Assignment of sigma factors of RNA polymerase to promoters in Corynebacterium glutamicum

Dostalova H, Holatko J, Busche T, et al. Assignment of sigma factors of RNA polymerase to promoters in Corynebacterium glutamicum. AMB EXPRESS. 2017;7(1): 133.Corynebacterium glutamicum is an important industrial producer of various amino acids and other metabolites. The C. glutamicum genome encodes seven sigma subunits (factors) of RNA polymerase: the primary sigma factor SigA (sA), the primary-like sigma(B) and five alternative sigma factors (sigma(C), sigma(D), sigma(E), sigma(H) and sigma(M)). We have developed in vitro and in vivo methods to assign particular sigma factors to individual promoters of different classes. In vitro transcription assays and measurements of promoter activity using the overexpression of a single sigma factor gene and the transcriptional fusion of the promoter to the gfpuv reporter gene enabled us to reliably define the sigma factor dependency of promoters. To document the strengths of these methods, we tested examples of respective promoters for each C. glutamicum sigma factor. Promoters of the rshA (anti-sigma for sH) and trxB1 (thioredoxin) genes were found to be sigma(H)-dependent, whereas the promoter of the sigB gene (sigma factor sigma(B)) was sigma(E)-and sigma(H)-dependent. It was confirmed that the promoter of the cg2556 gene (iron-regulated membrane protein) is sigma(C)-dependent as suggested recently by other authors. The promoter of cmt1 (trehalose corynemycolyl transferase) was found to be clearly sigma(D)-dependent. No sigma(M)-dependent promoter was identified. The typical housekeeping promoter P2sigA (sigma factor sigma(A)) was proven to be sigma(A)-dependent but also recognized by sigma(B). Similarly, the promoter of fba (fructose-1,6-bisphosphate aldolase) was confirmed to be sigma(B)-dependent but also functional with sigma(A). The study provided demonstrations of the broad applicability of the developed methods and produced original data on the analyzed promoters

Biotransformation of nitriles to amides using soluble and immobilized nitrile hydratase from Rhodococcus erythropolis A4

A semi-purified nitrile hydratase from Rhodococcus erythropolis A4 was applied to biotransformations of 3-oxonitriles 1a–4a, 3-hydroxy-2-methylenenitriles 5a–7a, 4-hydroxy-2-methylenenitriles 8a–9a, 3-hydroxynitriles 10a–12a and 3-acyloxynitrile 13a into amides 1b–13b. Cross-linked enzyme aggregates (CLEAs) with nitrile hydratase and amidase activities (88% and 77% of the initial activities, respectively) were prepared from cell-free extract of this microorganism and used for nitrile hydration in presence of ammonium sulfate, which selectively inhibited amidase activity. The genes nha1 and nha2 coding for α and β subunits of nitrile hydratase were cloned and sequenced

Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum

Busche T, Silar R, Picmanova M, Patek M, Kalinowski J. Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum. BMC Genomics. 2012;13(1): 445.Background The 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. Results Transcription 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. Conclusions The 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

Overlapping SigH and SigE sigma factor regulons in Corynebacterium glutamicum

Busche T, Dostalova H, Rucka L, et al. Overlapping SigH and SigE sigma factor regulons in Corynebacterium glutamicum. Frontiers in Microbiology . 2022;13: 1059649.The sigma H (sigmaEta) and sigma E (sigmaE) 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 sigmaEta regulon that are involved in heat and oxidative stress response have already been defined, whereas the genes of the sigmaE 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 DeltacseE with a deletion in the anti-sigmaEpsilon gene, differential gene expression was analyzed by RNA sequencing. We found 296 upregulated and 398 downregulated genes in C. glutamicum DeltacseE compared to C. glutamicum RES167. To confirm the functional link between sigmaEpsilon 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+sigmaEta and RNAP+sigmaEpsilon, which were previously shown to recognize similar promoters, proved that the sigmaEta and sigmaE regulons significantly overlap. The sigmaE-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 sigmaEta and sigmaEpsilon revealed that there are two groups of promoters: those which are exclusively sigmaEta-specific, and the other group of promoters, which are sigmaEta/sigmaE-dependent. No exclusively sigmaE-dependent promoter was detected. We defined the consensus sequences of exclusively sigmaEta-regulated promotors to be -35 GGAAt and-10 GTT and sigmaEta/sigmaE-regulated promoters to be -35 GGAAC and-10 cGTT. Fifteen genes were found to belong to the sigmaEta/sigmaEpsilon regulon. Homology modeling showed that there is a specific interaction between Met170 in sigmaEta and the nucleotides -31 and-30 within the non-coding strand (AT or CT) of the sigmaEta-dependent promoters. In sigmaE, Arg185 was found to interact with the nucleotides GA at the same positions in the sigmaE-dependent promoters. Copyright © 2023 Busche, Dostalova, Rucka, Holatko, Barvik, Stepanek, Patek and Kalinowski

Sigma regulatory network in Rhodococcus erythropolis CCM2595

Stepanek V, Dostalova H, Busche T, et al. Sigma regulatory network in Rhodococcus erythropolis CCM2595. FEMS Microbiology Letters. 2022;369(1): fnac014.The aim of this investigation was to discover the promoters that drive expression of the sig genes encoding sigma factors of RNA polymerase in Rhodococcus erythropolis CCM2595 and classify these promoters according to the sigma factors which control their activity. To analyze the regulation of major sigma factors, which control large regulons that also contain genes expressed under exponential growth and non-stressed conditions, we used the R. erythropolis CCM2595 culture, which grew rapidly in minimal medium. The transcriptional start sites (TSSs) of the genes sigA, sigB, sigD, sigE, sigG, sigH, sigJ, and sigK were detected by primary 5'-end-specific RNA sequencing. The promoters localized upstream of the detected TSSs were defined by their -35 and -10 elements, which were identical or closely similar to these sequences in the related species Corynebacterium glutamicum and Mycobacterium tuberculosis. Regulation of the promoter activities by different sigma factors was demonstrated by two independent techniques (in vivo and in vitro). All analyzed sig genes encoding the sigma factors with extracytoplasmic function (ECF) were found to be also driven from additional housekeeping promoters. Based on the classification of the sig gene promoters, a model of the basic sigma transcriptional regulatory network in R. erythropolis was designed. © The Author(s) 2022. Published by Oxford University Press on behalf of FEMS

Biocontrol of Soft Rot: Confocal Microscopy Highlights Virulent Pectobacterial Communication and Its Jamming by Rhodococcal Quorum-Quenching

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