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.

Nitrogen starvation-induced transcriptome alterations and influence of transcription regulator mutants in Mycobacterium smegmatis.

BACKGROUND: As other bacteria, Mycobacterium smegmatis needs adaption mechanisms to cope with changing nitrogen sources and to survive situations of nitrogen starvation. In the study presented here, transcriptome analyses were used to characterize the response of the bacterium to nitrogen starvation and to elucidate the role of specific transcriptional regulators. RESULTS: In response to nitrogen deprivation, a general starvation response is induced in M. smegmatis. This includes changes in the transcription of several hundred genes encoding e.g. transport proteins, proteins involved in nitrogen metabolism and regulation, energy generation and protein turnover. The specific nitrogen-related changes at the transcriptional level depend mainly on the presence of GlnR, while the AmtR protein controls only a small number of genes. CONCLUSIONS: M. smegmatis is able to metabolize a number of different nitrogen sources and nitrogen control in M. smegmatis is similar to control mechanisms characterized in streptomycetes, while the master regulator of nitrogen control in corynebacteria, AmtR, is plays a minor role in this regulatory network.

Common patterns - unique features: nitrogen metabolism and regulation in Gram-positive bacteria.

Gram-positive bacteria have developed elaborate mechanisms to control ammonium assimilation, at the levels of both transcription and enzyme activity. In this review, the common and specific mechanisms of nitrogen assimilation and regulation in Gram-positive bacteria are summarized and compared for the genera Bacillus, Clostridium, Streptomyces, Mycobacterium and Corynebacterium, with emphasis on the high G+C genera. Furthermore, the importance of nitrogen metabolism and control for the pathogenic lifestyle and virulence is discussed. In summary, the regulation of nitrogen metabolism in prokaryotes shows an impressive diversity. Virtually every phylum of bacteria evolved its own strategy to react to the changing conditions of nitrogen supply. Not only do the transcription factors differ between the phyla and sometimes even between families, but the genetic targets of a given regulon can also differ between closely related species.

A genomic view on nitrogen metabolism and nitrogen control in mycobacteria.

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.

Identification of a glucose permease from Mycobacterium smegmatis mc2 155.

We report here the molecular identification of a glucose permease from Mycobacterium smegmatis,a model organism for our understanding of the life patterns of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae. A computer-based search of the available genome of M. smegmatis mc(2) 155 with the sequences of well-characterized glucose transporters revealed the gene msmeg4187 as a possible candidate. The deduced protein belongs to the major facilitator superfamily of proton symporters and facilitators and exhibits up to 53% of amino acid identity to other members of this family. Heterologous expression of msmeg4187 in an Escherichia coli glucose-negative mutant led to the restoration of growth on glucose. The determination of the biochemical features characterize MSMEG4187 (GlcP) as a high affinity (K(m) of 19 microM), glucose-specific permease. The results represent the first molecular characterization of a sugar permease in mycobacteria, and thus supply fundamental data for further in-depth analysis on the nutritional lifestyle of these bacteria.

Sugar transport systems of Bifidobacterium longum NCC2705.

Here we present the complement of the carbohydrate uptake systems of the strictly anaerobic probiotic Bifidobacterium longum NCC2705. The genome analysis of this bacterium predicts that it has 19 permeases for the uptake of diverse carbohydrates. The majority belongs to the ATP-binding cassette transporter family with 13 systems identified. Among them are permeases for lactose, maltose, raffinose, and fructooligosaccharides, a commonly used prebiotic additive. We found genes that encode a complete phosphotransferase system (PTS) and genes for three permeases of the major facilitator superfamily. These systems could serve for the import of glucose, galactose, lactose, and sucrose. Growth analysis of NCC2705 cells combined with biochemical characterization and microarray data showed that the predicted substrates are consumed and that the corresponding transport and catabolic genes are expressed. Biochemical analysis of the PTS, in which proteins are central in regulation of carbon metabolism in many bacteria, revealed that B. longum has a glucose-specific PTS, while two other species (Bifidobacterium lactis and Bifidobacterium bifidum) have a fructose-6-phosphate-forming fructose-PTS instead. It became obvious that most carbohydrate systems are closely related to those from other actinomycetes, with a few exceptions. We hope that this report on B. longum carbohydrate transporter systems will serve as a guide for further in-depth analyses on the nutritional lifestyle of this beneficial bacterium.

A glucose kinase from Mycobacterium smegmatis.

Carbon metabolism and regulation is poorly understood in mycobacteria, a genus that includes some major pathogenic species like Mycobacterium tuberculosis and Mycobacterium leprae. Here, we report the identification of a glucose kinase from Mycobacterium smegmatis. This enzyme serves in glucose metabolism and global carbon catabolite repression in the related actinomycete Streptomyces coelicolor. The gene, msmeg1356 (glkA), was found by means of in silico screening. It was shown that it occurs in the same genetic context in all so far sequenced mycobacterial species, where it is located in a putative tricistronic operon together with a glycosyl hydrolase and a putative malonyl-CoA transacylase. Heterologous expression of glkA in an Escherichia coli glucose kinase mutant led to the restoration of glucose growth, which provided in vivo evidence for glucose kinase function. GlkA(Msm) was subsequently overproduced in order to study its enzymatic features. We found that it can form a dimer and that it efficiently phosphorylates glucose at the expense of ATP. The affinity constant for glucose was with 9 mM about eight times higher and the velocity was about tenfold slower when compared to the parallel measured glucose kinase of S. coelicolor. Both enzymes showed similar substrate specificity, which consists in an ATP-dependent phosphorylation of glucose and no, or very inefficient, phosphorylation of the glucose analogues 2-deoxyglucose and methyl alpha-glucoside. Hence, our data provide a basis for studying the role of mycobacterial glucose kinase in vivo to unravel possible catalytic and regulatory functions.

A genomic view of sugar transport in Mycobacterium smegmatis and Mycobacterium tuberculosis.

We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast- and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively.