242 (doubling time of 67 min) confiming the importance of the binding protein to P.  aeruginosa c e l l s growing in a l i m i t i n g environment. 6. Physical association between outer membrane protein P  and the periplasmic phosphate-binding protein. An association between maltose-binding protein and LamB porin protein of E. c o l i , demonstrated in v i t r o (Bavoil and Nikaido, 1981), has been suggested to be necessary in vivo for the e f f i c i e n t transport of maltose and maltodextrins across the E. c o l i outer membrane (Wandersmann et a l • , 1979). To determine i f this was the case for protein P-phosphate-binding protein-mediated phosphate uptake in P. aeruginosa the phosphate-binding protein and protein P were 103 Figure 15. Growth of a phosphate-binding protein-.deficient mutant and i t s wild-type parent in phosphate-limited medium. Overnight cultures of H242 (wild type parent strain) (0—0) and H585 (phosphate binding protein-deficient mutant) (X—X) grown in phosphate-sufficient medium were harvested, washed twice in phosphate-deficient medium and resuspended in phosphate-deficient medium at an absorbance at 600 nm of 0.20. Growth was followed by the time-dependent increase in A,-.,.. 104 examined for their a b i l i t i e s to associate in v i t r o . Using a modi fried ELISA procedure (see Methods) the phosphate-binding protein was immobilized on the bottom of the wells of mictotitre plates and examined for i t s a b i l i t y to s p e c i f i c a l l y retain protein P following incubation with protein P-containing extracts. The results in Table IX demonstrated that protein P and the phosphate-binding protein were apparently capable of associating in v i t r o . At least 3 ug/well of phosphate-binding protein was required to demonstrate protein P retention in the wells. At this concentration of binding protein, protein P retention in the wells could be detected when as l i t t l e as 20 ug/ml (approximately 0.14 uM) of protein P was added, to the binding protein-containing wells (Table IX). At a concentration of 20 ug/ml (0.14 uM), protein P binding was 16 % above background and this increased to 33 % above background at 100 ug/ml (0.69 uM) and 42 % above background at 250 ug/ml (1.74 uM) (Table IX). In contrast, protein P binding was <10 % above background in the absence of the phosphate-binding protein (Table IX). Increasing the amount of phosphate-binding protein in the wells did not increase the amount of protein P which bound (at a given concentration) suggesting that levels of binding protein > 3 ug were saturating the wells. The observation that phosphate-binding protein-dependent protein P binding was detectable at uM concentrations of protein P (Table IX) implied that the Kd for protein P-binding protein 106 Table IX. In v i t r o association of the phosphate-binding protein and outer membrane protein P [Protein P] Experiment ug/ml uMb + PBPC - PBPC 0.00 0.27+0.02 0.21+0.01 0.14 0.32+0.01 0.22+0.01 0.69 0.36+0.01 0.23+0.01 2 e 250 1.74 0.12+0.03 0.07+0.02 Absorbance at 405 nm + standard deviation of duplicate assays b Assuming a molecular weight of 144,000 [deduced from the protein P monomer molecular weight of 48,000 and the demonstrated trimer form of the native protein (Angus et a l , 1983)] Protein P binding to microtitre wells pre-coated with 3 ug of phosphate-binding protein (+PBP) or no phosphate-binding protein (-PBP) was detected using a protein P trimer-s p e c i f i c antiserum. Antibody binding, which was expected to be proportional to the amount of protein P present in the wells, was measured at A405 following incubation with an alkaline phosphatase-conjugated second antibody and a chromogenic substrate, para-nitrophenyl phosphate (pNPP). d After addition of the pNPP, colour developement was allowed to proceed for 18 h at room temperature (in the dark) before measuring absorbance at 405 nm e After addition of pNPg, colour development was allowed to proceed for 2 h at 37 C before measuring absorbance at 405 nm 1 A 4 0 5 1 d 0 20 100 107 association was in the uM range. This was consistent with data obtained for the association of maltose-binding protein and protein LamB (Rd = 0.15 uM) (Neuhaus et a l . , 1983). One of the problems with the ELISA method used above was the very low levels of protein P which were in fact being retained in the wells (in one experiment overnight incubation was necessary to detect protein P retention). Since protein P retention was apparently dependent upon the presence of phosphate-binding protein in the wells (Table IX) this could be attributed to saturation of the microtitre wells by phosphate-binding protein at low level s (> 3 ug). This was compounded by an observed decrease in the a f f i n i t y of the antiserum used to detect protein P binding in the presence of detergent (e.g. Triton X-100). As a result, i t was d i f f i c u l t to accurately and consistently measure binding between protein P and the binding protein since background levels often equalled or exceeded levels due to s p e c i f i c binding. Thus, in order to obtain higher levels of binding i t was deemed necessary to devise a method whereby larger amounts of the binding protein could be immobilized. Bavoil et a l . (1981) demonstrated an association between the maltose-binding protein and protein LamB by immobilizing large amounts. (10 mg) of the binding protein on Sepharose beads and using a f f i n i t y chromatography to demonstrate s p e c i f i c binding of the LamB protein. Using this methodology, a phosphate-binding protein Sepharose 4B column was constructed (see Methods) and examined for the 108 a b i l i t y to s p e c i f i c a l l y bind protein P. In a reciprocal experiment, protein P was immobilized on Sepharose beads and i t s a b i l i t y to s p e c i f i c a l l y bind the phosphate-binding protein was also examined. Unfortunately, these a f f i n i t y columns f a i l e d to bind any proteins, including the relevant phosphate transport proteins. The cross-linking of these proteins to Sepharose may well d i s t o r t the proteins s u f f i c i e n t l y to prevent the adoption of the necessary binding conformations. 7. Summary. A binding protein for inorganic phosphate was p u r i f i e d to apparent homogeneity from the shock f l u i d s of phosphate-limited Pseudomonas aeruginosa. The p u r i f i e d protein bound one molecule of phosphate per molecule of binding protein with a Kd of 0.34 + 0.05 uM. Arsenate, pyrophosphate and inorganic polyphosphates up to 15 units long could i n h i b i t the binding of phosphate to the binding protein, although organic phosphates such as glucose-6-phosphate, glycerol-3-phosphate and adenosine-5'-monophosphate could not. Mutants lacking the phosphate-binding protein were isolated and shown to be deficient in phosphate transport compared with wild-type c e l l s . Two k i n e t i c a l l y d i s t i n c t systems for phosphate uptake could be observed in wild-type c e l l s , with apparent Km values of 0.46 + 0.10 uM (high a f f i n i t y ) and 12.0 + 1.6 uM (low a f f i n i t y ) . In contrast, only a single low-affinity transport system was observable in mutants lacking the phosphate-binding protein 109 (Km apparent = 19.3 + 1.4 uM phosphate), suggesting the involvment of the binding protein in the h i g h - a f f i n i t y phosphate uptake system of P. aeruginosa. Mutants deficient in the binding protein were also defective in their a b i l i t y to grow in a phosphate-limiting medium consistent with the s p e c i f i c induction and requirement for the phosphate-binding protein-dependent h i g h - a f f i n i t y transport system under l i m i t i n g conditions. An apparent association between the phosphate-binding protein and the phosphate-limitation-inducible outer membrane protein P was demonstrated in v i t r o . 110 CHAPTER THREE Immunological cros s - r e a c t i v i t y of phosphate-starvation- induced outer membrane proteins of the families  Enterobacteriaceae and Pseudomonadaceae 1. Phosphate-starvation-induction of membrane proteins of  the Pseudomonaceae and the Enterobacteriaceae. Under conditions of phosphate-limitation, P. aeruginosa i s derepressed for the synthesis of protein P, a phosphate-selective (Hancock and Benz, manuscript submitted), channel-forming outer membrane protein. Growth of other Pseudomonads as well as members of the Enterobacteriaceae (Table X) in a phosphate-deficient medium resulted, in many of these strains, in the induction of novel membrane proteins (Fig. 16), many of which existed as the major c e l l envelope protein. The observation that these proteins were enriched in cation-aggregated membrane preparations demonstrated that they were probably outer membrane proteins. The phosphate starvation-induction of the PhoE outer membrane proteins of E. c o l i (Overbeeke and Lugtenberg, 1980) and S. typhimurium (Bauer et a l . , 1985) has been demonstrated previously. In experiments reported here, however, the PhoE protein of E. c o l i co-migrated with the OmpF protein of this strain (Fig. 16, lanes 17 and 18) making i t necessay to use an ompF mutant strain (JF700) (Table 1) to demonstrate PhoE induction (Fig. 16, lanes 15 and 16). New membrane proteins 111 Enriched, soluble preparations of the phosphate starvation-induced proteins were s o l u b i l i z e d at 23°C or 88°C prior to electrophoresis on SDS-polyacrylamide slab gels. Low molecular weight standards (Sigma Chemical Co, St. Louis, Mo.) were co-electrophoresed and a plot of log molecular weight vs Rf (measured as distance migrated in cm) for the standards was derived, from which molecular weights of the phosphate starvation-induced proteins were determined from their respective Rf values. Because the proteins s o l u b i l i z e d at 23°C occurred as smeared bands (see text) the distance migrated ( R f ) was determined for the midpoint of the area of densest s t a i n . 48K e.g. s i g n i f i e s a molecular weight of 48,000 b A, extractable from c e l l envelopes in 2 % (wt/vol) Triton X- 100/20 mM Tris-HCl pH 8.0/0.5 M EDTA; B, extractable from c e l l envelopes in 2 % (wt/vol) Triton X-100/20 mM Tris-HCl pH 8.0 after 30 min incubation at 37°C in the presence of 1 mg/ml of lysozyme; C, extractable from c e l l envelopes in 2 % (wt/vol) SDS/0.5 M NaCl Probable cross'-reactive protein (in the oligomer form); see text for discussion d not determined e The 37K protein of P. chlororaphis was extractable from c e l l envelopes in 2 % (wt/vol) Triton X-100 alone and is probably an inner membrane protein f The 37K and 24K proteins co-purified, using a l l methods tested, such that the resultant oligomers at 23° C could not be distinguished, but appeared as a high molecular smear of approximately 115K 1 1 2 Table X. Properties of phosphate starvation-induced membrane proteins of the Enterobacteriaceae and the Pseudomonadaceae Strain Apparent Molecular Weight 3 (in S o l u b i l i t y thousands) after s o l u b i l i z a t i o n at properties of 88°C 23°C native oligomers P. aeruginosa 48 97 c A p. fluorescens 50 102° A 22 22 N.D.d p. putida 45.5 97 c A p. chlororaphis 49.5 1 10C A 37 N.D. _e p. aureofaciens 48 107C A p. cepacia 37 1!5f C 24 1l5 f C 20.5 20.5 N.D. p. pseudomallei 39 104C C E. c o l i 37 83 C B,C S. typhimurium 36 82 c B,C K. pneumoniae 36.5 83 C B,C E. aerogenes 36 85 C B,C S. marcesens 37 87 C C 1 1 3 were not detected in c e l l envelopes of P. maltophilia (now Xanthomonas maltophilia), P. acidovorans and P. solanacearum strains grown in a phosphate-deficient medium, although these strains were derepressed for the synthesis of alkaline phosphatase in this medium (data not shown). P. s t u t z e r i , which apparently f a i l e d to produce a new membrane protein when grown in phosphate-deficient medium, grew extremely poorly in this medium, and P. syrinqae did not grow at a l l , although both of these strains grew quite well in phosphate-s u f f i c i e n t medium. While most strains expressed a single phosphate starvation-induced membrane protein band (Fig. 16), P. fluorescens and P. chlororaphis each apparently expressed two (Fig. 16, lanes 4 and 8, respectively), and P. cepacia apparently expressed three (Fig. 16, lane 12). With the exception of the 20.5K protein of P. cepacia (Fig. 16, lane 12) and the 22K protein of P. fluorescens (Fig. 16, lane 2), a l l of the phosphate starvation-induced membrane proteins were heat-modifiable (Table X). SDS-polyacrylamide gel electrophoretograms of soluble preparations of these proteins revealed that the native or unheated forms of the proteins ran as higher molecular weight oligomers which could be dissociated to monomers upon heating at temperatures greater than 60°C , a property shared by the majority of porins described to date (Lugtenberg and van Alphen, 1983). In addition, the monomer molecular weights ranged from 36R to 39K or from 46K to 50K (Table X), 1 14 Figure 16. SDS-polyacrylamide gel electrophoretogram of c e l l envelopes prepared from phosphate-deficient and phosphate-sufficient grown strains of the families Pseudomonadaceae and Enterobacteriaceae. C e l l envelopes (lanes 1 to 14) and Triton X-100 insoluble c e l l envelopes (lanes 15-27) were prepared from: lane 1, phosphate-sufficient and lane 2,phosphate-deficient P. aeruginosa; lane 3, phosphate-sufficient and lane 4, phosphate-deficient P. fluorescens; lane 5, phosphate-sufficient and lane 6, phosphate-deficient P. putida; lane 7, phosphate-sufficient and lane 8, phosphate-deficient P. chlororaphis; lane 9, phosphate-sufficient and lane 10, phosphate-deficient P. aureofaciens; lane 11, phosphate-sufficient and lane 12, phosphate-deficient P. cepacia; lane 13, phosphate-sufficient and lane 14, phosphate-deficient P. pseudomallei; lane 15, phosphate-sufficient and lane 16, phosphate-deficient E. c o l i K-12 st r a i n JF700; lane 17, L-broth grown E. c o l i K-12 st r a i n HMS174; lane 18, L-broth grown E. c o l i K-12 stra i n JF694; lane 19, phosphate-sufficient and lane 20, phosphate-deficient S. typhimurium LT2; lane 21, phosphate-sufficient and lane 22, phosphate-deficient 5. marcesens; lane 23, phosphate-sufficient and lane 24, phosphate-deficient K. pneumoniae; lane 25, phosphate-sufficient E. aerogenes; lanes 26 and 27, phosphate-deficient E. aerogenes. The arrows indicate the phosphate-regulated proteins. A l l samples were s o l u b i l i z e d at 88 °C prior to electrophoresis. The gel in lanes 15-27 contained urea at a f i n a l concentration of 6M. 1 15 1 1 6 c h a r a c t e r i s t i c of the major Enterobacterial porin proteins (Lugtenberg and van Alphen, 1983) and protein P of P.aeruginosa (see Chapter One), respectively. Typically, porin protein P can be extracted in i t s native state from peptidoglycan-associated c e l l envelopes or outer membranes with Triton X-100 in the presence of EDTA (Chapter One), in contrast to the major porin protein F of P. aeruginosa and the major porins of E. c o l i and S. typhimurium, which are s o l u b i l i z e d (in their native forms) in Triton X-100 only after lysozyme digestion of the peptidoglycan (Hancock et a l . , 1981) or in the presence of SDS and high salt ( > 0.4 M NaCl) (Nakamura and Mizushima, 1976; Tokunaga et a l . , 1979; Yoshimura et a l . , 1983). A number of the phosphate starvation-inducible proteins examined were soluble in Triton X-100-EDTA, including those of P. putida, P. fluorescens, P. aureofaciens and the 49.5K protein of P. chlororaphis (Table X). The 37K phosphate-starvation-induced protein of P. chlororaphis was extractable with Triton X-100 in the absence of EDTA. In contrast_to the other proteins described here, this protein may well be from the cytoplasmic membrane. The remainder of the phosphate-starvation-induced membrane proteins were insoluble in Triton X-100-Tris-EDTA but could be s o l u b i l i z e d in their oligomeric forms in SDS/0.5 M NaCl (Table X). Interestingly, lysozyme digestion of the peptidoglycan did not f a c i l i t a t e Triton X-100 s o l u b i l i z a t i o n of the phosphate starvation-induced membrane proteins of P. cepacia, 117 P. pseudo-mallei or S. marcesens, although such treatment did y i e l d Triton-soluble phosphate-starvation-induced proteins . in the cases of E. c o l i , S. typhimurium, K. pneumoniae and E. aerogenes (Table X). 2. Immunological c r o s s - r e a c t i v i t y of phosphate starvation- induced outer membrane proteins. a. Cross-reactivity of protein oligomers in phosphate- limited c e l l envelopes. To test for immunological cross-r e a c t i v i t y of the phosphate starvation-induced membrane proteins of the various strains examined , SDS-polyacrylamide gel electrophoretograms of phosphate-limited c e l l envelopes were elect r o p h o r e t i c a l l y transferred to nit r o c e l l u l o s e and probed with a protein P trimer-specific polyclonal antiserum (for s p e c i f i c i t y of the antiserum see Fig. , 17 lane 1; c f . lane 2 ) . This antiserum was demonstrated to s p e c i f i c a l l y detect protein P trimers in c e l l envelopes of phosphate-limited P. aeruginosa c e l l s (Fig. 17, lane 4). The protein P-specific antiserum was capable of reacting with a component present in the phosphate-limited c e l l envelopes of a l l strains which had produced a phosphate-starvation-induced membrane protein and with a component present in c e l l envelopes of E. c o l i K-12 s t r a i n JF694 which lacks the major porin proteins OmpF and OmpC but i s constitutive for PhoE (Fig. 18) . In each case smeared bands of high molecular weight reacted with the antiserum, suggesting that the native (unheated) oligomers 118 Figure 17. Interaction of protein P trimer-specific or monomer-specific antiserum with Western blots of p u r i f i e d protein P and Pseudomonas aeruginosa PA01 str a i n H103 c e l l envelopes. C e l l envelopes or p u r i f i e d proteins were electrophoretically transferred from SDS-polyacrylamide gel electrophoretograms to n i t r o c e l l u l o s e and incubated with a protein P trimer-s p e c i f i c (lanes 1 to 5) or monomer-specific (lanes 6 to 10) antiserum. Antibody binding was detected using an alkaline phosphatase-conjugated goat-anti-rabbit IgG antibody (for the trimer-specific antiserum) or an alkaline phosphatase-conjugated goat-anti-mouse IgG antibody (for the monomer-specific antiserum) and a histochemical alkaline phosphatase substrate. Lanes 1 and 7, p u r i f i e d protein P s o l u b i l i z e d at 23°C (trimer form); lanes 2 and 6, p u r i f i e d protein P s o l u b i l i z e d at 880c (monomer form); lane 3, c e l l envelope preparation of phosphate-sufficient P. aeruginosa s o l u b i l i z e d at 23°C; lanes 4 and 10, c e l l envelope preparation of phosphate-deficient P. aeruginosa s o l u b i l i z e d at 23°C; lanes 5 and 9, c e l l envelope preparation of phosphate-deficient P. aeruginosa s o l u b i l i z e d at 88°C; lane 8, c e l l envelope preparation of phosphate-sufficient P. aeruginosa s o l u b i l i z e d at 88°C. A small amount of monomer protein P can be seen in the trimer preparation in lane 7. The protein P oligomer (trimer) band observable in Coomassie-staihed gels (e.g. Fi g . 3, lanes 4 and 5) migrates at a position corresponding to the bottom of the smeared band in lane 1. 119 120 Figure 18. Interaction of protein P trimer-specific antiserum with Western blots of c e l l envelope preparations of dif f e r e n t bacteria grown under phosphate-deficient or s u f f i c i e n t conditions. Lane 1, phosphate-deficient and lane 2, phosphate-sufficient P. aeruginosa; lane 3, phosphate-deficient and lane 4, phosphate-sufficient P. fluorescens; lane 5, phosphate-deficient and lane 6, phosphate-sufficient, P. putida; lane 7, phosphate-deficient and lane 8, phosphate-sufficient P. chlororaphis; lane 9, phosphate-deficient and lane 10, phosphate-sufficient P. aureofaciens; lane 11, phosphate-deficient and lane 12, phosphate-sufficient P. cepacia; lane 13, phosphate-deficient P. cepacia; lane 14, phosphate-deficient P. cepacia c e l l envelopes s o l u b i l i z e d in 2% SDS/0.5 M NaCl to inactivate contaminating alkaline phosphatase; lane 15, phosphate-deficient and lane 16, phosphate-sufficient P. pseudomoallei; lane 17, phosphate-deficient and lane 18, phosphate-sufficient K. pneumoniae; lane 19, phosphate-deficient and lane 20, phosphate-sufficient E. aeroqenes; lane 21, phosphate-deficient and lane 22, phosphate-sufficient S. marcesens; lane 23, phosphate-deficient and lane 24, phosphate-sufficient S. typhimurium; lane 25, L-broth grown E. c o l i K-12 strain JF694; lane 26, L-broth grown E. c o l i K-12 stra i n HMS174; lane 27, phosphate-deficient and lane 28, phosphate-sufficient E. c o l i K-12 strain JF700. The c e l l envelopes were so l u b i l i z e d at 23 °C prior to electrophoresis. The blots, with the exception of lane 11, were developed with the protein P-trimer-specific antiserum as described in the legend to Fi g . 17. The blot in lane 13 was incubated d i r e c t l y with an alkaline phosphatase histochemical substrate to detect contaminating c e l l envelope bound alkaline phosphatase. Similar controls were negative for a l l other strains shown here. 121 1 i i L 12 3 4 5 6 7 8 910 11 12 13 14 1516 17 18 19 20 21 22 23 24 25 26 2728 1 22 of the phosphate starvation-induced proteins were reacting. In support of th i s , the non-heat-modified oligomeric forms of the phosphate starvation-induced membrane proteins were i d e n t i f i a b l e as high-molecular weight smeared bands in Coomassie stained SDS-polyacrylamide gel electrophoretograms of enriched, soluble preparations of these proteins. S o l u b i l i z a t i o n of c e l l envelopes at 8-8°C for 10 min, which converted oligomeric proteins to monomers (Table X), destroyed t h i s r e a c t i v i t y . This was consistent with the i n a b i l i t y of the antiserum to react with protein P monomers in heat treated, phosphate-limited c e l l envelopes (Fig.17, lane 5) or p u r i f i e d in detergent (Fig. 17, lane 2). These data excluded the non-heat-modifiable 20.5K and 22K proteins of P. cepacia and P. fluorescens, respectively, as the cross-reactive species in these s t r a i n s . In the case of P. cepacia, a strong c r o s s - r e a c t i v i t y o r i g i n a l l y seen (Fig. 18, lane 11) was demonstrated to be due, in part, to the presence of alkaline phosphatase associated with the c e l l envelope (Fig. 18, lane 13). Using a 2 % SDS/0.5 M NaCl soluble preparation of a phosphate-limited P. cepacia c e l l envelope, which contained the phosphate starvation-induced proteins but lacked alkaline phosphatase, a weak r e a c t i v i t y with the protein P trimer-specific antiserum was detected (Fig. 18, lane 14). No r e a c t i v i t y was observed with any c e l l envelopes derived from phosphate-sufficient c e l l s (Fig. 18). 123 b. Id e n t i f i c a t i o n of the cross-reactive proteins. To confirm that the cr o s s - r e a c t i v i t y seen in native phosphate-limited c e l l envelopes was indeed due to the oligomeric forms of the phosphate-starvation-induced protein in each case, we attempted to convert the material present in the cross-reactive smeared bands to the appropriate monomeric proteins by heating. Thus, SDS-polyacrylamide gel electrophoretograms of native (unheated) phosphate-limited c e l l envelopes ( f i r s t dimension) were heated at 88°C and electrophoresed on fresh SDS-polyacrylamide slab gels (second dimension). A t y p i c a l result is shown in F i g . 19. Proteins which were not-heat modifiable t y p i c a l l y (Russel, 1976) appeared on the diagonal of a 2-dimensional (unheated vs heated) SDS-polyacrylamide gel, since their molecular weights would remain unchanged in the second dimension after heating. Proteins which form native oligomers which dissociate in response to heating would t y p i c a l l y appear at a position to the l e f t of the diagonal at their appropriate monomer molecular weights. As expected, protein P occured to the l e f t of the diagonal (Fig. 19B) since i t ran as a trimer in the f i r s t dimension (unheated) and a monomer in the second dimension (heated). Furthermore, the protein P monomers ran as a broad band in the second dimension, consistent with the apparent heterogeneity of protein P trimers in phosphate-limited c e l l envelopes (Fig. 19B) or p u r i f i e d in detergent (Fig. 19A). 124 Figure 19. Two-dimensional (unheated x heated) SDS-polyacrylamide gel electrophoretogram of p u r i f i e d protein P and c e l l envelopes prepared from phosphate-limited strains of the fluorescent Pseudomonads. A) p u r i f i e d protein P; B) P. aeruginosa; C) P. chlororaphis; D) P. pseudomallei; E) §7 marcesens; and F) P. cepacia. SDS-polyacrylamide gel electrophoretograms of c e l l envelopes s o l u b i l i z e d at 23°C for 10 min prior to electrophoresis were excised (1st dimension) were heated at 88°C for 10 min, l a i d across the top of a second SDS-polyacrylamide slab gel with (4E) or without (4A,B,C,D,F) urea, and electrophoresed in the second dimension as described in Materials and Methods. Western immunoblots of f i r s t dimension gels (e.g. Fig 3) are included above the 2-D gels to indicate the position of the cross-reacting material in native c e l l envelopes prior to heating in the second dimension. The phosphate-starvation-induced protein monomers are indicated by arrows. 1 25 126 For a l l strains expressing a heat-modifiable phosphate starvation-induced protein, i t was possible to demonstrate the presence of phosphate-starvation-induced monomers to the l e f t of the diagonal in the second dimension of a two-dimensional unheated vs heated SDS-polyacrylamide slab gel. In a l l cases these monomeric proteins ran as broad bands, the positions of which corresponded with the position of the cross-reactive smeared oligomer bands present in native phosphate-limited c e l l envelopes in the f i r s t dimension (Fig. 19). Since the phosphate starvation-induced proteins of P. putida, P. fluorescens and P. aureofaciens represented the only heat-modifiable membrane proteins in these strains, demonstrating broad bands which appeared to the l e f t of the diagonal in the second dimension and which corresponded with the c r o s s - r e a c t i v i t y seen in .first dimension gels (e.g. F i g . 19B), they were readily confirmed as the cross-reactive species in these str a i n s . Similarly, the c o n s t i t u t i v e l y produced PhoE protein of the porin-deficient E. c o l i strain JF694 existed as the lone heat-modifiable, oligomeric protein in c e l l envelopes of t h i s s t r a i n (Fig. 16; Table X), occurring to the l e f t of the diagonal and thus accounting for the c r o s s - r e a c t i v i t y observed with c e l l envelopes of t h i s s t r a i n . Of the two heat-modifiable phosphate starvation-induced membrane proteins produced by P.  chlororaphis, only the 49.5K monomer protein ran as a broad band in the second dimension, whose position also corresponded with the c r o s s - r e a c t i v i t y seen in f i r s t 127 dimension gels (Fig. 19C), consistent with i t s being the cross-reactive membrane protein in this s t r a i n . The remaining strains, which included P. cepacia, P. pseudomallei, E. c o l i K-12, S. typhimurium, K. pneumoniae, E. aerogenes and S. marcesens, a l l produced a number of constitutive membrane proteins which, l i k e the various phosphate-starvation-induced membrane proteins, were heat-modifiable, appearing to the l e f t of the diagonal in the second dimension of a two-dimensional unheated vs heated SDS-polyacrylamide gel (e.g. Figs. 19D and 19E). Furthermore, they occurred as broad bands of monomer molecular weight in a position which also corresponded with the cross-reactive smears observed in f i r s t dimension gels of native phosphate-limited c e l l envelopes (e.g. Figs. 19D and 19E). However, the' expression of these constitutive proteins in the c e l l envelopes of phosphate s u f f i c i e n t c e l l s , which had previously f a i l e d to react with the protein P-trimer s p e c i f i c antiserum (Fig. 17), excluded these proteins as the cross-reactive components in these strains. Thus the single heat-modifiable phosphate starvation-inducible protein present in each strain (except P. cepacia) must be responsible for the cr o s s - r e a c t i v i t y observed with the protein P-specific antiserum. Nonetheless, this demonstrated that the smearing (heterogeneity) on SDS-polyacrylamide gels of native oligomers was not r e s t r i c t e d to phosphate-starvation-induced membrane proteins. Rather, i t may well be a property of oligomeric membrane proteins, 128 s p e c i f i c a l l y porins. The expression by P. cepacia of two heat-modifiable phosphate starvation-induced membrane proteins, both of which migrated as broad bands to the l e f t of the diagonal in the second dimension, and in a position which corresponded with the cross-reactive smeared band seen in the f i r s t dimension (Fig. 19F), made i t d i f f i c u l t to unambiguously identif y the cross-reactive species. Furthermore, the proteins were invariably co-purified by a l l tested methods, making i t impossible to i n d i v i d u a l l y examine their r e a c t i v i t i e s with the protein P-specific antiserum . Based on molecular weight, however, the l i k e l y candidate for the cross-reactive species was the 37K protein, whose monomer molecular weight was more t y p i c a l of porins in general than that of the 24K protein. c. Cross-reactivity of phosphate-starvation-induced  monomers. Porin monomers, obtained by heat denaturation of porin trimers, have been shown to exhibit an alpha-helical structure (Nakamura and Mizushima, 1976) very d i s t i n c t from the beta-structure of the native protein (Nakamura and Mizushima, 1976). Consistent with t h i s , the monomer and trimer forms of individual porins have been demonstrated to be immunologically non-cross-reactive (Hofstra and Dankert, 1981). The demonstration, then, that the porin monomers of d i f f e r e n t species of the family Enterobacteriaceae could immunologically cross-react (Hofstra and Dankert, 1980; 1 29 Overbeeke and Lugtenberg, 1980), although implying that linear epitopes present in porin monomers had been conserved during porin evolution, did not demonstrate the existence of conserved epitopes in the native oligomers. In order to determine whether the cr o s s - r e a c t i v i t y of phosphate starvation-induced membrane proteins could be attributed to conserved linear epitopes, the monomer and oligomer forms of the various phosphate-starvation-inducible membrane proteins were tested for their a b i l i t y to react with an antiserum raised against heat-dissociated protein P monomers. The antiserum was demonstrated to react s p e c i f i c a l l y with protein P monomers in heat denatured c e l l envelopes of phosphate-limited P. aeruginosa (Fig. 17, lane 9) or with p u r i f i e d protein P monomers (Fig. 17, lane 6), exhibiting no r e a c t i v i t y with the trimer form of the protein (Fig. 17, lanes 7 and 10) or with uninduced (phosphate-sufficient) c e l l envelopes (Fig. 17 lane 8). This antiserum f a i l e d to react with the phosphate-starvation-inducible membrane proteins, in monomer or oligomer form (data not shown), indicating that the phosphate-starvation-inducible membrane proteins do not cross-react immunologically with protein P monomers. 