JAC Advance Access originally published online on February 8, 2007
Journal of Antimicrobial Chemotherapy 2007 59(3):411-424; doi:10.1093/jac/dkl536
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Function of penicillin-binding protein 2 in viability and morphology of Pseudomonas aeruginosa
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1 Canada
* Corresponding author. Tel: + 1-519-824-4120; Fax: + 1-519-837-1802; E-mail: aclarke{at}uoguelph.ca
Received 6 October 2006; returned 20 November 2006; revised 8 December 2006; accepted 11 December 2006
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Objectives: To investigate the function of penicillin-binding protein 2 (PBP 2) in Pseudomonas aeruginosa PAO1.
Methods: The growth and morphology of P. aeruginosa cultured in the absence and presence of mecillinam was assessed. The gene encoding PBP 2, pbpA, was identified in the genome of P. aeruginosa PAO1 and both its full-length and an engineered truncated form were cloned and expressed in Escherichia coli. Site-directed mutagenesis was used to confirm Ser-327 as the catalytic nucleophile of its transpeptidase domain. Allelic exchange was used to construct a chromosomal mutant of pbpA in strain PAO1.
Results: PAO1 grew with a spherical morphology in the presence of mecillinam at concentrations as high as 2000 mg/L. Both wild-type and truncated, soluble forms of PBP 2 were shown to bind penicillins and a competition assay demonstrated their specificity for mecillinam. The PAO1 
pbpA insertional mutant also grew as spheres, and complementation with a plasmid encoding active pbpA, but not with an inactive Ser-327 
Ala derivative, restored rod-shape morphology. MIC values of a variety of ß-lactams were significantly lower for the insertional mutant compared with wild-type PAO1. The muropeptide profile of peptidoglycan from PAO1 pbpA analysed by HPLC/MALDI TOF MS indicated wild-type levels of cross-linking despite the loss of PBP 2 transpeptidase activity.
Conclusions: PBP 2 in P. aeruginosa is responsible for the rod-shape morphology of the cells and contributes significantly to ß-lactam resistance. The viability of cells lacking an active PBP 2 suggests that the organization of the peptidoglycan biosynthetic machinery is different in this pathogen compared with E. coli.
Keywords: peptidoglycan , P. aeruginosa , mecillinam
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Penicillin-binding proteins (PBPs) are essential for the growth and division of bacterial cells because they catalyse the final stages of peptidoglycan biosynthesis within the periplasm.1–4 They belong to the family of acyl serine transferases and they are broadly classified according to their molecular size as either high-molecular-weight (HMW) or low-molecular-weight (LMW) PBPs.5 All are associated with the outer leaflet of the cytoplasmic membrane, and all possess a penicillin-binding (PB) domain. The PB domain, which contains the catalytic serine residue, functions to catalyse either the transpeptidation (HMW-PBPs) or hydrolysis (LMW-PBPs) of the stem peptides associated with the muramoyl residues of the newly incorporated peptidoglycan subunits. The HMW-PBPs are further characterized by an N-terminal, non-penicillin binding (n-PB) domain which is anchored to the cytoplasmic membrane via a non-cleavable signal-sequence peptide. In some cases, the n-PB domain also catalyses the transglycosylation of bactoprenol-linked precursors into the growing peptidoglycan sacculus. These latter proteins are thus both multi-modular and bi-functional, and they have been classified as class A HMW-PBPs. The class B HMW-PBPs possess an N-terminal domain, but it does not appear to catalyse transglycosylase activity. For these HMW-PBPs, it has been suggested that the n-PB domain plays a role in directing the proper folding of the C-terminal PB domain and/or is involved in protein–protein interactions.6–8
In Escherichia coli, there are 13 known PBPs; three class A (PBPs 1a, 1b and 1c) and two class B (PBPs 2 and 3) HMW-PBPs, and eight LMW-PBPs (PBPs 4, 5, 6, 7 and 8, DacD, AmpC and AmpH).9 Inhibition experiments with mecillinam, a ß-lactam with specificity for PBP 2 (reviewed in ref. 10), and in vitro deletion studies targeting the gene encoding PBP 211–13 have shown that cells grow as enlarged spheres in the absence of this protein's activity suggesting that it functions in the maintenance of the rod shape of this bacterium. Upon continued exposure to mecillinam for 2 h or more, E. coli cells begin to lyse due to a blockage in cell division.14,15 In contrast, cells of the opportunistic pathogen Pseudomonas aeruginosa seem to be able to tolerate exposure to mecillinam and are viable in the presence of up to at least 400 mg/L.16,17
Using the conventional PBP assay, which involves SDS-PAGE analysis of proteins treated with radiolabelled penicillin and autoradiography,18 an earlier study suggested that P. aeruginosa produces six PBPs.17 Based on apparent molecular masses, these were proposed to be homologues of E. coli PBPs 1a, 1b, 2, 3, 4 and 5. Subsequently, the genes encoding PBPs 1a,19 3,20 a second form of 3 named 3x21 and 522 were cloned and characterized. In addition, the product of P. aeruginosa gene pbpG was shown to be a homologue of E. coli PBP 7, a DD-endopeptidase.23 Nothing else has been reported to date confirming the existence of other PBPs, including PBP 2, in this pathogen.
Given the importance of the ß-lactam antibiotics to combat bacterial infections over the past sixty years, a considerable amount of information is known about the structure and function relationship of the PB domain of the PBPs. However, the emergence of a number of ß-lactam-resistant pathogens has posed serious threats to the continued efficacy of this important class of antibiotics. In efforts to develop new therapeutic strategies, attention has been drawn to the n-PB domain of the HMW PBPs (e.g. ref. 24) about which little is currently known (reviewed in ref. 25). The determination of the three-dimensional structures at high resolution of the class B PBPs 2x from Streptococcus pneumoniae (PDB: 1QMF)26 and 2a from an MRSA strain of Staphylococcus aureus (PDB: 1VQQ)27 will greatly facilitate investigations of these enzymes, but enzymological studies are still lacking.
To further our understanding of the organization, activity and function of the class B PBPs, we have initiated studies with the enzymes of P. aeruginosa. Here, we investigate the effects of mecillinam on the growth and morphology of P. aeruginosa and describe the cloning and overexpression of the P. aeruginosa gene encoding PBP 2, pbpA, in both its native membrane-bound form and a genetically-engineered soluble form. We also describe the generation and phenotype of a mutant lacking a functional pbpA gene.
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Plasmids, bacterial strains and growth conditions
All plasmids used or constructed, together with bacterial strains used in this study are listed in Table 1. Cultures were routinely grown in Luria-Bertani (LB) broth [1% tryptone (Difco, Detroit, MI, USA), 0.5% yeast extract (Difco) and 0.5% NaCl] at 37°C with agitation. For overexpression of cloned genes, cultures were grown in rich medium containing 3.2% tryptone, 2% yeast extract and 1% NaCl. Strains harbouring resistance determinants were grown in the presence of ampicillin (100 mg/L), kanamycin (50 mg/L), chloramphenicol (34 mg/L) and/or tetracycline (15 mg/L for E. coli, 110 mg/L for P. aeruginosa). Unless otherwise stated, all reagents and chemicals were obtained from Sigma-Aldrich Canada Ltd., Oakville, ON, Canada.
