Genetic analysis of the β-lactamases of Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to β-lactam antibiotics

Flores, Anthony R.; Parsons, Linda M.; Pavelka, Martin S.

doi:10.1099/mic.0.27629-0

Cited by 190 publications

(190 citation statements)

References 39 publications

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“…Interestingly, failure to secrete BlaS confers ampicillin susceptibility upon strains with tatA, tatB, or tatC mutations. This is consistent with the observation that deletion of blaS confers ampicillin susceptibility (14), although M. smegmatis contains several lactamases (www.tigr.org.tdb /ofmg/). We analyzed the genome of M. smegmatis with TATFIND (11) and identified 49 putative TAT substrates, which represent approximately 0.7% of the encoded proteins.…”

Section: Discussionsupporting

confidence: 78%

Characterization of the Twin-Arginine Translocase Secretion System of Mycobacterium smegmatis

2006

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The twin-arginine translocation (TAT) system secretes fully folded proteins that contain a twin-arginine motif within their signal sequence across the cytoplasmic membrane in bacteria. Using a green fluorescent protein fused with a TAT signal sequence, we demonstrated that Mycobacterium smegmatis contains a TAT system. By inactivating individual genes, we showed that three genes (tatA, tatB, and tatC) are required for a functional TAT system in M. smegmatis. The tat mutants exhibited a decreased growth rate and altered colony morphology compared to the parent strain. Comparison of the secreted proteins of the ⌬tatC and parent strain by two-dimensional polyacrylamide gel electrophoresis revealed an alteration in the secretion of at least five proteins, and one of the major TAT-dependent secreted proteins was identified as ␤-lactamase (BlaS). The genome of M. smegmatis was analyzed with the TATFIND program, and 49 putative TAT substrates were identified, including the succinate transporter DctP. Because disruption of the TAT secretion system has a direct effect on the physiology of M. smegmatis and homologs of the TAT proteins are also present in the genome of Mycobacterium tuberculosis, the TAT secretion system or its substrates may be good candidates for drug or vaccine development.

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Section: Discussionsupporting

confidence: 78%

Characterization of the Twin-Arginine Translocase Secretion System of Mycobacterium smegmatis

2006

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“…We previously showed that the mutants have an increased susceptibility to most ␤-lactam antibiotics, particularly the penicillins. However, there is still a basal level of resistance in the mutants to certain penicillins, and the susceptibilities of the mutants to some cephalosporin-based ␤-lactams are essentially the same as those of the wild types (17). We found a minor cephalosporinase, BlaE, in M. smegmatis but showed that its contribution to ␤-lactam resistance is minimal (17).…”

mentioning

confidence: 74%

“…To eliminate this problem, our laboratory has constructed mutants of M. tuberculosis and M. smegmatis with deletions of the major ␤-lactamase genes, blaC and blaS, respectively (17). We previously showed that the mutants have an increased susceptibility to most ␤-lactam antibiotics, particularly the penicillins.…”

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confidence: 99%

Characterization of Novel Mycobacterium tuberculosis and Mycobacterium smegmatis Mutants Hypersusceptible to β-Lactam Antibiotics

