Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S RNAs in B. subtilis and Ms1 in M. smegmatis, we detected MTS2823, a homologue of Ms1, on RNAP in M. tuberculosis. In C. glutamicum, we discovered novel types of structured RNAs that associate with RNAP. Furthermore, we identified other species-specific RNAs including full-length mRNAs, revealing a previously unknown landscape of RNAs interacting with the bacterial transcription machinery.

• MeSH
• Bacillus subtilis genetics   metabolism   MeSH
• Bacterial Proteins * metabolism   genetics   MeSH
• RNA, Bacterial * metabolism   genetics   MeSH
• Corynebacterium glutamicum genetics   metabolism   MeSH
• DNA-Directed RNA Polymerases * metabolism   genetics   MeSH
• Transcription, Genetic MeSH
• Nucleic Acid Conformation MeSH
• Mycobacterium smegmatis genetics   metabolism   enzymology   MeSH
• Mycobacterium tuberculosis genetics   metabolism   MeSH
• RNA, Untranslated MeSH
• Gene Expression Regulation, Bacterial MeSH
• Sigma Factor * metabolism   genetics   MeSH
• Streptomyces coelicolor genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Vysokoškolská učebnice, která se zaměřuje na různé druhy mikroorganismů a jejich ekologii a patogenitu.

• MeSH
• Ecology MeSH
• Communicable Diseases MeSH
• Microbiological Phenomena MeSH
• Environmental Microbiology MeSH
• Molecular Biology MeSH
• Publication type
• Monograph MeSH

• Conspectus
• Mikrobiologie
• Učební osnovy. Vyučovací předměty. Učebnice
• NML Fields
• mikrobiologie, lékařská mikrobiologie
• NML Publication type
• učebnice vysokých škol

The sigma H (σΗ) and sigma E (σE) subunits of Corynebacterium glutamicum RNA polymerase belong to Group 4 of sigma factors, also called extracytoplasmic function (ECF) sigma factors. Genes of the C. glutamicum σΗ regulon that are involved in heat and oxidative stress response have already been defined, whereas the genes of the σE regulon, which is involved in cell surface stress response, have not been explored until now. Using the C. glutamicum RES167 strain and its derivative C. glutamicum ΔcseE with a deletion in the anti-σΕ gene, differential gene expression was analyzed by RNA sequencing. We found 296 upregulated and 398 downregulated genes in C. glutamicum ΔcseE compared to C. glutamicum RES167. To confirm the functional link between σΕ and the corresponding promoters, we tested selected promoters using the in vivo two-plasmid system with gfpuv as a reporter gene and by in vitro transcription. Analyses with RNAP+σΗ and RNAP+σΕ, which were previously shown to recognize similar promoters, proved that the σΗ and σE regulons significantly overlap. The σE-controlled genes were found to be involved for example in protein quality control (dnaK, dnaJ2, clpB, and clpC), the regulation of Clp proteases (clgR), and membrane integrity maintenance. The single-promoter analyses with σΗ and σΕ revealed that there are two groups of promoters: those which are exclusively σΗ-specific, and the other group of promoters, which are σΗ/σE-dependent. No exclusively σE-dependent promoter was detected. We defined the consensus sequences of exclusively σΗ-regulated promotors to be -35 GGAAt and - 10 GTT and σΗ/σE-regulated promoters to be -35 GGAAC and - 10 cGTT. Fifteen genes were found to belong to the σΗ/σΕ regulon. Homology modeling showed that there is a specific interaction between Met170 in σΗ and the nucleotides -31 and - 30 within the non-coding strand (AT or CT) of the σΗ-dependent promoters. In σE, Arg185 was found to interact with the nucleotides GA at the same positions in the σE-dependent promoters.

• Publication type
• Journal Article MeSH

Rhodococcus erythropolis CCM2595 is a bacterial strain, which has been studied for its capability to degrade phenol and other toxic aromatic compounds. Its cell wall contains mycolic acids, which are also an attribute of other bacteria of the Mycolata group, such as Corynebacterium and Mycobacterium species. We suppose that many genes upregulated by phenol stress in R. erythropolis are controlled by the alternative sigma factors of RNA polymerase, which are active in response to the cell envelope or oxidative stress. We developed in vitro and in vivo assays to examine the connection between the stress sigma factors and genes activated by various extreme conditions, e.g., heat, cell surface, and oxidative stress. These assays are based on the procedures of such tests carried out in the related species, Corynebacterium glutamicum. We showed that the R. erythropolis CCM2595 genes frmB1 and frmB2, which encode S-formylglutathione hydrolases (named corynomycolyl transferases in C. glutamicum), are controlled by SigD, just like the homologous genes cmt1 and cmt2 in C. glutamicum. The new protocol of the in vivo and in vitro assays will enable us to classify R. erythropolis promoters according to their connection to sigma factors and to assign the genes to the corresponding sigma regulons. The complex stress responses, such as that induced by phenol, could, thus, be analyzed with respect to the gene regulation by sigma factors.