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Determination of MICs
The MIC values for the ß-lactams mecillinam (Leo Pharmaceutical Products, Denmark), ampicillin, amoxicillin, carbenicillin, cefotaxime, cefoxitin, cefalotin and ticarcillin for P. aeruginosa PAO1 and E. coli DH5
were determined using LB broth containing doubling concentrations of the penicillin at 37°C with shaking at 200 rpm. MICs were defined as the lowest concentration of compound to inhibit visible growth after 18 h incubation.
Growth curves and viable counts
The growth characteristics of the E. coli and P. aeruginosa in the absence and presence of mecillinam were determined by incubating a 1/20 dilution of overnight cultures in 50 mL of LB containing the appropriate amount of mecillinam at 37°C with shaking (200 rpm). Growth of the cultures, in triplicate, was monitored by both OD600 and viable counts for at least 6 h. For viable counts, samples of the growing cultures were serially diluted in sterile saline (10–2, 10–3, 10–4, 10–6, 10–8) and 100 µL aliquots were plated onto LB plates containing 5% agar (Difco). After 16 h incubation at 37°C, the colonies were counted and viable counts determined.
Bacterial cell pellets were washed and resuspended in 0.07 M Sorensen's phosphate buffer, pH 6.8 (PBS), placed on a 13 mm carbon planchette (Canemoc & Marivac, Lakefield, Quebec, Canada) and fixed with 2% gluteraldehyde in PBS buffer for 1 h. The samples were then rinsed in several changes of buffer, dehydrated through a series of ethanol washes, critical point dried using carbon dioxide, and sputter coated with 20 nm of gold/palladium in a Hummer VII sputter coater (Anatech Corp. Alexandria, VA, USA). To visualize the bacteria, filters were scanned using a Hitachi S-570 SEM (Tokyo, Japan), and images were collected directly from the SEM using Quartz PCI software (Quartz Imaging Corp. Vancouver, BC, Canada).
Cloning and genetic engineering of pbpA
P. aeruginosa chromosomal DNA from strain PAO1 was prepared from an overnight culture using DNAzol (Invitrogen Canada Inc., Burlington, ON, Canada), according to the manufacturer's protocol. The isolated DNA was used to PCR amplify pbpA encoding PBP 2 using the Expand PCR kit (Roche Molecular Biochemicals, Laval, QC, Canada) according to the manufacturer's instructions. The oligonucleotide primers used were 5'-CCAGTCCATATGCCGCAGACCATCCACCTG-3' (NdeI) and 5'-GCGGTCGAAGCTTCTGTTCAAGGGCGGGCG-3' (HindIII). NdeI and HindIII restriction sites were used to facilitate the cloning into the vectors pGEM-T (Promega) and pET30a(+) (Novagen, Madison, WI, USA) to provide pACJW1 and pACJW2, respectively. In addition, the pET30a(+) construct was designed to express the full-length PBP 2 as a fusion with a C-terminal hexa-His tag
To facilitate its expression and purification, a truncated construct of pbpA was engineered by removing the 5' sequence coding for the hydrophobic leader peptide (amino acids 1 to 38) of PBP 2 (Figure 1). The open reading frame encoding PBP 2 starting at amino acid 39 (methionine) was PCR amplified as described above using the following oligonucleotide primers: 5'-TGGTGGCGCATATGTACCACCTG-3' (NdeI) and 5'-GCGGTCGAAGCTTCTGTTCAAGGG-3' (HindIII). The truncated pbpA was cloned into pET30a(+) yielding pACKD16 thereby encoding the truncated protein with a hexa-His tag. The nucleotide sequences of each of the constructs were confirmed prior to protein expression experiments.
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Site-specific replacement of Ser-327 in the full-length PBP 2 with an Ala residue was performed using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) resulting in construct pACDC38. The following primers were used for this procedure using the recommended protocol given by Stratagene: 5'-CTATCCTCCGGGCGCGACGGTGAAGCCG-3' and 5'-CGGCTTCACCGTCGCGCCCGGAGGATAG-3'.
Expression and purification of PBP 2
E. coli BL21(
DE3) codon plus (pLysS) harbouring either pET30a(+), pACJW2 or pACKD16 were grown in 1 L of rich medium at 37°C with agitation. To induce protein expression, 1 mM IPTG (Roche) was added to the culture when it reached an OD600 of 0.6, and growth was continued for a maximum of 3 h. The cells were harvested by centrifugation (6000 g, 15 min, 4°C), and re-suspended in either 10 mM Tris-HCl pH 8.0, containing 10 mM MgCl2 and Complete Mini, EDTA-free Protease Inhibitor Cocktail Tablets (Roche) for cell fractionation to obtain membrane-associated PBP 2-His6, or lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0 containing Complete Mini, EDTA-free Protease Inhibitor Cocktail Tablets) for direct purification of sPBP 2-His6.
For the isolation of membrane-bound PBP 2, the cell suspension was lysed by passage through a French Pressure Cell Press (American Instrument Co., Silver Spring, MD, USA). Unlysed cells were removed by centrifugation (6000 g, 10 min) and the membrane fraction was recovered by ultracentrifugation (100 000 g, 60 min, 4°C). This isolated membrane fraction was resuspended in 20 mM Tris-HCl, pH 8.0, containing 2% N-lauryl-sarcosine, stirred for 12 h at 4°C, and then unsolubilized matter was removed by ultracentrifugation (100 000 g, 60 min, 4°C). The membrane extract was diluted to 1% Sarkosyl with 200 mM sodium phosphate, 30 mM Tris buffer, pH 8.0, containing 1 M NaCl, 20% glycerol and subjected to affinity chromatography on His-Select Nickel Affinity Gel (Sigma) at 4°C. The resin was washed with 250 mL of wash buffer (100 mM sodium phosphate, 15 mM Tris, pH 8.0, containing 0.5 M NaCl, 10% glycerol and 0.5% Sarkosyl) and the bound PBP 2 was eluted with 2 x 1 mL volumes of wash buffer containing 250 mM imidazole.
For isolation and purification of sPBP 2, the cell suspension in lysis buffer was passed through a French Press and un-lysed cells and other particulates were removed by centrifugation (8000 g, 10 min, 4°C). The cell lysate was added to 1 mL His-Select agarose, previously equilibrated in lysis buffer. After gentle agitation for 1 h at 4°C, the slurry was transferred to a 25 mL column and washed with 100 mL of lysis buffer containing 20 mM imidazole, followed by 100 mL of lysis buffer containing 30 mM imidazole. sPBP 2-His6 was eluted from the column with 6 mL lysis buffer containing 250 mM imidazole.
Detection of expressed PBP 2 took advantage of the fusion of the C-terminal His6-tag and a commercially available mouse anti-His antibody (‘His Probe H-3’ from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Western immunoblotting was performed as previously described.28
PBP 2-His6 and sPBP 2-His6 were analysed for penicillin-binding activity by the SDS-PAGE-based assay using fluorescent Bocillin FL (Molecular Probes Inc., Eugene, OR, USA)29 and biotinylated ampicillin.30 For the fluorescent PBP assays, 19 µL samples of the purified protein (
5 µg) were incubated with 5 µL of Bocillin (final concentration of 50 µM) for 2 h at 35°C. SDS-PAGE sample buffer was added to protein samples and boiled for 10 min prior to electrophoresis. Detection of PBP–penicillin complexes was achieved by exposure of the gel to UV290 nm light.
