Mycobacterium tuberculosis can utilize various nutrients including nitrate as a source of nitrogen. Assimilation of nitrate requires the reduction of nitrate via nitrite to ammonium, which is then incorporated into metabolic pathways. This study was undertaken to define the molecular mechanism of nitrate assimilation in M. tuberculosis. Homologues to a narGHJI-encoded nitrate reductase and a nirBD-encoded nitrite reductase have been found on the chromosome of M. tuberculosis. Previous studies have implied a role for NarGHJI in nitrate respiration rather than nitrate assimilation. Here, we show that a narG mutant of M. tuberculosis failed to grow on nitrate. A nirB mutant of M. tuberculosis failed to grow on both nitrate and nitrite. Mutant strains of Mycobacterium smegmatis mc 2 155 that are unable to grow on nitrate were isolated. The mutants were rescued by screening a cosmid library from M. tuberculosis, and a gene with homology to the response regulator gene glnR of Streptomyces coelicolor was identified. A DglnR mutant of M. tuberculosis was generated, which also failed to grow on nitrate, but regained its ability to utilize nitrate when nirBD was expressed from a plasmid, suggesting a role of GlnR in regulating nirBD expression. A specific binding site for GlnR within the nirB promoter was identified and confirmed by electrophoretic mobility shift assay using purified recombinant GlnR. Semiquantitative reverse transcription PCR, as well as microarray analysis, demonstrated upregulation of nirBD expression in response to GlnR under nitrogen-limiting conditions. In summary, we conclude that NarGHJI and NirBD of M. tuberculosis mediate the assimilatory reduction of nitrate and nitrite, respectively, and that GlnR acts as a transcriptional activator of nirBD.
Mycobacterium tuberculosis generally is assumed to depend on lipids as a major carbon and energy source when persisting within the host. The utilization of fatty acids requires a functional glyoxylate cycle with the key enzymes isocitrate lyase (Icl) and malate synthase. The open reading frame Rv0465c of M. tuberculosis H37Rv encodes a protein with significant sequence similarity to the transcriptional regulator RamB, which in Corynebacterium glutamicum controls the expression of several genes involved in acetate metabolism, i.e., those encoding enzymes of acetate activation and the glyoxylate cycle. We show here that the M. tuberculosis Rv0465c protein can functionally complement RamB in C. glutamicum and that it binds to the promoter regions of M. tuberculosis icl1 and Rv0465c. Construction and subsequent transcriptional and enzymatic analysis of a defined Rv0465c deletion mutant in M. tuberculosis revealed that the Rv0465c protein, now designated RamB, represses icl1 expression during growth with glucose and negatively autoregulates the expression of its own operon. Whole-genome microarray analysis of the M. tuberculosis ramB (ramB MT ) mutant and the wild type furthermore showed that apart from icl1 and the ramB MT operon, the expression of all other M. tuberculosis genes involved in acetate metabolism remain unchanged in the mutant. Thus, RamB MT has a more specific regulatory function as RamB from C. glutamicum and is confined to expression control of icl1 and the ramB MT operon.
In many bacterial species, the translational GTPase TypA acts as a global stress- and virulence regulator and also mediates resistance to the antimicrobial peptide BPI. On the chromosome of M. tuberculosis, typA is located next to narGHJI, which plays a role in adaptation of the pathogen to various environmental conditions. Here, we show that Mycobacterium tuberculosis is sensitive to P2, a derivative of BPI. Using a typA mutant of M. tuberculosis, we found this phenotype to be independent of TypA. We further tested typA expression in M. tuberculosis under defined stress conditions, such as oxygen- and nutrient depletion, low pH, heat shock, antibiotic stress and the presence of P2, and found that typA expression remains unaffected by any of these conditions. Analysis of growth and whole-genome expression revealed similar growth kinetics and gene expression profiles of the wild type and the mutant under normal growth conditions as well as under stress conditions. Our results suggest that in contrast to the findings in other bacteria, TypA does not act as a global stress- and virulence regulator in M. tuberculosis.
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