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Characterization of the Pseudomonas aeruginosa penicillin-binding proteins 3 and 3x : gene cloning, expression… Liao, Xiaowen 1996

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Characterization of the Pseudomonas aeruginosa penicillin-binding proteins 3 and3x: gene cloning, expression and role in susceptibility to pMactam antibiotics by  XIAOWEN LIAO B.Sc. (Microbiology), Xiamen University, 1984 M.Sc. (Biology), Dalhousie University, 1991  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES DEPARTMENT OF MICROBIOLOGY  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH COLUMBIA October, 1996 © Xiaowen Liao, 1996  In  presenting this  degree at the  thesis  in  University  of  partial  fulfilment  British Columbia,  of  the  requirements  of  department  by  his  or  her  representatives.  It  is  by the  understood  that  publication of this thesis for financial gain shall not be allowed without permission.  Department of The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  advanced  permission for extensive  this thesis for scholarly purposes may be granted or  an  I agree that the Library shall make it  freely available for reference and study. I further agree that copying  for  head of my copying  or  my written  ABSTRACT Two degenerate oligonucleotides primers were synthesized based on the amino acid sequences found in the conserved S X X K and K T G motifs of Escherichia coli high-molecular-weight PBPs and Nesseria gonorrhoeae PBP2. The primers were subsequently used in a PCR amplification experiment using Pseudomonas aeruginosa P A O l chromosomal DNA as the template. Five of the resulting PCR products were cloned and sequenced: two products that translated to sequences with strong homology to E. coli PBP3 and N. gonorrhoeae PBP2 were subsequently used as probes to clone the completepbpB and pbpC genes; the other three PCR products were identified as the homologues of the E. coli sucClsucD, yhhF and cypH gene products. The derived amino acid sequence of pbpB gene had 45.1% identity to that of E. coli PBP3. The downstream sequence of pbpB encoded an amino acid sequence homologous to the E. coli murE gene product. These two genes mapped to the same region of the chromosome as did other cell division genes including ftsA,ftsZ and env A. Analyses of the translated sequence of the pbpC gene revealed that it had 40.7% identity to that of E. coli PBP3. The downstream sequence of pbpC encoded convergently transcribed homologues of the E. coli soxR and Mycobacterium bovis adh gene products. Its upstream sequence, about 370 bp in length, did not resemble any sequences in the GenBank database. The pbpC gene mapped 2 megabase pairs from the pbpB gene on the P. aeruginosa chromosome and apparently was not associated with genes involved in cell division. The upstream sequence of pbpC contained a potential a recognition site, suggesting that the expression of this gene s  may be growth or stress regulated. The pbpB and pbpC genes were expressed in E. coli by the T7 RNA polymerase and promoter system. The produced proteins were exported to the cytoplasmic membrane of E. coli cells and bound H-penicillin. They had an apparent 3  Ill  molecular mass of 60 and 58 kDa respectively, whereas the calculated molecular mass were 63.69 and 61.128 kDa. The N-terminal amino acid sequences of the proteins produced in E. coli were identical to those deduced from the nucleotide sequences of the pbpB and pbpC genes, suggesting that there was no N-terminal processing. The pbpB and pbpC genes were expressed in P. aeruginosa PAO4089 using a broad-host-range vector pUCP27. Results from the minimal inhibitory concentration testing and H-penicillin binding competition assays indicated that overproduction of 3  pbpB gene product led to increased resistance to the PBP3-targeted antibiotics aztreonam, cefepime, cefsulodin and ceftazidime whereas the presence of the pbpC gene product in PAO4089 did not have any effect on susceptibility to the tested PBP3targeted antibiotics. E. colipbpB gene was expressed in PAO4089 using pUCP27. Overproduction of the E. colipbpB product in PAO4089 resulted in increased resistance to aztreonam, cefepime and ceftazidime. The attempt to construct a PBP3-defective mutant using a gene replacement technique was not sucessful. This result could be due to the location of the pbpB gene at the proximal end of an operon containing a cluster of cell division genes, where the placement of a polar mutation would be lethal to the cells. Using the same approach, a PBP3x-defective mutant (strain HCI32) was obtained and confirmed by Southern blot analysis. The PBP profiles of wild type strain HI03 and mutant strain HCI32 were similar, suggesting that the pbpC gene was not visibly expressed under the physiological conditions tested. Furthermore, inactivation of PBP3x did not cause any changes in cell morphology or growth rate, suggesting that pbpC was not required for cell viability under normal laboratory growth conditions.  t  iv  TABLE OF CONTENTS ABSTRACT  ...ii  T A B L E OF CONTENTS  iv  LIST O F T A B L E S  Vm  LIST O F FIGURES  ix  ABBREVIATIONS  xii  ACKNOWLEDGMENTS INTRODUCTION  xi£ 1  1. Pseudomonas aeruginosa  1  2. Penicillin-binding proteins (PBPs)  2  2.1 General properties of PBPs  2  2.2 PBPs: enzymes involved in the final steps of peptidoglycan biosynthesis 2.3 Structures of PBPs  6 9  2.3.1 Conserved motifs  9  2.3.2 Domain structures  11  2.4 Role of PBPs in E. coli growth and cell morphology  12  2.5 PBPs in P. aeruginosa  14  3. (3-lactam susceptibility and resistance in Gram-negative bacteria  15  3.1 Antimicrobial activities of pMactam antibiotics  15  3.2 pMactam resistance in Gram-negative bacteria  19  4. Aims of this study  21  MATERIALS AND METHODS  23  I. Bacterial strains, plasmids and growth conditions  23  II. D N A manipulation  23  V  III. D N A sequencing  29  IV. D N A and amino acid sequence analyses  30  V . Transformation  30  VI. Construction of a PBP3 or PBP3x-defective mutant  30  6.1 Construction of plasmids pBPB::Km and pBPC::Km  31  6.2 Conjugation  32  VII. Production of the pbpB and pbpC DNA probes by PCR  32  VIII. Protein expression using the T7 RNA polymerase/promoter system  34  8.1 Construction of the recombinant clones  34  8.2 Analyses of protein expression  35  IX. SDS-PAGE  36  X. Whole cell lysates  36  XI. Determination of N-terminal amino acid sequences  36  XII. Membrane protein preparation  37  XIII. Protein assay  38  IVX. Antibiotic susceptibility testing  38  X V . Penicillin-binding protein assays  39  15.1 Direct assay  39  15.2 Competition assay  39  XVI. Growth experiment  40  XVII. Cell shape examination  40  RESULTS  41  C H A P T E R O N E Gene cloning and sequence analyses of the P. aeruginosa pbpB and pbpC genes  41  1. Introduction  41  2. Generation of D N A probes for cloning the pbpB and pbpC genes by degenerate primer PCR  41  vi  3. Cloning of the pbpB and pbpC genes  45  4. D N A sequence analyses of the pbpB and pbpC loci  48  4.1 pbpB  48  4.2 pbpC  49  5. Features of the pbpB and pbpC gene products and comparison with other PBPs  58  6. Chromosomal locations of the genes cloned in this study  60  7. Summary  62  CHAPTER TWO Expression of the P. aeruginosa pbpB gene product and mutational analysis of the pbpB gene  64  1. Introduction  64  2. Expression of the pbpB gene product in E. coli  64  2.1 Preliminary work  64  2.2 pT7-7 as the expression vector  65  2.3 pBBRlMCS as the expression vector  70  2.3 Processing of the pbpB gene product in E. coli  75  3. Overproduction of the P. aeruginosa pbpB and E. colipbpB gene products in P. aeruginosa  75  4. Effect of overproduction of th& pbpB gene products on the susceptibility of P. aeruginosa to pMactam antibiotics 5. Mutational analysis of the pbpB gene  78 80  5.1 Rationale for carrying out the mutational analysis  80  5.2 Mutagenesis of the pbpB gene  83  6. Summary  86  CHAPTER T H R E E Expression of the P. aeruginosa pbpC gene product and mutational analysis of the pbpC gene  88  vii  1. Introduction  88  2. Expression of the pbpC gene product in E. coli  89  2.1 Preliminary work  89  2.2 pT7-7 as the expression vector  89  2.3 pBBRlMCS as the expression vector  91  2.3 Processing of the pbpC gene product in E. coli.  95  3. Overproduction of the pbpC gene product in P. aeruginosa  99  4. Effect of overproduction of the pbpC gene product on the susceptibility of P. aeruginosa to (3-lactamantibiotics 5. Mutational analysis of the pbpC gene  99 101  5.1 Construction of a PBP3x-defective mutant  101  5.2 PBP profile of the PBP3x-defective mutant  106  5.3. Cell morphology and growth of the PBP3x-defective mutant  106  6. Summary  DISCUSSION  109  113  1. General  113  2. Cloning of the pbpB and pbpC genes  114  3. Structures of PBP3 and PBP3x  115  4. PBP3 maps to a cluster of conserved and essential cell division genes  118  5. PBP3x: a second E. coli PBP3-like gene product 6. Production of recombinant PBP3 and PBP3x  120 124  7. Correlation of (3-lactam antibiotic susceptibility and the overproduction of PBP3 and PBP3x  REFERENCES  126  134  LIST OF TABLES Table 1. Properties of the penicillin-binding proteins of Escherichia coli  5  Table 2. Bacterial strains  24  Table 3. Plasmids  25  Table 4. PCR products with significant homology to GenBank sequences  43  Table 5. Percent identities and total conservation of the amino acid sequences of PBP3 and PBP3x to those of other PBPs  53  Table 6. MICs of (3-lactam antibiotics against P. aeruginosa PAO4089 expressing the P. aeruginosa pbpB or E. coli pbpB gene product  79  Table 7. I of P-lactam antibiotics for PBPs from P. aeruginosa PAO4089 expressing the P. aeruginosa pbpB or E. colipbpB gene product  82  50  Table 8. MICs of P-lactam antibiotics against P. aeruginosa PAO4089 expressing the pbpC gene product  103  Table 9. I of P-lactam antibiotics for PBPs from P. aeruginosa PAO4089 expressing the pbpC gene product  105  50  ix  LIST OF FIGURES Figure 1. Structure of the D-alanyl-D-alanine and biochemical reaction of (3-lactam and the penicillin-interactive, active-site serine proteins Figure 2. Schematic representation of E. coli peptidoglycan structure  3 8  Figure 3. Schematic representation of a cross section of the cell envelope of Gram-negative bacterium  18  Figure 4. Restriction map of pbpB locus  46  Figure 5. Restriction map of pbpC locus  47  Figure 6. Nucleotide sequence of the pbpB region and the deduced amino acid sequence of two ORFs Figure 7. Nucleotide sequence of the pbpC region and the deduced amino acid sequence of three ORFs  52 57  Figure 8. Amino acid sequence alignments of P. aeruginosa PBP3, PBP3x and E. coli PBP3  59  Figure 9. Physical map of the 5.94 mb genome of P A O l  61  Figure 10. Diagram of pXL706  66  Figure 11. SDS-10% PAGE of whole cell lysates and SDS-8.5% P A G E of cell membrane proteins of K38/pGPl-2(pXL706)  68  Figure 12. Autoradiogram of cell membrane proteins of K38/pGPl-2(pXL706) after incubation with H-penicillin and separation by SDS-8.5% PAGE  69  Figure 13. Diagram of pXL608  71  Figure 14. SDS-8.5% PAGE of whole cell lysates of BL21(DE3)/pXL608  72  3  Figure 15. SDS-8.5% PAGE of whole cell lysates and cell membrane proteins of BL21(DE3)/pXL608 Figure 16. Autoradiogram of cell membrane proteins of BL21(DE3)/pXL608  73  X  after incubation with H-penicillin and separation by SDS-8.5% PAGE 3  Figure 17. Autoradiogram of cell membrane proteins of PAO4089(pXL506) and PAO4089(pXLK20) after incubation with H-penicillin and separation by SDS-8.5% PAGE  74  3  77  Figure 18. Autoradiogram of cell membrane proteins illustrating competition of ceftazidime with H-penicillin for the PBPs of PAO4089(pXL506)....81 3  Figure 19. Schematic summary of gene replacement procedure for constructing a PBP3-defective or PBP3x-defective mutant  84  Figure 20. Diagram of pBPB::Km  85  Figure 21. Diagram of pXL732  90  Figure 22. SDS-10% P A G E of whole cell lysates and SDS-8.5% P A G E of cell membrane proteins of K38/pGPl-2(pXL732)  92  Figure 23. Autoradiogram of cell membrane proteins of K38/pGPl-2(pXL732) after incubation with 3H-penicillin and separation by SDS-8.5% PAGE  93  Figure 24. Diagram of pXL629  94  Figure 25. SDS-8.5% P A G E of whole cell lysates of BL21(DE3)/pXL608 and BL21(DE3)/pXL629 after induction with 0.5 mM IPTG for 1, 2, 3 and 4 hrs  96  Figure 26. SDS-8.5% PAGE of whole cell lysates and cell membrane proteins of BL21(DE3)/pXL629  97  Figure 27. Autoradiogram of cell membrane proteins of BL21(DE3)/pXL629 after incubation with H-penicillin and separation by SDS-8.5% PAGE  98  3  Figure 28. Autoradiogram of cell membrane proteins of PAO4089(pXL506) and PAO4089(pXL519) after incubation with H-penicillin and separation by SDS-8.5% PAGE 3  100  xi  Figure 29. Autoradiogram of cell membrane proteins illustrating competition of ceftazidime with H-penicillin for the PBPs of PAO4089(pXL519) 3  103  Figure 30. Diagram of pBPC::Km  105  Figure 31. Southern hybridization demonstrating the interruption of the pbpC gene with a kanamycin resistance cartridge  107  Figure 32. Autoradiogram of cell membrane proteins of HC132, H103 and HC131 after incubation with H-penicillin and separation by SDS-7.5% PAGE 108 3  Figure 33. Cell morphology of H103 and PBP3x-defective mutant HC132  110  Figure 34. Growth of H103 and PBP3x-defective mutant HCI32  Ill  ABBREVIATIONS Ap  ampicillin  AZT  aztreonam  bp  base pair  CEPH  cephaloridine  CFPM  cefepime  CFS  cefsulodin  CFU  colony forming unit  Cm  chloramphenicol  CTZ  ceftazidime  dH 0  distilled water  DIG  digoxigenin  hr  hour  HPLC  high performance liquid chromatography  I50  concentration inhibiting H-penicillin binding by 50%  IMIP  imipenem  IPTG  isopropyl-(3-D-thiogalactoside  kb  kilobase  Km  kanamycin  kDa  kilodaltons  LB  Luria-Bertani  2  3  mb  megabase  MH  Mueller-Hinton  MIC  minimal inhibitory concentration  min  minute  OD oo  optical density at 600 nm  ORF  open reading frame  PAGE  polyacrylamide gel electrophoresis  PBP  penicillin binding protein  PCR  polymerase chain reaction  PVDF  polyvinylidene difluoride  RBS  ribosome binding site  SDS  sodium dodecyl sulfate  sec  second  Tc  tetracycline  6  xiv  ACKNOWLEDGMENTS  I would like to thank Dr. Bob Hancock for his guidance throughout this study. I would also like to thank my supervisory committee members Drs. Tom Beatty, Tony Chow and Julian Davies for their advice at various times during the course of this work. Thanks also to the Canadian Cystic Fibrosis Foundation for financial support. Lastly, my thanks go to members in Dr. Bob Hancock's laboratory, past and present, for their help and friendship.  1  INTRODUCTION  1. Pseudomonas aeruginosa Pseudomonas aeruginosa is a motile, rod-shaped, Gram negative bacterium. It is commonly found in the environment, especially in soil, water and sediments. The broad environmental distribution of this organism is afforded by its minimal nutritional requirements (Palleroni, 1981; Durack, 1989). P. aeruginosa has been recognized as an opportunistic pathogen that causes a variety of infections, usually in immunocompromised hosts such as severe burn patients, children with cystic fibrosis and patients treated with immunosuppresive drugs. Consequently, P. aeruginosa is almost exclusively a nosocomial pathogen and was found in nosocomial lung infections to cause the highest mortality rate of any bacterium (Bodey et al, 1983, Cross et al, 1983). It is intrinsically resistant to many commonly used antibiotics. Among the few effective agents are some newer p 4  lactams, aminoglycosides and quinolones (Durack, 1989; Korvick & Yu, 1991). Combination therapy with two or three antibiotics is frequently chosen for serious infections. Although new and supposedly more effective anti-pseudomonal antibiotics have been developed, P. aeruginosa quickly acquired resistance to these agents. Production of antibiotic-inactivating enzymes such as (3-lactamase, changes in the permeability of the cell envelope and reduction in the affinity of target proteins  2  such as penicillin-binding proteins (PBPs) are believed to be the major mechanisms responsible for the high antibiotic resistance among most P. aeruginosa isolates (see section 3).  2 . Penicillin-binding proteins (PBPs) 2.1 General properties of PBPs Early studies on the mechanism of action of P-lactam antibiotics revealed that this group of compounds exerted their functions by acting as analogues of the acyl-Dalanyl-D-alanine (Fig. 1) moiety of the lipid-linked disaccharide-pentapeptide, the substrate of the enzymes catalyzing the crosslinking of the peptide side chains of nascent peptidoglycan (Tipper & Strominger, 1965). Later Blumberg & Strominger (1974) demonstrated that penicillin bound covalently to the enzymes that it inhibited. . This discovery led to the development of a convenient autoradiographic method for the detection of penicillin-sensitive enzymes as penicillin-binding proteins (PBPs) (Spratt, 1977a). With this method, PBPs are readily detected and their relative amounts quantitated by incubation of bacterial membranes with radio-labeled penicillin G, followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography. The PBP assay proved to be very useful as it allowed the study of all of the penicillin-sensitive enzymes of bacterial cells and is still widely used by many researchers for PBP studies. It can also be used to assess the relative affinity of a PBP for a nonradiolabeled (3-lactam antibiotic by determining the concentration of  3  CH3  Rl-NH-  H  A  CH3  N  COOH  RL.  E-Ser-OH R2  1 RL  E-Ser-O-i O  N R2  ACYL PROTEIN' rapid breakdown  beta-lactama ses  1  very slow breakdown or complete inertness  penicillin-bindincf-proteins  Figure 1. (A) Structure of acyl-D-alanyl-D-alanine illustrating its similarity to (3lactam molecule as shown in (B). (B) Biochemical reaction of P-lactam and the penicillin-interactive, active-site serine proteins (E-ser-OH) (Ghuysen, 1991).  4  the (3-lactam antibiotic required to reduce the binding of radiolabeled penicillin G to the PBP by 50%, after preincubation with the unlabeled (3-lactam antibiotic. All bacteria possess multiple PBPs. The affinities of each PBP for penicillin and other pMactam antibiotics vary widely, which is presumably due to small differences in the structure of the ^-lactam compounds and to differences in the sequence (and thus presumably the configuration) of the amino acid residues around the active-site serine (see below) of the PBP. Each bacterial species has its own assortment of PBPs. In a given organism, PBPs are numbered in order of decreasing apparent molecular weight. Usually there is no equivalent relationship between identically numbered PBPs of two distantly related organisms, although more closely related bacteria show similar PBP patterns. The most extensive studies on the targets of pMactam antibiotics have been focused on the PBPs of E. coli. In E. coli cells, there are at least eight PBPs that can be detected by radiolabeled-penicillin G (Spratt, 1977a, Henderson et al. 1994), namely PBPla, lb, PBP2, PBP3, PBP4, PBP5, PBP6 and PBP7. Each of the genes coding for these proteins has been cloned and mapped on the E. coli chromosome (Table 1). The PBPs of E. coli are localized in the cytoplasmic membrane (Spratt, 1977a) and each of these proteins has unique structural features that result in distinct penicillin-sensitive enzymatic functions which enable the protein to function in cell growth and morphogenesis (see below). Based on their structural features and  5  Table 1. Properties of the penicillin-binding proteins of Escherichia coli PBP  la  Relative molecular mass  Gene / location on chromosome  Mutant phenotype  Physiological function  Enzymatic activities  93 500  ponA  Cell lysis  a  Cell elongation  Peptidoglycan transglycosylase & transpeptidase  Cell lysis  a  Cell elongation  Peptidoglycan transglycosylase & transpeptidase  Round cell  Cell shape maintenance  Peptidoglycan transglycosylase & transpeptidase  Filamentous cell  Cell division in association with the FtsW protein  Peptidoglycan transglycosylase & transpeptidase* D-ala-carboxypeptidase & D D endopeptidase  1  73.5 min  lb  94 100  b  ponB  1  3.3 min  2  70 867  pbpA  1  14 min  3  63 850  pbpBI  2 min  4  49 568  dacB  0  c  1  None  6  Cell wall lysis and cell wall maturation  None  6  Cell wall maturation D-ala-carboxypeptidase  None  6  Cell wall maturation D-ala-carboxypeptidase  None  6  Prevents autolysis in nongrowing cells  70 min  5  44 330  dacA  1  14 min 6  44 000  dacC  1  19 min 7  30 910  pbpG  1  48 min  1  nt  a. Lethal when both ponA and ponB are mutated. b. Four components of P B P l b were produced from the ponB gene, a (94 100 Da), p\ y and 5. c. Only temperature sensitive mutants could be obtained; deletion mutation is lethal. d. Measured in crude membrane preparations containing disproportionately large amounts of PBP3 and FtsW. e. Single mutations are non-lethal. Double mutations of PBP4 and PBP5 or PBP5 and PBP6 are also non-lethal, nt. not tested  6  enzyme activities, PBPs of E. coli are classified as the high-molecular-weight PBPs which include PBPla, PBPlb, PBP2 and PBP3, and the low-molecular-weight PBPs which include PBP4, PBP5, PBP6 and PBP7.  