The biotinylated ampicillin PBP assays were adapted from the protocols of Dargis and Malouin30 and Okamoto et al.31 To prepare biotinylated ampicillin, NHS-biotin was added at a 5:1 ratio to 110 mg/L of ampicillin in 100 mM sodium phosphate buffer, pH 7.2 and incubated at room temperature for 30 min. To block any unreacted NHS-biotin, a ten-fold molar excess of glycine was added and incubated for an additional 30 min. To label and detect PBPs, typically 5 µL of the biotin–ampicillin was added to 19 µL of PBP sample (2–10 µg) and incubated at 35°C for 2–3 h. The reaction was stopped by adding 6 µL of SDS-PAGE sample buffer and boiling for 3 min. Following electrophoresis in a 12.5% SDS-PAGE gel, the separated proteins were transferred to a nitrocellulose membrane, blocked with 3% BSA, and washed as previously described.28 A 1:50 000 dilution of streptavidin–HRP (Pierce) in blocking buffer was applied to the membrane and allowed to bind to biotin–ampicillin–PBP complexes for 1 h at room temperature. To remove unbound streptavidin conjugates, the membrane was washed several times in TTBS at 37°C. The membrane was incubated in HRP substrate (SuperSignal West Femto Maximum Sensitivity Substrate; Pierce) for 5 min. Excess reagent was drained and the membrane was exposed to Bioflex MSI film (Clonex, Markham) and developed.
Equivalent protein samples (5–10 µg) were pre-incubated with 5 µL of either mecillinam (1, 5, 10, 50, 500 and 1000 mg/L, final concentrations) or water (as the control) for 30 min at 35°C before applying either fluorescent Bocillin FL or biotinylated ampicillin for incubation in a PBP assay.
The protocol used was that recommended by the Biological Mass Spectrometry Facility at the University of Guelph, modified from the procedure by Shevchenko et al.32 Briefly, the protein was subjected to SDS-PAGE. The separating gel was fixed for 30 min in 5:4:1 methanol:H2O:acetic acid and then stained for 30 min in Coomassie Brilliant Blue R. The band of interest was excised from the gel, diced and washed three times for 5 min by vortexing in H2O. To destain, 100 mL of 50 mM NH4HCO3 in 50% acetonitrile was added and the suspension was vortexed for 10 min. The gel particles were dried with 100 mL of acetonitrile. Reduction and alkylation of cystine groups was not performed because PBP 2 is not predicted to have any cysteine residues. Sequencing grade trypsin (Sigma) was added to the gel particles in 50 mL of 100 mM NH4HCO3 and the digesting mixture was incubated 37°C overnight. The resulting digest was spotted onto a MALDI sample plate and was analysed on a Bruker Reflex III MALDI-TOF in reflectron mode using a 337 nm nitrogen laser set to 109–121 mJ output. Analysis of peptides was done using the on-line tool MS-Fit (http://prospector.ucsf.edu).
Analysis of peptidoglycan cross-linking
Cultures of each of the strains were grown in LB at 37°C with shaking for 3 to 5 h until mid-exponential phase (OD600 of 0.60 to 0.90). Cells were harvested by centrifugation at 8000 g for 15 min and washed twice in 10 mM sodium phosphate pH 7.0. Insoluble peptidoglycan was extracted from whole cells by boiling in 4% SDS procedure as previously described.33 Peptidoglycan insoluble in 4% SDS was collected by ultracentrifugation at 100 000 g for 60 min at 20°C. The pellet was washed with water several times. This peptidoglycan was further purified by treatment with -amylase, DNase, RNase and pronase as described previously.33
Analysis of peptidoglycan cross-linking associated with the different P. aeruginosa transformants was accomplished using the HPLC-based method of Glauner34 following the digestion of isolated peptidoglycan with 20 U/mL mutanolysin at 37°C for 16 h. The digested material was subjected to centrifugation (12 000 g, 10 min, RT) to remove any remaining insoluble material, and the soluble muropeptides in the supernatants were reduced with sodium borohydride in 0.5 M sodium borate buffer, pH 9, before being applied to a 4.6 x 250 mm Gemini C18 (5 µ) analytical column (Phenomenex Inc., Torrance, CA, USA). Detection of eluting muropeptides was achieved by monitoring A205.
Generation of a pbpA chromosomal insertion mutant
A gene replacement strategy for making chromosomal knockout mutants in P. aeruginosa was employed.35 The oligonucleotide primers used to amplify pbpA and flanking regions were 5'-CCGCTCTAGAAGGTCGGCCCTGGAG-3' (XbaI) and 5'-CCCTAAGCTTCGTGGCCGATCAC-3' (HindIII). The amplified DNA was ligated into the suicide vector pEX18Ap to produce the plasmid pACBL13. This plasmid was transformed into the E. coli strain SCS110 (lacking Dam methylation on DNA). pACBL13 was isolated from this strain and subsequently digested with the restriction enzyme BclI (which is unable to digest Dam methylated DNA). A non-polar 1088 bp GmR cassette was obtained from the plasmid pPS856 by digestion with BamHI. Since BclI and BamHI create compatible sticky ends, the GmR cassette was inserted within the pbpA gene located on pACBL13 to create plasmid pACBL15.
pACBL15 was transformed into the E. coli mobilizer strain SM10, and transferred to P. aeruginosa PAO1 via conjugation using a modification of the method by Simon et al.36 Briefly, E. coli SM10 (pACBL15) and P. aeruginosa PAO1 were grown until late exponential stage. The two cell lines were combined in varying ratios (1:9, 9:1 and 1:1), centrifuged to concentrate, and then spotted onto pre-warmed LB plates and incubated overnight at 37°C. P. aeruginosa conjugants were selected by resuspending spotted cells in LB and plating on Pseudomonas Isolation Agar with 150 mg/L gentamicin. The pEX18Ap vector contains the Bacillus subtilis sacB gene, and when expressed makes cells sensitive to sucrose. Thus, streaking cells on a 10% sucrose medium containing 150 mg/L gentamicin inhibited the growth of merodiploids and selected for true recombinants. Colonies that could grow on this medium and were also carbenicillin susceptible (600 mg/L) were further screened by PCR using the primers 5'-CTACCACAAATAGTCCCCAGTCCCG-3' and 5'-GTCTTCGTTGGACAGGGTGCGGTCG-3' to verify insertion of the GmR cassette.