2005

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Our laboratory previously constructed mutants of Mycobacterium tuberculosis and Mycobacterium smegmatis with deletions in the genes for their major ␤-lactamases, BlaC and BlaS, respectively, and showed that the mutants have increased susceptibilities to most ␤-lactam antibiotics, particularly the penicillins. However, there is still a basal level of resistance in the mutants to certain penicillins, and the susceptibilities of the mutants to some cephalosporin-based ␤-lactams are essentially the same as those of the wild types. We hypothesized that characterizing additional mutants (derived from ␤-lactamase deletion mutants) that are hypersusceptible to ␤-lactam antibiotics might reveal novel genes involved with other mechanisms of ␤-lactam resistance, peptidoglycan assembly, and cell envelope physiology. We report here the isolation and characterization of nine ␤-lactam antibiotic-hypersusceptible transposon mutants, two of which have insertions in genes known to be involved with peptidoglycan biosynthesis (ponA2 and dapB); the other seven mutants have insertions which affect novel genes. These genes can be classified into three groups: those involved with peptidoglycan biosynthesis, cell division, and other cell envelope processes. Two of the peptidoglycan-biosynthetic genes (ponA2 and pbpX) may encode ␤-lactam antibiotic-resistant enzymes proposed to be involved with the synthesis of the unusual diaminopimelyl linkages within the mycobacterial peptidoglycan.The mycobacteria consist of a diverse group of organisms representing saprophytic bacteria as well as important human pathogens. Organisms such as Mycobacterium chelonae, Mycobacterium smegmatis, and Mycobacterium fortuitum are environmental bacteria and rarely cause disease. On the other hand, Mycobacterium tuberculosis, the causative agent of the disease tuberculosis, has successfully infected one-third of the world's population and is responsible for over 2 million deaths annually (15). Interest in mycobacterial pathogenesis and physiology has been stimulated by the emergence of multidrugresistant strains of M. tuberculosis as well as the observation that the immunocompromised (e.g., those with AIDS) are at a higher risk for M. tuberculosis infection (20,21).The biosynthesis of the mycobacterial cell envelope is an area of considerable research interest, as several of the antibiotics used to treat mycobacterial infections target the synthesis pathways of several envelope components. The main feature of the mycobacterial cell envelope is a single, covalently linked structure composed of peptidoglycan, arabinogalactan, and mycolic acids (the mAGP complex) (5). The peptidoglycan is the innermost layer of the complex and is attached to the arabinogalactan layer via a rhamnose-N-acetylglucosamine linker. The arabinose residues are in turn esterified to mycolic acids, which are long-chain (60 to 90 carbon) ␣-alkyl, ␤-hydroxy fatty acids oriented perpendicularly to the cell membrane (36). Extractable lipids in association with the mycolic acids serve to form an...

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“…A mycobacterial AmpG homologue has not yet been identified and characterized, but some transport of the PG material back into the cytosol would be needed. It should be noted that mycobacteria do have ␤-lactamases, enzymes associated with cell wall monitoring and remodeling (136,137,271,434).…”

Section: Do Mycobacteria Recycle Cell Wall Material?mentioning

confidence: 99%

Bacterial Growth and Cell Division: a Mycobacterial Perspective

Hett

Rubín

2008

Microbiol Mol Biol Rev

332

293

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SUMMARY The genus Mycobacterium is best known for its two major pathogenic species, M. tuberculosis and M. leprae, the causative agents of two of the world's oldest diseases, tuberculosis and leprosy, respectively. M. tuberculosis kills approximately two million people each year and is thought to latently infect one-third of the world's population. One of the most remarkable features of the nonsporulating M. tuberculosis is its ability to remain dormant within an individual for decades before reactivating into active tuberculosis. Thus, control of cell division is a critical part of the disease. The mycobacterial cell wall has unique characteristics and is impermeable to a number of compounds, a feature in part responsible for inherent resistance to numerous drugs. The complexity of the cell wall represents a challenge to the organism, requiring specialized mechanisms to allow cell division to occur. Besides these mycobacterial specializations, all bacteria face some common challenges when they divide. First, they must maintain their normal architecture during and after cell division. In the case of mycobacteria, that means synthesizing the many layers of complex cell wall and maintaining their rod shape. Second, they need to coordinate synthesis and breakdown of cell wall components to maintain integrity throughout division. Finally, they need to regulate cell division in response to environmental stimuli. Here we discuss these challenges and the mechanisms that mycobacteria employ to meet them. Because these organisms are difficult to study, in many cases we extrapolate from information known for gram-negative bacteria or more closely related GC-rich gram-positive organisms.

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Genetic analysis of the β-lactamases of Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to β-lactam antibiotics

Cited by 190 publications

References 39 publications

Characterization of the Twin-Arginine Translocase Secretion System of Mycobacterium smegmatis

Characterization of the Twin-Arginine Translocase Secretion System of Mycobacterium smegmatis

Characterization of Novel Mycobacterium tuberculosis and Mycobacterium smegmatis Mutants Hypersusceptible to β-Lactam Antibiotics

Bacterial Growth and Cell Division: a Mycobacterial Perspective

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