2.2 PBPs: enzymes involved in the final steps of peptidoglycan biosynthesis Nearly all bacteria contain a peptidoglycan layer that surrounds the cytoplasmic membrane. This peptidoglycan layer is a network structure composed of similar building blocks, but with different crosslinking moieties among the bacteria. Alternating residues of A^-acetylglucosamine and A/-acetylmuramic acid are linked through the D-lactate of N-acetylmuramic acid with a tetrapeptide of the structure L Ala-D-Glu-L-Xaa-D-Ala, for which Xaa is often a diamino acid. Crosslinking varies extensively among bacteria. PBPs are the enzymes responsible for the final steps of peptidoglycan biosynthesis. These final steps, with E. coli as an example, consist of the polymerization of the repeating disaccharide unit (transglycosylation) and the formation of the peptide crosslinking the amino terminal of meso-diaminopimelic acid and the carboxyl terminal of the penultimate D-alanine residue of the pentapeptide side chains accompanied by the removal of the terminal D-alanine residue (transpeptidation). The extent of the peptide crosslinking is controlled by a third reaction, DD-carboxypeptidation, which involves the transfer of the carbonyl group of the penultimate D-alanine residue of the pentapeptide to water, rather than to  7  an amino group (Fig. 2). pMactam antibiotics, due to their structural similarity to the D-alanyl-D-alanine carboxyl terminal residues of the pentapeptide, inhibit the transpeptidation and DD-carboxypeptidation. The transglycosylation reaction is not penicillin-sensitive. The high-molecular-weight PBPs of E. coli are generally regarded as bifunctional enzymes, having transglycosylase and transpeptidase activities which are essential for the completion of peptidoglycan biosynthesis. The dual enzymatic activities of the high-molecular-weight PBPs of E. coli were demonstrated by in vitro catalysis reactions on lipid-linked disaccharide-pentapeptide precursors, using purified PBP preparations [PBPla (Tomioka et al, 1982), lb or PBP3 (Ishino et al, 1981)] or membrane preparations lacking PBPlb and containing disproportionately large amounts of PBP2 or PBP3 (Ishino et al, 1986). The low-molecular-weight PBPs of E. coli act mainly as DDcarboxypeptidases. PBP4 has a highly penicillin-sensitive DD-carboxypeptidase activity (Matsuhashi et al, 1977, Iwaya et al, 1977) and a DD-endopeptidase activity which might account, in part, for the turnover of peptide cross-bridges in peptidoglycan (Korat et al, 1991; Matsuhashi, 1994). PBP5 and PBP6 catalyze a DDcarboxypeptidation reaction that is moderately sensitive to penicillin (Amanuma et al, 1980; Matsuhashi et al, 1979). There are no reports on PBP7 enzymatic activity in peptidoglycan biosynthesis. The major functions of low-molecular weight PBPs possibly involve the maturation of peptidoglycans (Izaki et al, 1968) and  a  CM  C  i-J  CD  GlcNAc - MurNAc - GlcNAc -MurNAc - GlcNAc - MurNAc I  i  L-Ala GO  o  L-Ala  I  P* CP  D-GIu  D-Ala  B  I  I  o  m-A2pm-NH2  i-S  CP  CP  cn CP 13  sr. o =3 O  D-Ala  /  I  D-GIu  D-Ala  D-Ala  m-A2pm-NH2 i  D-Ala  m-A2pm-NH2  D-Ala  i  I  D  ,  D-GIu  G l u  I  L-Ala  L-Ala  I  I  GlcNAc - MurNAc-...GlcNAc - MurNAc - GlcNAc - MurNAc  •ff.s ao o f» p  co  c  1 0  t  Transglycosylase (PBPla, lb, 2, 3)  (PBP4, 5, 6)  m-A2pm-NH2 I  Transpeptidase (PBPla, lb, 2, 3)  i 4 — Carboxypeptidase  9  regulation of the availability of peptidoglycan precursors (Begg et al, 1990).  2.3 Structures of PBPs 2.3.1 Conserved motifs PBPs are penicillin-interactive, active-site serine enzymes (or proteases) (Ghuysen, 1991). The acyl enzyme mechanism was proposed following the studies on the mechanism of (3-lactam antibiotic action (Tipper & Strominger, 1965). Involvement of an essential serine at the active site was biochemically demonstrated for the reactions with low-molecular-weight PBP from Streptomyces sp. R61 (Frere et al, 1976) and several class A and class C p-lactamases (Knott-Hunziker et al, 1979; Cohen et al, 1980; Fisher et al, 1981). Site-directed mutagenesis experiments performed on the PBPs of E. coli and ^-lactamases have confirmed the active-site serine mechanism (Keck et al, 1985; Nicholas et al, 1988). Central to this  I mechanism is the transfer of the electrophilic group R-C=0 of the scissile (peptide or amide) bond to the hydroxyl group of the active-site serine reside (Fig. IB). The ester-linked acyl-enzymes formed by reaction between the ^-lactam antibiotics and the (3-lactamases are usually very short-lived. In contrast, those formed by reaction with the PBPs are usually very long-lived (Ghuysen, 1991). Consequently, the (3-lactam antibiotics are substrates of the (3-lactamases (thus being hydrolyzed) and covalent inactivators of transpeptidases and DD-carboxypeptidases, which can thus be detected  10  as penicillin-binding proteins. The penicillin-interactive, active-site serine protein family consists of three members, class A and class C P-lactamases, low-molecular-weight PBPs, and highmolecular-weight PBPs. The primary sequences of the proteins in this family are highly divergent (Spratt et al, 1988). However, they share certain common features in their primary and secondary structures. Common among this protein family are three conserved amino acid motifs: the tetrad active-site serine-x-x-lysine (SXXK), where x is a variable amino acid, the triad serine or tyrosine-x-asparagine [S(Y)XN] and the triad lysine or histine-threonine or serine-glycine [K(H)T(S)G]. When the protein folds, these motifs are brought close to each other, generating an active-site at the junction between an all-a domain, and an a-P domain whose five-stranded P-sheet is protected by additional a-helices on both faces. In this structure, the serine of the S X X K motif is in a central position in the cavity whereas the SXN and the K T G motifs are on either side (Ghuysen, 1991). When penicillin enters this cavity, the active-site serine covalently binds to the carbonyl moiety of the compound whereas the lysine or histidine of the K(H)T(S)G motif is in a position to promote the initial binding by providing a positive charge that can interact with the carboxylate moiety of the substrate (Malhotra et al, 1992). Other amino acid residues surround the cavity also play certain roles in facilitating the binding to the substrate by hydrogen bonding or electrostatic interactions.  11  2.3.2 Domain structures In the low-molecular weight PBPs and (3-lactamases, the active-site serines reside close to the amino terminus of the proteins (Frere et al, 1985). In the highmolecular weight 'bifunctional' PBPs, the acylated serine residues are located towards the middle of the sequences (Keck et al, 1985) and the penicillin-sensitive transpeptidase domains (or the penicillin-binding domains) are located towards the carboxy-terminus of the proteins (Hedge & Spratt, 1984). The penicillin-binding domain is assumed to start 60 residues upstream of the essential S X X K motif and to terminate 60 residues downstream from the K T G motif (Ghuysen, 1991). In most high molecular weight PBPs, the penicillin-binding domain has a "tail" in the form of an approximately 100 amino acid carboxyl terminal extension (Ghuysen, 1994). The amino-terminal region, which has no counterpart in the low-molecular-weight PBPs and [3-lactamases, is believed to contain the transglycosylase domain, although the evidence for this assignment is weak in most cases (Nakagawa et al, 1984). The high molecular weight PBPs are not synthesized as preproteins but have a highly hydrophobic region near the amino terminus that acts as a noncleaved signallike sequence. Thus, this sequence could act not only to transport the protein to the periplasm but also to anchor the protein to the cytoplasmic membrane. This is consistent with fusion studies of PBPlb and PBP3 (with ^-lactamase as a reporter),  12  which each has only a single transmembrane segment located near the amino-terminal end, with the remainder of the polypeptide located in the periplasm (Edelman et al., 1987; Bowler & Spratt 1989). Of note is the sequence that precedes the hydrophobic segment. PBPlb contains a highly charged segment that is 63 amino acids in length, while PBP2 and PBP3 contain segments of 20 and 23 residues, respectively. PBPla, an exception, contains a sequence that is only five residues in length (Ghuysen, 1994). Although PBP3 of E. coli contains the consensus sequence for modification and processing of lipoproteins (L27CGC30), only a small fraction appears to be modified along this pathway (Hayashi et ah, 1988). Instead PBP3 undergoes maturation by elimination of a 10-amino acid stretch from its carboxy-terminus (Nagasawa et al, 1989). This reaction is catalyzed by the product of the pre gene (Hara et al, 1991), a C-terminal-specific protease localized in the periplasm (Keiler et al, 1995). The function of this posttranslational modification remains to be elucidated. The low-molecular-weight PBPs contain a carboxy-terminal extension which is about 50 to 100 amino acids in length and starts approximately 60 residues down stream from the KT(S)G motif (Ghuysen, 1991). The end of the carboxy-terminal extension contains a signal-like peptide segment that serves as a membrane anchor. As a consequence, the bulk of the protein is on the outer surface of the cytoplasmic membrane (Ghuysen, 1991; van der Linden et al, 1993).  13  2.4 Role of PBPs in E. coli growth and cell morphology The role of PBPs in E. coli cell growth and morphogenesis has been elucidated by two approaches: i) morphological analyses of mutants with altered PBP patterns, and ii) correlation between the binding affinities of P-lactam antibiotics to particular PBPs and the morphological effects of the P-lactam antibiotics on E. coli cells (|3lactam antibiotics cause at least three morphological effects: rapid cell lysis, formation of spherical cells, filamentation). The four high-molecular-weight PBPs are essential for cell growth and are thought to be the lethal targets of the P-lactam antibiotics (Table 1). Rapid lysis of E. coli occurs when both PBPla and lb are bound. However, deletion of either the PBPla- or the PBPlb- encoding gene is tolerated, suggesting that PBPla and lb are redundant or at least capable of fulfilling compensatory roles in cell elongation (Spratt, 1975, 1977b; Tamaki etal, 1977; Suzuke etal, 1978). Inactivation of PBP2, either with mecillinam (a PBP2-targeted P-lactam antibiotic) or by growing a mutant that produces a thermolabile form of PBP2 at the restrictive temperature, results in the growth of E. coli cells as spherical shapes (Spratt, 1975,1978). Thus, PBP2 is essential for lateral cell wall elongation and maintenance of the rod shape. E. coli PBP3 is involved in the formation of the septum during cell division. Mutations affecting PBP3 (temperature sensitive mutants) or the selective binding of  14  the protein by (3-lactam antibiotics (e.g., treatment with cephalexin, a PBP3-targeted antibiotic) result in the inhibition of cell division and the growth of E. coli as filamentous cells, and eventual cell death (Spratt, 1975, 1977b). The PBP3-encoding gene,  or pbpB, is found proximal to a cluster of genes required for the synthesis of  the peptidoglycan precursors (e.g., murE, murF, murG, murC, ddl) or for cell division and septum formation (e.g.,ftsW,ftsQ,ftsAandftsZ) (Ayala et al, 1994). The pbpB gene is essential for E. coli cell growth. Only conditional mutants have been isolated (Haraefa/., 1992). The low-molecular-weight PBPs 4, 5 and 6 of E. coli were suggested to be nonessential for cell survival and were therefore not considered to be of major importance in the killing mechanism of P-lactam antibiotics. This hypothesis is supported by the observation that mutation of E. coli PBP4 or double deletions of PBP5 and PBP6, do not produce any significant morphological abnormalities (Matsuhashi et al, 1977; Broome-Smith & Spratt, 1985). Recently, it was observed that an insertional mutation of the E. colipbpG gene, which encodes PBP7, did not produce any obvious growth defects (Henderson et al, 1995). Overall, lowmolecular-weight PBPs are dispensable for E. coli cell viability under laboratory conditions.  2.5 PBPs in P.  aeruginosa  PBPs of P. aeruginosa show an electrophoretic pattern similar to that of E.  15  coli, but are not well studied and their genes have largely not been isolated. Binding between the P. aeruginosa PBPs and P-lactam antibiotics generally results in morphological changes similar to those observed in E. coli (Curtis et al., 1979a). Comparative studies of the binding affinities for various P-lactam antibiotics indicated that P. aeruginosa PBPla, lb, 2, 3, 4 and 5, respectively, corresponded to E. coli PBPlb, la, 2, 3, 4 and 5, and that P. aeruginosa PBP6 and PBP7 were not always detected (Noguchi et al, 1979). The enzyme activity study by Noguchi et al is the only report on P. aeruginosa PBP function in peptidoglycan biosynthesis (Noguchi et al, 1985). It indicated that PBP5 of P. aeruginosa had a moderately penicillinsensitive D-alanine carboxypeptidase activity and that a defect in this enzyme activity was not lethal, as was found for the equivalent E. coli enzyme. Overall, very little is known about P. aeruginosa PBPs.  3. P-lactam susceptibility and resistance in Gram-negative bacteria 3.1 Antimicrobial activities of P-lactam antibiotics Since penicillin G was first discovered 50 years ago, enormous numbers of mostly semi-synthetic P-lactam compounds have been produced. These compounds contain an essential four-membered lactam ring (see Figure 1, B) that can exist as an isolated ring, as depicted by the monobactams (e.g., aztreonam), or may be fused to a five-membered ring (e.g., penicillin, imipenem) or a six-membered ring (e.g.,  16  cephaloridine, ceftazidime, cefepime) to form bicyclic ring structures. [3-lactam antibiotics are effective antimicrobial agents of low toxicity to eukaryotes. These agents often represent the first line of the therapy for the treatment of bacterial infections and have been used successfully for more than 50 years. The activity of a particular P-lactam is influenced by the type of substitutions attached to the basic nucleus. PBPs from different bacterial species often have different binding affinities for P-lactam antibiotics. The inactivation of PBPla and lb, or PBP2, or PBP3, is lethal for E. coli. The P-lactam antibiotics can therefore kill by three completely different routes (rapid cell lysis, production of spherical cells and eventual cell death, or inhibition of cell division and eventual cell death). Some effective derivatives are known that kill exclusively by rapid lysis (e.g., cefsulodin), by production of spherical cells and eventual cell death (mecillinam), or by filamentation and eventual cell death (cephalexin) (Curtis etal, 1979a; Spratt, 1983). More typically, P-lactam antibiotics kill bacteria by two or all three of these mechanisms. Monobactams and many cephalosporins, which have structures containing the essential four-membered lactam ring and the adjacent six-membered ring, have very high binding affinity to E. coli PBP3 (Curtis etal, 1979b; Georgopapadakou etal, 1983; Hayes etal, 1983), with filamentation being the initial response of the cell on exposure to the antibiotics. However, they also bind weakly but significantly to the bifunctional PBPla and lb,  17  eventually causing lysis of the organism. Carbapenems, a group of (3-lactams containing the essential four-membered lactam ring and an adjacent five-membered ring, have very high binding affinity to PBP2 in E. coli and other enteric bacteria. Although this interaction initially results in the formation of spherical cells, lysis subsequently occurs resulting from additional binding to PBPla or lb, or both (Spratt et al, 1977a). In P. aeruginosa, PBP3 is a major target of most of the third and fourth generation cephalosporins (Hayes et al., 1983; Maejima et al, 1991; Watanabe et al, 1992). Gram-negative bacteria are surrounded by a complex envelope (Fig. 3). In addition to the cytoplasmic membrane, these organisms usually have a thin peptidoglycan layer covered by an outer membrane. Between the peptidoglycan layer and the cytoplasmic membrane lies the periplasmic space, the location of most of the (3-lactamases found in Gram-negative bacteria. The target proteins of (3-lactam antibiotics, PBPs, are located on the outer surface of the cytoplasmic membrane and the polypeptides extend into the periplasm. Therefore (3-lactam antibiotics need to traverse the outer membrane to gain their access to their target proteins. Thus, the antimicrobial activity of (3-lactam antibiotics depends on a combination of factors including not only their affinity with the target PBPs, but also their rate of passage through the outer membrane barrier and their resistance to hydrolysis by (3-lactamases.  Figure 3. Schematic representation of a cross section of the cell envelope of a Gramnegative bacterium  19  The outer membrane is an asymmetric bilayer consisting of lipopolysaccharide (LPS) (in the outer leaf) and phospholipid (in the inner leaf) bilayers studded with proteins (Fig. 3). The outer membrane is usually described as a semipermeablebarrier, in which hydrophilic molecules of sizes below a given exclusion limit can pass through protein channels called porins. Porins are water-filled, nonspecific, transmembrane diffusion channels (diameter 0.6 - 2.3 nm) (Nikaido & Vaara 1985). The permeability of the porin channels for a P-lactam antibiotic is a function of the size, charge, and hydrophobic nature of the [3-lactam molecule. P-lactam antibiotics differ considerably in charge, a factor that affects their rate of passage through porin channels. In E. coli and related Enterobacteriaceae, the porin channels favour hydrophilic, cationic molecules (Benz, Schmid & Hancock, 1985; Nikaido, 1989). The total area of channels per outer membrane influences antibiotic uptake. This explains the greater intrinsic resistance to antibiotics of P. aeruginosa compared to E. coli, since in P. aeruginosa only a small fraction of the porin molecules form open channels (Nicas & Hancock, 1983).  3.2 P-lactam resistance in Gram-negative bacteria Resistance of Gram-negative bacteria to P-lactam antibiotics can result from a decrease in diffusion across the outer membrane, from the destruction of the antibiotics by P-lactamases, or from a reduction in the affinity of the target penicillin-  20  binding proteins (Hancock et al., 1988; Malouin et al., 1986). Alteration in outer membrane permeability resulting from porin loss or modification have been described in Gram-negative bacteria including P. aeruginosa (Angus et al., 1982; Godfrey et al., 1987). However, most strains with altered permeability have been generated in the laboratory. These mutants confer only moderate increases in (3-lactam resistance (Harder et al, 1981; Jaffe et al., 1983). There are fewer reports of the isolation of similar mutants from clinical samples (Nikaido, 1989b). It is possible that there is a limit to the extent that the permeability of the outer membrane can be reduced without seriously impairing the growth of the bacteria by limiting the uptake of nutrients (especially for P. aeruginosa given its already low level permeability compared to E. coli). Reports on the mechanisms responsible for (3-lactam antibiotic resistance in clinical isolates often involve the production of (3-lactamases (Sanders et al., 1992), which wholly or partly account for the observed (3-lactam resistance (Livermore, 1993). High levels of resistance usually result from the combined effect of decreased permeability and the presence of a (3lactamase (Angus et al, 1982; Hancock et al, 1988) or the alteration of PBPs (Mirelman et al, 1981). The latter is found particularly in non-(3-lactamase-producing bacteria (Malouin et al, 1986). Clinical resistance to (3-lactam antibiotics in Gram-negative bacteria is not commonly associated with altered PBPs (Georgopapadakou, 1993). This is probably  due to the effectiveness of (3-lactamase, coupled with reduced outer membrane permeability, in producing resistance. There are no reports of PBP-mediated clinical resistance in E. coli. However, PBP-mediated resistance to P-lactam antibiotics is well documented in Haemophilus influenzae (Malouin etal. 1986; Spratt, 1994) and Neisseria gonorrhoeae (Spratt, 1994). Non-P-lactamase-mediated resistance to P lactams in clinical isolates and laboratory mutants of P. aeruginosa has been reported and is associated with a reduction in PBP3 binding affinity (Godfrey et al, 1981; Gotoh etal, 1990).  4. Aims of this study The initial aims of this study were to clone and characterize the P. aeruginosa gene encoding PBP3 and investigate the role of this protein in susceptibility to P lactam antibiotics. PBP3 was chosen for study since it is the primary target of newer generation P-lactams (Maejima et al, 1991, Watanabe et al, 1992). Furthermore, it has been reported that non-P-lactamase-mediated resistance to P-lactams in clinical isolates and laboratory mutants of P. aeruginosa was associated with a reduction in PBP3 binding affinity (Godfrey et al, 1981; Gotoh et al, 1990). Therefore, it seemed likely that P. aeruginosa PBP3 plays an important role in susceptibility to P-lactam antibiotics. Preliminary results from the experiments of cloning the PBP3-encoding gene  22  indicated that P. aeruginosa might contain two copies of a gene encoding proteins with similar functions to E. coli PBP3. To test this hypothesis, further research was proposed to clone and characterize both the P. aeruginosa genes encoding E. coli PBP3-like proteins. The final goal of this project also included the investigation of roles of these two proteins in cellular functions and susceptibility to (3-lactam antibiotics.  