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

ABSTRACT: 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.

SUBMITTER: Dostalova H

PROVIDER: S-EPMC6338062 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Dostálová Hana H Busche Tobias T Holátko Jiří J Rucká Lenka L Štěpánek Václav V Barvík Ivan I Nešvera Jan J Kalinowski Jörn J Pátek Miroslav M

Frontiers in microbiology 20190109

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 < ...[more]

PMID: 30687273

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Project description:BackgroundZinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. Bacteria therefore tightly regulate zinc metabolism. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators.ResultsThe cg2502 (zur) gene was deleted in the chromosome of C. glutamicum ATCC 13032 by an allelic exchange procedure to generate the zur-deficient mutant C. glutamicum JS2502. Whole-genome DNA microarray hybridizations and real-time RT-PCR assays comparing the gene expression in C. glutamicum JS2502 with that of the wild-type strain detected 18 genes with enhanced expression in the zur mutant. The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794). Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression. The zinc-dependent expression of the putative cg0042 and cg2911 operons was detected in vivo with a gfp reporter system. Moreover, the zinc-dependent binding of purified Zur protein to double-stranded 40-mer oligonucleotides containing candidate Zur-binding sites was demonstrated in vitro by DNA band shift assays.ConclusionWhole-genome expression profiling and DNA band shift assays demonstrated that Zur directly represses in a zinc-dependent manner the expression of nine genes organized in five transcription units. Accordingly, the Zur (Cg2502) protein is the key transcription regulator for genes involved in zinc homeostasis in C. glutamicum.

Project description:The demand for alternative sources of food proteins is increasing due to the limitations and challenges associated with conventional food production. Advances in biotechnology have enabled the production of proteins using microorganisms, thus prompting the exploration of attractive microbial hosts capable of producing functional proteins in high titers. Corynebacterium glutamicum is widely used in industry for the production of amino acids and has many advantages as a host organism for recombinant protein production. However, its performance in this area is limited by low yields of target proteins and high levels of native protein secretion. Despite representing a challenge for heterologous protein production, the C. glutamicum secretome has not been fully characterized. In this study, state-of-the-art mass spectrometry-based proteomics was used to identify and analyze the proteins secreted by C. glutamicum. Both the wild-type strain and a strain that produced and secreted a recombinant β-lactoglobulin protein were analyzed. A total of 427 proteins were identified in the culture supernatants, with 148 predicted to possess a secretion signal peptide. MS-based proteomics on the secretome enabled a comprehensive characterization and quantification (based on abundance) of the secreted proteins through label-free quantification (LFQ). The top 12 most abundant proteins accounted for almost 80% of the secretome. These are uncharacterized proteins of unknown function, resuscitation promoting factors, protein PS1, Porin B, ABC-type transporter protein and hypothetical membrane protein. The data can be leveraged for protein production by, e.g., utilizing the signal peptides of the most abundant proteins to improve secretion of heterologous proteins. In addition, secretory stress can potentially be alleviated by inactivating non-essential secreted proteins. Here we provide targets by identifying the most abundant, secreted proteins of which majority are of unknown function. The data from this study can thus provide valuable insight for researchers looking to improve protein secretion and optimize C. glutamicum as a host for secretory protein production.

Project description:BACKGROUND: Recent discoveries on bacterial transcriptomes gave evidence that small RNAs (sRNAs) have important regulatory roles in prokaryotic cells. Modern high-throughput sequencing approaches (RNA-Seq) enable the most detailed view on transcriptomes offering an unmatched comprehensiveness and single-base resolution. Whole transcriptome data obtained by RNA-Seq can be used to detect and characterize all transcript species, including small RNAs. Here, we describe an RNA-Seq approach for comprehensive detection and characterization of small RNAs from Corynebacterium glutamicum, an actinobacterium of high industrial relevance and model organism for medically important Corynebacterianeae, such as C. diphtheriae and Mycobacterium tuberculosis. RESULTS: In our RNA-Seq approach, total RNA from C. glutamicum ATCC 13032 was prepared from cultures grown in minimal medium at exponential growth or challenged by physical (heat shock, cold shock) or by chemical stresses (diamide, H2O2, NaCl) at this time point. Total RNA samples were pooled and sequencing libraries were prepared from the isolated small RNA fraction. High throughput short read sequencing and mapping yielded over 800 sRNA genes. By determining their 5'- and 3'-ends and inspection of their locations, these potential sRNA genes were classified into UTRs of mRNAs (316), cis-antisense sRNAs (543), and trans-encoded sRNAs (262). For 77 of trans-encoded sRNAs significant sequence and secondary structure conservation was found by a computational approach using a whole genome alignment with the closely related species C. efficiens YS-314 and C. diphtheriae NCTC 13129. Three selected trans-encoded sRNAs were characterized by Northern blot analysis and stress-specific transcript patterns were found. CONCLUSIONS: The study showed comparable numbers of sRNAs known from genome-wide surveys in other bacteria. In detail, our results give deep insight into the comprehensive equipment of sRNAs in C. glutamicum and provide a sound basis for further studies concerning the functions of these sRNAs.

Project description:Corynebacterium glutamicum belongs to the microbes of enormous biotechnological relevance. In particular, its strain ATCC 13032 is a widely used producer of L-amino acids at an industrial scale. Its apparent robustness also turns it into a favorable platform host for a wide range of further compounds, mainly because of emerging bio-based economies. A deep understanding of the biochemical processes in C. glutamicum is essential for a sustainable enhancement of the microbe's productivity. Computational systems biology has the potential to provide a valuable basis for driving metabolic engineering and biotechnological advances, such as increased yields of healthy producer strains based on genome-scale metabolic models (GEMs). Advanced reconstruction pipelines are now available that facilitate the reconstruction of GEMs and support their manual curation. This article presents iCGB21FR, an updated and unified GEM of C. glutamicum ATCC 13032 with high quality regarding comprehensiveness and data standards, built with the latest modeling techniques and advanced reconstruction pipelines. It comprises 1042 metabolites, 1539 reactions, and 805 genes with detailed annotations and database cross-references. The model validation took place using different media and resulted in realistic growth rate predictions under aerobic and anaerobic conditions. The new GEM produces all canonical amino acids, and its phenotypic predictions are consistent with laboratory data. The in silico model proved fruitful in adding knowledge to the metabolism of C. glutamicum: iCGB21FR still produces L-glutamate with the knock-out of the enzyme pyruvate carboxylase, despite the common belief to be relevant for the amino acid's production. We conclude that integrating high standards into the reconstruction of GEMs facilitates replicating validated knowledge, closing knowledge gaps, and making it a useful basis for metabolic engineering. The model is freely available from BioModels Database under identifier MODEL2102050001.

Dataset Information

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

Publications

Overlap of Promoter Recognition Specificity of Stress Response Sigma Factors SigD and SigH in <i>Corynebacterium glutamicum</i> ATCC 13032.

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