Nitrogen control in Mycobacterium smegmatis: nitrogen-dependent expression of ammonium transport and assimilation proteins depends on the OmpR-type regulator GlnR.

The effect of nitrogen regulation on the level of transcriptional control has been investigated in a variety of bacteria, such as Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, and Streptomyces coelicolor; however, until now there have been no data for mycobacteria. In this study, we found that the OmpR-type regulator protein GlnR controls nitrogen-dependent transcription regulation in Mycobacterium smegmatis. Based on RNA hybridization experiments with a wild-type strain and a corresponding mutant strain, real-time reverse transcription-PCR analyses, and DNA binding studies using cell extract and purified protein, the glnA (msmeg_4290) gene, which codes for glutamine synthetase, and the amtB (msmeg_2425) and amt1 (msmeg_6259) genes, which encode ammonium permeases, are controlled by GlnR. Furthermore, since glnK (msmeg_2426), encoding a PII-type signal transduction protein, and glnD (msmeg_2427), coding for a putative uridylyltransferase, are in an operon together with amtB, these genes are part of the GlnR regulon as well. The GlnR protein binds specifically to the corresponding promoter sequences and functions as an activator of transcription when cells are subjected to nitrogen starvation.

A combination of metabolome and transcriptome analyses reveals new targets of the Corynebacterium glutamicum nitrogen regulator AmtR.

The effects of a deletion of the amtR gene, encoding the master regulator of nitrogen control in Corynebacterium glutamicum, were investigated by metabolome and transcriptome analyses. Compared to the wild type, different metabolite patterns were observed in respect to glycolysis, pentose phosphate pathway, citric acid cycle, and most amino acid pools. Not all of these alterations could be attributed to changes at the level of mRNA and must be caused by posttranscriptional regulatory processes. However, subsequently carried out transcriptome analyses, which were confirmed by gel retardation experiments, revealed two new targets of AmtR, the dapD gene, encoding succinylase involved in m-diaminopimelate synthesis, and the mez gene, coding for malic enzyme. The regulation of dapD connects the AmtR-dependent nitrogen control with l-lysine biosynthesis, the regulation of mez with carbon metabolism. An increased l-glutamine pool in the amtR mutant compared to the wild type was correlated with deregulated expression of the AmtR-regulated glnA gene and an increased glutamine synthetase activity. The glutamate pool was decreased in the mutant and also glutamate excretion was impaired.

A game with many players: control of gdh transcription in Corynebacterium glutamicum.

Glutamate dehydrogenase is a central enzyme connecting nitrogen and carbon metabolism via its precursors ammonium and oxoglutarate and its product glutamate. In Corynebacterium glutamicum glutamate dehydrogenase is especially important, since it is a key enzyme for the biotechnological production of the flavour enhancer L-glutamate. In this study, the regulation of gdh transcription was investigated. While originally described as a constitutively expressed gene, we could show that gdh transcription is highly variable depending on environmental conditions. Regulation of the gdh gene by the C. glutamicum nitrogen control protein AmtR was analyzed in detail in this study.

Nitrogen metabolism and nitrogen control in corynebacteria: variations of a common theme.

The published genome sequences of Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium glutamicum and Corynebacterium jeikeium were screened for genes encoding central components of nitrogen source uptake, nitrogen assimilation and nitrogen control systems. Interestingly, the soil-living species C. efficiens and C. glutamicum exhibit a broader spectrum of genes for nitrogen transport and metabolism than the pathogenic species C. diphtheriae and C. jeikeium. The latter are characterized by gene decay and loss of functions like urea metabolism and nitrogen-dependent transcription control. The global regulator of nitrogen regulation AmtR and its DNA-binding motif are conserved in C. diphtheriae, C. efficiens and C. glutamicum, while in C. jeikeium, an AmtR-encoding gene as well as putative AmtR-binding motifs are missing.

FarR, a putative regulator of amino acid metabolism in Corynebacterium glutamicum.

With the publication of the Corynebacterium glutamicum genome sequence, a global characterization of genes controlled by functionally uncharacterized transcriptional regulators became possible. We used DNA microarrays in combination with gel retardation experiments to study gene regulation by FarR, a HutC/FarR-type regulator of the GntR family. Based on our results, FarR seems to be involved in the regulation of amino acid biosynthesis in C. glutamicum. Especially, transcript levels of the arg cluster and the gdh gene are influenced by deletion of farR.