23  MATERIALS AND METHODS  I. Bacterial strains, plasmids and growth conditions The bacterial strains and plasmids used in this study are described in Table 2 and Table 3. Bacterial strains were maintained as frozen stocks in L B broth plus 7% dimethyl sulphoxide at -70°C. E. coli strains were grown in LB broth (Difco, Detroit, MI). P. aeruginosa strains were grown in M H broth (Becton Dickinson, Cockeysbille, MD). Solid media were made by the addition of 2% Bacto-agar (Difco, Detroit, MI). V B M M (3 g/L sodium citrate, 2 g/L citric acid, 10 g/L K H P 0 , 3.5 g/L N a N H H P 0 4 H 0 , pH 7.0; 0.8 M MgS0 , 0.08 M CaCl ) (Vogel 2  4  4  4  2  4  2  & Bonner, 1956) was used to select growth of P. aeruginosa, since E. coli can not use citrate as a carbon source. Antibiotics were used in selective media at the following concentrations: for E. coli; ampicillin at 75 u.g/ml, chloramphenicol at 30 p.g/ml, kanamycin at 50 u.g/ml and tetracycline at 12 u.g/ml; for P. aeruginosa; carbenicillin at 500 Ltg/ml, kanamycin at 300 Ltg/ml and tetracycline at 100 Ltg/ml.  II. D N A manipulation D N A manipulations were performed essentially as described by Maniatis et al (1982). Chromosomal D N A was prepared by the method of Ausubel et al. (1987). D N A probes for Southern hybridization experiments were labeled with  24  Table 2. Bacterial strains  Strain  Relevant characteristics  References or sources  E. coli DH5oc  supE44 lacU169 (80lacZM15) hsdR17 recAl endAl gyrA96 thi-1 relAl  BRL  E. coli JM110  dam dcm supE44 thi leu rpsL lacY galK galT ara tonA thr tsx (lac-proAB)  BRL  F[traD36proAB+ lacIQ lacZM15] E. coli K38  Russel & Model (1984)  HfrC (k)  E. coli BL21(DE3) hsdS gal (talts857 indl Saml nin5 lac wv5-T7 gene 1)  Studier & Moffat (1986)  E. co//S17-1  thi pro hsdR recA; mobilizer strain  Simon et al. (1986)  P. aeruginosa HI 03  P A O l , prototroph  Hancock & Carey (1979)  P. aeruginosa PAO4089 P. aeruginosa HC132  met-9020 pro-9024 bM9\ 11 blaP9202 pbpC mutant of P A O l , Km  r  Gotoh et al. (1990) This study  25  Table 3. Plasmids  Plasmid  Relevant characteristics  References or sources  pTZ18U  General cloning vector, Ap  USB  r  pTZ19U  USB  General cloning vector, Ap pUCP27 Broad host-range vector, Tc pT7-7  Cloning vector containing the T7 R N A polymerase promoter and RBS sequence, A p  pGPl-2  pBBRlMCS  l  Schweizer (1994) Studier Moffat (1986)  r  Contains heat inducible gene for  Studier &  T7 R N A polymerase, Cm'  Moffat, (1986)  Broad-host-range vector containing  Kovach et al. (1994)  the (3-galactosidase gene and the T3 and T7 promoters, Cm" pUC4KPA  Gene cartridge vector containing a 1.3 kb fragment which encodes the enzyme conferring kanamycin resistance, Km  Pharmacia  pUC19 with 10 bp Ndel-Notl adapter in Ndel site, Ap  Schweizer (1992)  Derivative of pHSS21 containing oriT and sacB, Km , Cm  Schweizer (1992)  Analogue of pBR322 and pAT153  Spratt et al.  in which K m replaces A p , K m ,  (1986)  r  pNOT19  r  pMOB3  1  pPH125  r  Tc  r  1  26  Table 2-continued pPH115  E. coli pbpB gene on a 2.6 kb BamHl-EcoRl fragment cloned at  Spratt et al. (1986)  r  Hindlll site within the Tc gene ofpPH125, Km' pXL2  pXL3  pXL5  pXL8  A 580 bp PCR product corresponding to portion of pbpB gene cloned in pTZ18U, A p  This study r  A 580 bp PCR product corresponding to portion of pbpC gene cloned in pTZ18U, Ap A 380 bp PCR product corresponding to portion of yhhF gene cloned in pTZ18U, Ap  r  L  A 510 bp PCR product corresponding to portion of sucC/sucD genes cloned in pTZl 8U, Ap r  pXL12  pSPHl  A 460 bp PCR product corresponding to portion of cypH gene cloned in pTZ18U, Ap A 1.4 Kb Sphl fragment of P A O l D N A cloned in pTZ19U, Ap  pPST18  A 1.8 Kb Pst\ fragment of P A O l D N A cloned in pTZ19U, Ap  pXSml6  A 4.4 Kb Smal-Xhol fragment of P A O l D N A cloned in pTZ19U, Ap  pXLSH36  A 5.4 Kb Sphl-Xhol fragment of P A O l D N A containing the pbpB and murE cloned inpTZ19U, Ap  pXLBB  r  A 5.4 Kb Sphl-Xhol fragment of P A O l D N A containing the pbpB and murE cloned in pTZ18U, Ap  1  27  Table 2-continued pXL706  P. aeruginosa pbpB cloned between Ndel and BamHI sites of pT7-7, A p  pXL608  r  A 1.7 kb Xbal-BamHl fragment from pXL706 containing the RBS sequence and pbpB cloned in pBBRlMCS, Cm r  pXL506  A 1.7 kb Xbal-BamHl fragment from pXL706 containing the RBS sequence and pbpB cloned in pUCP27, Tc r  pXL546  A 300 bp of Sphl-BamHl fragment from pXL706 containing 3'end of the pbpB gene cloned in pUCP27, Tc l  pXL706::Km  A1.3 kb K m cartridge cloned at Smal site of the pbpB in pXL706, Ap r  1  pBPB::Km  A 5.8 kb Notl fragment containing the OriT, sacB and Cm sequences cloned in pBPBl::Km, Ap , Km , Cm r  r  pXL-Xh401  r  r  A 2.5 kb Xhol fragment of P A O l D N A cloned inpTZ19U, Ap r  pXL-PS406  A 4.0 kb Pstl-Sall fragment of P A O l D N A cloned in pTZ19U, Ap r  pXL-Ec405  A 2.0 kb EcoRI fragment of P A O l D N A cloned in pTZ19U,Ap r  pXL-KE24  A 3.7 kb Kpnl-EcoRl fragment of P A O l D N A containing the pbpC cloned in pTZ19U, Ap 1  pXL-Hd2  A 2.7 kb Hindlll-EcoRl fragment of P A O l D N A containing the pbpC cloned inpTZ19U, Ap 1  28  Table 2. continued pXL-Xb5  A 2.3 Xbal-EcoRl fragment of P A O l D N A containing the pbpC cloned in pTZ19U, Ap r  pXL732  P. aeruginosa pbpC cloned between Ndel and BamHI sites of pT7-7, Ap 1  pXL629  pXL519  A 1.7 kb Xbal-BamEI fragment from pXL732 containing the RBS sequence and pbpC cloned in pBBRlMCS, Cm  r  A 1.7 kb Xbal-BamHI fragment from pXL732 containing the RBS sequence and pbpC cloned in pUCP27, Tc r  pXL732::Km  A 1.3 kb Km cartridge cloned at Smal site of the pbpC gene in pXL732, Ap r  1  pBPC::Km  A 5.8 kb Noil fragment containing the OriT, sacB and Cm sequences cloned in pBPCl::Km, Ap , Km , Cm 1  r  pXLK20  r  r  A 2.6 kb EcoRI-BamtU fragment from pPHl 15 containing E. colipbpB cloned in pUCP27 behind the lac promoter, Tc l  29  32  P-dATP (Amersham, Canada) or digoxigenin-dUTP (Boehringer Mannheim) by  the random primer labeling method. Restriction enzymes and D N A modification enzymes were purchased from either BRL, Pharmacia Canada, BoehringerMannheim Canada or New England Biolabs Canada, and used according to the manufacturer's directions. Oligodeoxyribonucleotides were synthesized with an ABI model 392 D N A / R N A synthesizer (Applied Biosystems Inc., Foster City, CA). Synthesized oligonucleotides were purified by the method of Sawadogo and Dyke (1990).  III. DNA sequencing Plasmid D N A for sequencing was prepared by the Qiawell-8 plasmid purification system (Qiagen Inc., Chatsworth, CA) according to the manufacturer's directions. D N A concentrations were determined with a mini-fluorometer (model T K O 100 , Hoefer Scientific Instruments, San Francisco, CA). D N A sequencing was done with an ABI Model 373 automated D N A sequencer and dye terminator chemistry following the protocols from ABI using the universal forward and reverse primers. Both D N A strands were sequenced. Nested deletions were created with the Erase-a-base kit (Promega, Madison, Wl). Oligonucleotide primers were constructed to fill in gaps.  30  IV. DNA and amino acid sequence analyses D N A and amino acid sequences were analyzed with the PC Gene, ESEE and D N A M A N computer programs. Sequences were compared to the GenBank database with the BLASTN, BLASTP and B L A S T X programs (Altschul et al. 1990).  V. Transformation E. coli was transformed by the CaCl method (Maniatis et al. 1982). P. 2  aeruginosa competent cells were prepared by the procedure of Schweizer (1991). Briefly, cells from exponential phase cultures were washed with ice cold 100 mM MgCl , resuspended in 100 mM CaCl and incubated on ice for 30 min. 2  2  Subsequently, the cells were pelleted by centrifugation and resuspended in ice cold 100 mM CaCl plus 10% glycerol, aliquoted and stored at -70°C until needed. For 2  transformation, competent cells were thawed on ice and mixed with plasmid DNA. The cells were then incubated on ice for 30 to 60 min, heat shocked for 3 min at 37°C followed by incubation on ice for 5 min; 1 ml of LB broth was added and the cells incubated at 37°C with agitation for 1.5 to 2.5 hrs to allow for the expression of the antibiotic resistance gene. The cells were plated on selective media and incubated at 37°C for 24 to 48 hrs.  VI. Construction of a PBP3 or PBP3x-defective mutant An improved gene replacement technique described by Schweizer (1992) was  used to construct a PBP3-defective or PBP3x-defective mutant. Plasmids pNOT19, pMOB3 and pUC4KAPA were used. pNOT19 is derived from pUC19 with the unique Ndel site changed to a Notl site. pMOB3 contains the MOB3 cassette as a 5.8 kb Notl fragment which is composed of oriT, the Bacillus subtilis sacB gene as a counter-selectable marker, and a chloramphenicol resistance gene allowing positive selection of both the oriT and the sacB. pUC4KAPA contained a 1.3 kb fragment which was derived from Tn901, which encoded the enzyme conferring kanamycin resistance, flanked by identical restriction enzyme recognition sites.  6.1 Construction of plasmids pBPB: :Km and pBPC: :Km The pbpB and pbpC genes on the plasmid pXL706 (see section VII) and pXL732 (see section VII) were mutated by the insertion of a 1.3 kb blunt ended Hindi fragment of Km cartridge (isolated from pUC4KPA) at the unique Smal site 1  in the pbpB or pbpC gene. This procedure permitted the generation of plasmid pXL706::Km which had 0.96 kb and 0.82 kb of chromosomal D N A sequence on either side of the Km cartridge, and the plasmid pXL732::Km which had 1.1 kb and 1  0.5 kb of chromosomal D N A sequence on either side of the Km cartridge. The 3.0 1  kb fragment containing thepbpB::Km orpbpC::Km was isolated from r  T  pXL706::Km or pXL732::Km and cloned into pNOT19, respectively. Subsequently, the MOB3 cassette was isolated as a 5.8 kb Notl D N A fragment from pMOB3 and cloned into the unique Notl site on pNOT19 withpbpB/.Km  1  or pbpC::Km to r  32  generate plasmids pBPB::Km (Fig. 20, see Results Chapter Two) and pBPC::Km (Fig. 30, see Results Chapter Three) respectively, which were then separately transformed into an E. coli mobilizing strain S17-1.  6.2 Conjugation Transfer of plasmids from the E. coli mobilizing strain S17-1 to P. aeruginosa P A O l strain HI03 was achieved by biparental mating as follows. The donor (E. coli S17-1) was grown overnight in LB broth at 30°C. The recipient (P. aeruginosa H103) was grown overnight in LB broth at 42°C. Samples of the donor and recipient cultures (100 ul of each) were mixed, diluted into 2 ml L B broth and incubated at room temperature for 30 min. The cell mixture was then filtered onto a membrane (0.45 um; Nalgene, Rochester, NY). The filter was placed cell-side up on a LB agar plate and incubated at 30°C overnight. The cells were then washed off the membrane into 1 ml of sterile saline, diluted and spread onto V B M M agar plates containing carbenicillin, chloramphenicol and kanamycin to allow for the selection of plasmid integration. The transconjugates were then plated onto M H agar containing kanamycin and 5% sucrose to select for the cointegrates and deletion of plasmid sequences.  VII. Production of pbpB and pbpC DNA probes by degenerate P C R Degenerate PCR primers for the amplification of portions of the pbpB and  pbpC genes were designed based on amino acid sequences surrounding the conserved S X X K and K T G motifs found in amino acid alignments of E. coli PBP1 A, IB, 2, 3 and N. gonorrhoeae PBP2 proteins, after adjusting the sequences according to the codon usage of P. aeruginosa (West & Iglewski, 1988). The sequence of the degenerate upstream primer corresponding to the S X X K motif was 5'  -TTTGAATTCGG(C)CA(T)C(G)C(G)G(AC)C(AT)G(C)G(A)C(T)G(C)AAGCC-  3' which corresponded to the amino acid sequence G(A)ST(ANL)V(IAM) KP; the sequence of the degenerate downstream primer corresponding to the K T G motif was 5' - A A A G A ATTCG(CT)T(C)T(G)C(G)GT(C)C(G)GTGCCG (C)G(C)T(A)CTT-3' which corresponded to the amino acid sequence KT(S)GTT(A)K(QRN); the letters in parentheses represent alternative nucleotides or amino acids at given positions in the sequence corresponding to the preceding nucleotide or amino acid. An EcoRI restriction site sequence (underlined) was included at the 5' end of both primers to facilitate subsequent cloning of PCR amplification products. PCR amplification was performed in the presence of 5% formamide, 10% glycerol and 15 mM M g  2 +  under conditions whereby the first 5 cycles involved  temperature cycles of 94°C for 15 sec, 37°C for 30 sec and 72°C for 90 sec. The primer annealing temperature was raised from 37°C to 55°C for the remaining 25 cycles.  VIII. Protein expression using the T7 RNA polymerase/promoter system 8.1 Construction of recombinant clones The P. aeruginosa pbpB and pbpC genes cloned in pT7-7 were amplified by PCR. The upstream primer contained an Ndel recognition sequence and the sequences coding for the N-terminus of PBP3 or PBP3x. Their sequences were respectively, 5 ' - T A A A C A T A T G A A A C T G A A T T A T T T C C A G G G C G C C C T - 3 ' and 5' - A A A C A T A T G A G C A G T C A A C G C C G A A A C T A C C G C T T C A-3'. The downstream primer contained the sequences coding for the C-terminus of PBP3 or PBP3x followed by a stop codon and the sequence for a BamHI recognition site. Their sequences were 5' - A A A G G A T C C T C A G C C A C G C C C T C C T T T T G C G G GCGCA-3' and 5' - A A A G G A T C C T C A G C C G T G G T G C T G G C G G T C G G C G A - 3 ' respectively. The PCR was performed under conditions involving 25 temperature cycles of 96°C for 60 sec, 63°C for 60 sec and 72°C for 90 sec, followed by a 10 min primer extension at 72°C. Vent DNA polymerase was used for all PCR R  amplifications according to the manufacturer's recommendations. The resulting PCR product of 1.7 kb in length, corresponding to the size of the pbpB or pbpC gene, was digested with Ndel and BamHI and cloned into the NdellBamHl digested pT7-7. The resulting plasmids pXL706 (Fig. 10B, see Results Chapter Two) and pXL732 (Fig. 21B, see Results Chapter Three) were transformed into E. coli K38/pGPl-2. The following procedure was used for the construction of the recombinant clone containing the pbpB or pbpC gene in the broad-host range vector pBBRlMCS.  35  A 1.7 kb Xbal-BamUI fragment isolated from pXL706 or pXL732, which contained an RBS sequence from pT7-7 and the pbpB or pbpC gene, was cloned behind the T7 promoter on the vector pBBRlMCS to generate plasmids pXL608 (Fig. 13, see Results Chapter Two) or pXL629 (Fig. 24, see Results Chapter Three), which were then separately transformed into E. coli BL21(DE3).  8.2 Analyses of protein expression LB broth containing ampicillin and kanamycin was inoculated at a 1:40 ratio with overnight cultures of E. coli K38/pGPl-2 (pXL706), K38/pGPl-2 (pXL732) or K38/pGPl-2 (pT7-7) grown at 30°C in L B broth. The freshly inoculated cultures were grown at 30°C until they reached an OD  6  oo  of 0.45. The cultures were then  incubated at 42°C for 30 min to induce expression of T7 RNA polymerase from pGPl-2 and the resulting expression of pbpB or pbpC under the control of the T7 promoter. Cell cultures were grown for an additional 90 min at 37°C before harvesting. Examination of protein expression for the genes cloned in pBBRlMCS was done as follows. L B broth plus chloramphenicol was inoculated at a ratio of 1:50 with overnight cultures of BL21(DE3)/pXL608, BL21(DE3)/pXL629 or BL21(DE3)/pBBRlMCS. The freshly inoculated cultures were grown at 37°C to an OD o 6 0  of 0.6. IPTG was then added into the cultures to a final concentration of 0.5  mM to induce expression of T7 RNA polymerase and pbpB or pbpC under the  36  control of the T7 promoter. Cultures were further incubated at 37°C for 1 to 4 hrs before harvesting.  IX. SDS-PAGE Protein profiles were analyzed by SDS-PAGE as described previously (Hancock & Carey, 1979). Proteins were solubilized in Laemmli solubilization buffer for 5 min at 100°C before being separated by SDS-PAGE. Proteins were visualized by staining with Coomassie Brilliant Blue R250 (Bio-Rad, Richmond, CA).  X. Whole cell lysates Whole cell lysates were prepared by an SDS boiling method (Nicas & Hancock, 1980). Briefly, samples from E. coli cultures were taken at different time points of growth. Cells were collected by centrifugation and resuspended in solubilization sample buffer and the proteins were analysed by SDS-PAGE.  XI. N-terminal amino acid sequence determination Partially purified protein preparations were separated by SDS-PAGE and electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore, San Francisco, CA) (Matsudaria, 1990). Briefly, protein samples were solubilized in Laemmli solubilization buffer that was prepared without bromphenol blue.  37  Following SDS-PAGE, the proteins were electroblotted onto PVDF membrane using 10% methanol and 0.2% 3'-[cyclohexylamino]-l-propanesulfonic acid (CAPS) (Sigma), pH 11.0, as the transfer buffer in an electroblotting apparatus (Bio-Rad); electroblotting was performed at 100V for 60 min. The PVDF membrane was then washed twice with fresh transfer buffer and stained with Ponceau-S Red solution (0.5% Ponceau-S and 1% acetic acid) at room temperature for 20 min to visualize the electroblotted proteins. The membrane was then rinsed with d H 0 until the 2  protein bands became visible above the background. The membrane was air dried and the appropriate protein bands excised from the membrane and sent to S. Kielland, Department of Biochemistry and Microbiology, University of Victoria, for N-terminal sequence determination using a gas phase sequencer (ABI, 470A).  XII. Membrane protein preparation E. coli and P. aeruginosa cells were harvested in the mid-log phase of growth and washed in 10 mM Tris-HCl buffer, pH 8.0. The washed cells were lysed by passage through a French Press (14000 pounds / square inch) and the particulate fraction was collected by ultracentrifugation at 50,000 rpm (Beckman 70.1 Ti) at 4°C for 1 h. The membrane-containing pellet was resuspended in 10 mM Tris-HCl buffer, pH 8.0 and sonicated twice on ice at maximum power for 15 sec (Fisher Sonic Dismembrator 300 fitted with a microtip 3.5 mm in diameter). The sonicate was centrifuged at 50,000 rpm (Beckman 70.1 Ti) at 4°C for 1 h and the membrane-  38  containing pellet was retained. The membrane-containing pellet was washed once in 10 mM Tris-HCl buffer, pH 8.0 then resuspended in the same buffer and used immediately or stored at -70°C until needed.  XIII. Protein assay Protein concentrations were determined by a modified Lowry assay (Sandermann & Strominger, 1972) which contained 1% SDS. This method allowed the analysis of protein samples which contained detergents. Bovine serum albumin was used as a standard.  IVX. Antibiotic susceptibility testing (3-lactam antibiotics used in this study included cefsulodin (Ciba Ceigy, Basel, Switzerland); cefepime (Bristol-Myers-Squibb Inc., Wallingford, CT); ceftazidime (Glaxo Canada Inc.); aztreonam (E. R. Squibb & Sons, Inc.); imipenem (Merck Sharp & Dohme, division of Merck & Co. Inc.) and cephaloridine (Sigma). Minimal inhibitory concentrations (MICs) were determined by the agar dilution method on M H agar plates using two-fold serial dilutions of the antibiotics. Overnight cultures of the bacteria to be tested were diluted in L B broth to approximately 10 colony forming unit per ml. Five ul of this diluted culture was 5  inoculated onto the surface of the M H agar plates containing antibiotic, or in control experiments, without any antibiotics. The recorded MIC corresponded to the lowest  3 9  concentration of antibiotic causing greater than 90% inhibition of bacterial growth after 18 h incubation at 37°C. Final MIC values were the average of at least three determinations.  XV. Penicillin-binding protein assays 15.1 Direct assay PBPs were assayed essentially by the method of Spratt (1977b). Membrane proteins were incubated with H-penicillin G (3.7 Lig/ml, 22 Ci/mol) at 25°C for 10 3  min. The reaction was stopped by the addition of an excess (lOOOx) of nonradioactive penicillin G. The samples were separated by SDS-PAGE (7.5%, 8.5% or 10% polyacrylamine) and the gel treated with 1 M sodium salicylate, pH 6.0, at room temperature for 30 min, dried at 80°C under vacuum for 2 hrs. The dried gel was then exposed to X-ray film (X-Omat K XK-1, Kodak) for 3 to 30 days at -70°C to visualize the radiolabeled proteins.  15.2 Competition assay Membrane proteins were incubated with a (3-lactam antibiotic or for control experiments with dH 0, at 25°C for 10 min. Subsequently, 4 uCi of H-penicillin G 3  2  (3.7 p.g/ml, 22 Ci/mol, Amersham) was added. After incubation at 25°C for 10 min, the reaction was stopped by the addition of an excess (lOOOx) of nonradioactive  40  penicillin G. The proteins were separated by SDS-7.5% PAGE. The resulting gel was treated with 1M sodium salicylate, dried, and exposed to X-ray film (X-Omat K XK-1, Kodak) for 3 to 6 days at -70°C. The intensities of the bands on the fluorograms were quantitated with a scanning densitometer (Studio Scan II, AGFA) in combination with a Macintosh computer using the public domain NIH Image program. The software was used to integrate scan peaks for quantitation of PBP binding to H-penicillin. Binding inhibition was evaluated as concentration of the 3  antibiotic inhibiting binding by 50% relative to binding in the absence of competing antibiotic.  XVI. Growth experiment Overnight cultures of P. aeruginosa were inoculated into fresh M H broth at a ratio of 1:100. Cultures were then incubated at 37°C with shaking (180 rpm). The absorbance at 600 nm of the culture was determined at various times and a growth curve was plotted.  XVII. Cell shape examination Cell shape was examined by phase contrast microscopy (BH2-PC, Olympus BHT, Tokyo, Japan). Cell photograph was taken with an Axiophot photomicroscope (Zeiss, Germany).  41  RESULTS  CHAPTER ONE Gene Cloning and Sequence Analyses of P.  aeruginosa pbpB  and pbpC  1. Introduction Sequences surrounding the active-site motif S X X K and the K T G motif are highly conserved among E. coli high-molecular-weight PBPs and Nesseria gonorrhoeae PBP2 (Ghuysen, 1991). N. gonorrhoeae PBP2 is anis. coli PBP3-like protein responsible for cross-wall formation during cell division, and its overall amino acid sequence is 47.7% homologous to that of E. coli PBP3 (Spratt, 1988). I hypothesized that P. aeruginosa PBP3 also contained the conserved S X X K and K T G motifs. Based on this hypothesis, two degenerate oligonucleotides were synthesized according to the amino acid sequences found in the conserved motifs of E. coli highmolecular-weight PBPs and N. gonorrhoeae PBP2. The strategy for cloning the gene encoding P. aeruginosa PBP3 thus involved the use of PCR to amplify a portion of the gene which was then used as a probe to clone the complete gene.  2.  Generation of DNA probes for cloning the pbpB and pbpC genes by degenerate PCR A pool of degenerate primers was synthesized based on the sequences at and  42  surrounding the conserved functional motifs S X X K and K T G of E. coli PBP1A, IB, 2, 3 and N. gonorrhoeae PBP2, after adjustment to the codon usage of P. aeruginosa (for sequences, see Materials and Methods section VII). These primers were used for PCR amplifications using P. aeruginosa P A O l chromosomal D N A as the template. A mixture of PCR products ranging from 220 bp to 600 bp was amplified. Since the spacing between the S X X K and K T G motifs of E. coli PBP3 is 184 amino acid residues (Nakamura et al, 1983), the fragment of about 600 bp in length was likely the product amplified from the DNA sequence coding for P. aeruginosa PBP3. Nevertheless, all of the PCR products were gel-purified, digested with EcoRI and cloned into a vector pTZ18U. The resultant clones were grouped by insert size and representative clones from each group were sequenced. The resulting D N A sequences were used for B L A S T X homology searches of the GenBank database. The results of these searches are shown in Table 4. One of the PCR products, a 580 bp DNA fragment cloned in pXL2, was found to translate to a protein sequence with 63.2% and 52.8% conservation of amino acids compared with E. coli PBP3 and N. gonorrhoeae PBP2, respectively. This PCR product was subsequently used as a probe to clone the complete P. aeruginosa pbpB gene. A second PCR product, which was 580 bp in length and cloned in pXL3, was found to translate to a protein sequence with 62.5% and 56.6% conservation of  Table 4. P. aeruginosa PCR products with significant homology to GenBank sequences Plasmid  Size of PCR product(bp)  Homologous Function genes  Original organism  pXL2  580  pbpB  PBP3  E. coli  39.4  63.2  penA  PBP2  N. gonorrhoeae  35.4  52.8  pbpB  PBP3  E. coli  37.5  62.5  penA  PBP2  N. gonorrhoeae  38.1  56.6  pXL3  580  Homology %Identity %Conservation  pXL5  380  yhhF  hypothetical protein  E. coli  53.6  62.7  pXL8  510  sucC  P-subunit of succinyl coenzyme A synthetase  E. coli  87.3  91.4  sucD  a-subunit of succinyl coenzyme A synthetase  E. coli  73.7  89.5  cypH  periplasmic peptidyl-propylcis-tran isomerase  E. coli  63.4  76.0  pXL12  460  44  amino acid compared with E. coli PBP3 and N. gonorrhoeae PBP2, respectively. The nucleotide sequences of these PCR products were 64.5% identical. The second PCR fragment was likely the product amplified from another region of the P A O l chromosome. Thus it seemed likely that P. aeruginosa has two copies of a PBP3 likeencoding gene. The second PCR product was subsequently used as a probe to clone the complete gene named pbpC. Three other PCR products were amplified by the same primers. One of the sequences cloned in pXL8 was 87.3% identical to amino acid residues 239-388 of the E. coli (3-subunit of succinyl coenzyme A synthetase encoded by sucC. The last nucleotide of the T A A stop codon was the first nucleotide of the probable methionine start codon of a stretch encoding 19 amino acids, 14 of which were identical to the product of the E. coli sucD gene which encodes the oc-subunit of succinyl coenzyme A synthetase and is linked to the sucC gene in E. coli. The %G+C of the third position of codons was 84%, which is typical of a high G+C organism like P. aeruginosa (West & Iglewski, 1988). A second sequence cloned in pXL5 was 53.6% identical to amino acids 1-104 of a hypothetical E. coli protein encoded by yhhF (formerly called ftsS) (Sofia et al, 1994). A third sequence cloned in pXL12 was 63.4% identical to residues 24-176 of an E. coli periplasmic peptidyl-prolyl cis-trans isomerase ('rotamase') encoded by rot or cypH. These sequences also had a high G+C content (83 % and 90 % for yhhF and cypH, respectively) in the third codon position.  45  3. Cloning of the pbpB and pbpC genes P. aeruginosa P A O l chromosome DNA, which had been digested with various restriction enzymes individually or in combination, was resolved by agarose gel electrophoresis, transferred to a nylon membrane, and probed with the radioactive labeled PCR products (probes for pbpB and pbpQ, respectively. This procedure was used to create two restriction enzyme maps (Fig. 4A & 5A). The resulting restriction maps were used for the construction of various subgenomic libraries to facilitate the cloning of the P. aeruginosa pbpB and pbpC genes. To clone the pbpB gene, an attempt was made to construct a library using the 6.0 kb Xhol fragments of P A O l chromosomal DNA. However, this approach was not successful. The cloning strategy was then changed to isolate three restriction fragments containing three different portions of the P A O l chromosomal D N A corresponding to the 1.4 kb Sphl, 1.8 kb PstI and 4.4 kb Smal-Xhol fragments respectively. These fragments were then ligated with digested pTZ19U and transformed into E. coli DH5oc. Three positive clones pSPHl, pPST18 and pXSml6 (Fig. 4B) were obtained after colony hybridization. However, none of these clones contained the entire pbpB gene. Therefore a 1 kb Smal fragment from pSPHl was cloned into the Smal site of pXSml6 to generate pXLSH36 (Fig. 4B). This plasmid contained a 5.4 kb Sphl-Xhol fragment of the P A O l chromosome D N A cloned in the vector pTZ19U. The 5.4 kb Sphl-Xhol fragment was isolated from pXLSH36 and cloned into a vector pTZ18U to generate p X L B B . p X L B B contained the P.  46  1  Sm _l  5m S.\ ^ P S X h S p S S P I Sj|Sp I I I I II L _ l  pbpB  SmSp I  M  L  3  I  P  Sm  xh  I  L  IT  MurE —>  >  Sm  3  Kb  S  p  I  pSPHl  pPST18 Sm  x/Sa  pXSml6 Sm Sp  N  ^  X/Sa pXLSH36  Figure 4. (A) Restriction map of the P. aeruginosa pbpB region. The open box indicates the location of the 580 bp PCR product. The solid bar indicates the sequenced 2.7 kb Sphl-Pstl region. The dashed boxes indicate the locations of the pbpB and murE ORFs. The arrows indicate the directions of transcriptions. (B) Restriction map of the various pbpB subclones. The dashed boxes represent the P A O l chromosomal D N A cloned in the vector pTZ19U. Abbreviations: P, Pstl; S, Sail; Sm, Smal; Sp, Sphl; X, Xhol; X/Sa: Xhol-Sall.  47  1  Sm  Xb X h Sa _i  K l  H l  kb  Sa  PEP ,11,1 L P  II I I I I I l"l~lr  pbpC soxR adh  B Xh/Sa  Xh/Sa  pXL-Xh401  Xh p 1, , , 1  Sa E  p X L - P S 4 06  E  Xh  -i i J J1 1 l_l 1 1 1 1 1 1 E  U 1 1 1 1 1 1 1 1 1  H  1 1 1 1  L  _i_J 1 11  E  1 1 l_l 1 1 1 1 1 1 1 1 JL — 11 xb  1  E  pXL-E405 pXL-KE24 pXL-Hd2 pXL-Xb5  Figure 5. (A) Restriction map of the P. aeruginosa pbpC region. The open box indicates the location of the 580 bp PCR product. The solid bar indicates the sequenced 3.2 kb Hindlll-Xhol region. The dashed boxes indicate the locations of the pbpC, soxR and adh ORFs respectively. The arrows indicate the directions of transcriptions. (B) Restriction map of the various pbpC subclones. The dashed boxes represent the P A O l chromosomal D N A cloned into the vector pTZ19U. Abbreviations: E, EcoRI; Ft, #mdIH; K, Kpnl; P, Pstl; Sa, Sail; Sm, Smal; Xb: Xbal; Xh, Xhol; X/Sa: Xhol-Sall.  48 aeruginosa pbpB gene under the control of the lac promoter (Liao & Hancock, 1995). To clone the pbpC gene, an attempt was made to construct a library using the 6.0 kb Sail fragments of P A O l chromosomal DNA. However, this was not successful. The cloning strategy was therefore changed to isolate three restriction fragments containing three different portions of the P A O l chromosomal D N A corresponding to the 2.5 kb Xhol, 4.0 kb Pstl-Sall and 2.0 kb EcoRI fragments, respectively. These fragments were then ligated with the digested pTZ19U and transformed into E. coli DH5a. Three positive clones pXL-Xh401, pXL-PS406 and pXL-E405 (Fig. 5B) were obtained after colony hybridization. However, none of these clones contained the entire pbpC gene. Therefore, a 1.9 kb Kpnl-Xhol fragment from pXL-PS406 was ligated with a 1.8 kb Xhol-EcoRl fragment from pXL-E405 and the resultant fragment was cloned into the Kpnl and .EcoRI sites on the vector pTZ19U to create the plasmid pXL-KE24 (Fig. 5B). Two subclones pXL-Hd2 and pXL-Xb5 were subsequently created after the deletion of the 1 kb Kpnl-Hindlll fragment and the 1.4 kb Kpnl-Xbal fragment from pXL-KE24, respectively (Fig. 5B).  4. DNA sequence analyses of the pbpB and pbpC loci 4.1 pbpB Both strands of the 2.7 kb Sphl-Psil region were sequenced after a series of overlapping nested deletion clones were created from pSPHl and pPST18, respectively. Two putative open reading frames (ORFs) were found in this sequence  49 (Fig. 6). They were located at nucleotides 44 through 1783 and 1783 through 2757. The second ORF appeared incomplete at its 3' end. The first ORF (ORF1) encoded a 579 amino acid sequence with 45% identity and a total of 57.7% conserved amino acids compared to the E. coli PBP3 (Table 5). It was preceded by a putative RBS sequence A G G A at nucleotides 30-34, which matched reasonably to a consensus RBS sequence. The N-terminal amino acid sequence of the pbpB gene product was M K L N Y F (see below) which showed that the potential initiation codon A T G at nucleotides 116-118 was not used. The third position of codons comprised 85.8% G+C, typical of P. aeruginosa genes. No potential transcription terminator sequences were identified downstream of the ORF1. The second putative ORF (ORF2) was incomplete and encoded 325 amino acids. The first nucleotide of the putative A T G start codon for ORF2 is the last nucleotide of the T G A stop codon for the ORF1. The sequence of the ORF2 was 43% identical and 65% similar to amino acids 1-337 of the E. coli murE gene product, which encodes uridine diphosphate-N-acetyl muramic acid-tripeptide synthetase (Michand et al, 1990). A putative RBS sequence A G G A was located at nucleotides 1768-1771. The third position of codons comprised 86.8% G+C.  4.2 pbpC Both strands of the 3.2 kb Hindlll-Xhol region were sequenced using the nested deletion clones of pXL-Xh401 and pXL-Hd2. Three putative open reading  50  Fig. 6 Sphl 1  GCATGCGCGTTCCCGACCCGGCCGAGGTCAGGATGGTGGCGCCATGAAACTGAATTATTT  1 61 7  pbpB ->  M  K  L  N  Y  F  CCAGGGCGCCCTCTACCCATGGCGGTTCTGCGTGATCGTCGGCCTGCTGCTGGCGATGGT Q G A L Y P W R F C V I V G L L L A M V  Sail 12i 27  CGGCGCCATCGTCTGGCGAATCGTCGACCTGCACGTGATCGACCATGACTTCCTCAAGGG G A I V W R I V D L H V I D H D F L K G  181 47  CCAGGGCGACGCGCGTAGCGTGCGGCATATCGCCATCCCTGCGCACCGGGGGCTGATCAC Q G D A R S V R H I A I P A H R G L I T  241 67  TGACCGCAACGGCGAACCGCTGGCGGTGAGCACCCCGGTCACCACCCTGTGGGCCAACCC D R N G E P L A V S T P V T T L W A N P  3 01 87  CAAGGAGCTGATGACCGCCAAGGAACGCTGGCCGCAACTGGCGGCGGCGCTCGGGCAGGA K E L M T A K E R W P Q L A A A L G Q D  3 61 107  TACCAAGCTGTTCGCCGACCGCATCGAGCAGAACGCCGAGCGCGAGTTCATCTATCTGGT T K L F A D R I E Q N A E R E F I Y L V  421 127  CCGTGGGCTGACCCCGGAGCAGGGCGAAGGCGTGATCGCCCTGAAGGTGCCCGGCGTGTA R G L T P E Q G E G V I A L K V P G V Y  481 147  CTCCATCGAGGAGTTTCGGCGTTTCTACCCGGCTGGCGAAGTGGTGGCCCATGCGGTCGG S I E E F R R F Y P A G E V V A H A V G  541 167  CTTTACCGATGTCGACGACCGCGGTCGCGAAGGTATCGAGCTGGCTTTCGACGAATGGCT F T D V D D R G R E G I E L A F D E W L  601 187  GGCCGGCGTGCCGGGCAAGCGCCAGGTGCTCAAGGATCGCCGTGGCCGCGTGATCAAGGA A G V P G . K R Q V L K D R R G R V I K D  6 61 207  CGTGCAGGTCACCAAGAATGCCAAACCGGGCAAGACCCTTGCGCTGTCCATCGACCTGCG V Q V T K N A K P G K T L A L S I D L R  721 227  CCTGCAGTACCTGGCTCATCGCGAACTGCGCAACGCTCTGCTGGAAAACGGCGCCAAGGC L Q Y L A H R E L R N . A L L E N G A K A  7 81 247  CGGCAGCTTGGTGATCATGGACGTGAAGACCGGGGAGATCCTGGCCATGACCAACCAGCC G S L V I M D V K T G E I L A M T N Q P  841 267  CACCTACAACCCGAACAATCGTCGTAACCTGCAGCCGGCGGCCATGCGCAACCGGGCGAT T Y N P N N R R N L Q P A A M R N R A M  9 01 287  GATCGACGTGTTCGAGCCGGGCTCGACGGTCAAGCCGTTCTCGATGAGCGCGGCGCTGGC I D V F E P G S T V K P F S M S A A L A  9 61 307  CAGCGGGCGCTGGAAACCCAGCGATATCGTCGACGTCTACCCGGGCACCCTGCAGATCGG S G R W K P S D I V D V Y P G T L Q I G  1021 327  CCGCTACACCATTCGCGACGTATCGCGCAATTCGCGGCAACTCGATCTCACCGGCATCCT R Y T I R D V S R N S R Q L D L T G I L  1081 347  GATCAAGTCGAGCAACGTCGGCATCAGCAAGATCGCCTTCGACATCGGCGCCGAATCCAT I K S S N V G I S K I A F D I G A E S I  Sail  Pstl  •Sail  Steal  Pstl  51 Fig. 6 1141 3 67  CTACTCGGTCATGCAACAGGTCGGTCTCGGGCAGGACACGGGGTTGGGCTTCCCCGGCGA Y S V M Q Q V G L G Q D T G L G F P G E  12 01 3 87  GCGCGTCGGCAACCTGCCCAACCACCGCAAGTGGCCGAAGGCGGAAACCGCGACCCTGGC R V G N L P N H R K W P K A E T A - T L A SphI CTACGGCTACGGTCTCTCGGTAACCGCGATCCAGTTGGCGCATGCCTATGCGGCCCTGGC Y G Y G L S V T A I Q L A H A Y A A L A  12 61 407 13 21 427 13 81 447  CAACGACGGCAAGAGCGTGCCGCTGAGCATGACCCGAGTCGACCGCGTGCCGGATGGTGT N D G K S V P L S M T R V D R V P D G V SphI GCAGGTGATCTCGCCTGAAGTGGCTTCCACCGTGCAGGGCATGCTGCAACAAGTGGTCGA Q V I S P E V A S T V Q G M L Q Q V V E  1441 467  GGCCCAGGGCGGGGTGTTCCGCGCCCAGGTGCCGGGTTACCACGCCGCCGGCAAGAGCGG A Q G G V F R A Q V P G Y H A A G K S G  1501 487  GACCGCGCGCAAGGTCTCGGTCGGCACCAAGGGCTACCGGGAAAACGCCTATCGCTCGCT T A R K V S V G T K G Y R E N A Y R S L  1561 507  GTTCGCCGGTTTCGCCCCGGCCACCGATCCGCGCATCGCGATGGTCGTGGTGATCGACGA F A G F A P A T D P R I A M V V V I D E  1621 527  GCCGAGCAAGGCGGGCTACTTCGGCGGCCTGGTGTCGGCGCCGGTGTTCAGTAAGGTCAT P S K A G Y F G G L V S A P V F S K V M  1681 547  GGCTGGCGCGCTGCGCCTGATGAACGTGCCGCCGGATAACCTGCCGACGGCCACCGAACA A G A L R L M N V P P D N L P T A T E Q  1741 5 67  1861 27  GCAGCAGGTCAATGCTGCGCCCGCAAAAGGAGGGCGTGGCTGATGCCTATGAGCCTGAGC Q Q V N A A P A K G G R G * M P M S L S murE —» CAACTGTTTCCCCAGGCCGAGCGCGATCTGCTGATCCGCGAGCTGACCCTGGATAGCCAC Q L F P Q A E R D L L I R E L T L D S H Sail GGCGTTCGTCCGGTCGACCTGTTCCTGACGGTTCCGGGCGGGCACCAGGATGGTCGTGCG G V R P V D L F L T V P G G H Q D G R A  1921 47  CACATCGCCGATGCCCTGACCAAGGGCGCGACTGCCGTGGCTTACGAGGCGGAAGGCGCC H I A D A L T K G A T A V A Y E A E G A  1981 67  GGAGAGTTGCCGCCCAGCGATGCGCCGCTGATCGCGGTGAAGGGGCTGGCCGCGCAACTG G E L P P S D A P L I A V K G L A A Q L  2 041 87  TCGGCGGTCGCCGGGCGTTTCTACGGCGAGCCGAGCCGCGGGCTGGACCTGATCGGCGTC S A V A G R F Y G E P S R G L D L I G V  2101 107  ACCGGCACCAACGGCAAGACCAGCGTCAGCCAACTGGTGGCCCAGGCCCTGGATCTGCTC T G T N G K T S V S Q L V A Q A L D L L  2161 127  GGCGAGCGCTGCGGCATCGTCGGCACCCTCGGCACCGGTTTCTACGGCGCCCTGGAGAGC G E R C G I V G T L G T G F Y G A L E S  2221 147  GGCCGGCACACCACGCCGGACCCGCTCGCGGTGCAGGCCACGCTGGCCACGCTGAAGCAG G R H T T P D P L A V Q A T L A T L K Q  22 81 167  GCCGGCGCCCGCGCGGTAGCGATGGAAGTGTCTTCCCACGGCCTCGACCAGGGCCGCGTG A G A R A V A M E • V S S H G L D Q G R V  1801 7  52  2341 187  GCGGCGCTCGGCTTCGATATCGCGGTGTTCACCAATCTGTCCCGCGACCACCTCGACTAT A A L G F D I A V F T N L S R D H L D Y  2401 207  CACGGTTCGATGGAAGCCTATGCCGCCGCCAAGGCCAAGCTGTTCGCCTGGCCGGACCTG H G S M E A Y A A A K A K L F A W P D L  2461 227  CGCTGCCGGGTGATCAACCTGGACGACGATTTCGGCCGTCGACTGGCCGGCGAGGAGCAG R C R V I N L D D D F G R R L A G E E Q  2 521 2 47  GACTCGGAGCTGATCACCTACAGCCTCACCGACAGCTCGGCGTTCCTCTATTGCCGCGAA D S E L I T Y S L T D S S A F L Y C R E  2 581 267  GCGCGCTTCGGCGACGCCGGCATCGAGGCGGCGCTGGTCACTCCGCACGGCGAGGGCCTG A R F G D A G I E A A L V T P H G E G L  2 641 287  CTGCGCAGCCCGTTGCTCGGCCGCTTCAACCTGAGCAACCTGCTGGCGGCGGTCGGTGCG L R S P L L G R F N L S . N L L A A V G A Pstl TTGCTTGGCCTGGGTTATCCCCTGGGCGATATCCTCCGCACTTTGCCGCAGCTGCAG L L G L G Y P L G D I L R T L P Q L Q  2 701 307  Figure 6. Nucleotide sequence of the P. aeruginosa pbpB region and the deduced amino acid sequences of two ORFs. The numbers on the left designate the nucleotide or amino acid at the left end of each row. The conserved motifs of PBP3 are bold and underlined. The putative RBS sequences are underlined. The arrows indicate direction of transcription. Sequences recognized by the restriction enzymes are underlined and indicated by the name beside.  Table 5. Percent identities and total conservation of the amino acid sequences of PBP3 and PBP3x to those of other PBPs* Protein  Accession number  Number of amino acids  % PBP3x  % PBP3 identity  conservation  identity  conservation  PBP3  X84053  579  100  100  48.3  63.2  PBP3x  X95517  565  48.3  63.2  100  100  EcoliPBP3  K00137  588  45.1  57.7  40.7  52.9  NgorPBP2  X07469  583  37.7  52.6  35.8  49.6  HinfPBP3  L45768  610  32.6  53.7  31.9  45.4  BsubSPOVD  Z25865  645  25.7  39.0  26.7  38.9  HinfPBP2  L44676  651  23.5  36.3  21.9  35.4  EcoliPBP2  X04516  633  22.8  36.4  20.4  33.9  BsubPBP2B  L09703  716  21.8  34.4  23.0  35.9  * The protein sequences were analyzed by the PC gene program using the genetic-code matrix with an open gap cost of 6 and an unit gap cost of 20. Similar amino acids include: AST; DE; NQ; RX; ILMV and FYW. EcoliPBP3, E. coli PBP3; NgorPBP2, N. gonorrhoeae PBP2; HinfPBP3, H. influenzae PBP3; BsubSPOVD, B. subtilis SPOVD protein; HinfPBP2, H. influenzae PBP2; EcoliPBP2, E. coli PBP2; BsubPBP2B, B. subtilis PBP2B.  54 frames were found in this sequence (Fig. 7). They were located at nucleotides 373 through 2070 (ORF1), 2078 through 2548 (ORF2) and 3028 through 3274 (ORF3) respectively. The third ORF was incomplete at its 5' end. The first ORF would be transcribed in the opposite orientation to the second and third ORFs. ORF1 encoded a 565 amino acid sequence with 40.7% identity and a total of 52.9% conserved amino acids compared to E. coli PBP3 (Table 5). A putative RBS sequence and two promoter-like sequences potentially recognized by a and a , s  70  respectively, were identified in the upstream region (Fig. 7). The third position of codons comprised 93% G+C. This amino acid sequence showed 48.3% identity and 63.2% conserved amino acids relative to that of the pbpB gene product (Table 5). ORF2 encoded a protein sequence of 156 amino acids which had 62% identity and a total of 78% conserved amino acids relative to E. coli soxR gene product E. coli SoxR protein is activated by superoxide-generating agents or nitric oxide and is a transcriptional activator of the soxS gene. The soxS gene product activates approximately 10 other promoters (Hidalgo et al, 1994; L i et al, 1994). In E. coli soxR and soxSare adjacent to one another (Wu et al, 1991). However, between the P. aeruginosa soxR (ORF2) and the ORF3, no soxS-like gene product was identified and this sequence showed no significant homology to any sequences in the GenBank database. ORF3 was incomplete at its 5' end and encoded 82 amino acids. This sequence showed 57.3% identity and 76.8% conserved amino acids compared to amino acids  55  Fig. 7 1  TCCACCAACAACAGGGCCTGGGCCGCCGCCTGCTGGAGCGCGCGGTGACCTACGCCCACG  61  CCAGCCACTGCCGGGCGCTGACCCTGACGACCTTCTGCGACGTGCCCTGGAACGCACCGT  121  TCTACGCACGCCTGGGCTTCCAGCGGCTGACCTGGCAGGAAGCCGGCGAGCGCTTGCGCG  181 241  CGATCCTCGGCCACGAGCAGGAGATCGGCTTCGCCGCCGACAGCCGCTGCGCGATGCGCC Xbal TGGTGCTCTAGACGGCggCAAAGGGC TTGACCGGCGGGTGGCGGGTGACGGTACAGTTGC  3 01  CAACTGCAACAGGATGTTTCAGCACTGCCCCGGACGGGCTTCCGCTCTCCCTCTTCCCCA  3 61 1  GTGTGCCCTTGCATGAGCAGTCAACGCCGAAACTACCGCTTCATCCTTGTCGTCACCCTG pbpC -> M S S Q R R N Y R F I L V V T L  421 17 481 37  TTCGTCCTCGCCTCCCTGGCCGTCTCCGGACGGTTGGTCTATCTCCAGGTCCACGACCAC F V L A S L A V S G R L V Y L Q V H D H EcoRI GAATTCCTCGCCGACCAGGGCGACCTCCGCTCGATCCGCGACCTGCCGATCCCGGTCACC E F L A D Q G D L R S I R D L P I P V T  541 57  CGCGGCATGATCACCGACCGCAACGGCGAGCCGCTGGCGGTATCCACCGAAGTCGCGTCG R G M I T D R N G E P L A V S T E V A S  601 77  ATCTGGTGCAACCCCAGGGAAATGGCCGCCCACCTCGACGAGGTGCCGCGCCTGGCCGGC I W C N P R E M A A H L D E V P R . L A G  661 97  GCCCTGCACCGCCCCGCGGCGGCGCTGCTGGCCCAGCTCCAGGCCAACCCGAACAAGCGC A L H R P A A A L L A Q L Q A N P N K R  721 117  TTCCTCTACCTCGAGCGCGGCCTGTCGCCGATCGAGGCCAGCGAGGTGATGGCCCTGGGC F L Y L E R G L S P I E A S E V M A L G  781 137  ATAAC GGGGGTACAC CAGATCAAGGAATACAAGCGTTTC TAC C C CAGTTC C GAGC TGAC C I T G V H Q I K E Y K R F Y P S S E L T  841 157  GCGCAGTTGATCGGCCTGGTCAACATCGACGGCCGCGGCCAGGAAGGCACCGAACTGGGC A Q L I G L V N I D G R G Q E G T E L G  901 177  TTCAACGACTGGCTGAGCGGCAAGGACGGGGTACGCGAGGTGGCGATCAACCCGCGCGGC F N D W L S G K D G V