• MeSH
• Corynebacterium glutamicum genetics   MeSH
• DNA-Directed RNA Polymerases * genetics   MeSH
• Promoter Regions, Genetic * MeSH
• Rhodococcus * enzymology   genetics   MeSH
• Sigma Factor * genetics   MeSH
• Publication type
• Journal Article MeSH

Corynebacterium glutamicum ATCC 13032 harbors five sigma subunits of RNA polymerase belonging to Group IV, also called extracytoplasmic function (ECF) σ factors. These factors σC, σD, σE, σH, and σM are mostly involved in stress responses. The role of σD consists in the control of cell wall integrity. The σD regulon is involved in the synthesis of components of the mycomembrane which is part of the cell wall in C. glutamicum. RNA sequencing of the transcriptome from a strain overexpressing the sigD gene provided 29 potential σD-controlled genes and enabled us to precisely localize their transcriptional start sites. Analysis of the respective promoters by both in vitro transcription and the in vivo two-plasmid assay confirmed that transcription of 11 of the tested genes is directly σD-dependent. The key sequence elements of all these promoters were found to be identical or closely similar to the motifs -35 GTAACA/G and -10 GAT. Surprisingly, nearly all of these σD-dependent promoters were also active to a much lower extent with σHin vivo and one (Pcg0607) also in vitro, although the known highly conserved consensus sequence of the σH-dependent promoters is different (-35 GGAAT/C and -10 GTT). In addition to the activity of σH at the σD-controlled promoters, we discovered separated or overlapping σA- or σB-regulated or σH-regulated promoters within the upstream region of 8 genes of the σD-regulon. We found that phenol in the cultivation medium acts as a stress factor inducing expression of some σD-dependent genes. Computer modeling revealed that σH binds to the promoter DNA in a similar manner as σD to the analogous promoter elements. The homology models together with mutational analysis showed that the key amino acids, Ala 60 in σD and Lys 53 in σH, bind to the second nucleotide within the respective -10 promoter elements (GAT and GTT, respectively). The presented data obtained by integrating in vivo, in vitro and in silico approaches demonstrate that most of the σD-controlled genes also belong to the σH-regulon and are also transcribed from the overlapping or closely located housekeeping (σA-regulated) and/or general stress (σB-regulated) promoters.

• Publication type
• Journal Article MeSH

BACKGROUND: Sigma factors are one of the components of RNA polymerase holoenzymes, and an essential factor of transcription initiation in bacteria. Corynebacterium glutamicum possesses seven genes coding for sigma factors, most of which have been studied to some detail; however, the role of SigD in transcriptional regulation in C. glutamicum has been mostly unknown. RESULTS: In this work, pleiotropic effects of sigD overexpression at the level of phenotype, transcripts, proteins and metabolites were investigated. Overexpression of sigD decreased the growth rate of C. glutamicum cultures, and induced several physiological effects such as reduced culture foaming, turbid supernatant and cell aggregation. Upon overexpression of sigD, the level of Cmt1 (corynomycolyl transferase) in the supernatant was notably enhanced, and carbohydrate-containing compounds were excreted to the supernatant. The real-time PCR analysis revealed that sigD overexpression increased the expression of genes related to corynomycolic acid synthesis (fadD2, pks), genes encoding corynomycolyl transferases (cop1, cmt1, cmt2, cmt3), L, D-transpeptidase (lppS), a subunit of the major cell wall channel (porH), and the envelope lipid regulation factor (elrF). Furthermore, overexpression of sigD resulted in trehalose dicorynomycolate accumulation in the cell envelope. CONCLUSIONS: This study demonstrated that SigD regulates the synthesis of corynomycolate and related compounds, and expanded the knowledge of regulatory functions of sigma factors in C. glutamicum.

• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Corynebacterium glutamicum genetics   growth & development   metabolism   MeSH
• Mycolic Acids metabolism   MeSH
• Gene Expression Regulation, Bacterial MeSH
• Sigma Factor genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH

The 6C RNA family is a class of small RNAs highly conserved in Actinobacteria, including the genera Mycobacterium, Streptomyces and Corynebacterium whose physiological function has not yet been elucidated. We found that strong transcription of the cgb_03605 gene, which encodes 6C RNA in C. glutamicum, was driven by the SigA- and SigB-dependent promoter Pcgb_03605. 6C RNA was detected at high level during exponential growth phase (180 to 240 molcules per cell) which even increased at the entry of the stationary phase. 6C RNA level did not decrease within 240 min after transcription had been stopped with rifampicin, which suggests high 6C RNA stability. The expression of cgb_03605 further increased approximately twofold in the presence of DNA-damaging mitomycin C (MMC) and nearly threefold in the absence of LexA. Deletion of the 6C RNA gene cgb_03605 resulted in a higher sensitivity of C. glutamicum toward MMC and UV radiation. These results indicate that 6C RNA is involved in the DNA damage response. Both 6C RNA level-dependent pausing of cell growth and branched cell morphology in response to MMC suggest that 6C RNA may also be involved in a control of cell division.

