| Although mycobacteria produce beta-lactamases and are intrinsically resistant to beta-lactam antibiotics, it has been suggested that beta-lactam antibiotics could be helpful in the treatment of mycobacteria) infections if used with beta-lactamase inhibitors. Resistance to beta-lactams in mycobacteria is believed to derive from a combination of three mechanisms: (1) production of beta-lactamases, (2) inability of the drug to penetrate the mycobacteria) cell wall, and (3) low affinity of the beta-lactams for mycobacteria) penicillin-binding proteins. In this study, we have generated mutants of the major beta-lactamases, BlaC and BlaS, in the pathogenic M. tuberculosis strain H37Rv and the model organism M. smegmatis strain PM274, respectively. The mutants, M. tuberculosis PM638 (DeltablaC1) and M. smegmatis PM759 (DeltablaS1), showed an increase in susceptibility to beta-lactams as determined by disk diffusion and minimal inhibitory concentration (MIC) assays. An additional beta-lactamase. BlaE, was identified in M. smegmatis PM759 but it contributes minimally to resistance. The susceptibility of the mutants to penicillin-type beta-lactams was affected most as compared to the cephalosporin-type beta-lactams as assayed by disk diffusion tests and suggests there are other mechanisms contributing to the resistance to this type of beta-lactam. We reasoned that this difference in susceptibility could be exploited to isolate mutants, hypersusceptible to beta-lactams, which could reveal novel genes involved with cell envelope biosynthesis. To explore this possibility, we performed transposon mutagenesis of PM759 and PM638 and screened for mutants with increases susceptibility to cefoxitin, ceftriaxone, and oxacillin. We isolated and chararacterized 6 mutants of M. smegmatis PM759 and 3 mutants of M. tuberculosis PM638. Mapping of the transposon insertions revealed insertions in two genes (ponA2 and dapB) known to be involved in peptidoglycan biosynthesis. The remaining mutants contained insertions within previously unidentified genes. The 9 proteins can be grouped into three classes: those that are known, or are postulated, to be involved in peptidoglycan biosynthesis, those that are predicted to be involved in cell division, and those involved in other cell envelope processes. The contribution of these proteins to pepdtidoglycan synthesis, beta-lactam resistance, and cell envelope processes is the final focus of this work. |