Construction of the pbpA complementation vectors
The pbpA gene together with an upstream ribosome-binding site, but lacking the histidine tag was subcloned from the pET construct pACJW2 using PCR with the primers 5'-GAGCGGATAACAATTCCCCTCTAGA-3' (XbaI) and 5'-GAGTGCGGCCGAATTCTACTGTTCAAGG-3' (EcoR1). After PCR amplification, the product was ligated into the complementation vector pUCP2637 resulting in construct pACBL31. Cloning was performed in E. coli DH5 using repressing conditions (0.2% glucose) and using medium adjusted to pH 5.0 to minimize the lethality of PBP 2 expressed in E. coli. A derivative of pACBL31 (named pACBL34) was engineered using the QuickChange Site-Directed Mutagenesis Kit and the primers described above to encode the Ser-327 Ala PBP 2 derivative. Electrocompetent cells of P. aeruginosa for transformation with pACBL31 and pACBL34 were prepared using the method of Choi et al.38
Predictions of transmembrane helices (membrane anchor) were made with the programs DAS (http://www.sbc.su.se/~miklos/DAS/),39 PSORTb (http://www.psort.org), TMHMM40 and TMpred (http://www.ch.embnet.org/software/TMPRED_form.html), while SignalP (v. 3.0)41 was used to search for signal peptides. The peptide mass fingerprinting tool MS-Fit was used to confirm the identity of PBP 2 by fitting mass spectrometry data to a protein sequence in the database [available online at the Protein Prospector42 Web site (http://prospector.ucsf.edu)]. Agarose gel electrophoresis was performed using 1% agarose (OmniPur, Darmstadt, Germany), while SDS-PAGE43 was performed using 12.5% (w/v) acrylamide [Bio-Rad Laboratories (Canada) Ltd, Mississauga, ON, Canada]. DNA sequencing was performed by the Guelph Molecular Supercentre, University of Guelph. Concentrations of both PBP 2-His6 and sPBP 2-His6 were determined using a commercial BCA assay (Pierce, Rockford, IL, USA) with BSA (0.05–2 mg/mL) as the standard. The assay was performed according to the procedure outlined by the manufacturer except the absorbance was measured at 595 nm rather than 560 nm. N-terminal sequence analysis of purified PBP 2-His6 and sPBP 2-His6 that were subjected to SDS-PAGE and subsequently transferred to 0.45 µm polyvinylidene difluoride membrane was performed by David Watson, National Research Council, Ottawa, Canada.
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Effect of mecillinam on growth and morphology in P. aeruginosa
The effects of the ß-lactam antibiotic mecillinam on growth of P. aeruginosa and E. coli in LB at 37°C were determined by monitoring both turbidity at 600 nm and viable cell counts. As seen in Figure 2, the addition of 10 mg/L mecillinam to cultures of E. coli caused a cessation of growth and following a short stationary phase, the cells entered into a death phase with concomitant cell lysis. In contrast, cultures of P. aeruginosa remained viable in concentrations of mecillinam as high as 400 mg/L and they continued to grow at approximately the same rate as untreated cells.
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An attempt was made to determine the MIC of mecillinam for P. aeruginosa PAO1 growth in LB broth. Under the conditions employed, concentrations as high as 2000 mg/L did not prevent growth while the MIC of this ß-lactam for E. coli DH5
was determined to be 1.0 mg/L. These data are thus consistent with those previously documented by others where MICs for strains of E. coli were found to be 
1.0 mg/L while P. aeruginosa is reported to be resistant to mecillinam.10,16 Despite the fact that mecillinam did not appear to inhibit growth of P. aeruginosa, cells cultured in its presence were nonetheless examined by SEM. When treated with 10 mg/L mecillinam (i.e. 10 x MIC) E. coli formed enlarged spherical cells within 1.5 to 2 h of antibiotic addition (Figure 2). These spheres were, on average, approximately 1.7 µm in diameter. As expected, evidence of cell lysis was observed after prolonged incubation. With P. aeruginosa at this concentration of mecillinam, cells retained their normal rod shape but, surprisingly, a morphological change to spherical cells was observed when incubated at concentrations between 200 mg/L and 400 mg/L (Figure 2). Unlike E. coli, however, these spherical cells were not enlarged but rather retained the same diameter of approximately 0.5–0.7 µm as their former rod shape. Also, in contrast to E. coli, removal of the antibiotic by harvesting the cells, washing with PBS, and re-suspending them in fresh LB broth resulted in their reversion back to their original rod shape. No such reversion was observed with E. coli. Thus, while mecillinam causes the same morphological changes in the two bacterial species, its different effect on cell viability prompted an investigation of the role and function of PBP 2 in P. aeruginosa.
Identification and characterization of pbpA from Pseudomonas aeruginosa
The nucleotide sequence of pbpA was identified within the P. aeruginosa genome44 by amino acid sequence alignments using E. coli PBP 2 (NCBI accession no. P08150) as the probe. Whereas two homologues of PBP 3 have been identified in P. aeruginosa,20,21 and despite a careful search, the only homologue of PBP 2 on the P. aeruginosa chromosome was identified as ORF PA4003 (NCBI accession no. AAD32230 [GenBank] ). This ORF codes for 646 amino acid residues which provide hypothetical Mr and pI values of 72213 and 7.87, respectively. Its predicted amino acid sequence was found to have 43.1% identity and 74.4% similarity to E. coli PBP 2 (Figure 1). Further analysis of the P. aeruginosa PBP 2 hypothetical sequence using the algorithm PSORTb provided a score of 8.02 as a plasma membrane protein associated with the periplasm, while the DAS, TMPred and TMHMM programs led to the identification of an inside to outside transmembrane helix comprising residues Ile-22 to Arg-38, Val-21 to Arg-38 and Val-21 to Tyr-40, respectively. In addition, SignalP identified a potential transmembrane signal peptide involving residues Arg-20 to Ala-37 but no cleavage site was predicted.
Sequence alignment analysis of the hypothetical P. aeruginosa PBP 2 suggests it would be classified as a class B2 PBP, like E. coli PBP 2. Presumably this P. aeruginosa PBP 2 is a mono-functional enzyme which combines both a membrane-associated n-PB domain with an acyl serine transferase PB domain, as known for its E. coli homologue.5 Based on this alignment, and the analyses described above, the membrane anchor would involve residues Arg-19 to Ala-37, and the nPB domain would continue to Ser-250 (Figure 1). The remaining C-terminal sequence of Ile-251 to Gln-652 would constitute the PB domain, with Ser-327 predicted to serve as the catalytic nucleophile for transpeptidation (and penicillin-binding) activity.
Analysis of the PAO1 chromosome immediately upstream and downstream of pbpA indicated that, as with E. coli, the gene is located within a cluster that contains several other genes involved in peptidoglycan metabolism, including rodA (Figure 3). RodA has also been demonstrated to be essential for rod-shape morphology in E. coli45 and it is presumed to interact directly with PBP 2. Interestingly, the PAO1 cluster also contains a gene encoding soluble lytic-transglycosylase B1 (SltB1) which is not present in the respective E. coli cluster.
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Cloning of pbpA from P. aeruginosa
Based on its nucleotide sequence, oligonucleotide primers were designed to allow PCR amplification of P. aeruginosa pbpA from genomic DNA and subsequent cloning into the T7-based expression vector pET30a(+) for fusion of the protein to a His6-tag. One construct, designated pACJW2 was transformed into the expression strain E. coli BL21 (DE3) Codonplus (pLysS) to give E. coli AC224. Due to difficulties encountered with the production and purification of PBP 2 (described below), a soluble derivative of the protein (named sPBP 2) truncated by 38 amino acids was engineered which lacked the hypothetical membrane anchor located at its N-terminus (Figure 1). One clone, designated pACKD16 was subsequently used to overexpress the truncated protein in E. coli BL21 (DE3) CodonPlus (pLysS) generating E. coli AC252.
Expression of P. aeruginosa pbpA
Growth of E. coli AC224 in LB at 37°C and induction with IPTG led to the over-production of PBP 2-His6 as detected by SDS-PAGE with Coomassie Brilliant Blue staining and western immunoblotting (Figure 4). Protein was expressed at a relatively low level, and cell fractionation studies indicated it to be localized to the cytoplasmic membrane fraction, as expected (data not shown). Very little PBP 2-His6 was found in inclusion bodies, which can sometimes be a problem with membrane proteins such as the HMW PBPs. Growth was allowed to continue for a maximum of 3 h, as this appeared to provide optimal expression. Incubation for longer periods of time resulted in very poor yields which were found to be due to cell lysis suggesting that overexpression of PBP 2 is lethal for the cell.
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In contrast to full-length PBP-His6, high levels of the truncated sPBP 2-His6 could be obtained with E. coli AC252 without any apparent impairment of cell growth, even after extended periods of incubation following IPTG induction (data not shown). Most of the protein produced was recovered in the soluble fraction of lysed cells, but some was contained in insoluble protein aggregates.
Cells of E. coli AC224 overexpressing full-length PBP 2-His6 were ruptured using a French Press and isolated membranes were solubilized with 2% Sarkosyl. The solubilized cytoplasmic membrane proteins were subjected to affinity chromatography on His-Select agarose and PBP 2-His6 was eluted using imidazole. Using western immunoblotting with an anti-His antibody, it was found that most of the PBP 2-His6 did not bind the affinity column but instead was contained in the flow-through fraction (Figure 5a). Attempts to increase the binding efficiency of PBP 2-His6 using slower flow rates, decreased buffer and Sarkosyl concentrations, and/or different detergents (octyl-glucoside, CHAPS, Triton X-100) failed. The amount of PBP 2-His6 that bound the column and was subsequently purified by this method was estimated to be approximately 60 µg.
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As noted above, growth and IPTG induction of E. coli AC252 led to the high-level expression of truncated sPBP 2-His6. This protein could be subjected to affinity chromatography on His-Select agarose without the use of detergent. sPBP 2-His6 was recovered from the column with a yield of 800–1000 µg, more than ten times the amount compared with that of full-length PBP 2-His6 (Figure 5b).
To ensure that the proteins being investigated were indeed either the full-length or truncated PBP 2 from P. aeruginosa, N-terminal sequence analysis and mass-spectrometry fingerprinting were performed. The N-terminal amino acid sequences obtained corresponded to those expected for each protein (Figure 1), and the MS-Fit42 gave a MOWSE score of 3.3 x 107, thus confirming the identity of P. aeruginosa PBP 2.
Both PBP 2-His6 and sPBP 2-His6 were assayed for penicillin-binding activity to determine if they remained active following their isolation and purification. The purified full-length PBP 2-His6 and truncated sPBP 2-His6 were able to bind both Bocillin FL, a fluorescently labelled penicillin and biotinylated ampicillin (Figure 5), suggesting that the PBPs were functional and apparently not hindered in this capacity by the presence of the C-terminal His-tags.
To confirm the specificity of mecillinam for P. aeruginosa PBP 2, a competition assay was performed. Protein samples were pre-incubated with mecillinam and then subjected to both the fluorescent Bocillin FL- and biotinylated-ampicillin-based PBP assays. As demonstrated in Figure 6, binding of both Bocillin FL and biotinylated ampicillin was greatly inhibited by this pre-treatment in a concentration-dependent manner, whereas control samples pre-incubated in its absence were readily detected by the assays. Inhibition of the PBP assay was detected with as little as 1.0 mg/L mecillinam, and concentrations greater than 5.0 mg/L virtually abolished the binding of labelled ampicillin.
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Engineering of a non-functional mutant of pbpA
The sequence alignment studies described above identified residue 327 of the SxxK consensus sequence as the invariant Ser located in the PB-domain of the P. aeruginosa PBP 2, strongly suggesting its role as the catalytic nucleophile that covalently binds ß-lactams. Site-specific replacement of Ser-327 in the truncated sPBP 2 with an Ala residue was achieved using the QuickChange Site-Directed Mutagenesis kit to generate pACDC38. The mutated pbpA gene on pACDC38 was overexpressed in E. coli BL21 (E. coli AC253) and the protein generated was isolated by affinity chromatography on His-Select agarose (Figure 5c). Although not purified to apparent homogeneity, PBP assays conducted with the Ser-327 Ala PBP 2 preparation demonstrated its inability to bind penicillin (Figure 5), thus confirming the identification of Ser-327 as the catalytic nucleophile in the PB (transpeptidase) domain.
Generation of a pbpA chromosomal insertion knockout mutant
A chromosomal knockout mutant of pbpA was constructed by inserting a non-polar 1088 bp GmR cassette within the open reading frame of the chromosomal pbpA gene. A final PCR-based screen was used to verify insertion of the GmR cassette into the chromosomal copy of pbpA. Template DNA isolated from wild-type cells produced a PCR product of 1994 bp whereas that obtained from insertion mutants produced a PCR product of 3082 bp.
PAO1 pbpA mutants lacking a functional pbpA gene were observed to grow in LB broth at 37°C, albeit at an apparent slower rate compared with wild-type PAO1 cells (Figure 7). SEM analysis revealed that, like wild-type cells treated with mecillinam, the PAO1 pbpA mutant grows with a spherical morphology, although perhaps with a little less uniformity in appearance (Figure 7). Normal rod-shaped morphology and cell dimensions were restored upon transformation of the mutant with the complementation vector pACBL31 which contained a functional copy of pbpA. However, complementation of the PAO1 pbpA mutant with pACBL34 harbouring the inactive Ser-327 Ala PBP 2 derivative did not result in the restoration of wild-type cell morphology (data not shown).
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The MIC values of a select group of ß-lactams, including the anti-pseudomonal antibiotics carbenicillin, cefotaxime and ticarcillin, for the different P. aeruginosa strains are presented in Table 2. As expected, wild-type P. aeruginosa PAO1 was relatively resistant to amoxicillin, ampicillin, cefoxitin and cefalotin while it was susceptible to the anti-pseudomonal ß-lactams carbenicillin, cefotaxime and ticarcillin. Interestingly, the PAO1
pbpA mutant was significantly more susceptible to each of these ß-lactams. For the anti-pseudomonal compounds, the MICs were lower by approximately one order of magnitude while the values for the other ß-lactams were decreased by one to two orders of magnitude. As observed with the morphological phenotype, complementation of the insertional mutant with a functional copy of pbpA restored wild-type levels of the MIC values for these antibiotics.
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Analysis of peptidoglycan
With the PBPs being responsible for the final stages of peptidoglycan biosynthesis, a comparison of the peptidoglycans isolated from wild-type PAO1 grown in the absence and presence of 200 mg/L mecillinam with that of the insertional pbpA mutant cells was made. As expected, the overall compositional analysis of the peptidoglycans was the same and consistent with the A1
chemotype involving a stem peptide containing diaminopimelic acid (DAP) (data not shown). Muropeptide maps generated by analytical HPLC indicated that the overall distribution of the mutanolysin-derived oligomuropeptides was very similar for the three preparations (data not shown). Total cross-linking was determined based on these analyses and, unlike the situation with E. coli, 46 only a slight increase was observed in the peptidoglycan synthesized in the presence of mecillinam compared with that obtained from untreated cells (32.8% and 28.4%, respectively). Likewise, a similar level of 29.5% cross-linking was present in the peptidoglycan from the PAO1 pbpA mutant.
