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

Abstract

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 DpbpA 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 b-lactams were significantly lower for the insertional mutant compared with wild-type PAO1. The muropeptide profile of peptidoglycan from PAO1 DpbpA 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 b-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.

High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography

“…3B). As expected from a previous study (65), P. aeruginosa strains exhibited a small yet significant increase in cross-linking relative to E. coli (average across strains and two-sided t test: P. aeruginosa, 33 Ϯ 0.2%, E. coli, 31 Ϯ 0.3%, p Ͻ 0.001), and average glycan strand length (17 Ϯ 0.4) (Fig. 3B) was intermediate between those of E. coli (27 Ϯ 1) and V. cholera (12 Ϯ 0.6) (Fig.…”

“…Some strains are resistant to many antibiotics, and therefore P. aeruginosa is of significant clinical interest (61)(62)(63)(65)(66)(67). The peptidoglycan of P. aeruginosa is of the A1␥ chemotype, the same as E. coli (65,(67)(68)(69), and is characterized by a 4-3 cross-link involving the stem peptide diaminopimelic acid (24).…”

Interaction of Penicillin-Binding Protein 2 with Soluble Lytic Transglycosylase B1 inPseudomonas aeruginosa

“…1) (18,19), suggesting that they may represent binding partners. To test this possibility, recombinant SltB1 was used as the ligand in affinity chromatography of P. aeruginosa preparations.…”

“…Consequently, each of the collected fractions were assayed specifically from the presence of PBPs by using both biotinylated ampicillin as described previously (18,19) and [phenyl-4(n)- 3 H]benzylpenicillin (25 Ci/mmol and 1 mCi/ml; TRK 779; GE Healthcare) according to the method described by Spratt (30,31) as modified by Preston et al (25). Prior to autoradiography of the latter, the gels were soaked in Amplify Scintillant (GE Healthcare) for 15 to 30 min and dried in vacuo on Whatman filter paper.…”

“…1). Its coding sequence was cloned into pET30a(ϩ), thereby endowing the overproduced protein with a C-terminal six-His tag (19). The presence of the His tag is especially important for detection of the protein by Western immunoblot analysis using the anti-six-His tag antibody, because if the C-terminal PB module were lacking, PBP assays could not be used.…”