Analysis of amino acid sequences of penicillin-binding protein 2 in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone.

Neisseria gonorrhoeae strains with reduced susceptibility to cefixime and ceftriaxone, with minimum inhibitory concentrations (MICs) of cefixime of 0.125-0.25 microg/ml and ceftriaxone of 0.031-0.125 microg/ml, were isolated from male urethritis patients in Tokyo, Japan, in 2006. The amino acid sequences of PenA, penicillin-binding protein 2, in these strains were of two types: PenA mosaic and nonmosaic strains. In the PenA mosaic strain, some regions in the transpeptidase-encoding domain in PenA were similar to those of Neisseria perflava/sicca, Neisseria cinerea, Neisseria flavescens, Neisseria polysaccharea, and Neisseria meningitidis. In the PenA nonmosaic strain, there was a mutation of Ala-501 to Val in PenA. In addition, we performed homology modeling of PenA wild-type and mosaic strains and compared them. The results of the modeling studies suggested that reduced susceptibility to cephems such as cefixime and ceftriaxone is due to a conformational alteration of the beta-lactam-binding pocket. These results also indicated that the mosaic structures and the above point mutation in PenA make a major contribution to the reduced susceptibility to cephem antibiotics.

Transcriptional analysis of the Escherichia coli mreBCD genes responsible for morphogenesis and chromosome segregation.

The Escherichia coli mreB gene encodes an actin-like cytoskeletal protein and is required for rod shape formation of cells and chromosome segregation. Just downstream of mreB, the mreC and mreD genes are located. They are also required for rod shape formation, though their role in chromosome segregation is unclear. lacZ fusion analysis and Northern hybridization showed that the mreB, mreC, and mreD genes formed an operon. Most of the transcripts were expressed as a monocistronic mreB mRNA, and only 1-2% of the transcripts were expressed as a polycistronic mreBCD mRNA. Introduction of a frame-shift mutation in the mreB gene resulted in a significant decrease in the amount of polycistronic mreBCD mRNA but not in that of monocistronic mreB mRNA, suggesting that an attenuation-like regulation was involved in this transcriptional control. Primer extension analysis identified three transcriptional initiation sites. Three possible sigma(D)-dependent promoter-like sequences were found just upstream of these transcriptional initiation sites. lacZ fusion analysis confirmed that these three promoters contributed to the expression of mreBCD. On the basis of these findings, the essentiality of the mreB gene was confirmed.