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 multidrug-resistant 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
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) α-alkyl, β-hydroxy fatty acids oriented perpendicularly to the cell membrane (36
). Extractable lipids in association with the mycolic acids serve to form an outer membrane-like structure at the cell surface.
Our laboratory studies mycobacterial peptidoglycan biosynthesis and the role of the peptidoglycan in the physiology of the mycobacterial cell envelope. We are interested in using β-lactam antibiotics, which target peptidoglycan assembly enzymes, as tools to study peptidoglycan biosynthesis. However, mycobacteria are intrinsically resistant to β-lactam antibiotics, primarily due to the production of β-lactamases, although the permeability of the cell envelope, drug efflux pumps, low-affinity penicillin-binding proteins (PBPs), and β-lactam-insensitive peptidoglycan-biosynthetic enzymes may also play a role in resistance to this class of antibiotics (8
). The intrinsic resistance of these organisms has hampered efforts to use these antibiotics to study cell wall biosynthesis.
To eliminate this problem, our laboratory has constructed mutants of M. tuberculosis
and M. smegmatis
with deletions of the major β-lactamase genes, blaC
, respectively (17
). We previously showed that the mutants have an increased susceptibility to most β-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 β-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 β-lactam resistance is minimal (17
). Our data are consistent with the view that additional mechanisms contribute to β-lactam resistance in these two organisms.
We hypothesized that characterizing mutants derived from β-lactamase deletion mutants that are hypersusceptible to β-lactam antibiotics might reveal novel genes involved with other mechanisms of β-lactam resistance, peptidoglycan assembly, and cell envelope physiology. To this end, we performed transposon mutagenesis of the β-lactamase mutants of M. tuberculosis and M. smegmatis described above and screened for mutants hypersusceptible to the cephalosporin ceftriaxone. We report here the characterization (i) of seven β-lactam antibiotic-hypersusceptible mutants in which the transposon affects genes with no known function and (ii) of two mutants in which the insertions are in known peptidoglycan-biosynthetic genes. Our analyses suggest that the seven unknown proteins are involved with peptidoglycan biosynthesis, cell division, or other cell envelope processes.