Function of penicillin-binding protein 2 in viability and morphology of Pseudomonas aeruginosa

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. aerugin...

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Published in:Journal of antimicrobial chemotherapy 2007-03, Vol.59 (3), p.411-424
Main Authors: Legaree, Blaine A., Daniels, Kathy, Weadge, Joel T., Cockburn, Darrell, Clarke, Anthony J.
Format: Article
Language:English
Subjects:
Amdinocillin - pharmacology
Amino Acid Sequence
Antibiotics. Antiinfectious agents. Antiparasitic agents
Bacteria
Binding sites
Biological and medical sciences
Cloning, Molecular
E coli
Escherichia coli
Genomics
mecillinam
Medical sciences
Molecular Sequence Data
Mutation
P. aeruginosa
Penicillin
Penicillin-Binding Proteins - genetics
Penicillin-Binding Proteins - isolation & purification
Penicillin-Binding Proteins - physiology
peptidoglycan
Peptidoglycan - analysis
Pharmacology. Drug treatments
Proteins
Pseudomonas aeruginosa
Pseudomonas aeruginosa - cytology
Pseudomonas aeruginosa - drug effects
Pseudomonas aeruginosa - physiology
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container_start_page 411
container_title Journal of antimicrobial chemotherapy
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creator Legaree, Blaine A.
Daniels, Kathy
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Cockburn, Darrell
Clarke, Anthony J.
description 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 ΔpbpA 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 β-lactams were significantly lower for the insertional mutant compared with wild-type PAO1. The muropeptide profile of peptidoglycan from PAO1 ΔpbpA 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 β-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.
doi_str_mv 10.1093/jac/dkl536
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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 ΔpbpA 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 β-lactams were significantly lower for the insertional mutant compared with wild-type PAO1. The muropeptide profile of peptidoglycan from PAO1 ΔpbpA 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 β-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.</description><identifier>ISSN: 0305-7453</identifier><identifier>EISSN: 1460-2091</identifier><identifier>DOI: 10.1093/jac/dkl536</identifier><identifier>PMID: 17289762</identifier><identifier>CODEN: JACHDX</identifier><language>eng</language><publisher>Oxford: Oxford University Press</publisher><subject>Amdinocillin - pharmacology ; Amino Acid Sequence ; Antibiotics. Antiinfectious agents. Antiparasitic agents ; Bacteria ; Binding sites ; Biological and medical sciences ; Cloning, Molecular ; E coli ; Escherichia coli ; Genomics ; mecillinam ; Medical sciences ; Molecular Sequence Data ; Mutation ; P. aeruginosa ; Penicillin ; Penicillin-Binding Proteins - genetics ; Penicillin-Binding Proteins - isolation &amp; purification ; Penicillin-Binding Proteins - physiology ; peptidoglycan ; Peptidoglycan - analysis ; Pharmacology. Drug treatments ; Proteins ; Pseudomonas aeruginosa ; Pseudomonas aeruginosa - cytology ; Pseudomonas aeruginosa - drug effects ; Pseudomonas aeruginosa - physiology</subject><ispartof>Journal of antimicrobial chemotherapy, 2007-03, Vol.59 (3), p.411-424</ispartof><rights>The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org 2007</rights><rights>2007 INIST-CNRS</rights><rights>The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c477t-484b488d2b8d337e5948eef27360f51d32ee781b36179bc81ebe6f68c14ab6e53</citedby><cites>FETCH-LOGICAL-c477t-484b488d2b8d337e5948eef27360f51d32ee781b36179bc81ebe6f68c14ab6e53</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,786,790,1591,27957,27958</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&amp;idt=18626344$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17289762$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Legaree, Blaine A.</creatorcontrib><creatorcontrib>Daniels, Kathy</creatorcontrib><creatorcontrib>Weadge, Joel T.</creatorcontrib><creatorcontrib>Cockburn, Darrell</creatorcontrib><creatorcontrib>Clarke, Anthony J.</creatorcontrib><title>Function of penicillin-binding protein 2 in viability and morphology of Pseudomonas aeruginosa</title><title>Journal of antimicrobial chemotherapy</title><addtitle>J Antimicrob Chemother</addtitle><description>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 ΔpbpA 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 β-lactams were significantly lower for the insertional mutant compared with wild-type PAO1. The muropeptide profile of peptidoglycan from PAO1 ΔpbpA 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 β-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.</description><subject>Amdinocillin - pharmacology</subject><subject>Amino Acid Sequence</subject><subject>Antibiotics. Antiinfectious agents. Antiparasitic agents</subject><subject>Bacteria</subject><subject>Binding sites</subject><subject>Biological and medical sciences</subject><subject>Cloning, Molecular</subject><subject>E coli</subject><subject>Escherichia coli</subject><subject>Genomics</subject><subject>mecillinam</subject><subject>Medical sciences</subject><subject>Molecular Sequence Data</subject><subject>Mutation</subject><subject>P. aeruginosa</subject><subject>Penicillin</subject><subject>Penicillin-Binding Proteins - genetics</subject><subject>Penicillin-Binding Proteins - isolation &amp; purification</subject><subject>Penicillin-Binding Proteins - physiology</subject><subject>peptidoglycan</subject><subject>Peptidoglycan - analysis</subject><subject>Pharmacology. 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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 ΔpbpA 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 β-lactams were significantly lower for the insertional mutant compared with wild-type PAO1. The muropeptide profile of peptidoglycan from PAO1 ΔpbpA 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 β-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.</abstract><cop>Oxford</cop><pub>Oxford University Press</pub><pmid>17289762</pmid><doi>10.1093/jac/dkl536</doi><tpages>14</tpages><oa>free_for_read</oa></addata></record>
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subjects Amdinocillin - pharmacology
Amino Acid Sequence
Antibiotics. Antiinfectious agents. Antiparasitic agents
Bacteria
Binding sites
Biological and medical sciences
Cloning, Molecular
E coli
Escherichia coli
Genomics
mecillinam
Medical sciences
Molecular Sequence Data
Mutation
P. aeruginosa
Penicillin
Penicillin-Binding Proteins - genetics
Penicillin-Binding Proteins - isolation & purification
Penicillin-Binding Proteins - physiology
peptidoglycan
Peptidoglycan - analysis
Pharmacology. Drug treatments
Proteins
Pseudomonas aeruginosa
Pseudomonas aeruginosa - cytology
Pseudomonas aeruginosa - drug effects
Pseudomonas aeruginosa - physiology
title Function of penicillin-binding protein 2 in viability and morphology of Pseudomonas aeruginosa
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