R E V A I N P R G  9 61 197  TCGCTGGTCAACAGCATCAAGGTGCTGAAGACGCCCAAGGCCAGCCAGGACGTGGCCCTG S L V N S I K V L K T P K A S Q D V A L  1021 217  AGCATCGACCTGCGACTACAGTTCATCGCCTACAAGGCGCTGGAAAAGGCCGTGCTCAAG S I D L R L Q F I A Y K A L E K A V L K  1081 237  TTCGGCGCGCACTCCGGCTCGGCGGTCCTGGTGAACCCGAAGAGCGGGCAGATCCTGGCG F G A H S G S A V L V N P K S G Q I L A  1141 257  ATGGCCAACTTCCCCTCCTACAACCCGAACAACCGCGCCAGCTTCGCCCCGGCCTTCATG M A N F P S Y N P N N R A S F A P A F M  12 01 277  CGCAACCGCACCCTCACCGATACCTTCGAGCCGGGCTCGGTGATCAAGCCGTTCAGCATG R N R T L T D T F E P G S V I K P F S M  Xhol  56 Fig. 7 12 61 297  TCGGCGGCGCTGGCCTCCGGCAAGTTCGACGAGAACAGTCAAGTCAGCGTGGCACCGGGC S A A L A S G K F D E N S Q V S V A P G  13 21 317  TGGATGACCATCGACGGGCACACCATCCACGACGTCGCCCGGCGCGACGTACTGACCATG W M T I D G H T I H D V A R R D V L T M Pstl ACCGGGGTGCTGATCAACTCCTCGAACATCGGCATGAGCAAGGTCGCCCTGCAGATCGGA T G V L I N S S N I G M S K V A L Q I G  13 81 337 1441 357 1501 ' 377  CCCAAGCCGATCCTCGAACAGCTCGGCCGGGTCGGTTTCGGCGCGCCGCTGTCGCTGGGC P K P I L E Q L G R V G F G A P L S L G Smal TTCCCCGGCGAGAACCCGGGCTACCTGCCGTTCCACGAGAAATGGTCGAACATCGCCACC F P G E N P G Y L P F H E K W S N I A T  15 61 397  GCCAGCATGTCGTTCGGCTACAGCCTGGCGGTGAACACCGCCGAGCTGGCCCAGGCCTAC A S M S F G Y S L A V N T A E L A Q A Y  1621 417  TCGGTGTTCGCCAACGACGGCAAGCTGGTGCCGCTCAGCCTGCTCCGCGACAACCCGCAG S V F A N D G K L V P L S L L R D N P Q  1681 437  AACCAGGTGCGACAGGCGATGGACCCGCAGATCGCACGGCGCATCCGGGCGATGCTGCAA N Q V R Q A M D P Q I A R R I R A M L Q  1741 457  ACCGTGGTGGAAGACCCGAAGGGCGTGGTCCGCGCCCGCGTGCCGGGCTACCACGTGGCG T V V E D P K G V V R A R V P G Y H V A  1801 477  GGCAAGAGCGGCACCGCGCGCAAGGCCTCGGGCCGGGGCTACGCGGACAAGTCCTACCGT G K S G T A R K A S G R G Y A D K S Y R  1861 497  TCGCTGTTCGTCGGCATGGCGCCGGCGTCCGACCCGCAACTGGTGCTGGCGGTGATGATC S L F V G M A P A S D P Q L V L A V M I  1921 517 1981 537  GATTCGCCGACCAGGATCGGCTACTTCGGCGGCCTGGTCTCGGCGCCCACCTTCAACGAC D S P T R I G Y F G G L V S A P T F N D Pstl ATCATGGCCGGATCGCTACGCGCCCTGGCGATCCCGCCGGACAACCTGCAGGACAGCCCG I M A G S L R A L A I P P D N L Q D S P  2 041 557  GCCGTCGCCGACCGCCAGCACCACGGCTGACCGCTGCCTAGCCGTCGTGCTCGCGGCCCT A V A D R Q H H G * * G D H E R G E150  2101  CGGCGTCCAGCCAGTGCGCTCCCGGCCCCTCGGCGGAAAGCTGGTCGCCGGGGTTGCGCA A D L W H A G P G E A S L Q D G P N R L130  2161  ACGGGCAGGCCTGGAGCGACAGGCAGCCGCAACCGATGCAGCCGTCCAGTTGGTCGCGCA P C A Q L S L C G C G I C G D L Q D R L110  2221  ACAGCAGCAGCTTGTCGATGCGCTCGGTGAGATCCTCCTTCCACTGCGCCGACAGGCGCG L L L K D I R E T L D E K W Q A S L R A 9 0  2281  CCCAGTCCGCCGCGCTAGGGCTGCGCCCCGCCGGCAGGGTCTGCAGGGCGCGAGCGATCT W D A A S P S R G A P L T Q L A R A I E 7 0  2341  CCGCGAGGGGAATGCCGACCCGCTGGGCGACCTTGATCACCACCACCCGGCGTAGCGTCT A L P I G V R Q A V K I V V V R R L T E 5 0  2401  CGCGACTGAAGCGCCGCTGGTTGCCGGCGTTGCGCTGGCTGCTGATCAGCCCCTTGGTTT R S F R R Q N G A N R Q S S I L G K T E 3 0  57 Fig. 7 24 61 2 521  CGTAGAAATGCAGGGCGGAGACCGCCACGCCGGCACGCCTGGCCAGTTCGCCGACGCTCA Y F H L A S V A V G A R R A L E G V S L 1 0 EcoRI GTTCACGAGATGCGCAGGAATTCTTCATGATTGGCTTGACCTCAAGATTGCTTGAGGTTT E  R  S  A  C  S  N  K  M  <- soxR  2 581  TACCCTGGGCCGGCCCTCGACACAAGTCACCCGGAGTAACCCGCCATGCTCATCCCCTCC  2 641  TCCTACTGCTGCACCGACGATCAGTGCCCGGCCGCTAGCGAGCGGTCGCCGTGCTACGCG  27 01  CAACTCATCGATATCGAGGTGGAACCCGCCAGCCAGTTGTCGCTGGCGCTGGAACAGAAC •Smal GCGCACCTGCAACGCCTGGAACGCTGCTTCAGGAACTGCCCGGGCTACCTGTCCGCCAGC  2 7 61 2 821 2 881 2 941 3 001  CTGCACCCCAGCGAGGACGGGCAGCACGTGCTGAACTACACCTGCTGGCGTTCACGCGAG Smal GACTGCGAACGCGCCTGGCTGGCCCGGGAGGACGCGCAAGGCCCGCTGAGCGCGGGCGTC Sail TGGCGGCTGGGGGCGAAAAGCGTGCGTTTCGAGACCTTCCTGGTCGACGCCGAGGGCTGC TGAGCCCGCCGGCGCCGACGGCCCGTCTCAGAAGGACTTCTGCAGGGAGGCCATGTCGAT * F S K Q L S A M D I 73  3 0 61  CACGAAGCGATACTTCACGTCGCTGGCCAGCATCCGCTCGTAGGCCTGGTTGATCTGCTG V F R Y K V D S A L M R E Y A Q N I Q Q 5 3  3121  GATCTCGATCATCTCGATGTCGCAGGCGATCCCGTGGGCCGCGCAGAAATCCAGCATTTC I E I M E I D C A I G H A A C F D L M E 3 3  3181 3 2 41  1  CTGGGTCTCGGCGATGCCGCCGATGGCCGAGCCGGCGATCGAGCGGCGTCCCATCACCAG Q T E A I G G I A S G A I S R R G M V L 1 3 Xhol TTGCGCACCGTGCACCGCCGGCTCCAGCGGCTCGAG Q A G H V A P E L P E L <- adh 1  Figure 7. Nucleotide sequence of the P. aeruginosa pbpC region and the deduced amino acid sequences of three ORFs. The numbers on both the left and right designate the nucleotide or amino acid at the end of each row. The conserved motifs of PBP3x are bold and underlined. The putative RBS sequence is bold. The putative promoter sequences recognized by c are bolded italic and underlined. The putative promoter sequences recognized by a are italic and underlined. The arrows indicate the directions of transcriptions. The conserved motifs of PBP3x are bold and underlined. Sequences recognized by the restriction enzymes are underlined and indicated by the name beside. s  7 0  58  219-346 of the Mycobacterium bovis adh gene product, an NADP-dependent alcohol dehydrogenase (Stelandre etal., 1992).  5. Features of the pbpB and pbpC gene products and comparison with  other PBPs All of the high-molecular-weight PBPs studied to date are known to be cytoplasmic membrane proteins with their hydrophobic amino terminus anchored in the cytoplasmic membrane. Hydropathy analyses of the deduced amino acid sequences of both the P. aeruginosa PBP3 and PBP3x showed similar profiles to that of E. coli PBP3 (data not shown). Analysis of membrane spanning segments by the PC Gene computer program predicted that there was one transmembrane segment for each of the pbpB and pbpC gene product. The transmembrane domains were localized to residues 15 to 31 and 10 to 26 as the inner boundaries and residues 8 to 39 and 7 to 31 as the outer boundaries for the pbpB and pbpC gene products, respectively. These results suggested that P. aeruginosa PBP3 and PBP3x were cytoplasmic membrane-associated proteins. Nine conserved motifs or boxes within the amino acid sequences of the high molecular weight PBPs have been identified as being unique to the class B highmolecular-weight PBPs by Piras et al. (1993) and Ghuysen (1994). As shown in Fig. 8, P. aeruginosa PBP3, PBP3x and E. coli PBP3 were conserved in all of the nine boxes. The motifs SXXK, SXN and K T G were located in the C-terminal portion and  59  PBP3 PBP3x ECPBP3  M K L N Y F Q G A L Y P  PBP3 PBP3x ECPBP3  S V R H I A l P A H R G L I T D R N G E P L A V S T P V T T L W A N P K E L M T A K E R W P Q L A A A L G Q D T K L F A  PBP3 PBP3x ECPBP3  W R F C V I V G L L L A M V G A I V W R I V D L H V I D H D F L K G Q G D A R  -ssqrrnyrfil w t l fvlas-avsgrlvylqvh-hef lad 1--aaaktqkpkrqeehanfis allc-ci-lalafllg-vaw-q--sp-m-vke--m-i-dlp--vt--m -l-vqqvsts--m D R I E Q N A E R E  s-r Box 1  e-asi-c--r-maahldev-r--g--hrpaaall v--kai--d vhd-ggisvgdrwkalanalnip  F I Y L V R G L T P E Q G E G V I A L K V P G V Y S I E E F  RRF Y P A G E W A H A V G  aqlqa-pnkr - l - - e s-iease-m--git--hq-k-yk ss-lt-qlildqlsarinanpkgra-qvn-dmadyikk--l--ihlr--s--y--s t--liBox 2 :  PBP3 PBP3x ECPBP3  F T D V D D R G R E G I E L A F D E W L A G V P G K R Q V L K D R R G R V I K D V Q V T K N A K P G K T L A L S I D L R  PBP3 PBP3x ECPBP3  L Q Y L A H R E L R N A L L E N G A K S L G S L V I M D V K T G E I L A M T N Q P T Y N P N N R R N L Q P A A M R N R A M  PBP3 PBP3x ECPBP3  I D V F E P G S T V K P F S M S A A L A S G R W K P S D I V D V Y P G T L Q I G R Y T I R D V S R N S R Q L D L T G I L  PBP3 PBP3x ECPBP3  lvni-g--q--t—g-nd--s-kd-v-e-ainp--slvnsik-l-tp-asqdv --n--sq-i--v-ks--k--t-q--e-i-r y e-iss-dsqaah.n eBox 3 Box 4  —fi-yka-ek-v-kf--hs--a-lvnp-s-q a-f-s —a-vy n--vaf-k-es--a-lv--n v a-s-s Box 5 t-t t  asfa--f lsgtpke  kf densq-s-a--wmt-dgh--h--a-rdvltmtgvli mwmt--qr-wrensvlnti-yringheikdvaryselt. .. vBox 6  I K S S N V G I S K I A F D I G A E S I Y S V M Q Q V G L G Q D T G L G F P G E R V G N L P N H R K W P K A E T A T L A  np-y--f-e--snia--sms s-ly-qkqr-sdi-r--f s  PBP3 PBP3x ECPBP3  Y G Y G L S V T A I Q L A H A Y A A L A N D G K S V P L S M T R V D R V P D G V Q V I S P E V A S T V Q G M L Q Q W E  PBP3 PBP3x ECPBP3  A Q G G V F R A Q V P G Y H A A G K S G T A R K V S V G T K G Y R E N A Y R S L F A G F A P A T D P R I A M V W I D E  PBP3 PBP3x ECPBP3  P S K A G Y F G G L V S A P V F S K V M A G A L R L M N V P P D N L P T A T E Q Q Q V N A A P A K G G R G  f--s-a-ntae--q--svf 1 f m--pl rv--tigsy-iyr  11-dnpqnqvr-amd-qi-rrira 1 i-k--ppvp-er-fpesivr--vh-mes-al  dpk--v--r v a-grgyadks.. v-m s--qlvla-m--s pg--gvk-aik--ri-i-t k--gpdgryinkyiaytagv.. sq--f-l nd Box 8 Box 9 - t r i -qagk-y--a  t-ndi s--alai qdspavadrqhhg gai-g-v--t--ie--a-t-gdknef-inqgegt-grs  111 106 120 166 161 180 22 6 221 239  286 t l 281 t i 299  vi  n. i-m--v-lq--pkp-leqlgr--f-apls q v--l-lampssalvdtysrf ka-n--lv Box 7  51 46 60  346 341 356 40 6 400 416 466 460 47 6 52 6 518 534 579 565 588  Figure 8. Amino acid sequence alignments of P. aeruginosa PBP3, PBP3x and E. coli PBP3. The protein sequences were aligned by the D N A M A N software program. Gaps introduced into the sequences are indicated by periods. Positions at which identical residues are found are indicated by - . The residues corresponding to the conserved boxes are bold. The numbers on the right correspond to the last residue in that row for each protein. The sequences are as follows: PBP3, P. aeruginosa PBP3; PBP3x, P. aeruginosa PBP3x; ECPBP3, E. coli PBP3.  belonged to boxes 6, 7 and 8 respectively. The active-site serine residue that binds to penicillin and is typically a part of the S X X K motif (box 6), was presumably located at residues 294 and 289 of pbpB and pbpC gene products. The SXN motif (boxes 7) was located at residues 349 to 351 and 343 to 345, while the K T G motif (box 8) was found at residues 484 to 486 and 478 to 480 of pbpB and pbpC gene product, respectively. As observed for other class B high molecular weight PBPs, the spacings between these boxes in the protein sequences were highly conserved. Further analyses of the amino acid sequences of PBP3 and PBP3x with those of other PBPs indicated that both of these two proteins were more related to E. coli PBP3, N. gonorrhoeae PBP2 and H. influenzae PBP3, a group of PBPs functioning in cell-wall septum formation of Gram negative bacteria. The sequences of these two proteins were found to be similar, but to a lesser degree, to the E. coli PBP3-like proteins in B. subtilis, PBP2B and spoVD protein, and to the E. coli and H. influenzae PBP2 (Table 5).  6. Chromosomal locations of the genes cloned in this study All plasmids described in Table 4 were examined by Dr. R. Levesque, Laval University and Dr. J. Lam, University of Guelph, respectively, to assist in the P. aeruginosa genome sequencing project. As the result of these collaborations, the physical locations of the genes cloned in this study were mapped by hybridization to the P A O l chromosome (Fig. 9) (Liao et ah, 1996). Of note, pbpB and pbpC mapped  61  0  Spel  Dpnl  MB -  OriC  o" H  yhhF  W Fl  A  L H M  cypH 2— V  •pbpC,  soxR,  sucC,  sucD  adh  X  3—  N  4— A  I  pbpB, murE  J 0  F2 OriC  Figure 9. Physical map of the 5.94 Mb genome of P A O l . The locations of the genes are indicated by the horizontal lines. Dpnl and Spel fragments are identified in capital letters. The smaller Spel fragments are not indicated.  62 more than 2 Mb apart on the chromosome. The pbpB gene mapped to exactly the same genomic fragments as theftsA and envZ genes, suggesting that pbpB and murE may be part of a large cluster of genes involved in cell division as observed in E. coli.  7. Summary Two degenerate oligonucleotides were synthesized according to the amino acid sequences found in the conserved motifs of E. coli high-molecular-weight PBPs and N. gonorrhoeae PBP2 and subsequently used in a degenerate PCR amplification experiment using P. aeruginosa P A O l chromosomal D N A as the template. Five of the PCR products were cloned and sequenced: two were found to translate to sequences with strong homology to E. coli PBP3 and N. gonorrhoeae PBP2 and were subsequently used as probes to clone the complete pbpB and pbpC genes; the other three PCR products were identified as the homologues of the E. coli gene products sucC/sucD, yhhF and cypH respectively. The physical locations of these genes on the genome of P. aeruginosa P A O l were established in collaboration with others. Probing of chromosomal digests separately with the two PCR products allowed the pbpB and pbpC genes to be cloned in E. coli. D N A sequence analysis of the pbpB region confirmed the cloning and led to the identification of an ORF in the downstream sequence, which encoded an amino acid sequence homologous to the E. coli murE gene product. D N A sequence analysis of the pbpC region revealed that P. aeruginosa contains a second copy of the gene coding for PBP3. This was confirmed  63  by the physical locations of the pbpB and pbpC genes on the P A O l genome. Two putative ORFs were identified downstream of pbpC. These ORFs were found to be homologues of the E. coli soxR and M. bovis adh gene products, respectively. Analyses of the deduced amino acid sequences of the pbpB and pbpC gene products suggested they were both integral cytoplasmic membrane proteins containing the nine conserved amino acid sequence motifs which have been identified as common to the class B, high-molecular-weight PBPs.  64 CHAPTER TWO Expression of the P. aeruginosa pbpB Gene Product and  Mutational Analysis of the pbpB Gene  1. Introduction Evidence of similar amino acid sequences and modular design between the P. aeruginosa and E. colipbpB gene products strongly supported the hypothesis that the cloned P. aeruginosa pbpB gene encoded an E. coli PBP3-like protein. P. aeruginosa PBP3 is an important killing target for the newer generation P-lactam antibiotics (Watanabe et al., 1988; Maejima et al, 1991). It was observed by Godfrey et al. (1981) that PBP3 and /or PBP6 of some P-lactam-resistant clinical isolates of P. aeruginosa from cystic fibrosis patients had apparently lost their binding abilities to radiolabeled penicillin. Moreover, a laboratory mutant strain that is resistant to cefsulodin has been reported and its phenotype is associated with reduced PBP3 binding affinity (Gotoh et al, 1990). Therefore, it seems likely that P. aeruginosa PBP3 plays an important role in susceptibility to P-lactam antibiotics. This chapter describes the characterization of this protein and the effect of its overproduction on the susceptibility of P. aeruginosa to P-lactam antibiotics.  2. Expression of the P. aeruginosa pbpB gene product in E. coli  2.1. Preliminary work  65  E. coli DH5oc was transformed with p X L B B which contained the P. aeruginosa pbpB gene as a 5.4 kb Sphl-Xhol fragment cloned in the same orientation as the lac promoter on the vector pTZ18U (see Chapter One, section 3). Following 3  induction with IPTG, membrane protein samples were collected and used in H penicillin binding assays. However, no novel PBP protein corresponding to the molecular mass of P. aeruginosa PBP3 was detected (data not shown). This might have been due to weak expression of the pbpB gene product or instability of the mRNA or the translational product. Accordingly, a more efficient expression system based on the T7 RNA polymerase and the corresponding promoter (Tabor & Richardson, 1985) was used for the expression of the P. aeruginosa pbpB gene product in E. coli.  2.2 pT7-7 as the expression vector The expression vector pT7-7 contains the T7 RNA polymerase promoter and a RBS sequence followed by multiple cloning sites. The multiple cloning site contains an Ndel restriction site (CATATG, Fig. 10A). As part of the Ndel recognition sequence, A T G can be used as the translation start codon for appending in frame protein-encoding sequences. To permit cloning into this site, an Ndel site was engineered upstream of the pbpB gene. The pbpB gene was amplified by PCR using an upstream primer containing an Ndel recognition sequence in front of the sequence  66  A GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACT  T7 promoter  Xbal  TTAAGAAGGAGATATACATATGGCTAGAATTCGCGCGCCCGGGGATCCTCTAGAGTCGACC RBS Ndel EcoRI Smal BamHI Xbal  B  BamHI  Figure 10. (A) Nucleotide sequence of the region containing the T7 RNA polymerase promoter, RBS and multiple cloning site on the vector pT7-7. (B) Diagram of pXL706 used for the expression of the P. aeruginosa pbpB gene product. The regions shown include the ampicillin resistance gene (Ap), promoter and RBS of T7 RNA polymerase, and the P. aeruginosa pbpB gene (pbpB). The plasmid is not drawn to scale. Only selected restriction enzyme sites are shown.  67 coding for the N-terminus of PBP3, and a downstream primer encoding the Cterminus of PBP3 followed by a stop codon and the sequence for a BamHl recognition site (for the sequences utilized, see Materials and Methods section 8.1). The PCR product corresponding to the size of the pbpB gene was purified and cloned into the vector pT7-7 between the Ndel and BamHl sites to generate the plasmid pXL706 (Fig. 10B). The host strain for the expression of the pbpB gene product was E. coli K38 containing pGPl-2. pGPl-2 contains the gene for T7 R N A polymerase and the expression of this gene is temperature regulated. A novel protein was observed from the SDS-PAGE of whole cell lysate sample of E. coli K38/pGPl-2 (pXL706) after the induction of the T7 R N A polymerase expression at 42°C, indicating that the PBP3 was 3  expressed in E. coli (Fig. 11 A).  H-penicillin assay showed that the pbpB gene  product bound penicillin (Fig. 12). However, the penicillin binding ability of the 3  protein detected by the H-penicillin assay appeared to be lower than expected, given its abundance in the membrane protein sample (Fig. 1 IB). This could possibly be due to incomplete removal of the [^-lactamase produced by pT7-7, which might also 3  explain the weak reaction between the PBP5/6 (42 kDa) and the H-penicillin in the similar types of samples as demonstrated previously by Parr et al (1988).  68  Figure 11. (A) SDS-10% PAGE of whole cell lysates. Lane 1, Standard molecular mass markers. Lane 2, K38/pGPl-2(pXL706), containing the cloned P. aeruginosa pbpB gene. Lane 3, K38/pGPl-2(pT7-7), vector control for the lane 2. (B) SDS-8.5% PAGE of cell membrane proteins. Lane 1, Standard molecular mass markers. Lane 2, K38/pGPl-2(pT7-7), vector control for the lane 3. Lane 3, K38/pGPl-2(pXL706), containing the cloned P. aeruginosa pbpB gene. Numbers indicate molecular mass in kDa. PBP3 in lane 2 (A) and lane 3 (B) are indicated by the arrows.  69  1  2  PBP  Figure 12. Autoradiogram of cell membrane proteins after incubation with H penicillin and separation by SDS-8.5% P A G E . Lane 1, K38/pGPl-2(pT7-7), vector control for the lane 2. Lane 2, K38/pGPl-2(pXL706), containing the cloned P. aeruginosa pbpB gene. Numbers on the left indicate molecular mass in kDa. PBPs are indicated on the right. 3  70 2.3 p B B R l M C S as the expression vector To overcome the problem of the presence of the (3-lactamase, a broad-hostrange vector pBBRlMCS, which carries the T7 promoter (however, without the RBS sequence) and a chloramphenicol resistance gene, was used in combination with an E. coli strain BL21(DE3) in which the T7 RNA polymerase gene is present in the chromosome with its expression under the control of the lac promoter. The recombinant clone pXL608 (Fig. 13) was generated by cloning the 1.7 kb XbalBamHI fragment isolated from pXL706 into the XbaVBamHl digested pBBRlMCS. This strategy moved the RBS from the pT7-7 along with the pbpB gene. The resultant plasmid pXL608 contained the pbpB gene under the control of the T7 promoter and in the opposite orientation to the lac promoter. Upon induction with IPTG (0.5 mM), a novel protein was observed in the SDSP A G E of the whole cell lysate sample of E. coli BL21(DE3)/pXL608, indicating that PBP3 was efficiently expressed (Fig. 14). The protein cofractionated with cytoplasmic membrane proteins (Fig. 15), indicating that it was incorporated 3  efficiently into the membrane in the E. coli cells. A n H-penicillin assay showed that the pbpB gene product bound penicillin (Fig. 16). The protein migrated to a location similar to that of E. coli PBP3, which has an apparent molecular mass of 60 kDa.  71  Xbal  BamHI  Cm  Figure 13. Diagram of pXL608 used for the expression of the P. aeruginosa pbpB gene product. The regions shown include the T7 RNA polymerase promoter, RBS, pbpB gene (pbpB), chloramphenicol resistance gene (Cm), gene required for plasmid mobilization (mob) and the gene required for plasmid replication (rep). The plasmid is not drawn to scale. Only selected restriction enzyme sites are shown.  72  Figure 14. SDS-8.5% PAGE of whole cell lysates. Lane 1, Standard molecular mass markers. Lanes 2, BL21(DE3)/pXL608, containing the cloned P. aeruginosa pbpB gene. Lanes 3, BL21(DE3)/pBBRlMCS, the vector control for the lane 2. Numbers on the left indicate molecular mass in kDa. PBP3 present in the lane 2 is indicated by the arrow.  73  Figure 15. (A) SDS-8.5% PAGE of cell membrane proteins. Lane 1, Standard molecular mass markers. Lane 2, BL21(DE3)/pBBRlMCS, vector control for the lane 3. Lane 3, BL21(DE3)/pXL608, containing the cloned P. aeruginosa pbpB gene. (B) SDS-8.5% PAGE of cell membrane proteins. Lane 1, Standard molecular weight markers. Lanes 2, 3, 4 and 5, BL21(DE3)/pXL608 in 5 fold series dilutions (lane 2 contains 1/5 of the proteins in lane 3 [A]). Numbers on the left indicate molecular mass in kDa. PBP3 is indicated by the arrow.  