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Start Over Author "Václav Štěpánek" Journal frontiers in microbiology
4 results on '"Václav Štěpánek"'

1. Overlapping SigH and SigE sigma factor regulons in Corynebacterium glutamicum

Author

Tobias Busche, Hana Dostálová, Lenka Rucká, Jiří Holátko, Ivan Barvík, Václav Štěpánek, Miroslav Pátek, and Jörn Kalinowski

Subjects
Corynebacterium, sigma factor, regulon, promoter, stress, consensus sequence, Microbiology, QR1-502
Abstract

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.

Published
2023
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2. Overlap of Promoter Recognition Specificity of Stress Response Sigma Factors SigD and SigH in Corynebacterium glutamicum ATCC 13032

Author

Hana Dostálová, Tobias Busche, Jiří Holátko, Lenka Rucká, Václav Štěpánek, Ivan Barvík, Jan Nešvera, Jörn Kalinowski, and Miroslav Pátek

Subjects
Corynebacterium glutamicum, stress response, RNA-seq, in vitro transcription, sigma factor, promoter, Microbiology, QR1-502
Abstract

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.

Published
2019
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3. Overlap of Promoter Recognition Specificity of Stress Response Sigma Factors SigD and SigH in Corynebacterium glutamicum ATCC 13032

Author

Jan Nešvera, Hana Dostálová, Ivan Barvík, Tobias Busche, Miroslav Pátek, Jiří Holátko, Jörn Kalinowski, Lenka Rucká, and Václav Štěpánek

Subjects
Microbiology (medical), lcsh:QR1-502, Microbiology, lcsh:Microbiology, Corynebacterium glutamicum, 03 medical and health sciences, chemistry.chemical_compound, Transcription (biology), Sigma factor, RNA polymerase, Consensus sequence, Gene, 030304 developmental biology, Genetics, 0303 health sciences, promoter, 030306 microbiology, fungi, Promoter, stress response, Regulon, chemistry, bacteria, sigma factor, RNA-seq, in vitro transcription
Abstract

Corynebacterium glutamicum ATCC 13032 harbors five sigma subunits of RNA polymerase belonging to Group IV, also called extracytoplasmic function (ECF) sigma factors. These factors sigma(C), sigma(D), sigma(E), sigma(H), and sigma(M) are mostly involved in stress responses. The role of sigma(D) consists in the control of cell wall integrity. The sigma(D) 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 sigma(D)-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 sigma(D)-dependent. The key sequence elements of all these promoters were found to be identical or closely similar to the motifs -35 GTAAC(A)/G and -10 GAT. Surprisingly, nearly all of these sigma(D)-dependent promoters were also active to a much lower extent with sigma(H) in vivo and one (Pcg0607) also in vitro, although the known highly conserved consensus sequence of the sigma(H)-dependent promoters is different (-35 GGAA(T)/C and -10 GTT). In addition to the activity of sigma(H) at the sigma(D)-controlled promoters, we discovered separated or overlapping sigma(A) - or sigma(B)-regulated or sigma(H)-regulated promoters within the upstream region of 8 genes of the sigma(D)-regulon. We found that phenol in the cultivation medium acts as a stress factor inducing expression of some sigma(D)-dependent genes. Computer modeling revealed that sigma(H) binds to the promoter DNA in a similar manner as sigma(D) to the analogous promoter elements. The homology models together with mutational analysis showed that the key amino acids, Ala 60 in sigma(D) and Lys 53 in sigma(H), 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 sigma(D)-controlled genes also belong to the sigma(H)-regulon and are also transcribed from the overlapping or closely located housekeeping (sigma(A)-regulated) and/or general stress (sigma(B)-regulated) promoters.

Published
2019
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4. Overlap of Promoter Recognition Specificity of Stress Response Sigma Factors SigD and SigH in

Author

Hana, Dostálová, Tobias, Busche, Jiří, Holátko, Lenka, Rucká, Václav, Štěpánek, Ivan, Barvík, Jan, Nešvera, Jörn, Kalinowski, and Miroslav, Pátek

Subjects
Corynebacterium glutamicum, promoter, sigma factor, stress response, RNA-seq, Microbiology, in vitro transcription, Original Research
Abstract

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 σH in 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.

Published
2018

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