3. Summary. Bacteria from the families Enterobacteriaceae and Pseudomonadaceae were grown under phosphate-deficient (0.1 - 0.2 mM inorganic phosphate) conditions and examined for the production of novel membrane proteins. Twelve of 130 the seventeen strains examined expressed a phosphate-starvation-induced outer membrane protein which was heat-modifiable, in that after s o l u b i l i z a t i o n in SDS at low temperature the proteins ran on gels as diffuse bands of higher apparent molecular weight, presumably oligomer forms, which shifted to their apparent monomer forms after s o l u b i l i z a t i o n at high temperature. These proteins f e l l into two classes based on their monomer molecular weights and the detergent conditions required to release the proteins from the peptidoglycan. The f i r s t class, expressed by species of the P. fluorescens branch of the family Pseudomonadaceae, was similar to the phosphate-starvation-inducible channel-forming protein P of P. aeruginosa. The second class resembled the major Enterobacterial porin proteins and the phosphate-regulated PhoE protein of E. c o l i . Using a protein P trimer-specific polyclonal antiserum i t was possible to demonstrate c r o s s - r e a c t i v i t y of the oligomeric forms of both classes of these proteins on Western blots. However, this antiserum did not react with the monomeric forms of any of these proteins, including protein P monomers. Using a protein P monomer-specific antiserum, no r e a c t i v i t y was seen with any of the phosphate-starvation-inducible membrane proteins (in either oligomeric or monomeric form) with the exception of protein P monomers. These results suggest the presence of conserved antigenic determinants only in the native, functional proteins. 131 CHAPTER FOUR Characterization o§ protein P-like porins from the  fluorescent Pseudomonadacea 1. P u r i f i c a t i o n of the phosphate-starvation-inducible outer  membrane proteins of the fluorescent Pseudomonads. In the previous chapter a number of ba c t e r i a l strains were demonstrated to synthesize phosphate-starvation-inducible outer membrane proteins. Members of the fluorescent Pseudomonadaceae, including P. putida, P. fluorescens, P. aureofaciens and P. chlororaphis synthesized an oligomeric, heat-modifiable outer membrane protein which exhibited a number of properties in common with protein P of P. aeruginosa. Using the observed immunological cross-r e a c t i v i t y of these proteins with protein P, an attempt was made to purify these phosphate-regulated proteins by sp e c i f i c retention on an immunoadsorbent column constructed using the protein P trimer-specific antiserum described in Chapter one. Protein P, the o r i g i n a l antigen, was readily p u r i f i e d and in reasonable quantities by this method (Fig. 20, lanes 1 and 2). It was also possible to isolate the other phosphate-starvation-inducible outer membrane proteins using t h i s column, although the yields were substantially lower, requiring a sensitive s i l v e r staining procedure to detect the proteins in SDS-polyacrylamide gels (eg. F i g . 20, lanes 3 and 4). Apparently, the cross-reactive antibodies in the protein P trimer-specific 132 Figure 20. SDS-polyacrylamide gel electrophoretogram of p u r i f i e d phosphate-starvation-inducible outer membrane proteins of the fluorescent Pseudomonads. Phosphate-starvation-inducible proteins were p u r i f i e d from the outer membranes of lanes 1,2,11 and 12, P. aeruginosa ( i . e . protein P); lanes 3 and 4, P. fluorescens; lanes 5 and 6, P. putida; lanes 7 and 8, P. aureofacTens; lanes 9 and 10, P. chlororaphis. The proteins in lanes 3,4,11 and 12 were p u r i f i e d via a f f i n i t y chromatography using a rabbit anti-protein P immunoadsorbant column. The proteins in lanes 1,2 and 5-10 were p u r i f i e d by electroelution from polyacrylamide gels. Samples were s o l u b i l i z e d at 88°C (lanes 1,3,5,7,9,11) or 23°C (lanes 2,4,6,8,10,12) prior to electrophoresis. Lanes 3 and 4 were stained for protein using a sensitive s i l v e r staining procedure (Wray et a l 1981). A r t i f a c t bands v i s i b l e in these lanes are a product of the s i l v e r staining procedure. A l l other lanes were stained by Coomassie b r i l l i a n t blue. The faint continuous band seen in the middle of the gel i s an a r t i f a c t and was observable in lanes were no protein was loaded. 133 - f i 1 2 3 4 5 6 7 8 9 10 11 12 134 antiserum represent only a minor or low-affinity component of t h i s antiserum. The p u r i f i e d proteins occurred as higher molecular weight oligomers in SDS-polyacrylamide gels when so l u b i l i z e d at room temperature prior to electrophoresis (e.g. Fig. 20, lane 4), dissociating to lower molecular weight monomers when so l u b i l i z e d at 88°C (e.g. F i g . 20, lane 3). This was consistent with the observed properties of these proteins in phosphate-limited c e l l envelope preparations (see Chapter Three, F i g . 16) and with the properties of p u r i f i e d protein P (Fig. 20,- lanes 1 and 2). To improve yi e l d s , these proteins were also p u r i f i e d using a procedure for the electroelution of proteins out of SDS-polyacrylamide gels (Parr et a l . , 1986). Phosphate-starvation-induced protein-containing extracts prepared from each of the above strains were run on SDS-polyacrylamide gels, the relevant protein oligomer bands excised and the protein electroeluted from the gel. This method produced substantially increased yields of a l l proteins (Fig. 20, lanes 5-12) which were easily v i s i b l e in Coomassie stained gels. Again, the p u r i f i e d proteins retained their oligomeric structure, as attested by their resistance to SDS denaturation (Fig. 20, lanes 6,8,10,12) unless heated at high temperature (Fig. 20, lanes 5,7,9,11). 2. Single channel experiments. When the p u r i f i e d phosphate-starvation-inducible outer membrane proteins were added in small quantities (5-10 ng/ml) to the aqueous 1 35 solutions bathing a black l i p i d bilayer membrane, membrane conductance was seen to increase i s a stepwise fashion (e.g. Fig. 21), presumably due to the incorporation of individual protein oligomers into the membrane as suggested for other porins (Benz et a l . , 1978; Benz et a l . , 1979; Benz and Hancock, 1981). The observed single channel conductance increments were distributed about a mean (e.g. F i g . 22), although larger increments were also seen at 2 (Fig. 22), 3 and 4 (not shown) times-the average single channel conductance. These probably represented multiple insertions of the protein oligomers into the bilayer membrane as has been observed for other porins, including protein P (Hancock et a l . , 1982). The average single channel conductances in 1 M KC1 measured for a given protein p u r i f i e d by either a f f i n i t y chromatography or electroelution were not s i g n i f i c a n t l y d i f f e r e n t (Table XI), confirming both the u t i l i t y of the anti-protein P immunoadsorbent column in purifying functional cross-reactive molecules and the general a p p l i c a b i l i t y of the electroelution procedure in purifying functionally active porin proteins. In addition, the derived average single channel conductance values obtained for each of the phosphate-starvation-inducible proteins were not s i g n i f i c a n t l y d i f f e r e n t , f a l l i n g between 233 and 252 pS (Table XI). These values were substantially less than those obtained for the E. c o l i porins, including the phosphate-starvation-inducible PhoE porin of this st r a i n (approximately 2 nS) (Benz et a l . , 1985), and for the major 136 Figure 21. S t r i p chart recordings of stepwise increases in the conductance of a small (0.1 mm2) oxidized cholesterol membrane (1.5 % in n-decane) caused by the addition of 10 ng/ml of the phosphate-starvation-induced outer membrane protein from P. putida to the aqueous phase (1 M KC1, pH 6.0). The applied voltage was 50 mV and the temperature was 25°C. 1 37 I — 1 ho cn tn O -o ° > - ° CO 1 38 Figure 22. Histogram of the conductance fluctuations observed with membranes of oxidized cholesterol (1.5 % in n-decane) in the presence of the phosphate-starvation-induced outer membrane protein of P. putida and 1 M KC1 (pH 6.0) in the aqueous phase. The applied voltage was 50 mV and the temperature was 25°C. P(A) i s the probability of a given conductance increment A ~ taken from recorder tracings such as that shown in Figure 21. 1 39 A 1 = 247 pS n = 152 A 2 = 4 9 0 pS n=7 0 120 240 360 480 600 A / p S 140 Table XI. Channel-forming properties of a f f i n i t y - p u r i f i e d and electroeluted phosphate-starvation-inducible outer membrane oligomers of the fluorescent Pseudomonads A f f i n i t y P u r i f i e d Electroeluted Strain Single channel 3 n b Single channel n conductance (pS) conductance (pS) P. aeruginosa 239 317 234 224 p. putida 233 74 247 307 p. fluorescens 241 1 17 - -p. aureofaciens 237 54 252 198 p. chlororaphis 243 45 237 201 a Average value from n events b Number of single channel events measured 141 porin protein F of P. aeruginosa (5 nS) (Benz and Hancock, 1981). They were, however, in excellent agreement with the observed single channel conductance of protein P in 1 M KC1 (Table XI) suggesting that the phosphate-regulated porin proteins of the fluorescent Pseudomonads a l l form small channels t y p i c a l of protein P and in contrast to the majority of porins descibed to date (Benz et a l . , 1981), including other phosphate-starvation-inducible porins proteins (Benz et a l . , 1981; Verhoef et a l . , 1984; Bauer et a l . f 1985). 3. I o n - s e l e c t i v i t y . To examine the i o n - s e l e c t i v i t y of these channels, single channel conductance was. measured in salt s of varying cation or anion si z e . The anion-specific protein P channel has previously been demonstrated to y i e l d average single channel conductances, the magnitudes of which were dependent exclusively upon the size of the anion (Benz et a l . , 1983). Thus conductance through protein P channels was demonstrated to be inversely related to the size of the anion (Benz et a l . , 1983), while remaining ba s i c a l l y unaffected by changes in cation size of the s a l t bathing a protein P-containing l i p i d bilayer membrane (Hancock et a l . , 1982). By comparing the average single channel conductance values obtained in K+C1 with, for example, T r i s + C l and K+Hepes , in which cases the cation and anion sizes, respectively, are increased, i t should be possible to gain some idea of the ion s e l e c t i v i t y of each of 142 the phosphate-starvation-induced porin proteins. The results in Table XII suggested that in a l l cases the observed single channel conductance was dependent upon anion size only, such that increasing the anion size in the case of K+Hepes (anion dimensions of 1.4x0.6x0.5 nm compared with a radius of 0.181 nm for K+Cl ) resulted in no detectable conductance increments, while increasing the cation size in the case of T r i s + C l (cation radius of 0.67 nm compared with 0.133 nm for K +C1 ) yielded a single channel conductance which was not discernably d i f f e r e n t from that observed in K+C1 . These data were consistent with the formation of anion-selective, i f not s p e c i f i c , channels by these proteins. The an i o n - s p e c i f i c i t y of protein P has been shown to be due to the presence of an anion-binding s i t e within the channel (Benz et a l . , 1983). Thus conductance through protein P channels saturates at high salt concentrations (Benz et a l . , 1983), in contrast to porin proteins which lack binding si t e s and t y p i c a l l y reveal a linear dependence of single channel conductance on salt concentration (Benz and Hancock, 1981; Benz et a l . , 1984). To determine i f the anion-selectivity of the phosphate-regulated porin proteins could be attributed to binding si t e s within their respective channels, single channel conductance was measured as a function of salt (KC1) concentration. In every case, single channel conductance was seen to saturate at high salt concentrations (e.g. F i g . 23) consistent with the presence 143 Table XII. Single channel conductance of phosphate-starvation-inducible porin proteins of the fluorescent Pseudomonads in salt s of varying anion and cation size Average single channel conductance (pS) in Strain K + C l " b T r i s + C l " b K +Hepes" b P. aeruginosa 158 141 <25 p. putida 144 1 43 <25 p. fluorescens 156 149 <25 p. aureofaciens 164 169 <25 p. chlororaphis 166 1 67 <25 Average of at least 60 single channel events b Salts were employed at a concentration of 0.5 M. Ion r a d i i (in nm) are as follows: K , 0.133; C l " , 0.181; T r i s + , 0.670. Hepes -, an e l l i p s o i d molecule, has dimensions 1.4x0.6x0.5 nm. 144 of a binding s i t e within these channels. By pl o t t i n g the data as an Eadie-Hofstee plot (e.»g. F i g . 23 inset), Kd values for Cl binding were readily derived (Table XIII). While some v a r i a b i l i t y in the a f f i n i t y of Cl for each of the channels was observed, there was only a 2-fold range in Kd values for Cl binding for a l l channels, including protein P, demonstrating that the re l a t i v e a f f i n i t i e s of each of the channels for Cl were similar to protein P. 4. Phosphate i n h i b i t i o n of macroscopic conductance. Because the single channel conductance of protein P channels in phosphate i s low (6-9 pS in 1 M H 2P0 4 ) (Hancock et a l . , 1982), approaching the resolution l i m i t s of the black l i p i d bilayer apparatus, the presence of a phosphate-binding s i t e within protein P channels was supported by the a b i l i t y of orthophosphate to i n h i b i t Cl conductance through protein P channels. The derived I^Q value for phosphate (defined as the concentration of phosphate which yielded 50 % i n h i b i t i o n of chloride conductance) indicated that orthophosphate had a 60-100 fold higher a f f i n i t y for protein P channels than did Cl (Hancock and Benz, submitted) the anion previously exhibiting the highest a f f i n i t y for protein P channels (Benz et a l . , submitted). In order to identify potential phosphate-binding s i t e s in the phosphate-starvation-induced porin proteins of P. putida, P. fluorescens, P. aurefaciens and P. chlororaphis, this strategy was also applied. Thus, after formation of a l i p i d bilayer (indicated by the 145 Figure 23. Average single channel conductance of the phosphate-starvation-induced porin protein of P.  aureofaciens as a function of the KC1 concentration in the aqueous solution bathing an oxidized cholesterol (1.5 % in n-decane) membrane. The applied voltage was 50 mV and the temperature was 25°C. The aqueous phase contained approximately 10 ng/ml of porin protein at KC1 concentrations of 300 mM and higher. For KC1 concentrations below 300 mM, 100 ng/ml of protein had to be added to obtain a s u f f i c i e n t number of single channels. Inset. An Eadie-Hofstee plot of the data obtained from measurements of single channel conductance (V) as a function of the KC1 concentration (S). Binding constants (Table XIII) were obtained from such a plot using least squares analysis. 146 KCl Concentration (mM) 147 Table XIII. Binding a f f i n i t i e s of phosphate-starvation-inducible porin proteins of the fluorescent Pseudomonads for chloride and orthophosphate IgQ for phosphate (mM)b at Strain Kd for Cl a (mM) 40 mM C l " 1 M C l " P. aeruginosa 153 0.59 12.7 P. putida 192 1 .08 -P. fluorescens 220 - 9.7 P. aureofaciens 297 - 27.0 P. chlororaphis 204 2.40 -The average single channel conductance from at least 75 recorded events was determined at each of 5 concentrations of KC1 between 50 and 1000 mM. The data was plotted as an Eadie-Hofstee plot (Figure 23 inset) from which Kd values were obtained by least squares analysis. b Inhibition of macroscopic chloride conductance by phosphate was car r i e d out as described in Methods. The % i n h i b i t i o n of i n i t i a l conductance was measured for dif f e r e n t concentrations of phosphate and the data plotted as an Eadie-Hofstee plot (see F i g . 24 inset) from which I 5 Q values were obtained by least squares analysis 148 membrane's turning o p t i c a l l y black) a small amount of protein was added to the aqueous solution bathing the l i p i d membrane and conductance followed u n t i l the rate of increase had slowed considerably (usually 15-25 min). At this time membrane conductance had usually increased 2-4 orders of magnitude and > 1000 channels were present in the membrane. Aliquots of phosphate were added sequentially and the new conductance l e v e l measured after each addition. For each protein studied, phosphate addition was seen to decrease the l e v e l of conductance o r i g i n a l l y observed in the presence of KC1 alone, and the magnitude of this decrease was d i r e c t l y related to the concentration added (e.g. F i g . 