The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis
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 mc2155 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 ΔglnR 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.
BangeF. C.,
CollinsF. M.,
JacobsW. R.1999; Survival of mice infected with Mycobacterium smegmatis containing large DNA fragments from Mycobacterium tuberculosis . Tuber Lung Dis 79:171–180
DelgadoJ.,
ForstS.,
HarlockerS.,
InouyeM.1993; Identification of a phosphorylation site and functional analysis of conserved aspartic acid residues of OmpR, a transcriptional activator for ompF and ompC in Escherichia coli . Mol Microbiol 10:1037–1047
DeTurkW. E.,
BernheimF.1958; Effects of ammonia, methylamine and hydroxylamine on the adaptive assimilation of nitrite and nitrate by a mycobacterium. J Bacteriol 75:691–696
FinkD.,
WeissschuhN.,
ReutherJ.,
WohllebenW.,
EngelsA.2002; Two transcriptional regulators GlnR and GlnRII are involved in regulation of nitrogen metabolism in Streptomyces coelicolor A3(2. Mol Microbiol 46:331–347
GennisR. B.,
StewartV.others1996; Respiration. In Escherchia coli and Salmonella: Cellular and Molecular Biology , 2nd edn. pp 217–261 Edited by
NeidhardtF. C.
Washington, DC: America Society for Microbiology;
NishimuraT.,
VertesA. A.,
ShinodaY.,
InuiM.,
YukawaH.2007; Anaerobic growth of Corynebacterium glutamicum using nitrate as a terminal electron acceptor. Appl Microbiol Biotechnol 75:889–897
OgawaK.,
AkagawaE.,
YamaneK.,
SunZ. W.,
LaCelleM.,
ZuberP.,
NakanoM. M.1995; The nasB operon and nasA gene are required for nitrate/nitrite assimilation in Bacillus subtilis . J Bacteriol 177:1409–1413
PavelkaM. S.,
JacobsW. R.1999; Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis , Mycobacterium bovis bacillus Calmette-Guerin, and Mycobacterium tuberculosis H37Rv by allelic exchange. J Bacteriol 181:4780–4789
SohaskeyC. D.,
WayneL. G.2003; Role of narK2X and narGHJI in hypoxic upregulation of nitrate reduction by Mycobacterium tuberculosis . J Bacteriol 185:7247–7256
StermannM.,
BohrssenA.,
DiephausC.,
MaassS.,
BangeF. C.2003; Polymorphic nucleotide within the promoter of nitrate reductase (NarGHJI) is specific for Mycobacterium tuberculosis . J Clin Microbiol 41:3252–3259
StoverC. K.,
de la CruzV. F.,
BansalG. P.,
HansonM. S.,
FuerstT. R.,
JacobsW. R.Jr,
BloomB. R.1992; Use of recombinant BCG as a vaccine delivery vehicle. Adv Exp Med Biol 327:175–182
TakenoS.,
OhnishiJ.,
KomatsuT.,
MasakiT.,
SenK.,
IkedaM.2007; Anaerobic growth and potential for amino acid production by nitrate respiration in Corynebacterium glutamicum . Appl Microbiol Biotechnol 75:1173–1182
TiffertY.,
SupraP.,
WurmR.,
WohllebenW.,
WagnerR.,
ReutherJ.2008; The Streptomyces coelicolor GlnR regulon: identification of new GlnR targets and evidence for a central role of GlnR in nitrogen metabolism in actinomycetes. Mol Microbiol 67:861–880
VirtanenS.1960; A study of nitrate reduction by mycobacteria. The use of the nitrate reduction test in the identification of mycobacteria. Acta Tuberc Scand Suppl 48:1–119
WeberI.,
FritzC.,
RuttkowskiS.,
KreftA.,
BangeF. C.2000; Anaerobic nitrate reductase ( narGHJI ) activity of Mycobacterium bovis BCG in vitro and its contribution to virulence in immunodeficient mice. Mol Microbiol 35:1017–1025
WrayL. V.Jr,
AtkinsonM. R.,
FisherS. H.1991; Identification and cloning of the glnR locus, which is required for transcription of the glnA gene in Streptomyces coelicolor A3(2. J Bacteriol 173:7351–7360
The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis