title: The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis.
abstract: 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 DeltaglnR 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.
authors.affiliation: Department of Medical Microbiology and Hospital Epidemiology, Medical School Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Malm
authors.affiliation: Microbiology/Biotechnology, Microbiological Institute, Faculty of Biology, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Tiffert
authors.affiliation: Department of Medical Microbiology and Hospital Epidemiology, Medical School Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Micklinghoff
authors.affiliation: Department of Medical Microbiology and Hospital Epidemiology, Medical School Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Schultze
authors.affiliation: Department of Medical Microbiology and Hospital Epidemiology, Medical School Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
authors.forename: Insa
authors.initials: I
authors.lastname: Joost
authors.affiliation: Department of Medical Microbiology and Hospital Epidemiology, Medical School Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
authors.forename: Isabel
authors.initials: I
authors.lastname: Weber
authors.affiliation: Department of Medical Microbiology and Hospital Epidemiology, Medical School Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Horst
authors.affiliation: Department of Medical Microbiology and Hospital Epidemiology, Medical School Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Ackermann
authors.affiliation: Department of Medical Microbiology and Hospital Epidemiology, Medical School Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
authors.forename: Mascha
authors.initials: M
authors.lastname: Schmidt
authors.affiliation: Microbiology/Biotechnology, Microbiological Institute, Faculty of Biology, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wohlleben
authors.affiliation: Molecular Infection Biology, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, 23845 Borstel, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Ehlers
authors.affiliation: Department of Cell Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Geffers
authors.affiliation: Microbiology/Biotechnology, Microbiological Institute, Faculty of Biology, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Reuther
authors.affiliation: Department of Medical Microbiology and Hospital Epidemiology, Medical School Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
authors.forename: Franz-Christoph
authors.initials: FC
authors.lastname: Bange