Mycobacterium smegmatis d -Alanine Racemase Mutants Are Not Dependent on d -Alanine for Growth

Abstract

Mycobacterium smegmatis is a fast-growing nonpathogenic species particularly useful in studying basic cellular processes of relevance to pathogenic mycobacteria. This study focused on the D-alanine racemase gene (alrA), which is involved in the synthesis of D-alanine, a basic component of peptidoglycan that forms the backbone of the cell wall. M. smegmatis alrA null mutants were generated by homologous recombination using a kanamycin resistance marker for insertional inactivation. Mutants were selected on Middlebrook medium supplemented with 50 mM D-alanine and 20 g of kanamycin per ml. These mutants were also able to grow in standard and minimal media without D-alanine, giving rise to colonies with a drier appearance and moreraised borders than the wild-type strain. Mycobacteria include the facultative intracellular pathogensMycobacterium tuberculosis and Mycobacterium avium. It has been estimated that up to a third of the world's population is infected with M. tuberculosis (14). Microorganisms of the M. avium complex have achieved prominence as major opportunistic pathogens of AIDS patients. M. avium is naturally resistant to most first-line antituberculosis drugs (19). This threat to public health has been partially met by therapy with appropriate antimicrobial agents, but unfortunately drug-resistant M. avium and M. tuberculosis strains readily appear (6, 15), underscoring the need to develop new and more effective antimycobacterial agents.Biosynthesis of the mycobacterial cell wall has received considerable attention in the search for inhibitors useful for drug therapy (7). These cell walls display a complex architecture of glycolipids and proteins linked to the mycolyl-arabinogalactanpeptidoglycan backbone (26). This structure is a barrier that contributes to drug resistance (43), and many of its components have been found to play a major role in pathogenesis (11). The analysis of the M. tuberculosis genome sequence suggests that peptidoglycan biosynthesis in mycobacteria follows the general pathway of other bacteria, including the formation of the basic building block D-alanyl-D-alanine (2, 9, 44). DCS is particularly effective against mycobacteria albeit with marked side effects (10, 49). Moreover, overproduction of Alr in Mycobacterium smegmatis, Mycobacterium intracellulare, and Mycobacterium bovis BCG leads to a DCS-resistant phenotype. We have also shown that the M. smegmatis enzyme is inhibited by DCS in a concentration-dependent manner (5). Likewise, the M. avium and M. tuberculosis enzymes produced from E. coli recombinant clones are also inhibited by DCS (39). Nonetheless, the specific characteristics of the mycobacterial enzymes involved in peptidoglycan biosynthesis, including the essentiality of each of their functions, remain unknown. This knowledge is important to the design of specific inhibitors that would serve as novel bactericidal agents to treat M. tuberculosis and M. avium infections. Furthermore, the inactivation of the genes encoding for these enzymes may lead to the generation

Use of NMR Metabolomics To Analyze the Targets of d-Cycloserine in Mycobacteria: Role of d-Alanine Racemase

“…Surprisingly, this is not the case. 21 Wild-type M. smegmatis, TAM23 pTAMU3, DCS resistant strains (GPM14, GPM16) and TAM23 are able to grow both in the presence (Figure 2 B, E, H, K, N) and the absence of D-alanine (Figure 2 A, D, G, J, M). However, the growth is slower in the absence of D-alanine, where the cell wall of TAM23 appears to be weaker by its flaky appearance and irregular shaped borders.…”

“…21 The alr gene has been found in many other bacterial species such as E. coli and streptococcus species. 40 DCS is effective against mycobacterium because it disrupts cell wall synthesis by inhibiting peptidoglycan biosynthesis.…”

“…19,20 Similarly, an alr null mutant increases the susceptibility of mycobacteria to DCS, but surprisingly bacterial viability is not dependent on D-alanine. 21 Thus, the lethal cellular target (s) of DCS is still undetermined, but since mycobacteria sensitivity to DCS depends on Alr activity, this suggests Alr is still a potential in vivo target contributing to the overall effect of DCS. Potentially, the therapeutic activity of DCS may require inhibiting multiple proteins.…”

The Alanine Racemase of Mycobacterium smegmatis Is Essential for Growth in the Absence of d -Alanine

“…One study with Mycobacterium smegmatis suggested that even in the absence of D-alanine, alanine racemase was not required for growth (4). In that study the alanine racemase gene was insertionally inactivated.…”

“…Other bacterial species, such as Lactobacillus plantarum and Lactococcus lactis, have a single gene for alanine racemase, the deletion of which leads to a requirement for D-alanine for growth (18,24). Still other species, such as Listeria monocytogenes, express a D-amino acid transaminase that must be deleted along with the alr gene to yield D-alanine auxotrophy (29 (4,21). It is known that insertional inactivation can sometimes still allow for the production of active protein (13,31), which is why gene inactivation by deletion is a more definitive technique.…”