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Discussion |
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Previous studies had demonstrated that P. aeruginosa is resistant to the ß-lactam antibiotic mecillinam,10,16,17 a finding confirmed in the current investigation. This resistance was postulated to result from decreased entry of the antibiotic into the periplasm,10 a phenomenon common with this opportunistic pathogen for most ß-lactams and a variety of other antibiotics (recently reviewed in ref. 47). However, our finding that mecillinam induces conformational changes in P. aeruginosa similar to those observed with E. coli and other related bacteria (namely conversion of rod-shaped cells into spherical cells) clearly indicates that it is able to reach its target and affect peptidoglycan synthesis. Indeed, the resulting morphological changes preclude the possibility that the AmpC ß-lactamase encoded on the P. aeruginosa chromosome serves as the resistance factor because its activity would have rendered the hydrolysed mecillinam incapable of covalently binding to, and thereby inactivating, its target PBP. Likewise, efflux pumps can be excluded. This resistance thus suggested that, unlike the situation with E. coli,15,48 the target of mecillinam in P. aeruginosa is not essential for its viability.
Earlier investigations involving whole cell preparations of P. aeruginosa had shown that its class B HMW PBP 2 is the target of mecillinam.17,49 In this study, we demonstrated that PA4003 encodes PBP 2 and that it does indeed bind mecillinam covalently and with specificity. The consequent generation of spherical cells caused by mecillinam inhibition or insertional mutation of PBP 2 indicates that, like E. coli, the function of PBP 2 in P. aeruginosa relates directly to the biosynthesis and maintenance of the cylindrical wall in the peptidoglycan sacculus. Our ability to generate the viable insertional mutant is consistent with our proposal that PBP 2 is non-essential to P. aeruginosa. This distinguishing property could be accounted for if the bacterium produces a second PBP 2 homologue with reduced affinity for the ß-lactam in a manner analogous to the methicillin-resistant strains of Staphylococcus aureus which produce a second form of its PBP 2 (PBP 2a). Indeed, such a possibility is conceivable given that the genome size of P. aeruginosa is approximately double that of E. coli, and it is known that P. aeruginosa does encode and produce two homologues of PBP 320,21 in contrast to only one in E. coli. However, an extensive and careful search of the P. aeruginosa genome failed to find any evidence of a second copy of PBP 2. Likewise, a second P. aeruginosa PBP 2 homologue has not been detected experimentally using SDS-PAGE-based assays,22 a finding we have confirmed (data not presented).
The difference between the activities of PBPs 2 and 3 is thought to be caused by differences in substrate specificity, with PBP 2 requiring pentapeptide stem peptides to function while PBP 3 prefers tripeptide side chains for optimal activity.50,51 Unfortunately, the enzymatic properties of PBP 3x from P. aeruginosa have not been characterized, but it is conceivable the specificity of its transpeptidase domain is less stringent than PBP 3 thereby providing a compensatory activity for PBP 2 to retain the cells in a viable state, a phenomenon referred to as ‘equivalent substitution’.52 The observation that the average level of peptidoglycan cross-linking was similar for both the wild-type and mutant strains does suggest that at least one of the other HMW PBPs (namely PBP 3x and/or 1a, 1b and 3) accommodates for the loss of some transpeptidase activity associated with PBP 2. However, it is clear that the activity of these other HMW PBPs does not substitute for PBP 2 in the maintenance of the cylindrical cell wall. That the transpeptidase activity associated with PBP 2 is normally required for this purpose was unequivocally demonstrated by the inability of the plasmid harbouring the inactive Ser-237 Ala PBP 2 to complement the morphological change associated with the PAO1 pbpA insertional mutant.
In addition to providing compensation for the lack of PBP 2 in P. aeruginosa, equivalent substitution also appears to contribute to ß-lactam resistance involving the activity of this PBP. Cells lacking a functional copy of pbpA were observed to be significantly more susceptible to a variety of different ß-lactams, including those not considered to be anti-pseudomonal such as ampicillin. Indeed, the MIC of this ß-lactam for wild-type PAO1 was observed to decrease from over 3200 mg/L to 25 mg/L upon the insertional mutation of pbpA. These data suggest that P. aeruginosa PBP 2 is relatively unsusceptible to these ß-lactams and that, in both wild-type PAO1 and the pbpA insertional mutant harbouring pACBL31, the functional PBP 2 may provide the equivalent substitution for the other more susceptible PBPs that become inactivated. While more detailed studies are required, these preliminary findings do demonstrate the significance of PBP 2 to P. aeruginosa and suggest that it may represent an important specific target for the development of new antibiotics.
A second major distinction between P. aeruginosa and E. coli in the effect of inactivating PBP 2 concerns the size of the resulting cells which may, in fact, account for the continued viability of P. aeruginosa. That cells of P. aeruginosa lacking PBP 2 activity retain their original diameter is significant because the cell circumference represents an important factor for the process of cell division. The formation of the Z ring that has to extend around the complete circumference of a cell at its site of division requires both a critical concentration of FtsZ and appropriate proportions of the associated Z-ring stabilizing and destabilizing proteins (Figure 8). For this to occur, the division proteins are produced in both constant and balanced concentrations sufficient to maintain normal morphology (recently reviewed in ref. 53). In view of this, it has been proposed that the ever-enlarging spherical cells of E. coli lacking a functioning PBP 2 would have insufficient concentrations of these proteins to generate a complete Z ring, thus precluding their ability to divide and leading to their consequent death. This hypothesis is supported by studies demonstrating that inactivation of PBP 2 in E. coli is permitted conditional upon overexpression of the cell division proteins FtsZ, FtsA and FtsQ, either directly in engineered mutants11,15 or indirectly by increasing ppGpp concentrations.11,13,48 Whereas considerably less is known about the division process in P. aeruginosa, an analysis of its Mra operon suggests the clustering of fts and mur genes with an arrangement remarkably similar to that on the E. coli chromosome.54,55 Hence, it is generally assumed that division proceeds similarly to the process known for E. coli. Given this, the fact that the circumference of P. aeruginosa cells lacking a functioning PBP 2 does not significantly increase would permit complete Z rings to form thereby allowing their continued division. Why the diameter of P. aeruginosa lacking a functional PBP 2 does not change remains unknown, and given the complexity of both peptidoglycan structure and metabolism, it may prove extremely difficult to determine.
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Another significant finding of this study pertains to our observation that reproducing spherical cells of P. aeruginosa are capable of reversion back to rod shape morphology upon removal of inhibiting mecillinam. A model proposed for the propagation of the cylindrical wall region in E. coli invokes a strand of an existing peptidoglycan chain to serve as a guide and template for the biosynthetic complex of enzymes.56 This complex was thus proposed to copy the existing sacculus to transmit the shape from one generation to the next. However, the apparent capability of P. aeruginosa to revert back to its normal rod shape is in contradiction to this model, at least as it might apply to this bacterium. This view is further supported by our ability to complement the insertional genomic pbpA mutant (which grow and reproduce as cocci) with a functional copy of pbpA to restore the normal rod-shaped morphology. These observations clearly indicate that cell morphology as directed by the peptidoglycan sacculus is not dependent on any pre-existing structure to function as a template.