74  PBP  1 2  3  4  Figure 16. Autoradiogram of cell membrane proteins after incubation with Hpenicillin and separation by SDS-8.5% P A G E . Lane 1, B L 2 1 ( D E 3 ) / p B B R l M C S , containing same amount of membrane protein as in lane 2 Figure 15 (A). Lanes 2, 3 and 4, BL21(DE3)/pXL608, containing same amount of membrane proteins as lanes 2, 3 and 4 in Figure 15 (B) respectively. Numbers on the right indicate molecular mass in k D a . Numbers on the left indicate the locations of PBPs. P. aeruginosa PBP3 is indicated by the arrow.  75  2.4 Processing of the P. aeruginosa pbpB gene product in E. coli The derived amino acid sequence of the pbpB gene product containing 579 amino acids had a calculated molecular mass of 62.856 kDa. However, the protein produced in E. coli migrated with apparent molecular mass of 60 kDa. The PBP3 protein was thus subjected to N-terminal amino acid sequencing after being transferred to a PVDF membrane following SDS-PAGE separation of the membrane protein sample prepared from E. coli K38/pGPl-2 (pXL706) (see figure 11B). The first six N-terminal amino acids were M K L N Y F for PBP3, which was identical to that of the translated sequences. Therefore, the sequences at the N-terminus of PBP3 were apparently not removed, and did not appear to be characteristic of typical signal peptides. Nor did it contain a putative lipoprotein signal processing sequence as does the E. coli PBP3 (Hayashi et al, 1988). It is possible that the disparity in molecular weights may reflect post-translational C-terminal processing, as is known to occur with the E. coli PBP3 (Nagasawa et al, 1989; Hara et al, 1991).  3. Overproduction of the P. aeruginosa and E. colipbpB gene products in P. aeruginosa P. aeruginosa PAO4089, a strain deficient in production of chromosomal (3lactamase, was chosen to be the host for expression of the pbpB gene product. This 3  strain would reduce the problem of P-lactamase interference in the H-penicillin assay.  76 The broad-host-range vector pUCP27, which is a derivative of the vector pUC19 and contains a tetracycline resistance gene and a stabilizing fragment for maintenance in P. aeruginosa was used as the expression vector. The 1.7 kb Xbal-BamHl D N A fragment isolated from pXL706, which contained the RBS sequence from the pT7-7 and the pbpB gene, was cloned into pUCP27 behind the lac promoter to generate pXL506. This plasmid was transformed into PAO4089. To address whether E. coli PBP3 functions in P. aeruginosa, an attempt was made to express the E. colipbpB gene product in PAO4089. The 2.6 kb BamHlEcoRl fragment isolated from the plasmid pPH115 (obtained from Dr. B Spratt), which contained the E. coli pbpB gene and the upstream putative promoter sequence, was cloned into the vector pUCP27 behind the lac promoter to create pXLK20. This plasmid was then transformed into PAO4089. Both the P. aeruginosa and E. colipbpB gene products were expressed in PAO4089(pXL506) and PAO4089(pXLK20) with apparent molecular masses of 60 kDa, and could be detected by a H-penicillin binding assay of membrane protein 3  preparations (Fig. 17). The proteins were not visible by Coomassie Blue staining following SDS-PAGE of the membrane protein preparations. Using a scanning densitometer, the amounts of P. aeruginosa and E. coli PBP3 produced from the recombinant clones were estimated to be 7-fold that of the native PBP3 present in PAO4089(pXL546). pXL546, which contained a D N A fragment of 300 bp in length (corresponding to the 3' end of the P. aeruginosa pbpB gene ) cloned in the  77  1 2  3  PBP  Figure 17. Autoradiogram of cell membrane proteins after incubation with H penicillin and separation by SDS-8.5% PAGE. Lane 1, PAO4089(pXL506), containing the cloned P. aeruginosa pbpB gene. Lane 2, PAO4089(pXL546), containing the 300 bp at 3'end of the P. aeruginosa pbpB gene and used as the control for the lane 1 and lane 3. Lane 3, PAO4089(pXLK20), containing the cloned E. coli pbpB gene. Numbers on the left indicate molecular mass in kDa. PBPs are indicated on the right. 3  78  pUCP27, failed to express any novel product.  4. Effect of overproduction of the pbpB aeruginosa  gene products on the susceptibility of  P.  to pMactam antibiotics  To investigate the effect of overproduction of the P. aeruginosa and E. coli PBP3 on antibiotic susceptibility, several P-lactam antibiotics, including the PBP3targeted compounds cefsulodin (PBP1-targeted in E. coli), ceftazidime, cefepime and aztreonam, PBP2-targeted imipenem and PBP1-targeted cephaloridine, were tested for the MICs. Table 6 summarizes the MIC results for these antibiotics against P. aeruginosa PAO4089 producing P. aeruginosa or E. coli PBP3. Overproduction of PBP3 from the cloned P. aeruginosa pbpB gene led to 2-8 fold increased MICs of PBP3-targeted antibiotics. MICs of the PBP2-targeted and PBP1-targeted antibiotics were not influenced by the presence of the extra copy of this PBP3 protein. A similar result was observed in PAO4089 which overproduced the E. coli pbpB gene product, except that cefsulodin susceptibility was not affected, a result consistent with the fact that cefsulodin has a different target in E. coli as compared to P. aeruginosa. The finding that the overexpression of the P. aeruginosa or E. coli pbpB gene product led to increased MICs, was further verified by an indirect H-penicillin 3  binding assay, or competition assay. H-penicillin was used in a competition assay 3  because it is the only radioactive-labeled P-lactam commercially available. The  Table 6.  Strain  PAO4089  MICs of pMactam antibiotics against PAO4089 expressing the P. aeruginosa and E. coli pbpB gene products  MIC (ug/ml)  PBP gene expressed  /  PAO4089(pXL546)  ApbpB  PAO4089(pXL506)  pbpB  PAO4089(pXLK20)  pbpB  P  P  e  p, P. aeruginosa gene, e, E. coli gene.  CEPH  CTZ  CFS  CFPM  AZT  IMIP  1.25  0.78  0.5  4  0.25  16  1.25  0.78  0.5  4  0.25  16  10  6.25  2  8  0.25  16  5  0.78  2  8  0.25  16  80  parameter for evaluating the affinity of a PBP and a selected (3-lactam antibiotic was the I50, which is defined as the concentration of the (3-lactam antibiotic that reduces the H-penicillin binding by 50%. Therefore, the lower the measured I , the higher 3  50  the affinity. The results of the competition binding assays indicated that the PBP3targeted (3-lactams, ceftazidime (Fig. 18), cefepime and aztreonam reacted preferentially with the P. aeruginosa and E. coli PBP3s (Table 7). In contrast, cefsulodin bound preferentially to P. aeruginosa PBP3 but did not influence penicillin binding to E. coli PBP3, a result consistent with its known binding properties. The data presented in Table 7 were consistent with literature data and demonstrated an overall pattern that was consistent with the MIC testing. Thus, the competition study confirmed that increases of the MICs of the PBP3-targeted antibiotics were due to the overproduction of the P. aeruginosa and E. colipbpB gene products.  5. Mutational analysis of the pbpB gene 5.1 Rationale for carrying out the mutational analysis PBP3 and PBP3x of P. aeruginosa, having similar amino acid sequences and modular designs, were related to E. coli PBP3 (Chapter One). Genes encoding the PBP3 and PBP3x were cloned independently and they were mapped to different loci on the P. aeruginosa chromosome (Chapter One). Taken together, these data suggest that P. aeruginosa contains two copies of a gene encoding proteins having similar  81  PBP  1  2  3  4  5  6  Figure 18. Autoradiogram of cell membrane proteins illustrating competition of ceftazidime with H-penicillin for the PBPs of PAO4089 expressing the P. aeruginosa pbpB gene product. The ceftazidime concentration increases from 0.0125 to 3.2 ug/ml (4-fold increase/lane from lane 2 to 6) and lane 1 is the control containing no ceftazidime. Numbers on the right indicate molecular mass in kDa. PBPs are indicated on the left. 3  Table 7.  Strain  I of (3-lactam antibiotics for PBPs from PAO4089 expressing the P. aeruginosa and E. coli pbpB gene products 50  I50 (ug/ml)  PBPs CTZ  CFS  CFPM  AZT  CEPH  PAO4089 (pXL506)  IA IB 3 4 5/6  0.8 >3.2 0.4 >3.2 >3.2*  >9.6 1.6 0.6 >9.6 >9.6*  >0.0625 >0.0625 0.0125 >0.0625 >0.0625*  >1.25 >1.25 0.05 >1.25 >1.25*  0.5 0.2 2.5 0.05 >5.0*  PAO4089 (pXLK20)  IA IB 3 4 5/6  0.8 >1.5 0.3 >1.5 >1.5*  25 2.5 >25 >25 >25*  0.15 0.4 0.15 >0.75 >0.75*  >1.25 >1.25 0.25 >1.25 >1.25*  0.5 0.2 8.0 0.05 >25*  pXL506. P. aeruginosa pbpB gene cloned in the vector pUCP27; pXLK20, E. * indicating the highest concentration used in the competition assay. PBP2 was not observed in these experiments.  colipbpB  gene cloned in the vector pUCP27.  83  functions to E. coli PBP3. Subsequently, it was found that PBP3 and PBP3x had different affinities for various PBP3-targeted (3-lactam antibiotics (see Chapter Two, section 3 and next Chapter, section 3). This result suggested that these two proteins might have different roles in cell growth. To test if PBP3 and PBP3x were redundant in P. aeruginosa, whether they both functioned in concert, or if one of them was silent, an attempt was made to construct PBP3-defective or PBP3x-defective mutants (see below).  5.2 Mutagenesis of the pbpB gene The strategy for constructing a PBP3-defective mutant, as illustrated in Fig. 19, involved the use of a gene replacement procedure (Schweizer, 1992) which involved a ColEl-type plasmid pNOT19, which contains a unique Not\ site, a MOB3 cassette as a Not\ fragment containing oriT, and the B. subtilus sacB gene as a counter-selectable marker for promotion of gene replacement. The insertion plasmid pBPB::Km (Fig. 20) was generated by cloning the mutated chromosomal sequences of pbpB (disrupted by a kanamycin resistance cartridge) and the MOB3 cassette into pNOT19 (Fig. 19, step 1). The mobilization of the insertion plasmid into P. aeruginosa and subsequent growth of the transconjugants on V B M M medium (selective for P. aeruginosa growth) containing kanamycin and carbenicillin should select for plasmid integration into the chromosome, i.e. a tandem duplication of the wild-type and mutant alleles of the cloned gene with the plasmid sequence in between the two copies (Fig. 19, step 2).  84  Km ' ^  wild-type chromosome  Plasmid integration  Km I  S  acB Cm oriT  I Homologous recombination and excision • of plasmid sequence mutant chromosome Km  Figure 19. Schematic summary of gene replacement procedure for constructing a PBP3-defective or PBP3x-defective mutant. 1, Cloning of the mutated pbpB or pbpC gene and the MOB3 cassette into pNOT19 to generate an insertion plasmid. 2, Mobilization of the insertion plasmid into P. aeruginosa followed by growth on a selective medium for obtaining cointegrates. 3, Growth on sucrose containing medium to select homologous recombination and excision of plasmid sequences.  85  Smal/Xbal  Figure 20. Diagram of pBPB::Km used for allele replacement mutagenesis. The lightly shaded arrow between the Xbal and BamHI restriction site represents the P. aeruginosa pbpB gene and the open bar in the middle represents the 1.3 kb kanamycin resistance cartridge that was used to interrupt the pbpB gene. The fragment between two Notl site is the 5.8 kb MOB3 cassette including origin of transfer (oriT), chloramphenicol resistance gene (Cm) and the sacB loci from B. subtilus. Ori indicated the colEl origin of replication. The fragments are not drawn to scale. Only selected restriction sites are shown.  A subsequent shift to a sucrose-containing medium should promote the deletion of the plasmid sequence by homologous recombination, since the expression of the sacB gene is lethal to P. aeruginosa in the presence of sucrose at 37°C (Fig. 19, step 3). In an attempt to generate integration of the plasmid pBPB::Km into the chromosome of P A O l strain H103, it was found that in repeated attempts no transconjugant could grow on the V B M M medium containing kanamycin and carbenicillin. The lack of cointegrates did not prove the pbpB gene was essential, since at this stage one normal and one defective copy of the gene would be present. Instead this could possibly be due to the location of the pbpB gene within a cluster of cell division genes, likely causing a deleterious polar effect on the expression of the downstream genes.  6. Summary The P. aeruginosa pbpB gene product was expressed in E. coli using the T7 RNA polymerase and promoter system. The expressed protein was exported to the cytoplasmic membrane of E. coli cells and bound H-penicillin. It had an apparent 3  molecular mass of 60 kDa, whereas the calculated molecular mass was 62.856 kDa. The N-terminal amino acid sequence of the expressed protein was identical to that of PBP3 deduced from the nucleotide sequence of the pbpB gene, suggesting that there was no N-terminal processing.  87  The P. aeruginosa and E. coli pbpB gene products were expressed in P. aeruginosa PAO4089 using a broad-host-range vector pUCP27. Results from MIC testing and "H-penicillin binding competition assays indicated that overproduction of both the P. aeruginosa and E. colipbpB gene products in P. aeruginosa led to increased resistance to the PBP3-targeted compounds aztreonam, cefepime, cefsulodin (except for E. coli pbpB gene product) and ceftazidime. An attempt was made to construct a PBP3-defective mutant using a gene replacement technique. However, no PBP3-defective mutant was obtained. This could be due to the location of the pbpB gene at the proximal end of a cluster of cell division genes, where the placement of a polar mutation would be lethal to the cells.  88  CHAPTER THREE Expression of the P. aeruginosa pbpC Gene Product and Mutational Analysis of the pbpC Gene  1. Introduction  In Chapter two, it was reported that the P. aeruginosa pbpB gene was efficiently expressed in E. coli by the T7 RNA polymerase/promoter system and the expressed pbpB gene product was subsequently characterized by a H-penicillin 3  binding assay. It was found that the P. aeruginosa pbpB gene product produced in E. coli was not processed at its N-terminus, however, a disparity between the calculated and apparent molecular mass was observed. Overproduction of both the P. aeruginosa and E. colipbpB gene products in P. aeruginosa results in increased resistance to selected PBP3-targeted P-lactam antibiotics. The P. aeruginosa pbpC gene product has similar amino acid sequence and modular design to those of both the P. aeruginosa and E. colipbpB gene products (Chapter One). This chapter describes further characterization of the P. aeruginosa pbpC gene product by the approaches described in Chapter two. The influence of PBP3x overproduction on the susceptibility to P-lactam antibiotics was also investigated. To further test whether P. aeruginosa contains two genes encoding proteins having similar function to E. coli PBP3, a PBP3x-deficient mutant was characterized and is described in the last section of this chapter.  89  2. Expression of the P. aeruginosa pbpC gene product in E. coli 2.1. Preliminary work The membrane protein samples from E. coli DH5a (pXLHd2) were examined for the expression of the PBP3x protein. pXLHd2 (see figure 5B) had the pbpC gene and the upstream putative RBS and promoter sequences cloned in the opposite orientation to the lac promoter in the vector pTZ18U. However, no novel PBP protein 3  corresponding to the molecular mass of P. aeruginosa PBP3x was detected by the H penicillin assay (data not shown). Therefore, an expression system using the T7 RNA polymerase and promoter was applied to the expression of the P. aeruginosa pbpC in E. coli.  2.2 pT7-7 as the expression vector As described for the expression of pbpB gene, an Ndel site was engineered upstream of the pbpC gene by using an upstream PCR primer containing an Ndel recognition sequence in front of the sequence coding for the N-terminus of PBP3x. The A T G in the Ndel recognition sequence was the translational start codon for the pbpC gene, and a downstream primer containing the sequence coding for the Cterminus of PBP3x followed by a stop codon and the sequence for a BamHl recognition site (for sequences, see Materials and Methods section 8.1). The amplified pbpC gene was purified and cloned into the vector pT7-7 between the Ndel and BamHl sites, to generate pXL732 (Fig. 21).  90  A GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACT  T7 promoter  Xbal  TTAAGAAGGAGATATACATATGGCTAGAATTCGCGCGCCCGGGGATCCTCTAGAGTCGACC RBS Ndel EcoRI Smal BamHl Xbal  Figure 21. (A) Nucleotide sequence of the region containing the T7 RNA polymerase promoter, RBS and multiple cloning site on the vector pT7-7. (B) Diagram of pXL732 used for the expression of the P. aeruginosa pbpC gene product. The regions shown include the ampicillin resistance gene (Ap), T7 RNA polymerase promoter, RBS and the P. aeruginosa pbpC gene. The plasmid is not drawn to scale. Only selected restriction enzyme sites are shown.  91  The host strain for the expression of the pbpC gene was E. coli K38 containing pGPl-2. A novel protein was observed from the SDS-PAGE of whole cell lysate samples of E. coli K38/pGPl-2 (pXL732) after the induction of the T7 R N A polymerase expression at 42°C, indicating that the PBP3x was produced in E. coli 3  (Fig. 22A). An H-penicillin assay showed that the pbpC gene product bound penicillin (Fig. 23). As observed in the expression of the pbpB gene product using pT7-7 (Chapter Two), the penicillin binding ability of the PBP3x protein detected by 3  H-penicillin assay appeared to be lower than expected given its abundance in the membrane protein sample (Fig. 22B). This could possibly be due to incomplete removal of the (3-lactamase produced by pT7-7.  2.3 pBBRlMCS as the expression vector To avoid the problem of the presence of the [3-lactamase, pBBRlMCS was used as an alternative expression vector. The recombinant clone pXL629 (Fig. 24) was generated after ligation of pBBRlMCS with the 1.7 kb Xbal-BamHl fragment isolated from pXL732, which contained the pbpC gene and the RBS sequence from the vector pT7-7. The resultant plasmid pXL629 contained the pbpC gene under control of the T7 promoter and in the opposite orientation to the lac promoter. After induction with IPTG (0.5 mM), a novel protein was observed  92  Figure 22. (A) SDS-10% PAGE of whole cell lysates. Lane 1, Standard molecular mass markers. Lane 2, K38/pGPl-2(pXL506), containing the cloned P. aeruginosa pbpB gene, lane 3, K38/pGPl-2(pXL732), containing the cloned P. aeruginosa pbpC gene. (B) SDS-8.5%PAGE of cell membrane proteins. Lane 1, Standard molecular mass markers. Lane 2, K38/pGPl-2(pT7-7), vector control for the lane 3. Lane 3, K38/pGPl-2(pXL732), containing the cloned P. aeruginosa pbpC gene. Numbers on the left indicate molecular mass in kDa. PBP3x is indicated by the arrow.  93  Figure 23. Autoradiogram of cell membrane proteins after incubation with H penicillin and separation by SDS-8.5% PAGE. Lane 1, K38/pGPl-2(pT7-7), vector control for the lane 2. Lane 2, K38/pGPl-2(pXL732), containing the cloned P. aeruginosa pbpC gene. Numbers on the left indicate molecular mass in kDa. PBPs are indicated on the right. PBP3x is indicated by the arrow. 3  94  Xbal  Cm  Figure 24. Diagram of pXL629 used for the expression of the P. aeruginosa pbpC gene product. The regions shown include the T7 RNA polymerase promoter, RBS sequence, P. aeruginosa pbpC gene, chloramphenicol resistance gene (Cm), gene required for plasmid mobilization (mob) and the gene required for plasmid replication (rep). The plasmid is not drawn to scale. Only selected restriction enzyme sites are shown.  95  in the SDS-PAGE of the whole cell lysate sample of E. coli BL21(DE3)/pXL629, indicating that pbpC was efficiently expressed (Fig. 25). As with PBP3, protein PBP3x was produced efficiently after induction with IPTG for 1 hr, and levels of protein production increased with longer time (up to 4 hr examined) of IPTG induction. The protein cofractionated with the cytoplasmic membrane proteins (Fig. 26), indicating that it was incorporated efficiently into the membrane of the E. coli 3  cells. The H-penicillin assay confirmed that the pbpC gene product bound penicillin (Fig. 27). The PBP3x protein migrated to a gel location slightly lower than that of E. coli PBP3, with apparent molecular mass of 58 kDa.  2.4 Processing of the P. aeruginosa pbpC gene product in E. coli The amino acid sequence derived from the pbpC gene containing 565 amino acids had a calculated molecular mass of 61.128 kDa. However, the protein produced in E. coli migrated with apparent molecular mass of 58 kDa. PBP3x did not appear to contain an N-terminus characteristic of a typical signal peptide, nor did it contain a putative lipoprotein signal processing sequence as proposed for E. coli PBP3. To confirm the lack of the N-terminal processing, the PBP3x protein was subjected to Nterminal amino acid sequencing after transfer to a PVDF membrane following SDSP A G E separation of the membrane protein sample prepared from E. coli K38/pGPl-2 (pXL732) (see figure 22B). The six N-terminal amino acids analyzed were SSQRRN, which was identical to that of the derived sequence, except that the first residue  96  15 14 13 12 11 10 9 8 7  6 5 4  3 2 1  PBP3 PBP3x  Figure 25. SDS-8.5% PAGE of whole cell lysates. Lane 1, Standard molecular mass markers. Lanes 2, 5, 8, 11 and 14, BL21(DE3)/pXL608, containing the cloned P. aeruginosa pbpB gene. Lanes 3, 6, 9, 12 and 15, BL21(DE3)/pXL629, containing the cloned P. aeruginosa pbpC gene. Lanes 4, 7, 10 and 13, BL21(DE3)/pBBRlMCS, the vector control for the clones pXL608 and pXL629. Lanes 2 and 3, IPTG induction for 0 hr. Lanes 4, 5 and 6, IPTG induction for 1 hr. Lanes 7, 8 and 9, IPTG induction for 2 hr. Lanes 10, 11 and 12, IPTG induction for 3 hr. Lanes 13, 14 and 15, IPTG induction for 4 hr. Numbers on the right indicate molecular mass in kDa. PBP3 and PBP3x are indicated by the arrows.  