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Physiological roles of sigma factor SigD in Corynebacterium glutamicum.

ABSTRACT: 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.

SUBMITTER: Taniguchi H

PROVIDER: S-EPMC5508688 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Physiological roles of sigma factor SigD in Corynebacterium glutamicum.

Taniguchi Hironori H Busche Tobias T Patschkowski Thomas T Niehaus Karsten K Pátek Miroslav M Kalinowski Jörn J Wendisch Volker F VF

BMC microbiology 20170712 1

<h4>Background</h4>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.<h4>Results</h4>In this work, pleiotropic effects of sigD overexpression at the level of phenotype, transcripts, proteins and ...[more]

PMID: 28701150

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

Project description:BackgroundThe expression of genes in Corynebacterium glutamicum, a Gram-positive non-pathogenic bacterium used mainly for the industrial production of amino acids, is regulated by seven different sigma factors of RNA polymerase, including the stress-responsive ECF-sigma factor SigH. The sigH gene is located in a gene cluster together with the rshA gene, putatively encoding an anti-sigma factor. The aim of this study was to analyze the transcriptional regulation of the sigH and rshA gene cluster and the effects of RshA on the SigH regulon, in order to refine the model describing the role of SigH and RshA during stress response.ResultsTranscription analyses revealed that the sigH gene and rshA gene are cotranscribed from four sigH housekeeping promoters in C. glutamicum. In addition, a SigH-controlled rshA promoter was found to only drive the transcription of the rshA gene. To test the role of the putative anti-sigma factor gene rshA under normal growth conditions, a C. glutamicum rshA deletion strain was constructed and used for genome-wide transcription profiling with DNA microarrays. In total, 83 genes organized in 61 putative transcriptional units, including those previously detected using sigH mutant strains, exhibited increased transcript levels in the rshA deletion mutant compared to its parental strain. The genes encoding proteins related to disulphide stress response, heat stress proteins, components of the SOS-response to DNA damage and proteasome components were the most markedly upregulated gene groups. Altogether six SigH-dependent promoters upstream of the identified genes were determined by primer extension and a refined consensus promoter consisting of 45 original promoter sequences was constructed.ConclusionsThe rshA gene codes for an anti-sigma factor controlling the function of the stress-responsive sigma factor SigH in C. glutamicum. Transcription of rshA from a SigH-dependent promoter may serve to quickly shutdown the SigH-dependent stress response after the cells have overcome the stress condition. Here we propose a model of the regulation of oxidative and heat stress response including redox homeostasis by SigH, RshA and the thioredoxin system.

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Physiological roles of sigma factor SigD in Corynebacterium glutamicum.

Publications

Physiological roles of sigma factor SigD in Corynebacterium glutamicum.

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