24). By plot t i n g the data as % i n h i b i t i o n of chloride conductance as a function of % i n h i b i t i o n of chloride conductance/phosphate concentration (i.e an Eadie-Hofstee plot) (Fig. 24 inset) i t was possible to derive an apparent I^ Q value for phosphate i n h i b i t i o n of chloride conductance for each of the phosphate-regulated porins (Table X I I I ) . These data were consistent with the presence of a phosphate-binding s i t e within each of these channels. The apparent I ^ Q values varied with the concentration of KC1, ranging from 9.7 to 27 mM phosphate in 1 M KC1 and from 0.59 to 2.5 mM phosphate in 40 mM KC1 (at pH 7) (Table X I I I ) . At a given concentration of KC1, however, the variation in I ^ Q values obtained for a l l of the channels did not exceed 4-fold, indicating that the re l a t i v e a f f i n i t i e s of these channels for phosphate were quite si m i l a r . 149 Figure 24. Phosphate i n h i b i t i o n of chloride (Cl ) flux through protein P channels. Protein P (100 ng/ml) was added to the aqueous solution (40 mM KC1/ 1 mM T r i s -HCl, pH 7.0) bathing an oxidized cholesterol (1.5 % in n-decane) membrane and the membrane conductance allowed to increase u n t i l i t had s t a b i l i z e d (usually at a level 2-4 orders of magnitude higher than the i n i t i a l l e v e l ) . At this time aliquots of potassium phosphate buffer pH 7.0 were added to the aqueous phase on both sides of the membrane and the new conductance level recorded. The % decrease in conductance was calculated and plotted as a function of the aqueous phase phosphate concentration [ P i ] . The applied voltage was 20 mV and the temperature was 25 C. Inset. An Eadie-Hofstee plot of the data derived from measurements of the % i n h i b i t i o n of chloride conductance as a function of phosphate concentration. I 5 0 values for phosphate i n h i b i t i o n of chloride conductance (Table XIII) were calculated using least squares analysis. 150 100 10-OH 1 | 1 1 r 0 1 2 3 k 5 Phosphate Concentration ( m M ) 151 5. Summary. Phosphate-starvation induced oligomeric proteins from the outer membranes of P. fluorescens, P. putida, P. aureofaciens and P. chlororaphis were p u r i f i e d to homogeneity. The incorporation of p u r i f i e d proteins into planar l i p i d bilayer membranes resulted in stepwise increases in membrane conductance. Single channel conductance experiments demonstrated that these protein were a l l capable of forming small, protein P-like channels with an average single channel conductance in 1 M KC1 of between 233 and 252 pS. The conductance properties were not altered when the proteins were p u r i f i e d free of LPS prior to reconstitution in l i p i d bilayer membranes. Single channel conductance measurements made in sa l t s of varying cation or anion size indicated that the channels were uniformly anion-sel e c t i v e . The measurement of single channel conductance as a function of KC1 concentration revealed that a l l channels saturated at high s a l t concentrations, consistent with the presence of a binding s i t e in the channel. Apparent Kd values for Cl were calculated and shown to vary only two-fol d (180 - 297 pS) amongst a l l channels, including protein P channels. Phosphate was capable of i n h i b i t i n g chloride conductance through a l l channels, with apparent I^Q values of between 0.59 and 2.5 mM phosphate at 40 mM Cl , and between 9.7 and 27.0 mM phosphate at 1 M Cl . These data were consistent with the presence of a phosphate-binding site in the channels of these phosphate-regulated proteins. Furthermore, they indicated that these channels had at least 152 .a 20 to 80-fold higher a f f i n i t y for phosphate over chloride. 153 DISCUSSION The transport of inorganic phosphate in the gram-negative bacterium Pseudomonas aeruginosa involves translocation across two membranes. In this study only those constituents external to the cytoplasmic membrane were examined in d e t a i l , in an attempt to address the mechanism(s) by which phosphate overcomes the permeability barrier of the outer membrane under phosphate-limiting conditions. 1. A phosphate regulon in Pseudomonas aeruginosa. In response to phosphate-deficiency, wild type c e l l s of P.  aeruginosa were shown to be derepressed for the synthesis of the enzymes alkaline phosphatase and phospholipase C (Fig. 4), in addition to a periplasmic phosphate-binding protein and an outer membrane channel-forming protein, P (Figs. 3 and 10). The observation that c o l l e c t i v e l y these species were s i m i l a r l y c o n s t i t u t i v e l y produced or non-inducible in mutants of P. aeruginosa (Fig. 5) suggested that these constituents were indeed co-regulated, forming a phosphate regulon analogous to the pho regulon of Escherichia c o l i (Tommassen and Lugtenberg, 1982). Additional, as yet uncharacterized, orthophosphate-regulated proteins have been i d e n t i f i e d in phospholipase C regulatory mutants of P.  aeruginosa PAO (Gray et a l . , 1981, 1982) suggesting that a phosphate regulon in P. aeruginosa PAO may be s i g n i f i c a n t l y 1 54 more extensive than described here. The coordinate regulation of the constituent genes of operons or regulons usually r e f l e c t s the roles of their gene products in a common process. The maltose regulon in E. c o l i , for example, involves constituents of the transport and catabolism of maltose and maltodextrins (Bedouelle, 1984), while many of the components of the pho regulon in E. c o l i function in the acquisition of inorganic phosphate (Tommassen and Lugtenberg, 1982). Like E. c o l i , P. aeruginosa demonstrates two major uptake systems for inorganic phosphate, of low and high-a f f i n i t y , respectively (LaCoste et a l . , 1981; Fig. 14B). . The observation that the low-affinity system operates c o n s t i t u t i v e l y is consistent with a role in the transport of phosphate in a phosphate-rich medium. Its high capacity (Vmax = 12.1 nmol/min/mg c e l l protein) undoubtedly r e f l e c t s the ready a v a i l a b i l i t y of phosphate in a r i c h medium, as well as the growth potential of the organism under these conditions. In a d i l u t e environment, however, phosphate uptake via the low-affinity system w i l l be l i m i t i n g for growth (compare the rate of growth of wild-type P.  aeruginosa with that of a mutant expressing only the low-a f f i n i t y system (Fig. 13)). The derepression of a high-a f f i n i t y system permits e f f i c i e n t transport of phosphate from a d i l u t e environment. Thus, during phosphate li m i t a t i o n strains capable of expressing a h i g h - a f f i n i t y uptake system transport phosphate at s i g n i f i c a n t l y greater 155 rates than mutants def i c i e n t in this system (Fig. 12). The lower capacity of this system (Vmax =5.4 nmol/min/mg c e l l protein) compared with the low-affinity system undoubtedly r e f l e c t s the decreased a v a i l a b i l i t y of phosphate and a consequently reduced growth potential in a phosphate-limited environment (Fig. 2). Alkaline phosphatase (a phosphate monoesterase) may function to provide the inorganic orthophosphate (Pi) substrate for these transport systems by i t s action on phosphate-containing molecules. Its concerted action with phospholipase C, which releases the phosphoryl choline moiety from s p e c i f i c phospholipid molecules, probably functions to make phospholipids available as a phosphate source as well. The derepression of these enzymes in phosphate-limiting media suggests that in an orthophosphate-r i c h environment, larger phosphate-containing molecules may not normally be used as phosphate sources. However, upon depletion of the available orthophosphate supply, other sources are made available by the action of these hydrolytic enzymes. Similarly, the induction of phosphate-selective outer membrane porin protein P in a phosphate-limited environment is a response to the consequently low rate of d i f f u s i o n of phosphate across the outer membrane which would otherwise be l i m i t i n g for transport and growth (see below). Hig h - a f f i n i t y transport in P. aeruginosa was demonstrated to obligately require a periplasmic phosphate-binding protein, such transport being absent in a phosphate-156 binding protein-deficient mutant (Fig. 14A). The binding a f f i n i t y for phosphate (Kd=0.34 uM) was in good agreement with the observed kinetics of h i g h - a f f i n i t y phosphate transport (Km=0.46 uM). This i s c h a r a c t e r i s t i c of binding protein-dependent transport systems where the kinetics appear to be dictated by the binding proteins which function as the rate determining step. The binding a f f i n i t y and a b i l i t y to be released by cold-osmotic shock are t y p i c a l of binding proteins- isolated from P. aeruginosa (Stinson et a l . , 1977; Hoshino and Kageyama, 1 980; Eisenberg and Phibbs, 1982) and of binding proteins in general (Oxender and Quay, 1976). Binding protein-dependent transport systems are t y p i c a l l y energized by phosphate-bond energy (e.g. ATP) (Berger and Heppel, 1974). Thus, an inhibitory effect of arsenate on h i g h - a f f i n i t y phosphate transport in P.  aeruginosa (LaCoste et a l . , 1981) might be due to i t s affect on energization, although the competitive nature of the i n h i b i t i o n (Ki=0.24 mM; LaCoste et a l . , 1981) suggests that i t was acting at some component of the h i g h - a f f i n i t y transport system. The observation that arsenate was capable of i n h i b i t i n g the binding of phosphate to the binding protein at concentrations comparable to the. Ki (Table VII) implied that the effect of arsenate was at the binding protein stage of transport. It is noteworthy that, upon induction in phosphate-deficient medium, the enzymes alkaline phosphatase and phospholipase C were secreted from the c e l l (Fig. 4B). 157 Theories of extracellular protein release generally held that outer membrane breakdown was a means of releasing proteins from, for example, a periplasmic location. Obviously, i f such a mechanism were responsible for the observed enzyme release, the concomittant increase in outer membrane permeability could have functioned to increase the rate of d i f f u s i o n of phosphate across the outer membrane without the need for a s p e c i f i c channel. Nevertheless, enzyme secretion was s p e c i f i c , as demonstrated by the f a i l u r e to observe any concomittant release of the periplasmic beta-lactamase and phosphate-binding protein, and the barrier properties of the outer membrane were maintained (Fig. 6). This implied that the synthesis of a phosphate-selective outer membrane porin protein was not superfluous. Interestingly, i t has been suggested (Ingram et a l . , 1973; Bhatti and Ingram, 1981) that alkaline phosphatase release by P. aeruginosa i s associated with a change in outer membrane permeability in addition to LPS release. Unfortunately, their use of a Tris-buffered medium invalidated their results, given the observed a b i l i t y of T r i s to permeabilize and cause s i g n i f i c a n t structural reorganization of the outer membrane (Irvin et__al., 1981). 2. Properties of outer membrane protein P.. Outer membrane protein P of P. aeruginosa was demonstrated to form SDS-stable oligomers (trimers; Angus and Hancock, 1983) in polyacrylamide gels, a property shared by the majority of 158 porin proteins described to date. The monomer molecular weight of thi s protein, 48,000, was s i g n i f i c a n t l y greater than that of most Enterobacterial porins and porin protein F of P. aeruginosa (36-39,000) (Lugtenberg and van Alphen, 1983; Hancock and Carey, 1979). Attempts at determining the molecular weight of the native (trimer) form of the protein on SDS-polyacrylamide gels [from Ferguson plots of the electrophoretic mobility of the trimers as a function of the acrylamide concentration (Tokunaga et a l . , 1979)] have f a i l e d because the native protein migrates anomalously in this gel system. The anomalous migration of porin oligomers in SDS-polyacrylamide gels has been described previously (Tokunaga et a l . , 1979) and has been attributed to the high degree of betarstructure present in the native porins (Nikaido and Vaara, 1985), which results in the binding of less SDS (wt/wt) by these proteins (Rosenbusch, 1974). With few exceptions (e.g. proteins F and D1 of P.  aeruginosa; Hancock and Carey, 1979; Hancock and Carey, 1980), porins exist as undenatured trimers when extracted with SDS. The observation that protein P formed SDS-stable trimers, although t y p i c a l of porins in general, i s thus in contrast to the other porin proteins described in P.  aeruginosa. However, protein P was distinguishable from the majority of porins, including protein F, in that i t was readily s o l u b i l i z e d in a non-denaturing detergent (Triton X-100) in the presence of EDTA. Most porin proteins are soluble in Triton only after digestion of the peptidoglycan 159 with lysozyme, or in SDS and high salt (> 0.4 M NaCl) (Lugtenberg and van Alphen, 1983). This has been interpreted as indicating a strong non-covalent attachment of these porins to the peptidoglycan. In addition, the channels formed by protein P in l i p i d bilayer membranes (0.6 nm dia.) were s i g n i f i c a n t l y smaller than those formed by protein F (2.2 nm) and the major Enterobacterial porins (1-1.4 nm) (Benz et a l . , 1985). F i n a l l y , while the majority of porins studied to date, including the phosphate-starvation-inducible PhoE porin protein of E. c o l i , exhibit only weak ion s e l e c t i v i t y in planar l i p i d bilayer membranes (Benz et a l . , 1985), protein P channels are anion-specific (Hancock et a l . , 1982; Table I I I ) . As confirmed in thi s study, (Table XIII), the anion-s p e c i f i c i t y of protein P channels can be attributed to the presence of a binding s i t e for anions in the channel (Benz et a l . , 1983). Acetylation of available amino groups on protein P (see Methods), which did not affect the a b i l i t y of protein P to form SDS-stable oligomers (trimers) in polyacrylamide gels (not shown), resulted in the loss of the anion-binding s i t e and a concomittant loss of the anion-specif i c i t y of the channel (Hancock et a l . , 1983b). From these studies i t was concluded that the ani o n - s p e c i f i c i t y of protein P channels was a function of epsilon-amino groups of lysine residues present on the protein (Hancock et a l . , 1983b). Similarly, the phosphate-regulated PhoE porin of E.  c o l i has been demonstrated to possess acetylatable lysine 160 residues which were responsible for the observed, a l b e i t weaker, anion-selectivity of this channel in black l i p i d bilayer membranes (Darveau et a l . , 1984). The contamination of p u r i f i e d porin preparations with LPS is well documented (Nikaido and Vaara, 1985) and suggests a strong tendency for these molecules to associate. Indeed, an in vivo association of LPS with porins, suggested by the marked reduction in porin levels in the outer membranes of LPS-deficient mutants (e.g. Koplow and Goldfine, 1974), has been suggested to be necessary, not only for porin function (Schindler and Rosenbusch, 1978), but also for the modulation of porin a c t i v i t y (Kropinski e_t a l . , 1982). Although conventionally p u r i f i e d protein P preparations invariably contained s i g n i f i c a n t levels of LPS (Fig. 7; Table II), such an association was demonstrated to be dispensable in the formation of anion-specific channels by this protein (Table I I I ) . Recently, Korteland and Lugtenberg (1984) have reported that E. c o l i mutants with heptose-less LPS produce PhoE porins which f a c i l i t a t e a 6-to 8-fold more e f f i c i e n t permeation of anionic solutes. In contrast to results presented here with protein P, these data suggest that LPS may indeed influence porin a c t i v i t y . In addition to being co-regulated with components of a h i g h - a f f i n i t y phosphate uptake system in P. aeruginosas (see above), protein P channels have recently been demonstrated to possess a binding s i t e for inorganic phosphate (Hancock and Benz, submitted; Table XIII). The observation that 161 protein P channels exhibited a 60 to 80-fold higher a f f i n i t y for phosphate over chloride (Hancock and Benz, submitted; Table XIII) was consistent with the demonstrated role for protein P in phosphate uptake (Table V) (see below). 