97  Figure 26. (A) SDS-8.5% P A G E of cell membrane proteins. Lane 1, Standard molecular mass markers. Lane 2, B L 2 1 ( D E 3 ) / p B B R l M C S , vector control for the lane 3. Lane 3, BL21(DE3)/pXL629, containing the cloned P. aeruginosa pbpC gene. (B) SDS-8.5% P A G E of cell membrane proteins. Lane 1, Standard molecular mass markers. Lanes 2, 3, and 4, BL21(DE3)/pXL629 in 5 fold series dilutions (lane 2 contains 1/5 of the proteins in lane 3 [A]). Numbers on the left indicate molecular mass i n kDa. P B P 3 x is indicated by the arrow.  98  PBP  1 2  3  4  Figure 27. Autoradiogram of cell membrane proteins after incubation with H penicillin and separation by S D S - 8 . 5 % P A G E . Lane 1, B L 2 1 ( D E 3 ) / p B B R l M C S , containing same amount of membrane protein as in lane 2 Figure 15 (A). Lanes 2, 3 and 4, BL21(DE3)/pXL629, containing same amount of membrane proteins as lanes 2, 3 and 4 in Figure 26 (B) respectively. Numbers on the right indicate molecular mass in k D a . Numbers on the left indicate the locations of PBPs. P B P 3 x is indicated by the arrow. 3  9 9  methionine was cleaved from the gene product of pbpC. Similar to P. aeruginosa PBP3, the sequences at the N-terminus of PBP3x were not apparently removed. It is possible therefore that the disparity in molecular mass may reflect post translational C-terminal processing.  3. Overproduction of the pbpC gene product in P. aeruginosa P. aeruginosa PAO4089 was chosen as the host for expression of the pbpC gene product. The broad-host-range vector pUCP27 was used as the expression vector. The 1.7 kb Xbal-BamHl D N A fragments isolated from pXL732, which contained the RBS sequence from the pT7-7 and the pbpC gene, was cloned into pUCP27 behind the lac promoter to generate pXL519. This plasmid was transformed into PAO4089. A novel protein with a molecular mass of 58 kDa was detected by H-penicillin 3  assay of the membrane protein sample of PAO4089(pXL519) (Fig. 28). The protein was not visible from Coomassie blue staining on SDS-PAGE of the membrane protein preparations. The amount of the P. aeruginosa PBP3x produced from the recombinant clone was estimated to be about the same as the native PBP3 present in PAO4089(pXL546).  4. Effect of overproduction of the pbpC gene product on the susceptibility of P. aeruginosa to pMactam antibiotics  100  Figure 28. Autoradiogram of cell membrane proteins after incubation with H penicillin and separation by S D S - 8 . 5 % P A G E . Lane 1, PAO4089(pXL506), containing the cloned P. aeruginosa pbpB gene. Lane 2, PAO4089(pXL546), containing the 300 bp at 3'end of the P. aeruginosa pbpB gene and used as the control for the lane 1 and lane 3. Lane 3, PAO4089(pXL519), containing the cloned P. aeruginosa pbpC gene. Numbers on the left indicate molecular mass in kDa. PBPs are indicated on the right (PBP3x is indicated by C). 3  101  (3-lactam antibiotics, including the PBP3-targeted compounds cefsulodin (PBP 1-targeted in E. coli), ceftazidime, cefepime and aztreonam, PBP2-targeted imipenem and PBP1-targeted cephaloridine, were used to determine the MICs of P. aeruginosa PAO4089 expressing the pbpC gene product. It appeared that the presence of an extra P. aeruginosa pbpC gene had no effect on the susceptibility of PAO4089 to the (3-lactam antibiotics tested in this study (Table 8). The results of competition binding assays indicated that the PBP3-targeted (3-lactams, ceftazidime (Fig. 29), cefsulodin, cefepime and aztreonam, primarily reacted with the P. aeruginosa PBP3 (Table 9), and to a much lesser (i.e., 3 to >16 fold) extent with the P. aeruginosa PBP3x. This suggested that the pbpC gene product had lower affinity to the PBP3-targeted (3-lactams than did the PBP3 protein, and thus would not be expected to affect antibiotic susceptibility when PBP3 was present in the cells (for the antibiotics that primary bind to PBP3).  5. Mutational analysis of the pbpC gene 5.1 Construction of a PBP3x-defective mutant The strategy for constructing a PBP3x-defective mutant is illustrated in Fig. 19. The insertion plasmid pBPC::Km (Fig. 30) was generated by cloning the mutated chromosomal sequences of pbpC (disrupted by a kanamycin resistance cartridge) and the MOB3 cassette into pNOT19 (Fig. 19, step 1). This plasmid was introduced into  Table 8. Strain  PAO4089  MICs of pMactam antibiotics against PAO4089 expressing the P. aeruginosa pbpC gene product PBP gene expressed  /  MIC (ug/ml)  CTZ  CFS  CFPM  AZT  IMIP  CEPH  1.25  0.78  0.5  4  0.25  16  PAO4089(pXL546)  ApbpB  P  1.25  0.78  0.5  4  0.25  16  PAO4089(pXL519)  pbpC  V  1.25  0.78  0.5  4  0.25  16  p, P. aeruginosa gene.  103  PBP 1  2  3  4  5  6  Figure 29. Autoradiogram of cell membrane proteins illustrating competition of ceftazidime with H-penicillin for the PBPs of PAO4089 expressing the P. aeruginosa pbpC gene product. The ceftazidime concentration increases from 0.0125 to 3.2 ug/ml (4-fold increase/lane from lane 2 to 6) and lane 1 is the control containing no ceftazidime. Numbers on the right indicate molecular mass in kDa. PBPs are indicated on the left (PBP3x is indicated by C). 3  Table 9.  Strain  PAO4089 (pXL519)  I of P-lactam antibiotics for PBPs from PAO4089 expressing the P. aeruginosa pbpC gene product 50  PBPs  IA IB 3 C 4 5/6  I  50  (ug/ml)  CTZ  CFS  CFPM  AZT  CEPH  0.8 >3.2 0.2 0.6 >3.2 >3.2*  >9.6 2.8 0.6 >9.6 >9.6 >9.6*  0.14 >0.3125 0.0125 0.06 >0.3125 >0.3125*  >1.25 >1.25 0.05 0.32 >1.25 >1.25*  0.5 0.2 2.5 2.5 0.05 >5.0*  pXL519, P. aeruginosa pbpC gene cloned in the vector pUCP27. *, indicating the highest concentration used in the competition assay. PBP2 was not observed in these experiments.  105  EcoRI  Figure 30. Diagram of pBPC::Km used for allele replacement mutagenesis. The lightly shaded arrow between the EcoRI and BamHI restriction site represents the P. aeruginosa pbpC gene and the open bar in the middle represents the 1.3 kb kanamycin resistance cartridge that was used to interrupt the pbpC gene. The fragment between two Not! site is the 5.8 kb MOB3 cassette which includes origin of transfer (oriT), chloramphenicol resistance gene (Cm) and the sacB loci from B. subtilus. Ori indicates the colEl origin of replication. The fragments are not drawn to scale. Only selected restriction sites are shown.  106  P. aeruginosa P A O l strain H103 and plasmid cointegrates were selected on V B M M containing kanamycin and carbenicillin. An estimated 160 colonies were then patched onto LB agar containing kanamycin and carbenicillin to confirm the cointegration; a single colony was found to grow on this plate. Transfer to M H agar containing kanamycin and 5% sucrose resulted in growth, indicating that the plasmid sequences had possibly been eliminated from the chromosome. The chromosomal D N A of this strain, named HCI32, was subsequently isolated, digested with EcoRI and Xhol, and analyzed by Southern blot hybridization. A probe derived from the 0.78 kb XholSmal fragment within the 5' region of the pbpC gene hybridized to a 3.3 kb EcoRI fragment and a 1.9 kb Xhol fragment (Fig. 31). These results confirmed that the 1.3 kb kanamycin resistance cartridge had been inserted into the pbpC gene on the chromosome.  5.2 PBP profile of the PBP3x-defective mutant 3  H-penicillin binding assays of the membrane proteins of P A O l strain HI03  and the mutant strain HC132 showed the same PBP profile (Fig. 32). These data indicate that the pbpC gene product was not expressed under the conditions tested.  5.3 Cell morphology and growth of PBP3x-defective mutant Stationary phase cells of H103 and HC132 grown in M H broth were harvested  107  2 kb EcoRI  EcoRI Xhol  Xhol  2.5 kb probe  B  3.3 EcoRI Xhol  kb EcoRI Xhol  1.9  kb  >^  1.9  kb  Xhol >  Figure 31. Southern hybridization demonstrating the interruption of the pbpC gene (dotted box) with a kanamycin resistance cartridge (dark-dotted box). The physical maps of the wild-type (A) and the mutant (B) pbpC locus are shown. The chromosomal DNAs were digested (C) with EcoRI and Xhol respectively and were hybridized to the DIG-labelled 0.78 kb Xhol-Smal fragment shown in panel A. Lane 1, HC132 DNA digested with EcoRI. Lane 2, H103 DNA digested with EcoRI. Lane 3, HC132 DNA digested with Afcol. Lane 4, H103 DNA digested withAfcoI. The molecular sizes of the fragments in kilobases are indicated on the right.  1  2 PBPs  Figure 32. Autoradiogram of cell membrane proteins after incubation with H penicillin and separation by SDS-7.5%PAGE. Lane 1, HCI 32, PBP3x-defective mutant. Lane 2, H103, wild type. Numbers on the left indicate the molecular mass kDa. PBPs are indicated on the right. PBP2 was not observed in this experiment. 3  109  and observed under a light microscope. As shown in Fig. 33 A & B, mutation of the pbpC gene did not cause any major change of cell shape. Growth curve experiments using M H broth showed no difference between H103 and HC132 (Fig. 34). These results suggested that the pbpC gene product was not essential for normal cell morphology or viability under the conditions tested. Results of MIC testing indicated that pbpC deficient did not change the susceptibility of P. aeruginosa to the tested 0lactam antibiotics mentioned in the previous section.  6. Summary The P. aeruginosa pbpC gene was expressed in E. coli using the T7 R N A polymerase and promoter system. The protein was exported to the cytoplasmic membrane of E. coli cells and reacted with H-penicillin. The protein had the 3  apparent molecular mass of 58 kDa, whereas the calculated molecular mass was 61.128 kDa. The N-terminal amino acid sequence of the expressed protein was identical to that of PBP3x except that the N-terminal methionine was removed. The P. aeruginosa pbpC gene was expressed in P. aeruginosa PAO4089 using a broad-host-range vector pUCP27. Data from MIC testing and H-penicillin binding 3  competition assays indicated that the presence of the pbpC gene product in the PAO4089 did not have any effect on susceptibility to PBP3- targeted antibiotics, in contrast to the properties of PAO4089 overproducing the P. aeruginosa and E. coli pbpB gene products.  110  Figure 33. Cell morphology of P. aeruginosa. (A) H103, wild type strain. (B) HCI32, PBP3x-defective mutant strain. The bar markers represent 10 urn.  Ill  Time (hr) Figure 34. Growth of P. aeruginosa PBP3x-defective mutant. Strains were grown at 37°C in M H broth and the optical density was monitored. Solid line, H103, wild type strain. Dashed line, HC132, PBP3x-defective mutant strain.  112  A PBP3x-defective mutant (strain HCI32) was obtained by a gene replacement procedure and confirmed by Southern blot analysis. The PBP profiles of wild type strain H103 and mutant strain HC132 were similar, suggesting that the pbpC gene was not visibly expressed under the physiological conditions tested. Furthermore, inactivation of pbpC did not cause any changes in cell morphology or growth rate, indicating that pbpC was not required for cell viability under normal laboratory growth conditions.  113  DISCUSSION  1. General P. aeruginosa is a clinically important bacterium that is intrinsically resistant to a large number of (3-lactam antibiotics. In addition, mutation resulting in high levels of resistance to (3-lactam antibiotics is common with this species. Several hypotheses have been proposed to explain this property including low membrane permeability, the presence of ^-lactamase activity and alterations in the affinity of drug targets (i.e. PBPs). The analysis of the resistance of P. aeruginosa to P-lactam antibiotics requires information on how specific P. aeruginosa PBPs interact with P-lactam antibiotics. Knowledge on how the P. aeruginosa PBPs interact with P-lactam antibiotics will facilitate the design of more efficient drugs and regimes for the treatment of P. aeruginosa infections. Prior to the work presented here, PBPs in P. aeruginosa had not been well studied and none of the P. aeruginosa PBP genes had been cloned or sequenced, except for a partial sequence of PBPla-encoding gene which was published three years ago (Martin et al., 1993). Of particular interest was whether P. aeruginosa PBPs possess similar structures and functions to those in other bacterial species such as E. coli, and whether E. coli PBPs and P. aeruginosa PBPs could be functionally exchanged.  114  In previous reports it had been suggested that P. aeruginosa PBP3 was the primary target for the expanded-spectrum and fourth generation cephalosporins (Watanabe et al., 1988; Maejima etal., 1991). In addition, P. aeruginosa P-lactamresistant clinical isolates from cystic fibrosis patients had apparently lost PBP3 and /or PBP6 (Godfrey et a/., 1981). The work presented here was designed to address the hypothesis that P. aeruginosa PBP3 plays an important role in susceptibility to Plactam antibiotics. Here I describe the cloning and characterization of the P. aeruginosa pbpB and pbpC genes. This work demonstrated that overproduction of the P. aeruginosa or E. colipbpB gene product in P. aeruginosa could confer resistance to the PBP3-targeted P-lactam antibiotics aztreonam, cefepime, cefsulodin and ceftazidime. This study also revealed that the presence of the P. aeruginosa pbpC gene did not affect the susceptibility of P. aeruginosa to a number of PBP3-targeted antibiotics tested.  2 . Cloning of the P. aeruginosa pbpB and pbpC genes The strategy for cloning the P. aeruginosa PBP3 gene was based on constructing a pair of degenerate PCR primers according to the amino acid sequences found in the conserved S X X K and K T G motifs of the E. coli high-molecular-weight PBPs and N. gonorrhoeae PBP2. This pair of degenerate PCR primers were employed in PCR reactions using P. aeruginosa chromosomal D N A as template;  115  several PCR products were obtained and subsequently cloned. D N A sequencing and D N A sequence analyses of the cloned PCR products led to the identification of two independent and different clones that had significant similarity to E. coli PBP3 suggesting the presence of two E. coli PBP3-like genes in P. aeruginosa. Subsequently, the cloned PCR products were used as probes to clone both genes which were named pbpB and pbpC, respectively. The genes encoding P. aeruginosa PBP3 and PBP3x mapped to two different loci on the P. aeruginosa chromosome. The pbpB gene mapped to a region of the chromosome located upstream of an E. coli murE homologue and was linked to a cluster of other essential genes involved in cell division. This finding suggests that the P. aeruginosa pbpB gene product, like E. coli PBP3, may be essential for cell division. The pbpC gene mapped approximately 2 megabase pairs from the pbpB gene and the gene appeared to be dispensable under normal laboratory growth conditions.  3. Structures of PBP3 and PBP3x All of the high-molecular-weight PBPs examined to date are cytoplasmic membrane proteins with their hydrophobic amino-terminus traversing the cytoplasmic membrane and the reminder of the protein extending into the periplasm (Ghuysen, 1991). This type of membrane topology is not unexpected since the transglycosylation, transpeptidation and carboxypeptidation reactions, that are  116  catalyzed by PBPs, are known to occur either on the outer surface of the cytoplasmic membrane or within the periplasmic space (Bowler & Spratt, 1989; van Heijenoort, 1994). The membrane topology of E. coli PBP3 has been deduced from its primary amino acid sequence in combination with protein fusion studies (using [^-lactamase as a reporter) and proteolytic digestion experiments. The results of these experiments have consistently shown that E. coli PBP3 is embedded in the cytoplasmic membrane at its amino terminus only and that the bulk of the protein is localized to the periplasmic space (Bowler & Spratt, 1989). The amino acid sequence of the P. aeruginosa PBP3 and PBP3x proteins showed that they were 45.1% and 40.7% identical to the E. coli PBP3 protein and that alignment with E. coli PBP3 was good. In addition, the hydropathy profiles of the P. aeruginosa PBP3 and PBP3x proteins were similar to that of E. coli PBP3 suggesting that there were only single transmembrane segments near their amino termini. This is consistent with the observation that the recombinant P. aeruginosa pbpB and pbpC gene products cofractionated with the membrane protein fractions of E. coli and P. aeruginosa (Figures 15,17 and Figures 26, 28). Collectively, these data suggest that both the P. aeruginosa PBP3 and PBP3x proteins have similar tertiary structure and membrane topology as that of E. coli PBP3 protein. The N-terminus of E. coli PBP3 has a sequence similar to typical E. coli lipoprotein signal sequences (Nakamura et al., 1983). Modification and processing of lipoprotein signal sequences in E. coli occur at a conserved cysteine residue that is  117  modified by acetylation and becomes the new N-terminus. However, it is debatable whether the E. coli PBP3 is processed in this manner since only 15% of E. coli PBP3 was apparently lipid-modified when it was overexpressed (Hayashi et al., 1988). E. coli PBP3 is processed in vivo by C-terminal proteolytic processing that results in the removal of the C-terminal 11 amino acids of the protein (Hara et al., 1991). This Cterminal processing is mediated by a tail-specific periplasmic protease that cleaves the Val 77-Ile 78 bond of E. coli PBP3. 5  5  The N-termini of P. aeruginosa PBP3 and PBP3x proteins had no sequence similarity to E. coli lipoprotein signal sequences or other known signal sequences. This observation makes it unlikely that P. aeruginosa PBP3 and PBP3x were processed by a similar mechanism to other lipoproteins. N-terminal amino acid sequencing of P. aeruginosa PBP3 and PBP3x that were expressed in E. coli showed that neither of these proteins were post-translationally processed at their N-terminus (Chapter Two; Chapter Three). It is possible that the amino termini of the P. aeruginosa PBP3 and PBP3x, similar to E. coli PBP3, function as noncleaved signallike sequences to mediate translocation of the bulk of the protein into the periplasm and anchor the protein in the cytoplasmic membrane. However, there is a discrepancy between the apparent molecular weights of P. aeruginosa PBP3 and PBP3x proteins produced in E. coli (as determined by SDS-PAGE and calculated molecular mass). In addition, when both the P. aeruginosa and E. colipbpB genes were expressed in P. aeruginosa, a similar discrepancy in observed and calculated molecular masses was  118  observed (Chapter Two, section 3). The same was observed when the P. aeruginosa pbpC gene was expressed in P. aeruginosa (Chapter Three, section 3). It is possible that these discrepancies resulted from C-terminal processing of the P. aeruginosa PBP3 and PBP3x. However, the substrate for the E. coli tail-specific protease (Val-Ile pair) that is responsible for the processing of E. coli PBP3 is not present within the Ctermini of P. aeruginosa PBP3 or PBP3x. Studies on the substrate specificity of the C-terminal protease showed that it processes peptides with PI residues that are small and uncharged, e.g., alanine, serine or valine (Keiler et al., 1995). Examination of the sequences of PBP3 and PBP3x of P. aeruginosa indicated that there would be several potential cleavage sites in each of the proteins. Whether these two proteins indeed undergo post-translational C-terminal processing has yet to be determined.  4. PBP3 maps to a cluster of conserved and essential cell division genes PBP3 of E. coli functions in the formation of the septum during cell growth (Spratt, 1975, 1977a). The similarity of P. aeruginosa and E. coli PBP3 proteins strongly suggests that PBP3 of P. aeruginosa serves a comparable role in P. aeruginosa cell division. Supporting evidence for this hypothesis comes from the discovery that the P. aeruginosa pbpB gene is located upstream of an E. coli murE homologue (UDP-MurNAc-tripeptide synthetase) and that these two genes mapped to the same region of the chromosome as did other cell division genes including the ftsA, ftsZ and env A genes (Chapter One). Many genes involved in cell shape maintenance  119  and cell division are clustered in E. coli (van Heijenoort, 1994). The largest cluster at 2 min is referred to as the dew (division and cell wall) cluster. The dew cluster contains genes encoding proteins responsible for the cytoplasmic and periplasmic stages of peptidoglycan biosynthesis including pbpB, murE, murF, mraY, murD,ftsW, murG, murC and ddl (van Heijenoort, 1994; Vincente & Errington, 1996). Downstream of the ddl gene, are four additional cell division genes, ftsQ,ftsA, ftsZ and env A, followed by the sec A gene which is involved in protein export. A similar cluster of cell division genes is found in Bacillus subtilis (Buchanan et al., 1994). The fact that the pbpB, murE mdftsA, ftsZ, env A genes of P. aeruginosa all mapped to the same region is consistent with the possibility that P. aeruginosa, like E. coli and B. subtilis, may also have a conserved major cell division and cell wall synthesis gene cluster. D N A sequence analysis of the P. aeruginosa pbpB and murE genes suggests that they are cotranscribed from a promoter upstream of pbpB which is similar to the situation found in E. coli (Ayala et al., 1994); pbpB and murE are adjacent to each other in both E. coli and P. aeruginosa. Their spacing is only two nucleotides apart in P. aeruginosa, whereas in E. coli the coding region for murE overlaps the end of the pbpB coding region by 11 base pairs (Michaud et al., 1990); there were no obvious signals for transcriptional termination downstream of the P. aeruginosa pbpB gene. This finding may explain the failure to obtain cointegrates during gene replacement mutagenesis, since a polar mutation in the pbpB gene that effected the expression of  120  essential downstream cell division genes (i.e. murE) would be lethal. It should be noted that one cannot rule out the possibility that pbpB is itself an essential gene. Isolation of pbpB conditional lethal or nonsense mutants would answer this question.  