The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis
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E-Artikel
Titel:
The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis
Beschreibung:
<jats:p>
<jats:italic>Mycobacterium tuberculosis</jats:italic> 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 <jats:italic>M. tuberculosis</jats:italic>. Homologues to a <jats:italic>narGHJI</jats:italic>-encoded nitrate reductase and a <jats:italic>nirBD</jats:italic>-encoded nitrite reductase have been found on the chromosome of <jats:italic>M. tuberculosis</jats:italic>. Previous studies have implied a role for NarGHJI in nitrate respiration rather than nitrate assimilation. Here, we show that a <jats:italic>narG</jats:italic> mutant of <jats:italic>M. tuberculosis</jats:italic> failed to grow on nitrate. A <jats:italic>nirB</jats:italic> mutant of <jats:italic>M. tuberculosis</jats:italic> failed to grow on both nitrate and nitrite. Mutant strains of <jats:italic>Mycobacterium smegmatis</jats:italic> mc<jats:sup>2</jats:sup>155 that are unable to grow on nitrate were isolated. The mutants were rescued by screening a cosmid library from <jats:italic>M. tuberculosis</jats:italic>, and a gene with homology to the response regulator gene <jats:italic>glnR</jats:italic> of <jats:italic>Streptomyces coelicolor</jats:italic> was identified. A Δ<jats:italic>glnR</jats:italic> mutant of <jats:italic>M. tuberculosis</jats:italic> was generated, which also failed to grow on nitrate, but regained its ability to utilize nitrate when <jats:italic>nirBD</jats:italic> was expressed from a plasmid, suggesting a role of GlnR in regulating <jats:italic>nirBD</jats:italic> expression. A specific binding site for GlnR within the <jats:italic>nirB</jats:italic> 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 <jats:italic>nirBD</jats:italic> expression in response to GlnR under nitrogen-limiting conditions. In summary, we conclude that NarGHJI and NirBD of <jats:italic>M. tuberculosis</jats:italic> mediate the assimilatory reduction of nitrate and nitrite, respectively, and that GlnR acts as a transcriptional activator of <jats:italic>nirBD</jats:italic>.</jats:p>