3. The outer membrane of P. aeruginosa as a permeability  barrier to phosphate under phosphate-limiting conditions. The low i n t r i n s i c permeability of the P. aeruginosa outer membrane i s well documented, supported by observations of the increased c r y p t i c i t y of periplasmic enzymes in P.  aeruginosa compared with the analogous enzymes in, for example, E. c o l i (Yoshimura and Nikaido, 1982) as well as direct measurements of outer membrane permeability (Angus et a l . , 1982; Yoshimura and Nikaido, 1982; Nicas and Hancock, 1983). Thus, the apparent transport and growth Km values for given nutrient molecules are often higher in P.  aeruginosa than in E. c o l i . The results of this study demonstrated that the outer membrane of P. aeruginosa indeed functions as a permeability barrier to phosphate molecules under phosphate-limiting conditions. This was supported by the observed increase in the apparent Km for h i g h - a f f i n i t y phosphate transport in the protein P-deficient mutant (Table V), explainable by a decrease (at a given external phosphate concentration) in the rate of di f f u s i o n of phosphate molecules across the protein P-deficient outer membrane compared with the wild type outer membrane. The fact that the Vmax of h i g h - a f f i n i t y uptake remained unaltered in the 162 mutant (Table V) further indicated that the di f f u s i o n of phosphate across the protein P-deficient outer membrane was only r a t e - l i m i t i n g for transport at low concentrations of phosphate. At higher concentrations of phosphate (e.g. > 25 uM) the rate of phosphate transport was not detectably different in the mutant compared with the wild-type. Furthermore, a defect in the growth c a p a b i l i t i e s of the protein P-deficient mutant strain was only seen at a very low concentration of phosphate (50 uM) (Table VI), indicating that phosphate dif f u s i o n across the outer membrane was dependent upon protein P at low external phosphate concentrations only. At higher concentrations of phosphate, d i f f u s i o n across the outer membrane through the major porin protein F of P. aeruginosa i s apparently capable of s a t i s f y i n g the c e l l u l a r requirements for this nutrient. In addition, the small size and the s e l e c t i v i t y of the protein P channels would undoubtedly serve to maintain the low i n t r i n s i c permeability of this membrane to other constituents. Whether the protein P channels are s u f f i c i e n t to f a c i l i t a t e the e f f i c i e n t movement of phosphate from a d i l u t e environment across the outer membrane remains in question. The apparent a b i l i t y of the phosphate-binding protein to associate in v i t r o with protein P may have physiological relevance in vivo concerning the mechanism by which phosphate dif f u s i o n across the outer membrane is f a c i l i t a t e d in a d i l u t e environment. Certainly the a b i l i t y of the 163 periplasmic maltose-binding protein to associate with the coregulated LamB outer membrane porin protein has been documented (Bavoil and Nikaido, 1981). Such an association, demonstrated in v i t r o , has been suggested to be necessary in  vivo for the translocation, across the outer membrane, of maltose, when present at low concentrations, and maltodextrins (Wandersman et a l . , 1979; Luckey and Nikaido, 1983). From these studies i t was suggested that a physical association between a h i g h - a f f i n i t y binding protein and an outer membrane porin could function to bring a h i g h - a f f i n i t y binding s i t e near the outer surface of the outer membrane. Such an association might also ensure rapid binding of substrate once i t entered the periplasm, maintaining a concentration gradient across the outer membrane, even under d i l u t e conditions, by reducing the levels of free substrate in the periplasm. In the case of the phosphate-binding protein and protein P of P. aeruginosa, such an association would ce r t a i n l y not be obligatory given the a b i l i t y of the phosphate-binding protein-dependent h i g h - a f f i n i t y uptake system to operate in., the absence of protein P. The increase in the Km for h i g h - a f f i n i t y phosphate transport observed for the protein P-deficient mutant strain may r e f l e c t higher free periplasmic concentrations of phosphate and a subsequently smaller concentration gradient to drive d i f f u s i o n across the outer membrane owing to the i n a b i l i t y of the phosphate-binding protein and protein P to associate at the outer membrane. 164 A further analogy with the maltose transport system of E. c o l i i s seen in the a b i l i t y of long phosphate polymers to bind to the phosphate-binding protein. If polyphosphates are capable of being transported intact by the h i g h - a f f i n i t y phosphate transport system in vivo, without prior hydrolysis by alkaline phosphatase or other phosphatases, their permeation of protein P channels w i l l undoubtedly depend upon the proper linear orientation of the molecules at the channel mouth, since these molecules exceeed the exclusion l i m i t of protein P channels. Such orientation could be carried out by the phosphate-binding s i t e of protein P and/or the h i g h - a f f i n i t y phosphate-binding s i t e present on the phosphate-binding protein, in association with the outer membrane channel-forming protein. Similarly, the a b i l i t y of the LamB protein to allow the d i f f u s i o n of maltodextrins which exceed the apparent exclusion l i m i t of the LamB pore, has been postulated to depend upon binding si t e s both in the channel and on the maltose-binding protein (Von Meyenburg and Nikaido, 1977; Ferenci and Boos, 1980). Growth of P. aeruginosa on polyphosphates as the sole phosphate source has been demonstrated (Valette et a l . , 1966). Confirmed in thi s study, i t was further demonstrated that growth in media containing phosphate polymers of up to 5 units in length occurred without the derepression of alkaline phosphatase (data not shown). This suggested that polyphosphates could be transported intact, without prior hydrolysis to constituent orthophosphate molecules. As 165 such, phosphatase(s) must be present in the cytoplasm to convert these polymers to »the usual currency of inorganic phosphate, orthophosphate. In this regard, a number of gram-negative organisms, including E. c o l i , have been demonstrated to posses cytoplasmic polyphosphatases (Yagil, 1975). Interestingly, growth of P. aeruginosa in a medium containing a phosphate polymer of 15 units as the sole phosphate source was, in fact, accompanied by the induction of detectable alkaline phosphatase in both the periplasm and in the external medium (data not shown). Whether th i s r e f l e c t s an i n a b i l i t y of polyphosphate P15 to be transported intact or a rate of uptake so slow as to mimic phosphate-deficiency i s uncertain. It may be a moot point, however, since alkaline phosphatase hydrolysis of such large polyphosphates was apparently necessary for growth. In contrast to P. aeruginosa, the observed higher i n t r i n s i c permeability of the E. c o l i outer membrane leads one to conclude that the major porins themselves may be capable of satis f y i n g the phosphate requirements of the c e l l , without the need for a f a c i l i t a t e d d i f f u s i o n channel for phosphate. According to Fick's f i r s t law of d i f f u s i o n , the rate of phosphate transport across the E. c o l i outer membrane via the constitutive porin pathway w i l l be higher, at a given concentration of phosphate, than in P. aeruginosa This makes the need of a phosphate channel in E. c o l i less obvious. Despite the fact that E. c o l i c e l l s deprived of phosphate are derepressed for a channel-forming outer 166 membrane protein, PhoE (Overbeeke and Lugtenberg, 1980), a sp e c i f i c role in phosphate uptake has not been demonstrated and unlike protein P, PhoE does not bind phosphate (Benz et a l . , 1984). Thus, although the PhoE porin functions as a more e f f i c i e n t channel for phosphate than do the OmpF or OmpC porins (Rorteland et al.,1982), i t does not provide any advantage to c e l l s growing in medium containing orthophosphate as the sole phosphate source (Overbeeke and Lugtenberg, 1982). Furthermore, a 10-fold increase in the Km for phosphate transport reported for a PhoE-deficient mutant compared with a PhoE + s t r a i n (Korteland et a l . , 1982) was obtained in a background deficient in the major porins. Thus i t was possible that the increase in Km of the PhoE mutant simply reflected the overall porin-deficiency of the PhoE mutant strain such that e.g. an OmpF+PhoE strain might have transported phosphate as well as the above OmpF PhoE + s t r a i n . The E. c o l i PhoE channel does, however, provide a growth advantage to c e l l s growing in a medium containing polyphosphate as the sole phosphate source (Overbeeke and Lugtenberg, 1982), and i t i s also an e f f i c i e n t channel for the uptake of organic phosphates. Possibly i t s physiological role i s in the transport of larger phosphorylated molecules. 4. Protein P and protein PhoE as members of two d i s t i n c t  classes of phosphate-regulated porins. In addition to E. c o l i and P. aeruginosa, a number of other gram-negative 167 bacteria, including S, typhimurium (Bauer et a l . , 1985) and E. cloacae (Verhoef et a l . , 1984), have been demonstrated to synthesize novel outer membrane porin proteins in response to phosphate-limitation. These proteins form anion-selective channels in reconstituted l i p i d bilayer - membranes (Bauer et a l . , 1985; Verhoef et a l . , 1984; Benz et a l . , 1984) consistent with their presumed roles in phosphate ac q u i s i t i o n . The results of this study extend the l i s t of bacteria synthesizing phosphate-starvation-inducible outer membrane proteins. Existing as oligomers (probably trimers) in SDS, these proteins can be dissociated to monomers when subjected to temperatures above 60°C (Table X), a property c h a r a c t e r i s t i c of porins (Lugtenberg and van Alphen, 1983). Furthermore, based on the monomer molecular we-ight, peptidoglycan association and abundance of these proteins, they are l i k e l y to be porins. Two classes of these proteins were distinguishable, based on monomer molecular weight (36 to 39K or 45.5 to 50K) and detergent requirements to remove the proteins from the peptidoglycan (SDS-high salt or Triton-Tris-EDTA). The former cl a s s , t y p i f i e d by protein PhoE, included the phosphate-starvation-inducible proteins of P. cepacia, P. pseudomallei and the Enterobacteriaceae. The l a t t e r class included the phosphate-starvation-inducible proteins of the P. fluorescens branch of the family Pseudomonadaceae and was exemplified by protein P. These two proteins were also distinguishable from a functional point of view. Thus, 168 while protein PhoE forms large (1 nm), weakly anion-selective channels (Benz et a l . , 1985) which lack binding sit e s for anions and for phosphate (Benz et a l . , 1984), protein P channels were small (0.6 nm) (Hancock et a l . , 1982), possessing binding sites for anions and phosphate (Benz et a l . , 1983; Hancock and Benz, submitted; Table XIII) consistent with the observed anion-specif i c i t y (Hancock e_t a l . , 1982) and phosphate-selectivity (Hancock and Benz, submitted; Table XIII) of this channel. Furthermore, the observed channel-forming properties of the phosphate-starvation-inducible outer membrane proteins of the fluorescent Pseudomonads were indeed consistent with the formation of protein P type porins by these proteins. One can only speculate on the purpose of synthesizing one or the other of these phosphate-regulated porin proteins. It seems probable that the production of a small s p e c i f i c channel l i k e protein P, in P. aeruginosa and the fluorescent Pseudomonads, r e f l e c t s the need to maintain low outer membrane permeability. Because these fluorescent Pseudomonads occur naturally in the s o i l , where many antibiotic-producing microorganisms are found, i t may be necessary for their survival that they maintain a barrier to a n t i b i o t i c s . Indeed, the outer membrane of P. aeruginosa has been implicated in the high i n t r i n s i c resistance of this organism to a n t i b i o t i c s (Yoshimura and Nikaido, 1982; Nicas and Hancock, 1983). In contrast, E. c o l i demonstrates s i g n i f i c a n t l y higher outer membrane permeability, implying 169 that an outer membrane of extremely low permeability i s not essential for E. c o l i c e l l s . The production of a phosphate-starvation-inducible porin protein, PhoE, whose channel size is not s i g n i f i c a n t l y different from the major constitutive porins of E. c o l i (Benz et a l • , 1985) would obviously function to maintain the same degree of permeability, especially since t o t a l porin levels in the outer membrane are regulated in a stringent fashion (Lugtenberg and van Alphen, 1983). Unlike the protein P channel of P.  aeruginosa, which i s too small to act as a channel for larger phosphate-containing molecules [except perhaps linear polymers of inorganic phosphate (see above)], the E. c o l i PhoE channel has been shown to allow the passage of large organic phosphate molecules (Overbeeke and Lugtenberg, 1982). Given the predicted high i n t r i n s i c permeability of the E. c o l i outer membrane to inorganic phosphate, the transport of larger phosphorylated molecules may, in fact, be the most important function of the PhoE channel. Indeed, E. c o l i i s often isolated from sewage effluent where polyphosphates (and organic phosphates) w i l l be in abundance, as a result of the action of anaerobic organisms which t y p i c a l l y make and store large quantities of polyphosphate (Kulaev, 1975). A cytoplasmic polyphosphatase has been i d e n t i f i e d in E. c o l i (Yagil, 1975), suggesting that polyphosphates are a potential source of phosphate in thi s organism. Because protein P channels are too small to act as channels for large phosphate-containing molecules, 170 the a b i l i t y of P. aeruginosa to u t i l i z e large phosphorylated molecules under di l u t e conditions, where uptake across the outer membrane via the major porin(s) w i l l be l i m i t i n g for transport and growth, may be dependent upon prior hydrolysis of these molecules by alkaline phosphatase, to release orthophosphate. Interestingly, alkaline phosphatase has been demonstrated to be secreted from P. aeruginosa c e l l s into the external medium (Cheng et a l . , 1970; F i g . 