5. P B P 3 x :  a s e c o n d E. coli P B P 3 - l i k e g e n e p r o d u c t  In this study two E. coli PBP3-like P. aeruginosa genes were identified among the products from PCR studies using degenerate primers, suggesting that P. aeruginosa may contain a second copy of an E. coli PBP3-like gene. The first product pbpB was described in the preceding section. To determine whether the P. aeruginosa pbpC gene product was functionally equivalent to E. coli PBP3, the gene was cloned and characterized. Analyses of the translated sequence of the P. aeruginosa pbpC gene revealed that, like the pbpB gene product, it was very similar to E. coli PBP3 in its amino acid sequence and the spacing between the nine conserved motifs, the signature of class B high-molecular-weight PBPs (Piras et al., 1993; Ghuysen, 1994). This suggested that the pbpC might have been a duplicated pbpB gene. Interestingly, B. subtilis also has a second copy of its pbpB gene, known as spoVD. SpoVD is only expressed during sporulation in B. subtilis, whereas pbpB is expressed during both vegetative growth and sporulation (Yanouri et al., 1993; Daniel et al., 1994). Thus, the two PBP3-like genes in B. subtilis have unique functions in cell growth and their expression is differentially regulated. There are other examples of gene duplications for a variety of other genes including other PBPs. For example, E. coli contains two  121  copies of the gene encoding PBP1. E. coli PBPla and PBPlb appear to be redundant and capable of fulfilling compensatory roles in cell elongation (Tamake et al., 1977; Suzuki et al, 1978). Whether the P. aeruginosa pbpC gene encodes a differentially expressed and functional equivalent of the P. aeruginosa pbpB homologue is difficult to determine without further studies. However, there is a potential a recognition s  sequence upstream of the P. aeruginosa pbpC gene and in addition, mutation of the P. aeruginosa pbpC had no discernible effect on the growth rate or cell morphology of the bacterium under the laboratory conditions tested. The finding of a potential a  s  recognition site, upstream of the pbpC gene suggests the possibility that the expression of pbpC may be growth rate or stress regulated (see below). Gene duplication has previously been demonstrated in P. aeruginosa. For example OprO and OprP, encoding outer membrane proteins which are adjacent to one another (Siehnel et al., 1988; Siehnel et al., 1992). OprO and OprP have 74% amino acids identity. These two proteins have certain functional similarities in anion specific binding, and yet also have distinct functions since OprO has a distinctly higher affinity for pyrophosphate than orthophosphate whereas for OprP the affinities are reversed (Hancock et al, 1992). P. aeruginosa PBP3 and PBP3x are similar to one another with 48.3% amino acid identity. Their C-terminal regions, or the penicillin-binding domains appear to be more conserved with 51.8% amino acids identity. However, this does not prove that these two proteins have closely related transpeptidase and yet distinct transglycosylase  122  functions, especially in the.absence of direct enzymatic assays for these activities. Indeed, such assays would not even be definitive since all the high-molecular-weight PBPs have similar enzymatic functions. A more useful approach involves mutational studies, which proved to be of limited value in these studies. The results presented in this study suggest that P. aeruginosa PBP3 and PBP3x may have different penicillinbinding domain structures (thus resulting in distinct transpeptidase activities) since binding studies indicated that they had different affinities for various (3-lactam antibiotics. These two proteins may have similar biochemical functions (transpeptidase and transglycosylase), but play different physiological roles during different stages of cell growth (see below). Based on analyses of the nucleotide sequences flanking the pbpC gene, it appears to be located at the end of an operon or exists as a single gene. Its upstream sequence, about 370 bp in length, did not resemble any sequences in the GenBank database. The sequence downstream of the pbpC gene encoded convergently transcribed homologues of the E. coli soxR and M. bovis adh gene products. Thus, the second copy of the PBP3 gene in P. aeruginosa was apparently not associated with genes involved in cell division. In an attempt to determine the function of the pbpC gene product, a PBP3xdefective mutant was constructed by the insertion of a Km cartridge. In this mutant r  strain, no phenotypic difference from the wild type strain could be detected. Failure to detect the loss of the pbpC gene product in the insertion mutant led to the tentative  123  conclusion that the pbpC gene product was not expressed under the conditions tested. The inability to prove the disappearance of the pbpC gene product in the insertion mutant by H-penicillin binding studies could have been the result of a number of 3  factors. For example it may be that this protein was not expressed under the physiological conditions tested and thus did not play any significant role in cell growth under the conditions tested. Alternatively, it may be that this protein comigrated with another PBP during SDS-PAGE, thus masking the disappearance of PBP3x in the insertion mutant, especially if the cell compensates for the loss of one PBP by elevating the production of another. It may also be that the pbpC gene product was labeled poorly by H-penicillin, given the observation that it had a lower 3  affinity for P-lactam antibiotics compared to PBP3 (Chapter Three, section 4) and that the overproduced PBP3x in P. aeruginosa PAO4089 (labeled by H-penicillin) 3  appeared to be only at the same level as native PBP3 (Chapter Three, section 3). It could also be that PBP3x was present in extremely low amounts because of low expression level or instability, especially when one considers that E. coli has been shown to have only 50 molecules / cell of PBP3 and 20 molecules / cell of PBP2 (Spratt, 1977a). It has been reported that & downregulates the expression of E. coli PBP3 when cells enter stationary phase and cell division ceases (Dougherty & Pucci, 1994). It may thus be significant that the upstream sequence of the pbpC gene contains a  124  consensus sequence recognized by a (Chapter One, section 4.2). Therefore it is s  possible that the expression of the pbpC gene is regulated by growth rate or nutrient conditions. When cells reach stationary phase, o might downregulate the production s  of the conventional PBP3 and produce the alternative PBP3, i.e., PBP3x. Future studies on the expression of the P. aeruginosa pbpC gene under a variety of growth conditions and at various growth stages should provide insight into the possible functions of this gene.  6. Production of recombinant PBP3 and PBP3x In preliminary studies, p X L B B and pXLFfd2 were used for the expression of pbpB and pbpC respectively but the corresponding proteins could not be detected by 3  H-penicillin binding assay. p X L B B contained the pbpB gene and its putative  upstream RBS cloned in the same orientation as the lac promoter on the high copy number vector pTZ18U. Failure to detect the pbpB gene product could possibly be explained by the low efficacy of the putative RBS of the pbpB gene. This is strongly similar to the RBS of the weakly expressed E. colipbpB gene. A lacZ transcriptional fusion study has confirmed that the efficacy of the RBS of E. colipbpB gene is very low (Ayala et al, 199»4). This could be one of the mechanisms by which E. coli and P. aeruginosa control PBP3 production at low levels. pXLHd2 had the pbpC gene, the upstream putative RBS, and the promoter sequence cloned in the opposite orientation to the lac promoter in the vector pTZ18U. Lack of expression in this  125  construction could be due to poor functioning of the RBS and promoter sequences in E. coli; the putative promoter sequence of the pbpC gene might be recognized by an alternative sigma factor o (see above) and /or the RBS and promoter sequences not s  functioning under normal laboratory conditions. In this study, recombinant P. aeruginosa PBP3 and PBP3x were efficiently produced using a T7 RNA polymerase system involving the promoter and RBS recognized by the T7 RNA polymerase. The optimization of translation was also attributed to the cloning of the pbpB or pbpC gene behind the RBS at the Ndel site on the vector pT7-7 such that the translational start signal for the T7 gene 10 was replaced by that for the pbpB or pbpC. Subsequently, the D N A fragment containing the T7 RBS and the pbpB or pbpC gene was cloned into a broad-host range vector p B B R l M C S which contains the T7 promoter. This cloning strategy led to the productions of the pbpB and pbpC gene products. PBP3 produced in P. aeruginosa PAO4089(pXL506), as detected by H 3  penicillin binding, was estimated to be at a 7-fold higher level than the native PBP3, whereas production of PBP3x in PAO4089(pXL519) was estimated to be nearly the same level as native PBP3. This may suggest that expression of the pbpC gene was under tight control. However, low production of the recombinant PBP3x as detected by H-penicillin could also have been due to limitations of the detection method 3  (given that PBP3x has a low binding affinity to P-lactam antibiotics) rather than that truly reflecting its low expression level.  126  7. Correlation of P-lactam antibiotic susceptibility and the overproduction of PBP3 and PBP3x The MIC of an antibiotic for a given bacterium is the lowest concentration of the antibiotic present in the growth medium that results in the inhibition of bacterial growth. In the case of the P-lactam antibiotics and Gram-negative bacteria, the susceptibility to P-lactams, as measured by the MIC, is dependent upon at least three parameters, outer membrane permeability, hydrolysis rate by P-lactamase, and interaction with the target proteins (i.e., PBPs). The alteration of either of the first two parameters can result in resistance to P-lactam antibiotics (Hancock et al., 1988; Spratt, 1989; Livermore, 1993), which is experimentally observed by an increase in the MIC of the tested antibiotic against that bacterial strain. However, the interaction with the target PBPs has been expressed as a factor Si (the periplasmic concentration of P-lactam antibiotics at the MIC) (Nikaido & Normark, 1987; Bellido et al, 1991a). The relationship between this factor and the kinetic constants of P-lactam and PBP interactions, as well as their influence on MIC is unknown (see below). The major impediments to P-lactam therapy have been the high level of resistance of some organisms, including P. aeruginosa, due to the intrinsic barrier properties of their outer membranes and the acquisition of high levels of P-lactamase due to either the presence of a plasmid or the derepression of a formerly inducible,  127  chromosomally encoded P-lactamase (Hancock et al, 1988; Philippon et al, 1989). The simple and convenient assay methods for P-lactamase activity have resulted in the generation of a substantial body of literature on P-lactamase-mediated resistance (Livermore, 1993; Moellering, 1993; Bush etal, 1995). Mutational alternations in the P-lactam targets, PBPs, have also been observed. Bacterial strains with altered PBPs have been generated in the laboratory and also found in clinical isolates (Malouin & Bryan, 1986; Georgopapadakou, 1993). It has been demonstrated that alteration of PBPs can result from either reduced affinities of PBPs for P-lactam antibiotics (Godfrey etal, 1981; Hedge & Spratt, 1985; Gotoh et al, 1990) or from acquisition of a resistant PBP by lateral gene transfer and homologous recombination (Spratt et al, 1989; Spratt, 1994). However, reports on bacterial resistance mediated by PBP alteration seem to occur with somewhat less frequency as compared with ones on the resistance mediated by P-lactamase production. The rarity of finding this type of resistance mechanism in clinical isolates could also be due to the lack of sufficient biochemical investigation. In addition, the experimental demonstration of PBP binding affinity for most bacteria is difficult due to a lack of biochemical and genetic information about the PBPs of the bacterium in question and the lack of a convenient and simple assay method. Currently, radiolabeled penicillin G is the only commercially available probe for assaying alterations in the properties of PBPs.  128  As mentioned above, the efficacy of (3-lactam antibiotics against Gramnegative bacteria, as measured by their MICs, depends on then: rate of penetration across the outer membrane, their degree of resistance to (3-lactiimase inactivation, and their ability to inhibit the target proteins, the PBPs. Zimmermann and Rosselet (1977) first described outer membrane permeability to (3-lactams in terms of Fick's first law of diffusion, i.e., V = P A (S -S ), where V is the rate of diffusion across the outer 0  p  membrane, S is the external and S the periplasmic concentration of the (3-lactam, A G  p  is the area of the membrane per unit weight of cells (calculated by electron microscopy) and P the permeability coefficient. As noted by Zimmermann & Rosselet (1977), the outer membrane permeability is rate-limiting for hydrolysis of (3-lactams by periplasmic (3-lactamase, which follows the Michaelis-Menten equation V = V S / (S + K ). p  p  m a x  At the steady state, the rate of hydrolysis of P-lactam is limited and  m  thus balanced by the rate of permeation, i.e., V = V . This gives V = P A (S -S ) = 0  V  m a x  S / (S + K ). p  p  m  p  The equation was modified by Nikaido and Normark (1987) to  permit the prediction of the factors involved in P-lactam efficacy, i.e., S = S [1 + G  V  m a x  p  / ( K + S ) P A]. When S is equivalent to MIC, S corresponds to Si which is m  p  0  p  the actual concentration of P-lactams in the periplasm at an external concentration equal to the MIC, assumed to be the target PBP inhibitory concentration. To predict the Si, Nikaido and Normark (1987) used literature estimates of the I  50  to one of the  essential PBPs, i.e., PBPlb, PBP2 or PBP3 to obtain a global estimate. However, this  129  might not be accurate. According to the rearrangement of the equation, S is related to p  the kinetic constants of the P-lactamase ( V  max  , K ) , the surface area of the bacterial m  cell (A) and the permeability coefficient of the outer membrane (P). The determination of the permeability coefficient of the outer membrane (P) can be influenced by the experimental approaches and has been a controversial issue. Bellido et al. (1991a) presented a direct method for assessing outer membrane permeability by using intact bacterial cells and high-performance-liquid-chromatography (HPLC), which avoids certain possible artifacts in those methods utilizing extrapolations from model membrane systems (Zimmermann & Rosselet, 1977; Nikaido et al., 1983). With the HPLC method, the rate (V) of antibiotic uptake by intact cells (i.e., disappearance from the medium) can be measured directly, and since S = V K / ( V p  m  m a x  - V), the coefficient P can then be assessed accurately according to V = P A (S - S ), Q  p  where S is known as the P-lactam concentration initially used for measuring V . 0  Using Enterobacter cloacae strain R l , Bellido et al (1991b) tested the use of the formula for predicting MICs, i.e. MIC = Si [1 + V calculation of P, V  m a x  m a x  / ( K + S ) P A], after m  p  and K and estimation of Si. Using variants of strain R l with m  different levels of P-lactamase or different outer membrane permeability, the general reliability of this predictive equation was tested. In all cases calculated MICs were the same as measured MICs. However the influence of changes in the target PBPs on MIC was not tested.  130  In this study, the influence of overproduction of P. aeruginosa PBP3 on the susceptibility of P. aeruginosa to P-lactam antibiotics was investigated. It was found that increased levels of the P. aeruginosa pbpB gene product in P. aeruginosa resulted in increased levels of resistance to certain PBP3-targeted antibiotics. It was observed that a 7-fold increase in the amount of PBP3 in P. aeruginosa resulted in a 2-fold increase in the level of resistance to aztreonam, a 4-fold increase in resistance to cefepime, and 8-fold increases in resistance to ceftazidime and cefsulodin. The increased MICs in the strains that overproduced PBP3 suggests that the PBP is biologically functional. P. aeruginosa PAO4089, used in this study, is a P-lactamase non-inducible mutant; use of this strain eliminated complications due to the induction of chromosomal P-lactamase expression. The reaction of a P-lactam antibiotic and a PBP involves the initial formation of a reversibly bound enzyme-inhibitor complex followed by covalent modification (acylation) and hence irreversible inhibition. The reaction scheme is analogous to that of the Michaelis-Menten mechanism, E + I = E I —> E-I* and should show saturation kinetics with increasing inhibitor concentrations. Increased expression of the PBPs in the PAO4089 cells would therefore require a corresponding increase in the amount of periplasmic P-lactam antibiotic to effect the inhibition of peptidoglycan biosynthesis to a level sufficient to result in growth inhibition. Thus, the overexpression of PBP3 should result in increased S to PBP3 A  targeted antibiotic, which consequently resulted in increased concentration of the  131  antibiotic in the medium that is required to inhibit cell growth. This corresponded to the observed increase in the MIC. The differential increases in MIC among the four different PBP3-targeted antibiotics may have been due to their different binding affinities for PBP3, as indicated by their different I values (Table 7). Ceftazidime 50  and cefsulodin had relatively lower affinities (i.e., higher I ) for PBP3 as compared to 50  cefepime and aztreonam, and higher concentrations of these two antibiotics were thus required to inhibit the overproduced PBP3 (i.e., higher MICs). Alternatively, it must be noted that MIC is not a simple function of Si (see above) and that other parameters in the periplasm would influence MIC. Furthermore, binding to PBPla, lb or PBP2 might have played some role. In any event, the results presented in this study demonstrate that the interaction of P-lactam antibiotics and their targeted PBPs are influenced by the level of PBP production and support the notion that regulation of PBP expression is a possible mechanism for resistance to P-lac tarn antibiotics in P. aeruginosa. To my knowledge this is the first demonstration of PBP-overexpressionmediated resistance to P-lactam antibiotics. In addition to the overproduction of P. aeruginosa PBP3, it was found that overproduction of the E. colipbpB gene product in P. aeruginosa PAO4089 resulted in 2-fold increase in the level of resistance to aztreonam and 4 fold increases in resistance to ceftazidime and cefepime, which suggests that the E. coli PBP3 retained its biological function in P. aeruginosa. In addition, these results support the  132  assertion discussed in the preceding section that the P. aeruginosa PBP3 was a functional homologue of the E. coli PBP3. Increased resistance to P-lactam antibiotics due to elevated expression of penicillin-sensitive PBPs has not been found in clinical isolates of Gram-negative bacteria and to my knowledge very little, if any, effort has been put forth to search for such mutants. The failure to identify this type of resistance mechanism in clinical isolates could have been due to the lack of an appropriate assay. Increased expression of PBPs alone probably would not contribute much to the overall resistance of a bacterium to P-lactam antibiotics. However, increased PBP expression coupled with reduced outer membrane permeability is potentially an effective mechanism for producing resistance. This work also revealed that overproduction of the P. aeruginosa pbpC gene product did not alter the susceptibility of the overexpressing strain to the P-lactam antibiotics tested. This probably resulted from the lower affinity of PBP3x for the PBP3-targeted antibiotics as compared to PBP3. This could result from the nature of the various amino acid residues making up and surrounding the active binding-site serine, which are known to play roles in facilitating the binding to the substrates (Ghuysen, 1991; Malhotra etal., 1992; Petrosino etal., 1996). 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