Investigation of the Essentiality of Glutamate Racemase in Mycobacterium smegmatis

Abstract

The mycobacterial cell wall frequently has been used as a target for drug development, and D-glutamate, synthesized by glutamate racemase (MurI), is an important component of peptidoglycan. While the essentiality of the murI gene has been shown in several bacterial species, including Escherichia coli, Bacillus anthracis, and Streptococcus pneumoniae, studies in mycobacteria have not yet provided definitive results. This study aimed to determine whether murI is indeed essential and can serve as a possible target for structure-aided drug design. We have achieved this goal by creating a ⌬murI strain of Mycobacterium smegmatis, a close relative of Mycobacterium tuberculosis. The deletion of the murI gene in M. smegmatis could be achieved only in minimal medium supplemented with D-glutamate, demonstrating that MurI is essential for growth and that glutamate racemase is the only source of D-glutamate for peptidoglycan synthesis in M. smegmatis. D-Glutamate is an essential component of the bacterial cell wall, and there are two enzymes in bacteria capable of synthesizing D-glutamate, glutamate racemase (MurI) and D-amino acid aminotransferase (DaaT). In Escherichia coli and Streptococcus pneumoniae, whose genomes contain only murI, genetic deletion studies have shown that MurI is essential for growth in the absence of external D-glutamate supplementation (1-4). Other bacteria, which contain DaaT, such as Staphylococcus haemolyticus and Bacillus sphaericus, are able to produce sufficient D-glutamate in the absence of glutamate racemase (5). There are also bacteria, including some other Bacillus species, that contain two copies of murI, as well as a copy of daaT, yet, at least in B. subtilis, murI is required for these bacteria to grow in both rich and minimal medium (6-8). MurI also has been implicated in several species as having a role in moderating the activity of DNA gyrase, which itself is an important drug target and an essential enzyme in bacteria (9-11). Therefore, the essentiality of murI is a complex question that appears to be species specific.In mycobacteria, where the identification and validation of new targets for drug design is an ongoing priority, the essentiality of glutamate racemase is unclear. The inspection of the genome of Mycobacterium tuberculosis reveals only one copy of murI and no copy of daaT (12). Therefore, it seems likely that MurI is essential, as it is the only enzyme capable of D-glutamate biosynthesis in this organism. However, in a transposon mutagenesis study carried out in M. tuberculosis, murI was not identified as likely to be essential (13). However, further investigation of that particular strain revealed the transposon had integrated at the penultimate position of the gene sequence and may not have inactivated the gene. In support of this interpretation, a more recent investigation of this issue employing transposon mutagenesis coupled with deep sequencing did conclude that murI was likely to be an essential gene (14). Moreover, a different set of experiments investigating glu...

Alanine racemase is essential for the growth and interspecies competitiveness of Streptococcus mutans

“…29, 30 The murl mutant of E. coli lysed in the absence of D-glutamic acid, 31 and the murl mutant of M ycobaterium smegmatis also showed aberrant cell shapes. 32 Although the precise mechanisms remain unknown, the deletion of alr not only led to growth arrest but also to cell death, indicating that alr might be a potential bactericidal target for the inhibition of S. mutans .…”

Overexpression of a newly identified d‐amino acid transaminase in Mycobacterium smegmatis complements glutamate racemase deletion

“…Previous work from our group demonstrated that glutamate racemase deletion mutants of M. smegmatis mc 2 155 were unable to grow in the absence of D-glutamate (Li et al, 2014). To investigate this further, we set up growth experiments in which the incubation period was extended to > 100 h, both in the absence and presence of D-glutamate ( Fig.…”

“…The murI gene has been reported as essential in E. coli (Doublet et al ., ), Bacillus anthracis (Oh et al ., ), B. subtilis (Kimura et al ., ), Helicobacter pylori (Lundqvist et al ., ) and Streptococcus pneumoniae (de Dios et al ., ), among others. Recent work has confirmed its essentiality in both M. smegmatis (Li et al ., ) and M. tuberculosis (Morayya et al ., ; Griffin et al ., ), contradicting earlier studies (Sassetti et al ., ; Harth et al ., ).…”

“…Glutamate racemase (MurI) has been proposed recently as a potential new target for inhibition by antituberculosis therapies (Li et al, 2014;Morayya et al, 2015;Poen et al, 2016;Prosser et al, 2016). MurI is an essential enzyme in most bacteria and it is needed to provide D-glutamate for completion of the peptidoglycan layer of the bacterial cell wall (Doublet et al, 1993;Geng et al, 2008).…”

“…All recent studies support the conditional essentiality of MurI in mycobacteria (Li et al, 2014;Morayya et al, 2015), but it is unknown if its deletion results in a bactericidal or bacteriostatic effect. Further, it is unknown how readily resistance can develop to this deletion (Leekha et al, 2011).…”