4B) where i t could, by cleavage of organic phosphates, provide a source of inorganic phosphate for transport. E. c o l i , on the other hand, synthesizes a porin protein in phosphate-l i m i t i n g media (PhoE) which i s capable of transporting these larger organic phosphates, and t h i s might explain why alkaline phosphatase i s exclusively located in the periplasm of t h i s bacterium (Malamy and Horecker, 1961). The production of phosphate-starvation-induced porins by the fluorescent Pseudomonads, a l l of which form protein P-like channels, suggests an evolutionary/taxonomic relationship. This is borne out by several pieces of data. A l l of these strains produce a major, constitutive protein (porin) which does not form SDS-stable oligomers. This is in contrast to the Enterobacteriaceae and other Pseudomonads, including P. cepacia and P. pseudomallei, which produce major porin proteins which are stable to SDS denaturation at temperatures below 60°C (Lugtenberg and van Alphen, 1983). This "heat-unmodifiable" phenotype i s ty p i c a l of porin protein F of P. aeruginosa (Hancock and 171 Carey, 1979). In addition, these fluorescent Pseudomonads also produce a lipoprotein which cross-reacts immunologically with outer membrane protein H2 of P. aeruginosa PA01 (Mutharia and Hancock, 1985). Furthermore, based on the results of DNA and rRNA hybridization studies, these strains were s i m i l a r l y c l a s s i f i e d in the same homolgy group and were c l e a r l y d i s t i n c t from other species of the family Pseudomonadaceae (De Vos and De Ley, 1983). These data suggest that outer membrane protein p r o f i l e s and immunological relationships may be important indicators of evolutionary l i n k s . Because a number of d i s t i n c t b a c t e r i a l species have been demonstrated to produce a protein P-like porin, the opportunity exists to. study the biogenesis of the protein and to identify functional domains. Given the s i m i l a r i t i e s of the functional properties of these proteins in d i f f e r e n t strains, which were quite d i s t i n c t from other phosphate-regulated porins, i t should be possible to i d e n t i f y regions of the protein involved in, for example, anion/phosphate-s e l e c t i v i t y as regions of close homology in the genes encoding these proteins. Such data w i l l undoubtedly contribute to elucidating the topology of t h i s protein in P. aeruginosa. Furthermore, by examining the expression of these foreign genes in P. aeruginosa PA01 we can identify regions of the protein important in synthesis, secretion and assembly. 172 5. Conserved antigenic determinants in phosphate- starvation-inducible outer membrane (porin) proteins. The demonstration here that phosphate-starvation-induced membrane oligomers cross-reacted immunologically (Fig. 18), indicated that these proteins express conserved antigenic s i t e s (regions of structural homology). The observed destruction of this c r o s s - r e a c t i v i t y by the heat-dissociation of oligomers to monomers, which also destroys porin function, and the f a i l u r e of phosphate starvation-induced monomers to cross-react with a protein P monomer-sp e c i f i c antiserum, suggested that the conserved regions of homology were maintained in the native, functional proteins only. Furthermore, the i n a b i l i t y of the protein P trimer-s p e c i f i c antiserum to react with the major porin proteins in these strains (Fig. 18) indicated that the c r o s s - r e a c t i v i t y was d i s t i n c t from any homologies r e l a t i n g to porin structure in general. Such homologies do, in fact, exist as indicated by the observed immunological c r o s s - r e a c t i v i t y of OmpF, OmpC and PhoE porins of E. c o l i (Overbeeke et a l . , 1980) and of porin proteins F and P of P. aeruginosa (K. Poole, unpublished r e s u l t ) . The c r o s s - r e a c t i v i t y of phosphate starvation-induced oligomeric proteins may well relate to spe c i f i c functional properties of these phosphate-regulated proteins. In this vein, a number of the phosphate starvation-induced membrane proteins have been demonstrated to form anion-selective channels (Hancock et a l . , 1982; Benz et a l . , 1984; Verhoef 1 73 et a l . , 1984; Bauer et a l . , 1985; Table XIII), in contrast with the major Enterobacterial porin proteins and the major porin protein F of P. aeruginosa, which are weakly cation-selective (Benz et a l . , 1985). The anion-selectivity of some of these proteins has been attributed to fixed positive charges, possibly epsilon-amino groups of lysine residues, in or near the mouth of the respective channels (Hancock et a l . , 1983b; Darveau et a l . , 1984). The demonstration of at least 14 exposed/accessible lysine residues in the protein P and PhoE channels (R.E.W. Hancock, unpublished result) i s consistent with this idea. By comparison, porin protein OmpF contains 5 accessible lysine residues (R.E.W. Hancock, unpublished r e s u l t ) . It is tempting to hypothesize that exposed lysine residues present in these, and perhaps other, phosphate-regulated porin proteins may be involved in the observed c r o s s - r e a c t i v i t y of phosphate-starvation-induced membrane proteins, perhaps forming part of the conserved antigenic s i t e ( s ) . In support of t h i s , protein PhoE channels are functionally indistinguishable from OmpF and OmpC channels upon acetylation of available lysine residues. The observed immunological c r o s s - r e a c t i v i t y of these proteins (Overbeeke et a l . , 1980), the 60-70 % amino acid homology (Tommassen et a l . , 1982; Mizuno et a l . , 1983), and the a b i l i t y of the ompF and phoE genes to hybridize in regions along their entire lengths (Tommassen et a l . , 1982) implies that the property of anion-selectivity as a function of lysine residues in the channel may well be the major 174 discriminating feature of PhoE compared with the other major porins in E. c o l i . If indeed the cross-reactive determinant(s) are involved in the ani o n - s e l e c t i v i t y of these proteins, then the question remains as to whether these phosphate-regulated porins evolved d i r e c t l y from the same ancestral phosphate porin gene, or whether they evolved from the various porin genes in these bacteria. Clearly, to answer t h i s question i t w i l l be necessary to obtain sequence information for each of the porins concerned. 175 LITERATURE CITED Angus, B.L., A.M. Carey, D.A. Caron, A.M.B. Kropinski, and R.E.W. Hancock. 1982. Outer membrane permeability in Pseudolmonas aeruginosa: comparison of a wild-type with an antibiotic-supersusceptible mutant. Antimicrobiol. Agents Chemother. 21: 299-309. Angus, B.L., and R.E.W. Hancock. 1983. Outer membrane porin proteins F, P and D1 of Pseudomonas aeruginosa and PhoE of Escherichia c o l i : chemical cross-linking to reveal native oligomers. J. Ba c t e r i o l . 155: 1042-1051. Argast, M., and W. Boos. 1980. Co-regulation in Escherichia  c o l i of a novel transport system for sn-glycerol-3-phosphate and outer membrane protein Ic (e,E) with a l k a l i n e phosphatase and the phosphate-binding protein. J . Bac t e r i o l . 143: 142-150. Bagdasarian M., M.M. Bagdasarian, S. Coleman, and K.N. Timmis. 1979. New vector plasmids for gene cloning in Pseudomonas, p. 411-422. I_n K.N. Timmis and A. Puhler (eds.), Plasmids of medical, environmental and commercial importance. Elsevier/North Holland, Amsterdam. Bauer, K., R. Benz, J. Brass, and W. Boos. 1985. Salmonella  typhimurium contains an anion-selective outer membrane porin protein induced by phosphate starvation. J . Bac t e r i o l . 161: 813-816. Bavoil, P. and H. Nikaido. 1981. Physical interaction between the lambda receptor protein and the c a r r i e r -immobilized maltose-binding protein of Escherichia c o l i . J . B i o l . Chem. 256: 11385-11388. Bedouelle, H. 1984. Controle de 1 ' u t i l i s a t i o n du maltose et des maltodextrines par Escherichia c o l i . B u l l e t i n de L' i n s t i t u t Pasteur 82: 91-145 Bennet, R.C. and M.H. Malamy. 1970. Arsenate resistant mutants of Escherichia c o l i and phosphate transport. Biochem. Biophys. Res. Commun.40: 496-503. 176 Benz, R. , K. Janko, W. Boos, and P. Lauger. 1979. Formation of large ion-permeable channels by the matrix protein (porin) of Escherichia c o l i . Biochim. Biophys. Acta 511: 309-315. Benz, R., A. Boehler-Kohler, R. Diertele and W. Boos. 1978. Porin a c t i v i t y in the osmotic shock f l u i d of Escherichia  c o l i . J . B a c t e r i d . 135: 1080-1090. Benz, R. and R.E.W. Hancock. 1981. Properties of the large ion-permeable pores formed from protein F of Pseudomonas  aeruginosa in l i p i d bilayer membranes. Biochim. Biophys. Acata. 646: 298-308. Benz, R., R.E.W. Hancock, and T. Nakae. 1982. Porins from gram-negative bacteria in l i p i d bilayer membranes, p. 123-134. In R. A n t o l i n i , A. G l i o z z i , and A. Goreo (eds.), Transport in biomembranes: model systems and reconstitution. Raven Press, New York. Benz, R., M. Gimple, K. Poole, and R.E.W. Hancock. 1983. An anion-selective channel from the Pseudomonas aeruginosa outer membrane. Biochim. Biophys. Acta 730: 387-390. Benz, R. 1984. Structure and s e l e c t i v i t y of porin channels, p. 199-219. I_n F. Bronner and W.D. Stein (eds.), Current topics in membranes and transport, v o l . 21. Academic Press, New York. Benz, R., R.P. Darveau, and R.E.W. Hancock. 1984. Outer membrane protein PhoE from Escherichia c o l i forms anion-selective pores in l i p i d bilayer membranes. Eur. J. Biochem. 140: 319-324. Benz, R., A. Schmid, and R.E.W. Hancock. 1985. Ion s e l e c t i v i t y of gram-negative bacterial pores. J . Bac t e r i o l . 162: 722-727. Berger, E.A., and L.A. Heppel. 1974. Different mechanisms of energy coupling for the shock-sensitive and shock-resistant amino acid permeases of Escherichia c o l i . J. B i o l . Chem. 249: 7747-7755. 177 Bhatti, A.R., and J.M. Ingram. 1981. The binding and secretion of alkaline phosphatase by Pseudomonas aeruginosa. FEMS Microbiol. Lett. 13: 353-356. Boehler-Kohler, B.A., W. Boos, R. Dieterle and R. Benz. 1979. Receptor for bacteriophage lambda of Escherichia c o l i forms larger pores in black l i p i d membranes than the matrix protein (porin). J. B a c t e r i d . 138: 33-39. Boos, W. and A.L. Staehlin. 1981. Ult r a s t r u c t u r a l • l o c a l i z a t i o n of the maltose-binding protein within the c e l l envelope of Escherichia c o l i . Arch. Microbiol. 129: 240-246. Bracha, M. and E. Yagi l . 1973. A new type of alkaline phosphatase-negative mutant in Escherichia c o l i K-12. Mol. Gen. Genet. 122: 53-60. Bragg, P.D. and C. Hou. 1972. Organization of proteins in the native and reformed outer membrane of Escherichia c o l i . Biochim. Biophys. Acta. 274: 478-488. Braun, V., H. Gnirke, U. Henning and K. Rehn. 1973. Model for the structure of the shape maintaining layer of Escherichia c o l i c e l l envelope. J . Ba c t e r i o l . 114: 1264-1270. Braun, V. 1975. Covalent lipoprotein from the outer membrane of Escherichia c o l i . Biochim. Bipophys. Acta. 415: 335-337. Braun, v., R.E.W. Hancock, K. Hantke and A. Hartman. 1976. Functional organization of the outer membrane of Escherichia  c o l i . Phage and c o l i c i n receptors as components of iron uptake systems. J. Supramol. Struct., v o l . 5. Braun, V. and K. Hantke. 1977. Bacterial receptors for phages and c o l i c i n s as constituents of s p e c i f i c transport systems, p. 99-137. I_n J.L. Reissig (ed.), Microbial interactions, series B, vol . 3. Chapman and Hul l , London. Braun, V. and J.H. Krieger-Bauer. 1977. Interrelationship of the phage lambda receptor protein and maltose transport in mutants of Escherichia c o l i K12. Biochim. Biophys. Acta. 469: 89-98. 178 Braun, V. and K. Hantke. 1982. Receptor-dependent transport systems in Echerichia c o l i for iron complexes and vitamin B12, p. 107-114. I_n A.N. Matonosi (ed.), Membranes and transport, v o l . 2. Plenum Press, New York. Brickman, E. and J. Beckwith. 1975. Analysis of the regulation of Escherichia c o l i alkaline phosphatase synthesis using deletions and 080-transducing phages. J. Mol. B i o l . 96: 307-316. Brown, M.R.W., H. Anwar and P.A. Lambert. 1984. Evidence that mucoid Pseudomonas aeruginosa in the c y s t i c f i b r o s i s lung grows under iron r e s t r i c t e d conditions. FEMS Microbiol. Lett. 21: 113-117. Ce l i s , R.T.F. 1984. Phosphorylation in vivo and in v i t r o of the arginine-ornithine periplasmic transport protein of Escherichia c o l i K12. Biochem. J. 178:133-137. Chai, T.-J. and J. Foulds. 1979. Interaction of bacteriophages by protein E, a new major outer membrane protein from an Escherichia c o l i mutant. J. B a c t e r i o l . 137: 226-233. Chai, T.-J., V. Wu and J. Foulds. 1982. C o l i c i n A receptor: role of two Escherichia c o l i outer membrane proteins (OmpF protein and btuB gene product) and lipopolysaccharide. J. Bac t e r i o l . 151: 983-988. Cheng, K.J., J.M. Ingram and J.W. Costerton. 1970. Release of alkaline phosphatase from c e l l s of Pseudomonas aeruginosa by manipulation of cation concentration and pH. J. B a c t e r i o l . 104: 748-753. Costerton, J.W. 1970. The structure and function of the c e l l envelope of gram-negative bacteria. Rev. Can. B i o l . 29: 299-316. Darveau, R.P., R.E.W. Hancock, and R. Benz. 1984. Chemical modification of the a n i o n - s e l e c t i v i t y of the PhoE porin from the Escherichia c o l i outer membrane. Biochim. Biophys. Acta 774: 67-74. 179 De Vos, P., and J. De Ley. 1983. Intra- and intergeneric s i m i l a r i t i e s of Pseudomonas and Xanthomonas ribosomal. ribonucleic acid cistrons. Int. J. Syst. B a c t e r i o l . 33:487-509. D i e t z e l , I., V. Kolb and W. Boos. 1978. Pole cap formation in Escherichia c o l i following induction of the maltose-binding protein. Arch. Microbiol. 118: 207-218. DiGirolamo, P.M. and C. Bradbeer. 1971. Transport of vitamin B12 in Escherichia c o l i . J . B a c t e r i o l . 106: 745-750. DiGirolamo, P.M., R.J. Kadner and C. Bradbeer. 1971. Isolation of vitamin B12 transport mutants of Escherichia c o l i . J . B a c t e r i o l . 106: 751-757. DiMasi, R.D., J.C. White, CA. Schnaitman and C. Bradbeer. 1973. Transport of vitamin B12 in Escherichia c o l i : common receptor s i t e s for vitamin B12 and the E c o l i c i n s on the outer membrane of the c e l l envelope. J. B a c t e r i o l . 115: 506-513. Echols, H., A. Garen, S. Garen and A. T o r r i a n i . 1961. Genetic control of repression of alkaline phosphatase in Escherichia c o l i . J. Mol. B i o l . 3: 425-438. Eisenberg, R.C. and P.V. Phibbs, J r . 1982. Characterization of an inducible mannitol-binding protein form Pseudomonas  aeruginosa. Current Microbiology. 7: 229-234. Ernst, J.F., R.L. Bennet and L.I. Rothfield. 1978. Constitutive expression of the iron-enterochelin and ferrichrome uptake systems in a mutant strain of Salmonella  typhimurium. J. B a c t e r i o l . 135: 928-934. Ferenci, T. 1980. The recognition of maltodextrins by Escherichia c o l i . Eur. J. Biochem. 108: 631-636. Ferenci, T. and W. Boos. 1980. The role of the Escherichia  c o l i lambda receptor in the transport of maltose and maltodextrins. J. Supramol. Struct.13:101-116. 180 Ferenci, T., Schwentorat, S. U l l r i c h and J. Vilmart. 1980. Lambda receptor in the outer membrane of Escherichia c o l i as a binding protein for maltodextrins and starch polysaccharides. J. B a c t e r i d . 142: 521-526. Figurski, D.H., and D.R. Helinski. 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acaf. S c i . U.S.A. 76: 1648-1652. Foulds, J., and T. - J . Chai. 1978. New major outer membrane protein found in an Escherichia c o l i t olF mutant resistant to bacteriophage Tulb. J. B a c t e r i d . 133: 1478-1483. Foulds, J., and T. - J . Chai. 1979. Isolation and characterization of isogenic E. c o l i strains with alterations in the l e v e l of one or more major outer membrane protein. Can. J. Microbiol. 2_5: 4230427. Friedberg, I. 1977. Phosphate transport in Micrococcus  lysodeikticus. Biochim. Biophys. Acta. 466: 451-460. Funahara, Y. and H. Nikaido. 1980. Assymetric l o c a l i z a t i o n of lipopolysaccharide on the outer membrane of Salmonella  typhimuriurn. J . B a c t e r i d . 141: 1463-1 465. Furukawa, H., H. Ymada and S. Mizushima. 1979. Interaction of bacteriophage T4 with reconstituted c e l l envelopes of Escherichia c o l i K-12. J. B a c t e r i d . 1 40: 1071-1080. Glauert, A.M. and M.J. Thornley. 1969. The topography of the bac t e r i a l c e l l wall. Annu. Rev. Microbiol. 23: 159-198. Gerdes, R.G. and H. Rosenberg. 1974. The relationship between the phosphate-binding protein and a regulator gene product fron Escherichia c o l i . Biochim. Biophys. Acta (Amstr.) 351: 77-86. Gerdes, R.G., K.P. Strickland and H. Rosenberg. 1977. Restoration of phosphate transport by the phosphate-binding protein in spheroplasts of Escherichia c o l i . J. B a c t e r i d . 131: 512-518. 181 Gray, G.L., R.M. Berka and M.L. V a s i l . 1981. A Pseudomonas  aeruginosa mutant non-derepressible for orthophosphate-regulated proteins. J. Bac t e r i o l . 