Identification of the Mycobacterium tuberculosis GlnE promoter and its response to nitrogen availability

Pashley, Carey A.; Brown, Alexander; Robertson, Dina; Parish, Tanya

doi:10.1099/mic.0.28942-0

Cited by 20 publications

(15 citation statements)

References 24 publications

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“…Since glutamine synthetase in M. tuberculosis is essential for this bacterium and consequently an important drug target [Harth et al, 1994[Harth et al, , 2005Nordqvist et al, 2008;Tullius et al, 2003], work concentrated first on the glnE gene product adenylyltransferase, which is involved in posttranslational modification and regulation of this enzyme. It was shown that glnE is essential in this organism [Parish and Stoker, 2000], an observation that is in agreement with the crucial function of adenylyltransferase in regulation of GS activity [Carroll et al, 2008]; furthermore, the M. tuberculosis glnE promoter is upregulated in ammonia-or glutamine-containing media, at least in the heterologous host M. smegmatis [Pashley et al, 2006], while the transcriptional organization of the glnA1 -glnEglnA2 gene cluster remains to be elucidated further [Hotter et al, 2008].…”

Section: Signal Transductionmentioning

confidence: 78%

A Genomic View on Nitrogen Metabolism and Nitrogen Control in Mycobacteria

2008

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Knowledge about nitrogen metabolism and control in the genus Mycobacterium is sparse, especially compared to the state of knowledge in related actinomycetes like Streptomyces coelicolor or the close relative Corynebacterium glutamicum. Therefore, we screened the published genome sequences of Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium ssp. paratuberculosis and Mycobacterium leprae for genes encoding proteins for uptake of nitrogen sources, nitrogen assimilation and nitrogen control systems, resulting in a detailed comparative genomic analysis of nitrogen metabolism-related genes for all completely sequenced members of the genus. Transporters for ammonium, nitrate, and urea could be identified, as well as enzymes crucial for assimilation of these nitrogen sources, i.e. glutamine synthetase, glutamate dehydrogenase, glutamate synthase, nitrate reductase, nitrite reductase, and urease proteins. A reduction of genes encoding proteins for nitrogen transport and metabolism was observed for the pathogenic mycobacteria, especially for M. leprae. Signal transduction components identified for the different species include adenylyl- and uridylyltransferase and a P_II-type signal transduction protein. Exclusively for M. smegmatis, two homologs of putative nitrogen regulatory proteins were found, namely GlnR and AmtR, while in other mycobacteria, AmtR was absent and GlnR seems to be the nitrogen transcription regulator protein.

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Section: Signal Transductionmentioning

confidence: 78%

A Genomic View on Nitrogen Metabolism and Nitrogen Control in Mycobacteria

2008

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“…We have previously shown that GlnE is essential in M. tuberculosis and that its expression is regulated by nitrogen availability (24,26,27). The essentiality of GlnE is surprising, given that it is not essential in other bacteria, including E. coli and streptomycetes (6).…”

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confidence: 99%

Functional Analysis of GlnE, an Essential Adenylyl Transferase in Mycobacterium tuberculosis

2008

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Glutamine synthetase (GS) plays an important role in nitrogen assimilation. The major GS of Mycobacterium tuberculosis is GlnA1, a type I GS whose activity is controlled by posttranscriptional modification by GlnE. GlnE is an adenylyl transferase comprised of an adenylylating domain and a deadenylylating domain which modulate GS activity. We previously demonstrated that GlnE is essential in M. tuberculosis in normal growth medium. In this study, we further show that GlnE is required under multiple medium conditions, including in nitrogen-limited medium. We demonstrate that adenylylation is the critical activity for M. tuberculosis survival, since we were able to delete the deadenylylation domain with no apparent effect on growth or GS activity. Furthermore, we identified a critical aspartate residue in the proposed nucleotidyltransferase motif. Temperature-sensitive mutants of GlnE were generated and shown to have a defect in growth and GS activity in nitrogen-limited medium. Finally, we were able to generate a GlnE null mutant in the presence of L-methionine sulfoximine, a GS inhibitor, and glutamine supplementation. In the presence of these supplements, the null mutant was able to grow similarly to the wild type. Surprisingly, the GlnE mutant was able to survive and grow for extended periods in liquid medium, but not on solid medium, in the absence of GS inhibition. Thus, we have confirmed that the unusual requirement of M. tuberculosis for GlnE adenylylation activity is linked to the activity of GS in the cell.

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“…Previous work on nitrogen uptake and assimilation concentrated mainly on the major pathogenic member of the genus, Mycobacterium tuberculosis. While extensive data are available for glutamine synthetases, especially the physiologically crucial GlnA1 enzyme (12), the associated adenylyltransferase GlnE (7,18), and the uridylyl transferase GlnD (20), not much information is available for the transcriptional regulation of nitrogen metabolism in mycobacteria (19). In Mycobacterium smegmatis, homologs of all previously characterized genes encoding glutamine synthetases in M. tuberculosis are present in the genome (GlnA1, MSMEG_ 4290; GlnA2, MSMEG_4294; GlnA3, MSMEG_3561; GlnA4, MSMEG_2595), as are open reading frames encoding additional glutamine synthetase-like proteins with unknown physiological functions (e.g., MSMEG_1116, MSMEG_3827, MSMEG_5374, and MSMEG_6693).…”

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confidence: 99%

Nitrogen Control in Mycobacterium smegmatis : Nitrogen-Dependent Expression of Ammonium Transport and Assimilation Proteins Depends on the OmpR-Type Regulator GlnR

et al. 2008

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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.

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Identification of the Mycobacterium tuberculosis GlnE promoter and its response to nitrogen availability

Cited by 20 publications

References 24 publications

A Genomic View on Nitrogen Metabolism and Nitrogen Control in Mycobacteria

A Genomic View on Nitrogen Metabolism and Nitrogen Control in Mycobacteria

Functional Analysis of GlnE, an Essential Adenylyl Transferase in Mycobacterium tuberculosis

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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