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

ABSTRACT: 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.

SUBMITTER: Amon J

PROVIDER: S-EPMC2580704 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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

Amon Johannes J Bräu Tanja T Grimrath Aletta A Hänssler Eva E Hasselt Kristin K Höller Martina M Jessberger Nadja N Ott Lisa L Szököl Juraj J Titgemeyer Fritz F Burkovski Andreas A

Journal of bacteriology 20080808 21

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 ...[more]

PMID: 18689485

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Project description:Cobalamin is an essential cofactor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase that catalyzes the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme use-MetH versus MetE-is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro We also demonstrate that M. smegmatis can transport and assimilate exogenous cyanocobalamin (CNCbl; also known as vitamin B12) and its precursor, dicyanocobinamide ([CN]2Cbi). However, the uptake of CNCbl and (CN)2Cbi in this organism is restricted and seems dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity.IMPORTANCE Alterations in cobalamin-dependent metabolism have marked the evolution of Mycobacterium tuberculosis into a human pathogen. However, the role(s) of cobalamin in mycobacterial physiology remains poorly understood. Using the nonpathogenic saprophyte M. smegmatis, we investigated the production of cobalamin, transport and assimilation of cobalamin precursors, and the role of cobalamin in regulating methionine biosynthesis. We confirm constitutive de novo cobalamin biosynthesis in M. smegmatis, in contrast with M. tuberculosis, which appears to lack de novo cobalamin biosynthetic capacity. We also show that uptake of cyanocobalamin (vitamin B12) and its precursors is restricted in M. smegmatis, apparently depending on the cofactor requirements of the cobalamin-dependent methionine synthase. These observations establish M. smegmatis as an informative foil to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.

Project description:Total transcriptome analyses were carried out to investigate changes in transcript patterns in the Mycobacterium smegmatis wild type strain SMR5 due to nitrogen starvation and to expand the knowledge about the role of the two transcriptional regulators of nitrogen metabolism, namely GlnR and AmtR, in these processes. A first experiment revealed enhanced transcript levels of 284 genes and reduced transcripts of 231 genes in the wild type under nitrogen starvation compared to nitrogen surplus. When glnR deletion strain MH1 was compared to the wild type under nitrogen starvation, decreased transcript levels of 125 genes were detected, indicating that these are activated by GlnR due to nitrogen limitation. Comparing amtR deletion strain YL1 to the wild type under nitrogen starvation, enhanced transcript levels of 2 genes were found, indicating that they are repressed by AmtR under nitrogen surplus. A comparison of YL1 and wild type under surplus, as well as a comparison of YL1 under nitrogen surplus and starvation and of MH1 under nitrogen surplus and starvation were carried out as additional control experiments. It can be concluded that GlnR is the master regulator of nitrogen control in M. smegmatis and that AmtR fulfills only a small, subordinate role in the regulation of an operon. This SuperSeries is composed of the following subset Series: GSE30033: Mycobacterium smegmatis - Comparison of glnR deletion strain MH1 under nitrogen surplus and starvation GSE30231: Mycobacterium smegmatis - Comparison of wild type SMR5 and amtR deletion strain YL1 under nitrogen starvation GSE30232: Mycobacterium smegmatis - Comparison of amtR deletion strain YL1 under nitrogen surplus and starvation GSE30233: Mycobacterium smegmatis - Comparison of wild type SMR5 under nitrogen surplus and starvation GSE30234: Mycobacterium smegmatis - Comparison of wild type SMR5 and glnR deletion strain MH1 under nitrogen starvation GSE30235: Mycobacterium smegmatis - Comparison of wild type SMR5 and amtR deletion strain YL1 under nitrogen surplus Refer to individual Series

Dataset Information

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

Publications

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

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