147: 675-678. Gray, G.L., R.M. Berka, and M.L. V a s i l . 1982. Phospholipase C regulatory mutation of Pseudomonas aeruginosa that results in constitutive synthesis of several phosphate-repressible proteins. J. Bact e r i o l . 150; 1221-1226. Haas, D., J. Watson, R. Krieg, and T. Leisenger. 1981. Isolation of an Hfr donor of Pseudomonas aeruginosa PAO by insertion of a plasmid RP1 into the trytophan synthase gene. Mol. Gen. Genet. 182: 240-244. Hague, M. and A.D. Russel. 1974. Effects of chelating agents on the s u s c e p t i b i l i t y of some strains of gram-negative bacyeria to some an t i b a c t e r i a l agents. Antimicrob. Agents Chemother. 6: 200-206. Hancock, R.E.W. and V. Braun. 1976. The c o l i c i n I receptor has a role in enterochelin-mediated iron transport. FEBS Letters 65: 208-210. Hancock, R.E.W. and H. Nikaido. 1978. Outer membrane of gram-negative bacteria. XIX. Isolation from Pseudomonas  aeruginosa PA01 and use in reconstitution and d e f i n i t i o n of the permeability barrier. J. Ba c t e r i o l . Hancock, R.E.W., G.M. Decad. and H. Nikaido. 1979. Ide n t i f i c a t i o n of the protein producing transmembrane di f f u s i o n pores in the outer membrane of Pseudomonas  aeruginosa PA01. Biochim. Biophys. Acta 554: 323-331. Hancock, R.E.W., and A.M. Carey. 1979. Outer membrane of Pseudomonas aeruginosa: heat- and 2-mercaptoethanol-modifiable proteins. J. Ba c t e r i o l . 140: 902-910. Hancock, R.E.W. and A.M. Carey. 1980. Protein D1-a glucose-inducible, pore-forming protein from the outer membrane of Pseudomonas aeruginosa. FEMS Microbiol. Lett. 8: 105-109. Hancock, R.E.W., R.T. Irvin, J.W. Costerton, and A,M. Carey. 1981a. Pseudomonas aeruginosa outer membrane: peptidoglycan associated proteins. J. Ba c t e r i o l . 145: 628-631 . 182 Hancock, R.E.W., V.J. Raffle and T.I. Nicas. 1981b. Involvement of the outer membrane in gentamicin and streptomycin uptake and k i l l i n g in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. j_9: 777-785. Hancock, R.E.W., K. Poole, and R. Benz. 1982. Outer membrane protein P of Pseudomonas aeruginosa: formation of small anion-specific channels in l i p i d bilayer membranes. J. Bacter i o l . 150: 730-738. Hancock, R.E.W., L.M. Mutharia, L. Chan, R.P. Darveau, D.P. Speert, and G.B. Pier. 1983a. Pseudomonas aeruginosa isolates from patients with cystic f i b r o s i s : a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide 0 side chains. Infect. Immun. 42: 170-177. Hancock, R.E.W., K. Poole, and R. Benz. 1983b. Modification of the conductance, s e l e c t i v i t y and concentrationrdependent saturation of Pseudomonas aeruginosa protein P channels by chemical acetylation. Biochim. Biophys. Acta 735: 137-144. Hantke, K. and V. Braun. 1975. Membrane-receptor-dependent iron transport in Escherichia c o l i . FEBS Letters 49: 301-305. Hantke, K. 1976. Phage T6-coli c i n K receptor and nucleoside transport in Escherichia c o l i . FEBS Letters 70: 109-112. Harold, F.M. and J.M. Baarda. 1966. Interaction of arsenate with a phosphate transport system in wild type and mutant Streptococcus f a e c a l i s . J . Bac t e r i o l . 91: 2257-2262. Harold, F.M. 1977. Membranes and energy transduction in bacteria, p. 83-141. I_n D.R. Sanadi (ed.), Current topics in bioenergetics, v o l . 6. Academic Press, New York. Hayashi, S.-L., J.P. Koch and E.C.C. Lin. 1964. Active transport of L-alpha-glycerophosphate in Escherichia c o l i . J. B i o l . Chem. 239: 3098-3105. 183 Helfman, D.M., J.R. Feramisco, J.C. Fiddes, G.P. Thomas, and S.H. Hughes. 1983. I d e n t i f i c a t i o n of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression l i b r a r y . Proc. Natl. Acad. S c i . U.S.A. 80; 31-35. Hengge, R. and W. Boos. 1983. Maltose and lactose transport in Escherichia c o l i . Examples of two d i f f e r e n t types of concentrative transport systems. Biochim. Biophys. Acta. 737: 443-478. Henning, U. Schmidmayer and I. Hindennach. 1977. Major proteins of the outer c e l l envelope membrane of Escherichia  c o l i K-12: multiple species of protein I. Mol. Gen. Genet. 154: 293-298. Hobot, J.A., E. Carlemalm, W. V i l l i g e r , and E. Kellenberger. 1984. Periplasmic gel: new concept resulting from the reinvestigation of b a c t e r i a l c e l l envelope ultrastructure by new methods. J. Bacteriol. 160: 143-152. Hofstra, H., and J. Dankert. 1980. Major outer membrane proteins: common antigens in Enterobacteriaceae species. J. Gen. Microbiol. 119: 123-131. Hofstra, H., and J. Dankert. 1981. Porin from the outer membrane of Escherichis c o l i : immunological characterization of native and heat-dissociated forms. J . Gen. Microbiol. 125: 285-292. Hong, J.-S., A,G, Hunt, P.S. Masters, and M.A. Lieberman. 1979. Requirement of acetylphosphate for the binding protein-dependent transport systems in Escherichia c o l i . Proc. Natl. Acad. S c i . U.S.A. 76: 1213-1217. Hoshino, T., and M. Kageyama. 1980. P u r i f i c a t i o n and properties of a binding protein for branched-chain amino acids in Pseudomonas aeruginosa. J. B a c t e r i o l . 141: 1055-1063. Hoshino, T. and K. Nishio. 1982. Isolation and characterization of a Pseudomonas aeruginosa mutant defective in the structural gene for the LIVAT-binding protein. J. B a c t e r i o l . 151:729-736. 184 Hou, C.I., A.F. Gronlund and J.J.R. Campbell. 1966. Influence of phosphate starvation on cultures of Pseudomonas  aeruginosa. J. B a c t e r i o l . 92: 851-855, Ichihara, S. and S. Mizushima. 1978. I d e n t i f i c a t i o n of an outer membrane protein responsible for the binding of the iron-enterochelin complex to Escherichia c o l i c e l l s . J. Biochem. 83: 137-140. Ingram, J.M., K.-J. Cheng, and J.W. Costerton. 1973. Alkaline phosphatase of Pseudomonas aeruginosa: the mechanism of secretion and release of the enzyme from whole c e l l s . Can. J . Microbiol. 19: 1407-1415. Irv i n , R.J., T.J. MacAlister, and J.W. Costerton. 1981. Tris(hydroxymethyl)aminomethane buffer modification of Escherichia c o l i outer membrane permeability. J . Bacteriol. 145: 1397-1403. Kadner, R.J. and G.L. Liggins. 1973. Transport of vitamin B12 in Escherichia c o l i : genetic studies. J. B a c t e r i o l . 115: 514-521 . Koch, A.L. 1971. The adaptive response of Escherichia c o l i to a feast and famine existence. Adv. Microb. Physiol. 6: 147-217. Koplow, J., and H. Goldfine. 1974. Alterations in the outer membrane of the c e l l envelope of heptose-deficient mutants of Escherichia c o l i . J . B a c t e r i o l . 117: 527-543. Romberg, H.L. and J. Smith. 1969. Genetic control of hexose phosphate uptake by Escherichia c o l i . Nature. 224: 1261-1262. Rorteland, J., J. Tommassen, and B. Lugtenberg. 1982. PhoE protein pore of the outer membrane of Escherichia c o l i R 12 i s a p a r t i c u l a r l y e f f i c i e n t channel for organic and inorganic phosphate. Biochim. Biophys. Acta 690: 282-289. Korteland, J., and B. Lugtenberg. 1984. Increased e f f i c i e n c y of the outer membrane PhoE protein pore in Escherichia c o l i K-12 mutants with heptose-deficient lipopolysaccharide. Biochim. Biophys. Acta 774: 119-126. 185 Kropinski, A.M., J. Kuzio, B.L. Angus and R.E.W. Hancock. 1982. Chemical and chromatographic analysis of lipopolysaccharide from an antibiotic-supersensitive mutant of Pseudomobnas aeruginosa. Antimicrobiol. Agents Chemother. 21: 310-319. Kulaev, I.S. 1975. Biochemistry of inorganic polyphosphates. Rev. Physiol. Biochem. and Pharmacol. 73: 136-158. Lacoste, A.-M., A. Cassaigne and E. Neuzil. 1981. Transport of inorganic phosphate in Pseudomonas aeruginosa. Current Microbiol. 6:115-120. Lee, D.R., CA. Schnaitman, and A.P. Pugsley. 1979. Chemical heterogeneity of major outer membrane pore proteins of Escherichia c o l i . J . B a c t e r i o l . 138: 861-870. Lever, J.E. 1972. Quantitative assay of the binding of small molecules to protein: comparison of d i a l y s i s and membrane f i l t e r assays. Anal. Biochem. 5_0: 73-83. Levitz, R., A. Klar, N. Sar and E. Yagil . 1984..A new locus in the phosphate-specific transport (Pst) region of Escherichia c o l i . Mol. Gen. Genet. 197: 98-103. Lin, E.C.C. 1976. Glycerol d i s s i m i l a t i o n and i t s regulation in bacteria. Annu. Rev. Microbiol. 30: 535-578. Luckey, M. and H. Nikaido. 1980a. Specificty of di f f u s i o n channels produced by lambda phage receptor protein of Escherichia c o l i . Proc. Natl. Acad. S c i . U.S.A. 77: 167-171. Luckey, M. and H. Nikaido. 1980b. Diffusion of solutes through channels produced by phage lambda receptor protein of Escherichia c o l i : i n h i b i t i o n by higher oligosaccharides of maltose series. Biochem. Biophys. Res. Commun. 9_3: 166-171 . Luckey, M. and H. Nikaido. 1983. Bacteriophage lambda receptor protein in Escherichia c o l i K-12: lowered a f f i n i t y of some mutant proteins for maltose-binding protein in v i t r o . J. Bact e r i o l . 153: 1056-1059. 186 Luderitz, 0., M.A. Freudenberg, C. Galanos, V. Lehmann, E.T.H. Rietschel and D.H. Shaw. 1982. Lipopolysaccharides of gram-negative bacteria, p. 79-151. I_n S. Razin and S. Rottem (ed.), Current topics in membranes and transport, v o l . 17. Academic Press, New York. Ludtke, D., J. Bernstein, C. Hamilton and A. T o r r i a n i . 1984. Id e n t i f i c a t i o n of the phoM gene product and i t s regulation in Escherichia c o l i K-12. J. B a c t e r i o l . 159: 19-25. Lugtenberg, B., H. Bronstein, N. van Selm and R. Peters. 1977. Peptidoglycan-associated outer membrane proteins in gram-negative bacteria. Biochim. Biophys. Acta. 465: 571-578. Lugtenberg, B., R. van Boxtel, C. Verhoef, and W. van Alphen. 1978. Pore protein e of the outer membrane of Escherichia c o l i K 12. FEBS Lett. 96: 99-105. Lugtenberg, B., and L. van Alphen. 1983. Molecular architecture and functioning of the outer membrane of Escherichia c o l i and other gram-negative bacteria. Biochim. Biophys. Acta 737: 51-115. Lutkenhaus, J.F. 1977. Role of a major outer membrane protein in Escherichia c o l i . J. B a c t e r i o l . 131: 631-637. Machtiger, N.A., and C F . Fox. 1973. Biochemistry of bacterial membranes. Annu. Rev. Biochem. 42: 575-600. Maezawa, S., Y. Hayashi, T. Nakae, J . I s h i i , K. Kameyama, and T. Takagi. 1983. Determination of molecular weight of membrane proteins by the use of low angle laser l i g h t scattering combined with high-performance gel chromatography in the presence of non-ionic surfactant. Biochim. Biophys. Acta 747: 291-297. Makino, K., H. Shinagawa and A. Nakata. 1982. Cloning and characterization of the alkaline phosphatase positive regulator gene (phoB) of Escherichia c o l i . Mol. Gen. Genet. 187: 181-186. 187 Malamy, M., and B. Horecker. 1961. The l o c a l i z a t i o n of alkaline phosphatase in Escherichia c o l i K-12. Biochem. Biophys. Res. Commun. 5: 104-108. Mcintosh, M.A. and C F . Earhart. 1977. Coordinate regulation by iron of the synthesis of phenolate compounds and three outer membrane proteins in Escherichia c o l i . J . Bact e r i o l . 131: 331-339. Meadow, P. 1975. Wall and membrane structure in the genus. Pseodomonas, p. 67-98. I_n P.H. Clarke and M.H. Richmond (eds.), Genetics and biochemistry of Pseudomonas. John Wiley and Sons, Toronto. Medveczky, N. and H. Rosenberg. 1970. The phosphate-binding protein of Escherichia c o l i . Biochim. Biophys. Acta. 211: 158-168. Medveczky, N. and H. Rosenberg. 1971. Phosphate transport in Escherichia c o l i . Biochim. Biophys. Acta. 241: 494-506. Midgley, M. and E.A. Dawes. 1973. The regulation of transport of glucose and methyl-alpha-glucoside in Pseudomonas aeruginosa. Biochem. J. 132: 141-154. Mi t c h e l l , P. 1954. Transport of phosphate across the osmotic barrier of Micrococcus pyogenes: s p e c i f i c i t y and kinetic s . J. Gen. Microbiol. 11: 73-82. Mi t c h e l l , P. 1961. , p. 581-603. In T.W. Goodwin and 0. Lindberg (eds.), B i o l o g i c a l structure and function. Academic Press, New York. Mizuno, T., M.-Y. Chin and M. Inouye. 1983. A comparative study on the genes for three porins of the Escherichia c o l i outer membrane: DNA sequence of the osmoregulated ompC gene. J. B i o l . Chem. 258: 6932-6940. Muhlradt, P.P. and J.R. Golecki. 1975. Assymetric d i s t r i b u t i o n and a r t i f a c t u a l reorientation of lipopolysaccharide in the outer membrane bilayer of Salmonella typhimurium. Eur. J. Biochem. 51: 343-352. 188 Mutharia, L.M., and R.E.W. Hancock. 1983. Surface l o c a l i z a t i o n of Pseudomonas aeruginosa porin protein F by using monoclonal antibodies. Infect. Immun. 42_: 1027-1033. •Mutharia, L.M., and R.E.W. Hancock. 1985. Monoclonal antibody for an outer membrane lipoprotein of the Pseudomonas fluorescens group of the family Pseudomonadaceae. Int. J. Syst. B a c t e r i o l . 3_5: 530-532. Nakae, T. and H. Nikaido. 1975. Outer membrane as a dif f u s i o n barrier in Salmonella typhimurium: penetration of oligo- and polysaccharides'into isolated outer membrane vesicles and c e l l s with degraded peptidoglycan layer. J. B i o l . Chem. 250: 7359-7365. Nakae, T. 1 9 7 6 . I d e n t i f i c a t i o n of the major outer membrane protein of Escherichia c o l i that produces transmembrane channels in reconstituted vesicle membranes. Biochem. Biophys. Res. Commun. 7J_: 8 7 7 - 8 8 4 . Nakae, T. 1979. A porin a c t i v i t y of p u r i f i e d lambda-receptor proteins from Escherichia c o l i in reconstituted membrane ve s i c l e s . Biochem. Biophys. Res. Commun. 88: 774-781. Nakae, T. and J. I s h i i . 1980. Permeability properties of Escherichia c o l i outer membrane containing pore-forming proteins: comparison between lambda receptor.protein and porin for saccharide permeation. J . Ba c t e r i o l . 142: 735-740. Nakamura, K., and S. Mizushima. 1976. Effects of heating in dodecly sulfate on the conformation and electrophoretic mobility of isolated major outer membrane proteins from Escherichia c o l i K-12. J . Biochem. 80: 1411-1422. Neuhaus, J.-M., H. Schindler and J . Rosenbusch. 1983. The periplasmic maltose-binding protein modifies the channel-forming c h a r a c t e r i s t i c s of maltoporin. EMBO J. 2: 1987-1991. Nicas, T.I., and R.E.W. Hancock. 1980. Outer membrane protein H1 of Pseudomonas aeruginosa: involvememnt in adaptive and mutational resistance to ethylenediaminetetraacetate, polymyxin B and gentamicin. J. Bact e r i o l . 143: 872-878. 189 Nicas, T.I., and R.E.W. Hancock. 1983. Pseodomonas  aeruginosa outer membrane permeability: i s o l a t i o n of a porin protein F-deficient mutant. J. Ba c t e r i o l . 153: 281-285. Nikaido, H. 1976. Outer membrane of Salmonella typhimurium. Transmembrane d i f f u s i o n of some hydrophobic substances. Biochim. Biophys. Acta. 433: 118-132. Nikaido, H. 1979. Nonspecific transport through the outer, membrane, p. 361-407. I_n M. Inouye (ed.), Bacterial outer membranes. Wiley-Interscience, New York. Nikaido, H., and M. Vaara. 1985. Molecular basis of bac t e r i a l outer membrane permeability. Microbiol. Rev. 49: 1-32. Norqvist, A., J. Davies, L. Norlander and S. Normark. 1978. The effect of iron starvation on the outer membrane protein composition of Neisseria gonorrheae. FEMS Microbiol. Lett. 4: 71-75. Osborn, M.J. 1963. Studies on the gram-negative c e l l wall. I. Evidence for the role of 2-keto-3-deoxyoctonate in the lipopolysaccharide of Salmonella typhimurium. Proc. Natl. Acad. S c i . U.S.A. 50: 499-507. Overbeeke, N., G. van Scharrenburg, and B. Lugtenberg. 1980. Antigenic relationships between pore proteins of Escherichia c o l i K 12. Eur. J. Biochem. 110: 247-254. Overbeeke, N., and B. Lugtenberg. 1980. Expression of outer membrane protein e of Escherichia c o l i K 12 by phosphate l i m i t a t i o n . FEBS Lett. 112: 229-232. Overbeeke, N., and B. Lugtenberg. 1982. Recognition s i t e for phosphorus-containing compounds and other negatively charged solutes on the PhoE protein pore of the outer membrane of Escherichia c o l i K 12. Eur. J. Biochem. 126: 113-118. Oxender, D.L. 1972. Membrane transport. Annu. Rev. Biochem. 41: 777-814. 190 Oxender, D.L., and S.C. Quay. 1976. Isolation and characterization of membrane binding proteins, p. 183-242. In E.D. Korn (ed.), Methods in membrane biology, v o l . 