Function of penicillin-binding protein 2 in viability and morphology of Pseudomonas aeruginosa.
OBJECTIVE
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 DeltapbpA 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 beta-lactams were significantly lower for the insertional mutant compared with wild-type PAO1. The muropeptide profile of peptidoglycan from PAO1 DeltapbpA 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 beta-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.
Legaree
Blaine A
BA
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1 Canada.
Daniels
Kathy
K
Weadge
Joel T
JT
Cockburn
Darrell
D
Clarke
Anthony J
AJ
D000560:Amdinocillin / Q000494:pharmacology; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D046915:Penicillin-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D010457:Peptidoglycan / Q000032:analysis; D011550:Pseudomonas aeruginosa / Q000166:cytology / Q000187:drug effects / Q000502:physiology*
D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan; D000560:Amdinocillin