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.

Centrum für Biotechnologie Universität Bielefeld Bielefeld Germany

Institute of Microbiology of the CAS v v i Prague Czechia

Institute of Physics Faculty of Mathematics and Physics Charles University Prague Czechia

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Albersmeier A., Pfeifer-Sancar K., Rückert C., Kalinowski J. (2017). Genome-wide determination of transcription start sites reveals new insights into promoter structures in the actinomycete Corynebacterium glutamicum. J. Biotechnol. 257 99–109. 10.1016/j.jbiotec.2017.04.008 PubMed DOI

Ao W., Gaudet J., Kent W. J., Muttumu S., Mango S. E. (2004). Environmentally induced foregut remodeling by PHA-4/FoxA and DAF-12/NHR. Science 305 1743–1746. 10.1126/science.1102216 PubMed DOI

Binder S. C., Eckweiler D., Schulz S., Bielecka A., Nicolai T., Franke R., et al. (2016). Functional modules of sigma factor regulons guarantee adaptability and evolvability. Sci. Rep. 6:22212. 10.1038/srep22212 PubMed DOI PMC

Busche T., Šilar R., Pičmanová M., Pátek M., Kalinowski J. (2012). 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 13:445. 10.1186/1471-2164-13-445 PubMed DOI PMC

Calamita H., Ko C., Tyagi S., Yoshimatsu T., Morrison N. E., Bishai W. R. (2005). The Mycobacterium tuberculosis SigD sigma factor controls the expression of ribosome-associated gene products in stationary phase and is required for full virulence. Cell. Microbiol. 7 233–244. 10.1111/j.1462-5822.2004.00454.x PubMed DOI

Campagne S., Marsh M. E., Capitani G., Vorholt J. A., Allain F. H. (2014). Structural basis for -10 promoter element melting by environmentally induced sigma factors. Nat. Struct. Mol. Biol. 21 269–276. 10.1038/nsmb.2777 PubMed DOI

Chaturongakul S., Raengpradub S., Palmer M. E., Bergholz T. M., Orsi R. H., Hu Y., et al. (2011). Transcriptomic and phenotypic analyses identify coregulated, overlapping regulons among PrfA, CtsR, HrcA, and the alternative sigma factors σB, σC, σH, and σL in Listeria monocytogenes. Appl. Environ. Microbiol. 77 187–200. 10.1128/AEM.00952-10 PubMed DOI PMC

Chen C., Pan J., Yang X., Guo C., Ding W., Si M., et al. (2016). Global transcriptomic analysis of the response of Corynebacterium glutamicum to vanillin. PLoS One 11:e0164955. 10.1371/journal.pone.0164955 PubMed DOI PMC

Chen C., Pan J., Yang X., Xiao H., Zhang Y., Si M., et al. (2017). Global transcriptomic analysis of the response of Corynebacterium glutamicum to ferulic acid. Arch. Microbiol. 199 325–334. 10.1007/s00203-016-1306-5 PubMed DOI

Chen C., Zhang Y., Xu L., Zhu K., Feng Y., Pan J., et al. (2018). Transcriptional control of the phenol hydroxylase gene phe of Corynebacterium glutamicum by the AraC-type regulator PheR. Microbiol. Res. 209 14–20. 10.1016/j.micres.2018.02.001 PubMed DOI

Cho B. K., Kim D., Knight E. M., Zengler K., Palsson B. O. (2014). Genome-scale reconstruction of the sigma factor network in Escherichia coli: topology and functional states. BMC Biol. 12:4. 10.1186/1741-7007-12-4 PubMed DOI PMC

Crooks G. E., Hon G., Chandonia J. M., Brenner S. E. (2004). WebLogo: a sequence logo generator. Genome Res. 14 1188–1190. 10.1101/gr.849004 PubMed DOI PMC

Dainese E., Rodrigue S., Delogu G., Provvedi R., Laflamme L., Brzezinski R., et al. (2006). Posttranslational regulation of Mycobacterium tuberculosis extracytoplasmic-function sigma factor sigma L and roles in virulence and in global regulation of gene expression. Infect. Immun. 74 2457–2461. 10.1128/IAI.74.4.2457-2461.2006 PubMed DOI PMC

Daly M. J., Gaidamakova E. K., Matrosova V. Y., Vasilenko A., Zhai M., Leapman R. D., et al. (2007). Protein oxidation implicated as the primary determinant of bacterial radioresistance. PLoS Biol. 5:e92. 10.1371/journal.pbio.0050092 PubMed DOI PMC

Denyer S. P. (1995). Mechanisms of action of antibacterial biocides. Int. Biodeterior. Biodegr. 36 221–225. 10.1016/0964-8305(96)00015-7 DOI

Dostálová H., Holátko J., Busche T., Rucká L., Rapoport A., Halada P., et al. (2017). Assignment of sigma factors of RNA polymerase to promoters in Corynebacterium glutamicum. AMB Express 7:133. 10.1186/s13568-017-0436-8 PubMed DOI PMC

