The NnaR orphan response regulator is essential for the utilization of nitrate and nitrite as sole nitrogen sources in mycobacteria

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.

Insights into the Metabolism of Oleaginous Rhodococcus spp

“…NnaR works as a coactivator together with GlnR of its own gene cluster during ammonium limitation, as has been reported by Amin et al (54). NnaR and GlnR cooperate in the regulation of gene expression associated with nitrate/nitrite assimilation also in Mycobacterium smegmatis (55). The orthologous of NnaR (best hit protein) is also found in the genome of oleaginous rhodococci located in the same gene cluster as nitrite/nitrate reduction.…”

“…As has been reported for Streptomyces and Mycobacterium, this regulatory protein also controls the expression of nitrate/nitrite assimilation genes in R. jostii RHA1. However, this protein additionally contributes to the modulation of lipogenesis and TAG accumulation in R. jostii and R. opacus (56), in contrast to Mycobacterium smegmatis (55). Since this transcriptional regulator modulates simultaneously nitrogen and lipid metabolisms in response to ammonium limitation, the protein was named NlpR (nitrogen lipid regulator) in oleaginous Rhodococcus (56).…”

“…In this context, transcriptomic studies recently revealed that this regulatory protein modulates the expression of a small set of genes critical for nitrate/nitrite assimilation in M. smegmatis (42). After comparison of an nnaR-disrupted mutant of M. smegmatis to the wild-type strain during cultivation under nitrogen-limiting conditions, only seven transcripts, all involved in nitrogen utilization pathways, were downregulated in the mutant strain (55). The possible amplification of NlpR/NnaR regulon in oleaginous rhodococci in comparison to other actinobacteria with a lower ability to accumulate TAG could be an interesting innovative feature acquired during the evolution of these microorganisms.…”

Mycobacterium smegmatis does not display functional redundancy in nitrate reductase enzymes

“…However, further sequence analysis revealed three additional putative NR-encoding genes: MSMEG_2237 , MSMEG_6816 and MSMEG_4206 . Although annotated as a pseudogene, MSMEG_4206 appeared to be the most promising candidate based on (i) its unique domain architecture, (ii) the fact that it forms part of the GlnR regulon, which when disrupted under nitrogen starvation leads to fold changes of up to 16-fold [ 29 , 33 ] and (iii) the annotation as a pseudogene has been attributed to a sequencing error [ 15 ]. The growth analysis and nitrate utilization data generated during this study confirms that MSMEG_4206 is the assimilatory NR.…”

Biobank Lodz – population based biobank at the University of Lodz, Lodz, Poland

“…DNA, RNA tests (DNA and RNA isolation from various biological materials), study of genetic variation using classical methods of molecular biology such as: PCR, real-time PCR, Sanger sequencing [18], high-resolution melting of PCR products (HRM) [3-4, 17, 21, 26, 44-45], Taq-Man probes, measurements of selected parameters of the tested material (fluorometric testing of the DNA concentration), separation of nucleic acids using horizontal, vertical, capillary electrophoresis, genomic DNA testing using microarrays (e.g. GWAS) [15-16, 20, 22-23], next generation sequencing (Illumina Platform, Nanopore Sequencing), gene expression testing (RNA-seq, microarray and qPCR) [46][47][48][49][50][51], epigenetic analysis (Chip-Seq, DNA methylation analysis, methylation RNA), microorganism genome analysis (de novo sequencing, variant calling) [44,[52][53][54][55][56][57], eukaryotes whole genome and targeting sequencing, aDNA analysis [58][59], human genome analysis (variant calling, NGS Target Enrichment, whole exome sequencing), amplicon sequencing, metagenomics and metabarcoding, bioinformatics analysis.…”