More recent studies with E. coli have demonstrated the relationship between FtsZ and cell shape extends to include the ‘cytoskeletal’ proteins MreB, MreC and MreD. A mutual interdependency between the formation of the MreB cables that extend below the cytoplasmic membrane and cell shape has been proposed.57 Interestingly, depletion of any one of these cytoskeletal proteins also leads to the formation of large spherical cells which eventually lyse. As with PBP 2 inactivation, this effect is likewise suppressed by overexpression of the FtsQAZ proteins. These morphogenic MreBCD proteins were shown to form a multi-protein, transmembrane complex with RodA and PBP 2 to direct longitudinal cell wall synthesis.57,58 With P. aeruginosa, our preliminary studies have indicated that a multi-protein complex involving PBP 2 also forms.59 This, together with the observations that pbpA encoding PBP 2 is clustered similarly with the rodA gene as on E. coli chromosome and that the mreB, mreC and mreD genes are arranged identically, would suggest that a similar interplay between PBP 2 and the MreBCD proteins might exist in P. aeruginosa. Hence, our findings tend to support the proposal that a morphogenic apparatus associated with peptidoglycan metabolism and not pre-existing structure is fully responsible for shape determination. However, delineating the true composition and mechanism of action of this putative morphogenic apparatus will certainly prove to be challenging recognizing that most of the proteins associated with it are membrane-bound, and they function on a totally insoluble complex heteropolymer. Given the clinical relevance of P. aeruginosa, it is possible that our understanding of its differences compared with the ‘model’ E. coli bacterium will be important for future approaches in antibacterial therapy.
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None to declare.
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Acknowledgements |
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We thank Leo Pharmaceutical Products, Denmark for the gift of mecillinam, and Herbert P. Schweizer (Colorado State University) for the Pseudomonas insertion vectors pEX18Ap, pPS856 and pUCP26. David Watson, National Research Council of Canada, Ottawa, is thanked for performing the N-terminal sequencing analyses, as are Sandy Smith and Dyanne Brewer, both of the University of Guelph, for the SEM and MS analyses, respectively. We also thank Edie Schuerwater, Kevin Bauer and Chelsea Clarke for expert technical assistance at different stages of this research. These studies were supported by an operating grant to A. J. C. from the Canadian Institutes of Health Research (MOP-49623), and a post-graduate scholarship (PGS B) to B. A. L. from the Natural Sciences and Engineering Research Council of Canada.
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1 Cabeen MT and Jacobs-Wagner C. (2005) Bacterial cell shape. Nat Rev Microbiol 3:601–10.[CrossRef][Web of Science][Medline]
2 Macheboeuf P, Contreras-Martel C, Job V, et al. (2006) Penicillin Binding Proteins: key players in bacterial cell cycle and drug resistance processes. FEMS Microbiol Rev 30:673–91.[CrossRef][Web of Science][Medline]
3
Scheffers DJ and Pinho MG. (2005) Bacterial cell wall synthesis: new insights from localization studies. Microbiol Mol Biol Rev 69:585–607.
4 Young KD. (2003) Bacterial shape. Mol Microbiol 49:571–80.[CrossRef][Web of Science][Medline]
5
Goffin C and Ghuysen J-M. (1998) Multimodular penicillin-binding proteins: an enigmatic family of orthologs and paralogs. Microbiol Mol Biol Rev 62:1079–93.
6
Goffin C, Fraipont C, Ayala J, et al. (1996) The non-penicillin-binding module of the tripartite penicillin-binding protein 3 of Escherichia coli is required for folding and/or stability of the penicillin-binding module and the membrane-anchoring module confers cell septation activity on the folded structure. J Bacteriol 178:5402–9.
7 Marrec-Fairley M, Pietter A, Gallet X, et al. (2000) Differential functionalities of amphiphilic peptide segments of the cell-septation penicillin-binding protein 3 of Escherichia coli. Mol Microbiol 37:1019–31.[CrossRef][Web of Science][Medline]
8 Nguyen-Disteche M, Fraipont C, Buddelmeijer N, et al. (1998) The structure and function of E. coli penicillin-binding protein 3. Cell Mol. Life Sci. 54:309–16.[CrossRef][Web of Science][Medline]
9
Denome SA, Elf PK, Henderson TA, et al. (1999) Escherichia coli mutants lacking all possible combinations of eight penicillin binding proteins: viability, characteristics, and implications for peptidoglycan synthesis. J Bacteriol 181:3981–93.
10 Neu HC. (1983) Penicillin-binding proteins and the role of amdinocillin in causing bacterial cell death. Am J Med 75:9–20.[Web of Science][Medline]
11 Navarro F, Robin A, D'Ari R, et al. (1998) Analysis of the effect of ppGpp on the ftsQAZ operon in Escherichia coli. Mol Microbiol 29:815–23.[CrossRef][Web of Science][Medline]
12
Vinella D, Cashel M, D'Ari R. (2000) Selected amplification of the cell division genes ftsQ-ftsA-ftsZ in Escherichia coli. Genetics 156:1483–92.
13 Vinella D, D'Ari R, Bouloc P. (1992) Penicillin binding protein 2 is dispensable in Escherichia coli when ppGpp synthesis is induced. EMBO J 11:1493–1501.[Web of Science][Medline]
14 Melchior NH, Blom J, Tybring L, et al. (1973) Light and electron microscopy of the early response of Escherichia coli to a 6-ß-amidinopenicillanic acid (FL 1060). Acta Pathol Microbiol Scand [B] Microbiol Immunol 81:393–407.[Medline]
15
Vinella D, Joseleau-Petit D, Thévenet D, et al. (1993) Penicillin-binding protein 2 inactivation in Escherichia coli results in cell division inhibition, which is relieved by FtsZ overexpression. J Bacteriol 175:6704–10.
16 Eng RH, Liu R, Smith SM, et al. (1988) Amdinocillin: interaction with other ß-lactam antibiotics for Gram-negative bacteria. Chemotherapy 34:18–26.[Web of Science][Medline]
17 Noguchi J, Matsuhashi M, Matsuhashi S. (1979) Comparative studies of penicillin-binding proteins in Pseudomonas aeruginosa and Escherichia coli. Eur J Biochem 100:41–9.[CrossRef][Web of Science][Medline]
18 Spratt BG. (1977) Properties of the penicillin-binding proteins of Escherichia coli K12. Eur J Biochem 72:341–52.[Web of Science][Medline]
19 Handfield J, Gagnon L, Dargis M, et al. (1997) Sequence of the ponA gene and characterization of the penicillin-binding protein 1A of Pseudomonas aeruginosa PAO1. Gene 199:49–56.[CrossRef][Web of Science][Medline]
20 Liao X and Hancock REW. (1995) Cloning and characterization of the Pseudomonas aeruginosa pbpA gene encoding penicillin-binding protein 3. Antimicrob Agents Chemother 39:1871–4.[Abstract]
21
Liao X and Hancock REW. (1997) Identification of a penicillin-binding protein 3 homolog, PBP3 x , in Pseudomonas aeruginosa: gene cloning and growth phase-dependent expression. J Bacteriol 179:1490–6.
22
Noguchi H, Fukasawa M, Komatsu T, et al. (1985) Mutation in Pseudomonas aeruginosa causing simultaneous defects in penicillin-binding protein 5 and in enzyme activities of penicillin release and D-alanine carboxypeptidase. J Bacteriol 162:849–51.
23
Song J, Xie G, Elf PK, et al. (1998) Comparative analysis of Pseudomonas aeruginosa penicillin-binding protein 7 in the context of its membership in the family of low-molecular-mass PBPs. Microbiology 144:975–83.
24 Goldman RC and Gange D. (2000) Inhibition of transglycosylation involved in bacterial peptidoglycan synthesis. Curr Med Chem 7:801–20.[Web of Science][Medline]
25
Van Heijenoort J. (2001) Formation of the glycan chains in the synthesis of bacterial peptidoglycan. Glycobiology 11:25R–36R.
26 Gordon E, Mouz N, Duee E, et al. (2000) The crystal structure of the penicillin-binding protein 2x from Streptococcus pneumoniae and its acyl-enzyme form: implication in drug resistance. J Mol Biol 299:477–85.[CrossRef][Web of Science][Medline]
27 Lim D and Strynadka NC. (2002) Structural basis for the ß-lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus. Nat Struct Biol 9:870–6.[Web of Science][Medline]
28 Blackburn NT and Clarke AJ. (2002) Characterization of soluble and membrane-bound family 3 lytic transglycosylases from Pseudomonas aeruginosa. Biochemistry 41:1001–13.[CrossRef][Medline]
29
Zhao G, Meier TM, Kahl SD, et al. (1999) BOCILLIN FL, a sensitive and commercially available reagent for detection of penicillin binding proteins. Antimicrob Agents Chemother 43:1124–8.
30
Dargis M and Malouin F. (1994) Use of biotinylated ß-lactams and chemiluminescence for study and purification of penicillin-binding proteins in bacteria. Antimicrob Agents Chemother 38:973–80.
31
Okamoto T, Yoshiyama H, Nakazawa T, et al. (2002) A change in PBP 1 is involved in amoxicillin resistance of clinical isolates of Helicobacter pylori. J Antimicrob Chemother 50:849–56.
32 Shevchenko A, Wilm M, Vorm O, et al. (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–8.[Medline]
33
Clarke AJ. (1993) Extent of peptidoglycan O-acetylation in the tribe Proteeae. J Bacteriol 175:4550–3.
34 Glauner B. (1998) Separation and quantification of muropeptides with high performance liquid chromatography. Anal Biochem 172:451–64.
35 Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, et al. (1998) A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86.[CrossRef][Web of Science][Medline]
36 Simon R, Priefer U, Pühler A. (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Biotechnology (NY) 1:784–91.[CrossRef]
37 West SE, Schweizer HP, Dall C, et al. (1994) Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in P. aeruginosa. Gene 148:81–6.[CrossRef][Web of Science][Medline]
38 Choi KH, Kumar A, Schweizer HP. (2006) A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: Application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64:391–7.[CrossRef][Web of Science][Medline]
39
Cserzo M, Wallin E, Simon I, et al. (1997) Prediction of transmembrane alpha-helices in procariotic membrane proteins: the Dense Alignment Surface method. Prot Eng 10:673–6.
40 Krogh A, Larsson B, von Heijne G, et al. (2001) Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J Mol Biol 305:567–80.[CrossRef][Web of Science][Medline]
41 Bendtsen JD, Nielsen H, von Heijne G, et al. (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–95.[CrossRef][Web of Science][Medline]
42 Clauser KR, Baker PR, Burlingame AL. (1999) Role of accurate mass measurement (+/–10 ppm) in protein identification strategies employing MS or MS/MS and database searching. Anal Chem 71:2871–82.[Medline]
43 Laemmeli UK. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–5.[CrossRef][Medline]
44 Stover KC, Pham XQ, Erwin AL, et al. (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1: an opportunistic pathogen. Nature 406:959–64.[CrossRef][Medline]
45
de Pedro MA, Donachie WD, Höltje J-V, et al. (2001) Constitutive septal murein synthesis in Escherichia coli with impaired activity of the morphogenic proteins RodA and penicillin-binding protein 2. J Bacteriol 183:4115–26.
46 Essig P, Martin HH, Gmeiner J. (1982) Murein and lipopolysaccharide biosynthesis in synchronized cells of Escherichia coli K12 and the effect of penicillin G, mecillinam and nalidixic acid. Arch Microbiol 132:245–50.[CrossRef][Web of Science][Medline]
47 Thomson JM and Bonomo RA. (2005) The threat of antibiotic resistance in Gram-negative pathogenic bacteria: ß-lactams in peril!. Curr Opin Microbiol 8:518–24.[CrossRef][Web of Science][Medline]
48
Ogura T, Bouloc P, Niki H, et al. (1989) Penicillin-binding protein 2 is essential in wild-type Escherichia coli but not in lov or cya mutants. J Bacteriol 171:3025–30.
49
Curtis NA, Orr D, Ross GW, et al. (1979) Competition of ß-lactam antibiotics for the penicillin-binding proteins of Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella aerogenes, Proteus rettgeri, and Escherichia coli: comparison with antibacterial activity and effects upon bacterial morphology. Antimicrob Agents Chemother 16:325–8.
50
Botta GA and Park JT. (1981) Evidence for involvement of penicillin-binding protein 3 in murein synthesis during septation but not during cell elongation. J Bacteriol 145:333–40.
51
Pisabarro AG, Prats R, Vaquez D, et al. (1986) Activity of pencillin-binding protein 3 from Escherichia coli. J Bacteriol 168:199–206.
52 Young KD. (2001) Approaching the physiological functions of penicillin-binding proteins in E. coli. Biochimie 83:99–102.[Medline]
53
Young KD. (2006) The selective value of bacterial shape. Microbiol Mol Biol Rev 70:660–703.
54 Azzolina BA, Yuan X, Anderson MS, et al. (2001) The cell wall and cell division gene cluster in the Mra operon of Pseudomonas aeruginosa: cloning, production, and purification of active enzymes. Protein Expr Purif 21:393–400.[CrossRef][Web of Science][Medline]
55 El Zoeiby A, Sanschagrin F, Havugimana PC, et al. (2001) In vitro reconstruction of the biosynthetic pathway of peptidoglycan cytoplasmic precursor in Pseudomonas aeruginosa. FEMS Microbiol Lett 201:229–35.[CrossRef][Web of Science][Medline]
56
Höltje J-V. (1996) A hypothetical holoenyzme involved in the replication of the murein sacculus of Escherichia coli. Microbiology 142:1911–8.
57 Kruse T, Bork-Jensen J, Gerdes K. (2005) The morphogenetic MreBCD proteins of Escherichia coli form an essential membrane-bound complex. Mol Microbiol 55:78–89.[CrossRef][Web of Science][Medline]
58
Varma A and Young KD. (2004) FtsZ collaborates with penicillin binding proteins to generate bacterial cell shape in Escherichia coli. J Bacteriol 186:6768–74.
59 Legaree BA and Clarke AJ. (2006) Using affinity chromatography to investigate protein-protein interactions involved in peptidoglycan synthesis in Pseudomonas aeruginosa.. Abstracts of the 56th Annual Conference of the Canadian Society of MicrobiologistsLondon, Ontario Abstract C20.
60
Hancock REW and Carey AM. (1979) Outer membrane of Pseudomonas aeruginosa: heat- 2-mercaptoethanol-modifiable proteins. J. Bacteriol. 140:902–10.
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