UBC Theses and Dissertations
Characterization of the Pseudomonas aeruginosa penicillin-binding proteins 3 and 3x : gene cloning, expression and role in susceptibility to [beta]-lactam antibiotics Liao, Xiaowen
Two degenerate oligonucleotides primers were synthesized based on the amino acid sequences found in the conserved SXXK and KTG 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 PAOl 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 s recognition site, suggesting that the expression of this gene 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 3H-penicillin. They had an apparent 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 3H-penicillin binding competition assays indicated that overproduction of 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 PBP3- targeted 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.