6. Plenum Press, New York. Parr, T.R., J r . , K. Poole, G.W.K. Crockford and R.E.W. Hancock. 1986. Lipopolysaccharide-free Escherichia c o l i OmpF and Pseudomonas aeruginosa protein P porins are functionally active in l i p i d bilayer membranes. J . Bacteriol., in press. Pollack, L.R. 1973. A sim p l i f i e d method for the quantitative assay of small amounts of protein in b i o l o g i c a l material. Anal. Biochem. 51: 654-655. Pugsley, A.P. and P. Reeves. 1976. Increased production of the outer membrane receptors for c o l i c i n s B, D and M by Escherichia c o l i under iron starvation. Biochem Biophys. Res. Commun. 70: 846-853. Pugsley, A.P., and CA. Schnaitman. 1978. Ide n t i f i c a t i o n of the three genes c o n t r o l l i n g production of new membrane pore proteins in Escherichia c o l i K-12. J. Ba c t e r i o l . 135: 1118-1 129. Rella, M., A. Mercenier, and D. Haas. 1985. Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cl u s t e r . Gene 33: 293-303. Rosenberg, H., Medveczky, N. and J.M. LaNauze. 1969. Phosphate transport in Ba c i l l u s cereus. Biochim. Biophys. Acta. 193: 159-167. Rosenberg, H., R.G. Gerdes and K. Chegwidden. 1977. Two systems for the uptake of phosphate in Escherichia c o l i . J. Bac t e r i o l . 131: 505-511. Rosenberg, H., R.G. Gerdes and F.M. Harold. 1979. Energy coupling to the transport of inorganic phosphate in Escherichia c o l i K12. Biochem. J. 178: 133-137. 191 Rosenbusch, J.P. 1974. Characterization of the major envelope protein from Escherichia c o l i . Regular arrangement on the peptidoglycan and unusual dodecylsulfate binding. J. B i o l . Chem. 249: 8019-8029. Ruitenberg, E.J., P.A. Steerenberg, B.J.M. Brosi and J . Buys. 1974. Serogiagnosis of T r i c h i n e l l a s p i r a l i i nfection in pigs by enzyme linked immunoadsorbent assays. B u l l . W.H.O. 51: 108-109. Russel, R.R.B. 1976. Two-dimensional SDS-polyacrylamide gel electrophoresis of heat-modifiable outer membrane proteins. Can. J . Microbiol. 22: 83-91. Sato, M., B.J. Staskawicz, N.J. Panopoulos, S. Peters, and M. Honma. 1981. A host-dependent hybrid plasmid suitable as a suicide c a r r i e r for transposable elements. Plasmid 6: 325-331 . Schacterle, G.R., and L.R. Pollack. 1973. A si m p l i f i e d method for the quantitative assay of small amounts of protein in b i o l o g i c a l material. Anal. Biochem. 5J_: 654-655. Schindler, H., and J.P. Rosenbusch. 1978. Matrix protein from Escherichia c o l i outer membrane forms voltage-gated channels in l i p i d b i l a y e r s . Proc. Natl. Acad. S c i . U.S.A. 75: 3751-3755. Schleifer, K.H., and 0. Handler. 1972. Peptidoglycan types of b a c t e r i a l c e l l walls and their taxanomic implications. B a c t e r i o l . Rev. 36: 407-477. Schleytr, U.B. 1978. Regular arrays of macromolecules on bacterial c e l l walls: structure, chemistry, assembly, and function. Intern. Rev. of Cytology 5_3: 1-64. Schweizer, H., T. Grussenmeyer and W. Boos. 1982. Mapping of two ugp genes coding for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia c o l i . J. B a c t e r i o l . 150: 1164-1171. Sciortino, C.V. and R.A. Fin k e l s t e i n . 1983. Vibrio cholorae expresses iron-regulated outer membrane proteins in vivo. Infect. Immun. 42: 990-996. 192 Siegel, L.S. and P.V. Phibbs, J r . 1979. Glycerol and L-alpha-glycerol-3-phosphate uptake by Pseudomonas aeruginosa. Current Microbiol. 2: 251-256. Sprague, G.F., J r . , R.M. B e l l and J.E. Cronan, J r . 1975. A mutant of Escherichia c o l i auxotrophic for organic phosphates: evidence for two deffects in inorganic phosphate transport. Mol. Gen. Genet. 143: 71-77. Sterkenburg, A., E. Vlegels, and J.T.M. Wouters. 1984. Influence of nutrient l i m i t a t i o n and growth rate on the outer membrane proteins of K l e b s i e l l a aerogenes NCTC 418. J. Gen. Microbiol. 130: 2347-2355. Stinson, M.W., M.A. Cohen and J.M. Merrick. 1977. P u r i f i c a t i o n and properties of the periplasmic glucose-binding protein of Pseudomonas aeruginosa. J. b a c t e r i o l . 131 : 672-681 . Stinson, M.W. and C. Hayden. 1979. Secretion of phospholipase C by Pseudomonas aeruginosa. Infect. Immun. 25: 558-564. Stock, J.B., B. Rauch and S. Roseman. 1977. Periplasmic space in Salmonella typhimurium and Escherichia c o l i . J. B i o l . Chem. 252: 7850-7861. Stocker, B.A.D., M. Nurminen, and P.H. Makela. 1979. Mutants defective in the 33K outer membrane protein of Salmonella  typhimurium. J. B a c t e r i o l . 139: 376-383. Szmelcman, S. and M. Hofnung. 1975. Maltose transport in Escherichia c o l i K12: involvement of the bacteriophage lambda receptor. J. B a c t e r i o l . 124: 112-118. Szmelcman, S., M. Schwartz, T.J. Silhavy and W. Boos. 1976. Maltose transport in Escherichia c o l i . Eur. J. Biochem. 65: 13-19. Teuber, M. and I.R. M i l l e r . 1977. Selective binding of polymyxin B to negatively charged l i p i d monolayers. Biochim. Biophys. Acta. 467: 280-289. 193 Tokunaga, M., H. Tokunaga, Y. Okajima, and T. Nakae. 1979. Characterization of porins from the outer membrane of Salmonella typhimurium. 2. physical properties of the functional oligomeric aggregates. ' Eur. J. Biochem. 95: 441-448. Tokunaga, H., M. Tokunaga and T. Nakae. 1981. Permeability properties of chemically modified porin trimers from Escherichia c o l i B. J. B i o l . Chem. 256: 8024-8029. Tommassen, J., P. van der Ley, A. van der Ende, H. Bergmans and B. Lugtenberg. 1982. Cloning of ompF, the structural gene for an outer membrane por protein of Escherichia c o l i K12: physiological l o c a l i z a t i o n and homology with the phoE gene. Mol. Gen. Genet. 185: 105-110. Tommassen, J. and B. Lugtenberg. 1982. Pho-regulon of Escherichia c o l i K12: a minireview. Ann. Microbiol. (Inst. Pasteur). 133A: 243-249. Tommassen, J., P. DeGeus and B. Lugtenberg. 1982. Regulation of the pho regulon of Escherichia c o l i K-12. Cloning of the regulatory genes phoB and phoR and i d e n t i f i c a t i o n of their gene products. J. Mol. B i o l . 157: 265-274. T o r r i a n i , A. 1960. Influence of inorganic phosphate on the formation of phosphatases by Escherichia c o l i . Biochim. Biophys. Acta (Amst.) 3_8: 460-470. Towbin, H., T. Staehlin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to n i t r o c e l l u l o s e sheets: procedure and some applications. Proc. Natl. Acad. S c i . U.S.A. 76: 4350-4354. Troy, F.A. 1979. The chemistry and biosynthesis of selected b a c t e r i a l capsular polymers. Annu. Rev. Microbiol. 33: 519-560. ' Tsai, CM. and C E . Frasch. 1982. A sensitive s i l v e r stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119: 115-119. 1 94 Tsuda, M., S. Harayama, and T. l i n o . 1984. Tn501 insertion mutagenesis in Pseudomonas aeruginosa PAO. Mol. Gen. Genet. 196: 494-500. Valette, J.P., A.M. Lacoste, S. Labeyrie, and E. Neuzil. 1966. C. R. Seances Soc. B i o l . F i i . 160: 1562-1567. Van Alphen, W., N. van Selm, and B. Lugtenberg. 1978. Pores in the outer membrane of Escherichis c o l i K-12. Involvement of proteins b and e in the functioning of pores for nucleotides. Mol. Gen. Genet. 159: 75-83. Van Golde, L.M.G., H. Schulman and E.P. Kennedy. 1973. Metabolism of membrane phospholipids and i t s relation to a novel class of oligosaccharides in Escherichia c o l i . Proc. Natl. Acad. S c i . U.S.A. 70: 1368-1372. Verhoef, C , C. van Koppen, P. Overduin, B. Lugtenberg, J. Korteland, and J. Tommassen. 1984. Cloning and expression in Escherichia c o l i K-12 of the structural gene for outer membrane PhoE protein from Enterobacter clocae.. Gene 32: 107-1 15. Von Meyenburg, K. 1971. Transport-limited growth rates in a mutant of Escherichia c o l i . J . B a c t e r i o l . 107: 878-888. Von Meyenburg, K. and H. Nikaido. 1977. Outer membranes of gram-negative bacteria. XVII. S p e c i f i c i t y of transport process catalyzed by the lambda-receptor protein in Escherichia c o l i . Biochem. Biophys. Res. Commun. 78: 1100-1 107. Wandersmann, C. M. Schwartz and T. Ferenci. 1979. Escherichia c o l i mutants impaired in maltodextrin transport. J. B a c t e r i o l . 140: 1-13. Wanner, B.L. and P. L a t t e r e l l . 1980. Mutants affected in alkaline phosphatase expression: evidence for multiple regulators of the phosphate regulon in Escherichia c o l i . Genetics 96: 353-366. 195 Wanner, B.L., S. Weider and R. McSharry. 1981. Use of the bacteriophage Mud1 to determine the orientation for three proC-linked phosphate-starvation-inducible (psi) genes in Escherichia c o l i K12. J . Bac t e r i o l . 146:93-101. Warburg, 0., and W. Chri s t i a n . 1941. Biochem. Z. 310: 384-421 . Warren, R.A.J. 1979. Amber suppressor mutations in Pseudomonas acidovorans. J. Bac t e r i o l . 137: 1 053-1055. Wayne, R. and J.B. Neilands. 1975. Evidence for common binding s i t e s for ferrichrome compounds and bacteriophage 080 in the c e l l envelope of Escherichia c o l i . J. Ba c t e r i o l . 121: 497-503. Weiss, A.A., and S. Falkow. 1983. Transposon insertion and subsequent donor formation promoted by Tn501 in Bordetella  pertussis. J . Ba c t e r i o l . 153: 304-309. Weiss, A.A., E.L. Hewlett,. G.A. Meyers, and S. Falkow. 1983. Tn5_-induced mutations a f f e c t i n g virulence factors of Bordetella pertussis. Infect. Immun. 42: 33-41. - White, J . C , P.M. DiGirolamo, M.L. Fu, Y.A. Preston and C. .Bradbeer. 1973. Transport of vitamin B12 in Escherichia  c o l i . Location and properties of the i n i t i a l Bl2-binding s i t e . J. B i o l . Chem. 248: 3978-3986. Williams, P., M.R.W. Brown and P. Lambert. 1984. Effect of iron deprivation on the production of siderophores and outer membrane proteins in K l e b s i e l l a aerogenes. J. Gen. Microbiol. 130: 2357-2365. Willsky, G.R., R.L. Bennet and M.H. Malamy. 1973. Inorganic phosphate transport in Escherichia c o l i : involvement of two genes which play a role in alkaline phosphatase regulation. J. B a c t e r i o l . 113: 529-539. Willsky, G.R. and M.H. Malamy. 1974. The loss of the phoS periplasmic protein leads to a change in the s p e c i f i c i t y of a constitutive inorganic phosphate transport system in Escherichia c o l i . Biochem. Biophys. Res. Commun. 60: 226-233. 196 Willsky, G.R. and M.H. Malamy. 1980. Effect of arsenate on inorganic phosphate transport in Escherichia c o l i . J. Ba c t e r i o l . 144: 366-374. Willsky, G.R. and M.H. Malamy. 1980. Characterization of two genetically separable inorganic phosphate transport systems in Escherichia c o l i . J. Bac t e r i o l . 144: 356-365. Wilson, D.B. 1978. Ce l l u l a r transport mechanisms. Annu.. Rev. Biochem. 47: 933-965. Wilson, D.B. and J.B. Smith. 1978. Bacterial transport proteins, p. 495-557. I_n B.P. Rosen (ed.), Bacterial transport. Marcel Dekker, New York. Wookey, P. and H. Rosenberg. 1978. Involvement of inner and outer membrane components in the transport of iron and in c o l i c i n B action in Escherichia c o l i . J. B a c t e r i o l . 133: 661-666. Wray, W., T. Boulikas, V.P. Wray and R. Hancock. 1981. Sil v e r staining of proteins in polyacrylamide gels. Anal. Biochem. 118: 197-203. Yagil, E. 1975. Derepression of polyphosphatase in Escherichia c o l i by starvation for inorganic phosphate. FEBS Lett. 55: 124-127. Yag i l , E., N. S i l b e r s t e i n and R.G. Gerdes. 1976. Co-regulation of the phosphate-binding protein and alkaline phosphatase synthesis in Escherichia c o l i . J. B a c t e r i o l . 127: 656-659. Yoshimura, F. and H. Nikaido. 1982. Permeability of Pseudomonas aeruginosa to hydrophilic solutes. J . Bac t e r i o l . 152: 636-642. Yoshimura, F., S.L. Zalman, and H. Nikaido. 1983. Pu r i f i c a t i o n and properties of Pseudomonas aeruginosa porin. J. B i o l . Chem. 258: 2308-2314. 1 97 Zimmermann, W., and A. Rosselet. 1977. The function of the outer membrane of Escherichia c o l i as a permeability barrier to beta-lactam a n t i b i o t i c s . Antimicrob. Agents Chemother. 12: 368-372. 198 P u b l i c a t i o n s 1. H a n c o c k , R . E . W . , K . P o o l e and R. B e n z . 1982 . O u t e r membrane p r o t e i n P of Pseudomonas a e r u g i n o s a : r e g u l a t i o n by p h o s p h a t e d e f i c i e n c y and f o r m a t i o n o f s m a l l a n i o n - s p e c i f i c c h a n n e l s i n l i p i d b i l a y e r m e m b r a n e s . J . B a c t e r i o l . 150 : 7 3 0 - 7 3 8 . 2 . H a n c o c k , R . E . W . , A . A . W i e c z o r e k , L . M . M u t h a r i a and K . P o o l e . 1982 . M o n o c l o n a l a n t i b o d i e s a g a i n s t Pseudomonas a e r u g i n o s a o u t e r membrane a n t i g e n s : i s o l a t i o n and c h a r a c t e r i z a t i o n . I n f e c t . Immun. 3_7: 1 6 6 - 1 7 1 . 3 . P o o l e , K . and R . E . W . H a n c o c k . 1983 . S e c r e t i o n o f a l k a l i n e p h o s p h a t a s e and p h o s p h a o l i p a s e C i n Pseudomonas a e r u g i n o s a i s s p e c i f i c and does n o t i n v o l v e an i n c r e a s e i n o u t e r membrane p e r m e a b i l i t y . FEMS M i c r o b i o l . L e t t . 1_6: 25-29 . 4 . B e n z , R . , K . P o o l e and R . E . W . H a n c o c k . 1983 . An a n i o n - s e l e c t i v e c h a n n e l f r o m the Pseudomonas a e r u g i n o s a o u t e r membrane . B i o c h i m . B i o p h y s . A c t a 7 3 0 : 3 8 7 - 3 9 0 . 5. B e n z , R . , M . G i m p l e , K . P o o l e and R . E . W . H a n c o c k . 1983 . C h a r a c t e r i z a t i o n ' and c h e m i c a l m o d i f i c a t i o n o f s m a l l a n i o n s p e c i f i c c h a n n e l s f o r m e d i n l i p i d b i l a y e r membranes by o u t e r membrane p r o t e i n P o f Pseudomonas a e r u g i n o s a . B i o p h y s . J . 4_5: 8 1 - 8 2 . 6 . H a n c o c k , R . E . W . , K . P o o l e , M . G i m p l e and R. B e n z . 1983 . M o d i f i c a t i o n o f the c o n d u c t a n c e , s e l e c t i v i t y and c o n c e n t r a t i o n -d e p e n d e n t s a t u r a t i o n o f Pseudomonas a e r u g i n o s a p r o t e i n P c h a n n e l s by c h e m i c a l a c e t y l a t i o n . B i o c h i m . B i o p h y s . A c t a 7 35 : 1 3 7 - 1 4 4 . 7 . B e n z , R . , K . P o o l e and R . E . W . H a n c o c k . 1984. C h a r a c t e r i z a t i o n and c h e m i c a l m o d i f i c a t i o n od s m a l l a n i o n s p e c i f i c c h a n n e l s f o r m e d i n l i p i d b i l a y e r membranes by o u t e r membrane p r o t e i n P o f Pseudomonas  a e r u g i n o s a . I n I o n i c c h a n n e l s i n membranes ( P a r s e g i a n , V . A . , e d . ) , p p . 8 1 - 8 2 . R o c k e f e l l e r U n i v e r s i t y P r e s s . 8 . P o o l e , K . and R . E . W . H a n c o c k . 1984 . P h o s p h a t e t r a n s p o r t i n Pseudomonas a e r u g i n o s a : i n v o l v e m e n t o f a p e r i p l a s m i c p h o s p h a t e -b i n d i n g p r o t e i n . E u r . J . B i o c h e m . 144: 6 0 7 - 6 1 2 . P u b l i c a t i o n s c o n t ' d 9 . P a r r . T . R . , J r . , K. P o o l e , G . W . K . C r o c k f o r d , and R . E . W . H a n c o c k . 1986 . L i p o p o l y s a c c h a r i d e - f r e e E s c h e r i c h i a c o l l OmpF and Pseudomonas a e r u g i n o s a p r o t e i n P p o r i n s a r e f u n c t i o n a l l y a c t i v e i n l i p i d b i l a y e r m e m b r a n e s . J . B a c t e r i o l . 165 : 5 2 3 - 5 2 6 . 10. P o o l e , K . , and R . E . W . H a n c o c k . 1986 . . I s o l a t i o n o f a Tn501 i n s e r t i o n m u t a n t l a c k i n g p o r i n p r o t e i n P o f P seudomonas  a e r u g i n o s a . M o l . G e n . G e n e t . , i n p r e s s . 11 . P o o l e , K . , and R . E . W . H a n c o c k . 1986 . P h o s p h a t e - s t a r v a t i o n -i n d u c e d o u t e r membrane p r o t e i n s o f members o f t h e f a m i l i e s E n t e r o b a c t e r i a c e a e and P s e u d o m o n a d a c e a e : d e m o n s t r a t i o n o f i m m u n o l o g i c a l c r o s s - r e a c t i v i t y w i t h an a n t i s e r u m s p e c i f i c f o r p o r i n p r o t e i n P o f P seudomonas a e r u g i n o s a . J . B a c t e r i o l . 1 6 5 : 9 8 7 - 9 9 3 . * 

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