• MeSH
• RNA, Bacterial genetics   MeSH
• Corynebacterium glutamicum genetics   growth & development   metabolism   MeSH
• Transcription, Genetic MeSH
• RNA, Small Untranslated chemistry   genetics   MeSH
• Promoter Regions, Genetic MeSH
• Gene Expression Regulation, Bacterial MeSH
• Base Sequence MeSH
• Sigma Factor metabolism   MeSH
• SOS Response, Genetics genetics   MeSH
• RNA Stability MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Promoter activities in Corynebacterium glutamicum strains with deletions of genes encoding sigma factors of RNA polymerase suggested that transcription from some promoters is controlled by two sigma factors. To prove that different sigma factors are involved in the recognition of selected Corynebacterium glutamicum promoters, in vitro transcription system was applied. It was found that a typical housekeeping promoter Pper interacts with the alternative sigma factor σ(B) in addition to the primary sigma factor σ(A). On the other way round, the σ(B)-dependent promoter of the pqo gene that is expressed mainly in the stationary growth phase was active also with σ(A). Some promoters of genes involved in stress responses (P1clgR, P2dnaK, and P2dnaJ2) were found to be recognized by two stress-responding sigma factors, σ(H) and σ(E). In vitro transcription system thus proved to be a useful direct technique for demonstrating the overlap of different sigma factors in recognition of individual promoters in C. glutamicum.

• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Corynebacterium glutamicum genetics   metabolism   MeSH
• Transcription, Genetic * MeSH
• Promoter Regions, Genetic * MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Sigma Factor genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH

Sucrose and molasses are attractive raw materials for industrial fermentation. Although Corynebacterium glutamicum shows sucrose-utilizing activity, sucrose or molasses is only a fraction of carbon source used in the fermentation medium in most works. An engineered C. glutamicum strain was constructed for producing L-ornithine with sucrose or molasses as a sole carbon source by transferring Mannheimia succiniciproducens β-fructofuranosidase gene (sacC). The engineered strain, C. glutamicum ΔAPE6937R42 (pEC-sacC), produced 22.0 g/L of L-ornithine with sucrose as the sole carbon source, which is on par with that obtained by the parent strain C. glutamicum ΔAPE6937R42 with glucose as the sole carbon. The resulting strain C. glutamicum ΔAPE6937R42 (pEC-sacC) produced 27.0 g/L of L-ornithine with molasses as the sole carbon source, which is higher than that obtained by the parent strain C. glutamicum ΔAPE6937R42 with glucose as the sole carbon. This strategy can be applied for developing sucrose- or molasses-utilizing industrial strains.

• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Corynebacterium glutamicum genetics   metabolism   MeSH
• Fermentation MeSH
• Molasses analysis   microbiology   MeSH
• Ornithine biosynthesis   MeSH
• Industrial Microbiology MeSH
• Sucrose metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Transcription initiation is the key step in gene expression in bacteria, and it is therefore studied for both theoretical and practical reasons. Promoters, the traffic lights of transcription initiation, are used as construction elements in biotechnological efforts to coordinate 'green waves' in the metabolic pathways leading to the desired metabolites. Detailed analyses of Corynebacterium glutamicum promoters have already provided large amounts of data on their structures, regulatory mechanisms and practical capabilities in metabolic engineering. In this minireview the main aspects of promoter studies, the methods developed for their analysis and their practical use in C. glutamicum are discussed. These include definitions of the consensus sequences of the distinct promoter classes, promoter localization and characterization, activity measurements, the functions of transcriptional regulators and examples of practical uses of constitutive, inducible and modified promoters in biotechnology. The implications of the introduction of novel techniques, such as in vitro transcription and RNA sequencing, to C. glutamicum promoter studies are outlined.

• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Biotechnology methods   MeSH
• Corynebacterium glutamicum genetics   metabolism   MeSH
• Transcription, Genetic MeSH
• Consensus Sequence MeSH
• Metabolic Networks and Pathways MeSH
• Molecular Sequence Data MeSH
• Promoter Regions, Genetic genetics   MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Base Sequence MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH