Abstract
Nitrogen is an essential component of biological molecules and an indispensable microelement required for the growth of cells. Nitrogen metabolism of Mycobacterium smegmatis is regulated by a number of transcription factors, with the glnR gene product playing a major role. Under nitrogen-depletion conditions, GlnR controls the expression of many genes involved in nitrogen assimilation, including the msmeg_0432 gene encoding NnaR, the homologue of a nitrite/nitrate transport regulator from Streptomyces coelicolor. In the present study, the role of NnaR in the nitrogen metabolism of M. smegmatis was evaluated. The ∆glnR and ∆nnaR mutant strains were generated and cultured under nitrogen-depletion conditions. Total RNA profiling was used to investigate the potential role of NnaR in the GlnR regulon under nitrogen-depletion and in nitrogen-rich media. We found that disruption of MSMEG_0432 affected the expression of genes involved in nitrite/nitrate uptake, and its removal rendered mycobacteria unable to assimilate nitrogen from those sources, leading to cell death. RNA-Seq results were validated using quantitative real-time polymerase chain reaction (qRT-PCR) and electrophoretic mobility shift assays (EMSAs). The ability of mutants to grow on various nitrogen sources was evaluated using the BIOLOG Phenotype screening platform and confirmed on minimal Sauton’s medium containing various sources of nitrogen. The ∆glnR mutant was not able to convert nitrates to nitrites. Interestingly, NnaR required active GlnR to prevent nitrogen starvation, and both proteins cooperated in the regulation of gene expression associated with nitrate/nitrite assimilation. The ∆nnaR mutant was able to convert nitrates to nitrites, but it could not assimilate the products of this conversion. Importantly, NnaR was the key regulator of the expression of the truncated haemoglobin trHbN, which is required to improve the survival of bacteria under nitrosative stress.
Highlights
The genus Mycobacterium contains obligatory pathogens known to cause serious diseases in mammals, including tuberculosis (Mycobacterium tuberculosis, Mtb) and leprosy (M. leprae) in humans, as well as a large number of opportunistic pathogens and/or free-living saprophytes, such as Mycobacterium smegmatis (M. smegmatis)[1]
GlnR controls the expression of ammonium transporters, signal transduction components, glutamine synthetase in M. smegmatis[10] and nirBD expression in M. tuberculosis[11]
We analysed the kinetics of growth by measuring the OD600 (Supplementary Fig. S2A) and viability (CFU/mL) (Fig. 1A), which revealed no significant differences in growth or survival between tested mutant strains (ΔnnaR; ΔglnR; Δ(nnaR, glnR); ΔnnaR-attB::phsp60nnaR; ΔglnR-attB::phsp60nnaR) and wild-type when propagated in 7H9/OADC liquid media
Summary
The genus Mycobacterium contains obligatory pathogens known to cause serious diseases in mammals, including tuberculosis (Mycobacterium tuberculosis, Mtb) and leprosy (M. leprae) in humans, as well as a large number of opportunistic pathogens and/or free-living saprophytes, such as Mycobacterium smegmatis (M. smegmatis)[1]. GlnR controls the expression of ammonium transporters (amt[1], amtB), signal transduction components (glnK, glnD), glutamine synthetase (glnA) in M. smegmatis[10] and nirBD expression in M. tuberculosis[11]. The GlnR-dependent regulation of more than 100 genes under nitrogen depletion has been demonstrated using ChIP-seq technology in M. smegmatis[3] Among them are those involved in ammonium, nitrate/nitrite, amino acid/peptide and urea uptake, genes encoding the nitrite reductase NirBD, amine oxidase, urea amidolyase, deaminase and hydrolases acting on carbon-nitrogen bonds, as well as regulatory genes, including msmeg_0432, the homologue of the nitrite/nitrate transport regulator (NnaR) of S. coelicolor[14] present in M. tuberculosis[5]. We have engineered ΔnnaR and ΔglnR M. smegmatis mutants to evaluate systematically the role of NnaR as a regulator of nitrogen metabolism in M. smegmatis by using phenotypic microarray technology and RNA-Seq analysis and by monitoring the kinetics of growth and viability in the presence of various nitrogen sources
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