Ehira S., Teramoto H., Inui M., Yukawa H. (2009). Regulation of Corynebacterium glutamicum heat shock response by the extracytoplasmic-function sigma factor SigH and transcriptional regulators HspR and HrcA. J. Bacteriol. 191 2964–2972. 10.1128/JB.00112-09 PubMed DOI PMC

Engels S., Schweitzer J. E., Ludwig C., Bott M., Schaffer S. (2004). Clpc and clpP1P2 gene expression in Corynebacterium glutamicum is controlled by a regulatory network involving the transcriptional regulators ClgR and HspR as well as the ECF sigma factor σH. Mol. Microbiol. 52 285–302. 10.1111/j.1365-2958.2003.03979.x PubMed DOI

Green M. R., Sambrook J. (2012). Molecular Cloning: A Laboratory Manual, Fourth Edn Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Gruber T. M., Gross C. A. (2003). Multiple sigma subunits and the partitioning of bacterial transcription space. Annu. Rev. Microbiol. 57 441–466. 10.1146/annurev.micro.57.030502.090913 PubMed DOI

Hanahan D. (1985). “Techniques for transformation of E. coli,” in DNA Cloning. A Practical Approach Vol. 1 ed. Glover D. M. (Oxford: IRL; ),109–135.

Hilker R., Stadermann K. B., Doppmeier D., Kalinowski J., Stoye J., Straube J., et al. (2014). ReadXplorer–visualization and analysis of mapped sequences. Bioinformatics 30 2247–2254. 10.1093/bioinformatics/btu205 PubMed DOI PMC

Hilker R., Stadermann K. B., Schwengers O., Anisiforov E., Jaenicke S., Weisshaar B., et al. (2016). ReadXplorer 2-detailed read mapping analysis and visualization from one single source. Bioinformatics 32 3702–3708. 10.1093/bioinformatics/btw541 PubMed DOI PMC

Holátko J., Šilar R., Rabatinová A., Šanderová H., Halada P., Nešvera J., et al. (2012). Construction of in vitro transcription system for Corynebacterium glutamicum and its use in the recognition of promoters of different classes. Appl. Microbiol. Biotechnol. 96 521–529. 10.1007/s00253-012-4336-1 PubMed DOI

Jordan S., Hutchings M. I., Mascher T. (2008). Cell envelope stress response in Gram-positive bacteria. FEMS Microbiol. Rev. 32 107–146. 10.1111/j.1574-6976.2007.00091.x PubMed DOI

Kalinowski J., Bathe J., Bartels D., Bischoff N., Bott M., Burkovski A., et al. (2003). The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins. J. Biotechnol. 104 5–25. 10.1016/S0168-1656(03)00154-8 PubMed DOI

Keilhauer C., Eggeling L., Sahm H. (1993). Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J. Bacteriol. 175 5595–5603. 10.1128/jb.175.17.5595-5603.1993 PubMed DOI PMC

Kirchner O., Tauch A. (2003). Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum. J. Biotechnol. 104 287–299. 10.1016/S0168-1656(03)00148-2 PubMed DOI

Knoppová M., Phensaijai M., Veselý M., Zemanová M., Nešvera J., Pátek M. (2007). Plasmid vectors for testing in vivo promoter activities in Corynebacterium glutamicum and Rhodococcus erythropolis. Curr. Microbiol. 55 234–239. 10.1007/s00284-007-0106-1 PubMed DOI

Kranz A., Busche T., Vogel A., Usadel B., Kalinowski J., Bott M., et al. (2018). RNAseq analysis of alpha-proteobacterium Gluconobacter oxydans 621H. BMC Genomics 19:24. 10.1186/s12864-017-4415-x PubMed DOI PMC

Krisko A., Copic T., Gabaldon T., Lehner B., Supek F. (2014). Inferring gene function from evolutionary change in signatures of translation efficiency. Genome Biol. 15:R44. 10.1186/gb-2014-15-3-r44 PubMed DOI PMC

Lane W. J., Darst S. A. (2006). The structural basis for promoter -35 element recognition by the group IV sigma factors. PLoS Biol. 4:e269. 10.1371/journal.pbio.0040269 PubMed DOI PMC

Langmead B., Salzberg S. L. (2012). Fast gapped-read alignment with Bowtie 2. Nat. Methods 9 357–359. 10.1038/nmeth.1923 PubMed DOI PMC

Love M. I., Huber W., Anders S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15:550. 10.1186/s13059-014-0550-8 PubMed DOI PMC

Luo Y., Helmann J. D. (2009). Extracytoplasmic function sigma factors with overlapping promoter specificity regulate sublancin production in Bacillus subtilis. J. Bacteriol. 191 4951–4958. 10.1128/JB.00549-09 PubMed DOI PMC

Nakunst D., Larisch C., Hüser A. T., Tauch A., Pühler A., Kalinowski J. (2007). The extracytoplasmic function-type sigma factor SigM of Corynebacterium glutamicum ATCC 13032 is involved in transcription of disulfide stress-related genes. J. Bacteriol. 189 4696–4707. 10.1128/JB.00382-07 PubMed DOI PMC

