Most mycobacterial species possess a full complement of genes for the biosynthesis of molybdenum cofactor (MoCo). However, a distinguishing feature of members of the Mycobacterium tuberculosis complex is their possession of multiple homologs associated with the first two steps of the MoCo biosynthetic pathway. A mutant of M. tuberculosis lacking the moaA1-moaD1 gene cluster and a derivative in which moaD2 was also deleted were significantly impaired for growth in media containing nitrate as a sole nitrogen source, indicating a reduced availability of MoCo to support the assimilatory function of the MoCo-dependent nitrate reductase, NarGHI. However, the double mutant displayed residual respiratory nitrate reductase activity, suggesting that it retains the capacity to produce MoCo. The M. tuberculosis moaD and moaE homologs were further analyzed by expressing these genes in mutant strains of M. smegmatis that lacked one or both of the sole molybdopterin (MPT) synthase-encoding genes, moaD2 and moaE2, and were unable to grow on nitrate, presumably as a result of the loss of MoCo-dependent nitrate assimilatory activity. Expression of M. tuberculosis moaD2 in the M. smegmatis moaD2 mutant and of M. tuberculosis moaE1 or moaE2 in the M. smegmatis moaE2 mutant restored nitrate assimilation, confirming the functionality of these genes in MPT synthesis. Expression of M. tuberculosis moaX also restored MoCo biosynthesis in M. smegmatis mutants lacking moaD2, moaE2, or both, thus identifying MoaX as a fused MPT synthase. By implicating multiple synthase-encoding homologs in MoCo biosynthesis, these results suggest that important cellular functions may be served by their expansion in M. tuberculosis.Mycobacterium tuberculosis, the causative agent of tuberculosis, accounts for approximately 1.7 million deaths each year (13). The success of M. tuberculosis as a pathogen is attributable, at least in part, to flexibility in its metabolism, which allows the organism to adapt to the diverse conditions encountered during transmission, infection, and pathogenesis (54). Since the virulence of M. tuberculosis is inextricably linked to its physiology (54), understanding the metabolism and metabolic flexibility of this pathogen under conditions relevant to disease is of paramount importance. A key environmental condition to which M. tuberculosis must adapt in vivo is hypoxia (2, 40, 52). Enzymes that utilize molybdenum cofactor (MoCo) harness the redox properties of molybdenum to catalyze redox reactions in carbon, nitrogen, and sulfur metabolism and to reduce terminal electron acceptors for anaerobic respiration. A search of the M. tuberculosis H37Rv proteome revealed nine proteins with recognizable MoCo-associated domains (see Table S1 in the supplemental material). One of these is NarG, the catalytic subunit of the narGHJI-encoded, membrane-bound respiratory nitrate reductase (NR) (2, 48), suggesting a potentially important role for MoCo in the metabolism of M. tuberculosis in vivo (52).The M. tuberculosis genome contains multiple g...
c Ergothioneine (ERG) and mycothiol (MSH) are two low-molecular-weight thiols synthesized by mycobacteria. The role of MSH has been extensively investigated in mycobacteria; however, little is known about the role of ERG in mycobacterial physiology. In this study, quantification of ERG at various points in the growth cycle of Mycobacterium smegmatis revealed that a significant portion of ERG is found in the culture media, suggesting that it is actively secreted. A mutant of M. smegmatis lacking egtD (MSMEG_6247) was unable to synthesize ERG, confirming its role in ERG biosynthesis. Deletion of egtD from wild-type M. smegmatis and an MSH-deficient mutant did not affect their susceptibility to antibiotics tested in this study. The ERG-and MSH-deficient double mutant was significantly more sensitive to peroxide than either of the single mutants lacking either ERG or MSH, suggesting that both thiols play a role in protecting M. smegmatis against oxidative stress and that ERG is able to partly compensate for the loss of MSH. G lutathione (GSH) is a thiol known for its efficient detoxification of reactive oxygen species, reactive nitrogen species, and free radicals in eukaryotes. Mycobacteria do not synthesize GSH but produce two low-molecular-weight thiols,and ergothioneine (2-mercaptohistidine trimethylbetaine) (ERG) (4, 5). Four genes are involved in MSH biosynthesis in mycobacteria, namely, mshA, mshB, mshC, and mshD, and mutants harboring deletions in mshB, mshC, and mshD produce different levels of MSH due to the ability of other enzymes to partially compensate for their loss (6, 7). MSH-deficient mutants of Mycobacterium smegmatis show increased sensitivity to oxidative stress, alkylating agents, and a range of antibiotics, including erythromycin, azithromycin, vancomycin, penicillin G, streptomycin, and rifampin, but exhibit increased resistance to isoniazid (INH) and ethionamide (ETH) (8, 9). The MSH-deficient ⌬mshA mutant of Mycobacterium tuberculosis requires catalase during in vitro growth, implicating MSH in detoxifying reactive oxygen species (10).ERG biosynthetic genes (egtA, egtB, egtC, egtD, and egtE) were recently identified in M. smegmatis (11). Although several lines of evidence support the cytoprotective and antioxidative role of ERG in eukaryotes (12), bacteria (13), and, recently, fungi (14), nothing is known of its role in mycobacteria. ERG has also been implicated in modulating the immune response (15) and in the inhibition of metalloenzymes, preventing the copper-induced oxidation of DNA and protein due to its metal-chelating properties (16,17). Eukaryotes obtain ERG from their diet, and its accumulation in cells is dependent on the activity of a highly specific transporter, OCTN1, since the zwitterionic nature of ERG prevents it from crossing the plasma membrane (18,19). In an M. smegmatis ⌬mshA mutant, which is MSH deficient, the levels of ERG and the organic hydroperoxide resistance (Ohr) protein are elevated, suggesting that ERG may partly compensate for the loss of MSH (20). This may exp...
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