Pátek M., Nešvera J. (2011). Sigma factors and promoters in Corynebacterium glutamicum. J. Biotechnol. 154 101–113. 10.1016/j.jbiotec.2011.01.017 PubMed DOI

Pfeifer-Sancar K., Mentz A., Rückert C., Kalinowski J. (2013). Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique. BMC Genomics 14:888. 10.1186/1471-2164-14-888 PubMed DOI PMC

Raman S., Hazra R., Dascher C. C., Husson R. N. (2004). Transcription regulation by the Mycobacterium tuberculosis alternative sigma factor SigD and its role in virulence. J. Bacteriol. 186 6605–6616. 10.1128/JB.186.19.6605-6616.2004 PubMed DOI PMC

Rezuchova B., Kormanec J. (2001). A two-plasmid system for identification of promoters recognized by RNA polymerase containing extracytoplasmic stress response σE in Escherichia coli. J. Microbiol. Methods 45 103–111. 10.1016/S0167-7012(01)00237-8 PubMed DOI

Ross W., Thompson J. F., Newlands J. T., Gourse R. L. (1990). E. coli Fis protein activates ribosomal RNA transcription in vitro and in vivo. EMBO J. 9 3733–3742. 10.1002/j.1460-2075.1990.tb07586.x PubMed DOI PMC

Salomon-Ferrer R., Case D. A., Walker R. C. (2013). An overview of the amber biomolecular simulation package. WIREs Comput. Mol. Sci. 3 198–210. 10.1002/wcms.1121 DOI

Schröder J., Tauch A. (2010). Transcriptional regulation of gene expression in Corynebacterium glutamicum: the role of global, master and local regulators in the modular and hierarchical gene regulatory network. FEMS Microbiol. Rev. 34 685–737. 10.1111/j.1574-6976.2010.00228.x PubMed DOI

Schulz S., Eckweiler D., Bielecka A., Nicolai T., Franke R., Dotsch A., et al. (2015). Elucidation of sigma factor-associated networks in Pseudomonas aeruginosa reveals a modular architecture with limited and function-specific crosstalk. PLoS Pathog. 11:e1004744. 10.1371/journal.ppat.1004744 PubMed DOI PMC

Seo J. H., Hong J. S., Kim D., Cho B. K., Huang T. W., Tsai S. F., et al. (2012). Multiple-omic data analysis of Klebsiella pneumoniae MGH 78578 reveals its transcriptional architecture and regulatory features. BMC Genomics 13:679. 10.1186/1471-2164-13-679 PubMed DOI PMC

Šilar R., Holátko J., Rucká L., Rapoport A., Dostálová H., Kadeøabková P., et al. (2016). Use of in vitro transcription system for analysis of Corynebacterium glutamicum promoters recognized by two sigma factors. Curr. Microbiol. 73 401–408. 10.1007/s00284-016-1077-x PubMed DOI

Taniguchi H., Busche T., Patschkowski T., Niehaus K., Pátek M., Kalinowski J., et al. (2017). Physiological roles of sigma factor SigD in Corynebacterium glutamicum. BMC Microbiol. 17:158. 10.1186/s12866-017-1067-6 PubMed DOI PMC

Toyoda K., Inui M. (2015). Regulons of global transcription factors in Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 100 45–60. 10.1007/s00253-015-7074-3 PubMed DOI

Toyoda K., Inui M. (2016). The extracytoplasmic function sigma factor σC regulates expression of a branched quinol oxidation pathway in Corynebacterium glutamicum. Mol. Microbiol. 100 486–509. 10.1111/mmi.13330 PubMed DOI

Toyoda K., Inui M. (2018). Extracytoplasmic function sigma factor σD confers resistance to environmental stress by enhancing mycolate synthesis and modifying peptidoglycan structures in Corynebacterium glutamicum. Mol. Microbiol. 107 312–329. 10.1111/mmi.13883 PubMed DOI

van der Rest M. E., Lange C., Molenaar D. (1999). A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl. Microbiol. Biotechnol. 52 541–545. 10.1007/s002530051557 PubMed DOI

Waterhouse A., Bertoni M., Bienert S., Studer G., Tauriello G., Gumienny R., et al. (2018). SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46 W296–W303. 10.1093/nar/gky427 PubMed DOI PMC

Wittchen M., Busche T., Gaspar A. H., Lee J. H., Ton-That H., Kalinowski J. (2018). Transcriptome sequencing of the human pathogen Corynebacterium diphtheriae NCTC 13129 provides detailed insights into its transcriptional landscape and into DtxR mediated transcriptional regulation. BMC Genomics 19:82. 10.1186/s12864-018-4481-8 PubMed DOI PMC

Promoter recognition specificity of Corynebacterium glutamicum stress response sigma factors σD and σH deciphered using computer modeling and point mutagenesis

Overlapping SigH and SigE sigma factor regulons in Corynebacterium glutamicum

Identification of Rhodococcus erythropolis Promoters Controlled by Alternative Sigma Factors Using In Vivo and In Vitro Systems and Heterologous RNA Polymerase