HrdB in streptomycetes is a principal sigma factor whose deletion is lethal. This is also the reason why its regulon has not been investigated so far. To overcome experimental obstacles, for investigating the HrdB regulon, we constructed a strain whose HrdB protein was tagged by an HA epitope. ChIP-seq experiment, done in 3 repeats, identified 2137 protein-coding genes organized in 337 operons, 75 small RNAs, 62 tRNAs, 6 rRNAs and 3 miscellaneous RNAs. Subsequent kinetic modeling of regulation of protein-coding genes with HrdB alone and with a complex of HrdB and a transcriptional cofactor RbpA, using gene expression time series, identified 1694 genes that were under their direct control. When using the HrdB-RbpA complex in the model, an increase of the model fidelity was found for 322 genes. Functional analysis revealed that HrdB controls the majority of gene groups essential for the primary metabolism and the vegetative growth. Particularly, almost all ribosomal protein-coding genes were found in the HrdB regulon. Analysis of promoter binding sites revealed binding motif at the -10 region and suggested the possible role of mono- or di-nucleotides upstream of the -10 element.

1st Faculty of Medicine Institute of Immunology and Microbiology Charles University 12800 Prague Czechia

Chemistry Department Faculty of Science J E Purkinje University 40096 Ústí nad Labem Czechia

Institute of Microbiology Academy of Sciences of the Czech Republic 14220 Prague Czechia

See more in PubMed

Paget M.S., Helmann J.D.. The sigma70 family of sigma factors. Genome Biol. 2003; 4:203. PubMed PMC

Gruber T.M., Gross C.A.. Multiple sigma subunits and the partitioning of bacterial transcription space. Annu. Rev. Microbiol. 2003; 57:441–466. PubMed

Fraser C.M., Gocayne J.D., White O., Adams M.D., Clayton R.A., Fleischmann R.D., Bult C.J., Kerlavage A.R., Sutton G., Kelley J.M. et al. . The minimal gene complement of Mycoplasma genitalium. Science. 1995; 270:397–403. PubMed

Bentley S.D., Chater K.F., Cerdeno-Tarraga A.M., Challis G.L., Thomson N.R., James K.D., Harris D.E., Quail M.A., Kieser H., Harper D. et al. . Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature. 2002; 417:141–147. PubMed

Hahn M.Y., Bae J.B., Park J.H., Roe J.H.. Isolation and characterization of Streptomyces coelicolor RNA polymerase, its sigma, and antisigma factors. Methods Enzymol. 2003; 370:73–82. PubMed

Kang J.G., Hahn M.Y., Ishihama A., Roe J.H.. Identification of sigma factors for growth phase-related promoter selectivity of RNA polymerases from Streptomyces coelicolor A3(2). Nucleic Acids Res. 1997; 25:2566–2573. PubMed PMC

Buttner M.J. RNA polymerase heterogeneity in Streptomyces coelicolor A3(2). Mol. Microbiol. 1989; 3:1653–1659. PubMed

Shiina T., Tanaka K., Takahashi H.. Sequence of hrdB, an essential gene encoding sigma-like transcription factor of Streptomyces coelicolor A3(2): homology to principal sigma factors. Gene. 1991; 107:145–148. PubMed

Tanaka K., Shiina T., Takahashi H.. Multiple principal sigma factor homologs in eubacteria: identification of the “rpoD box”. Science. 1988; 242:1040–1042. PubMed

Tanaka K., Shiina T., Takahashi H.. Nucleotide sequence of genes hrdA, hrdC, and hrdD from Streptomyces coelicolor A3(2) having similarity to rpoD genes. Mol. Gen. Genet. 1991; 229:334–340. PubMed

Tabib-Salazar A., Liu B., Doughty P., Lewis R.A., Ghosh S., Parsy M.L., Simpson P.J., O’Dwyer K., Matthews S.J., Paget M.S.. The actinobacterial transcription factor RbpA binds to the principal sigma subunit of RNA polymerase. Nucleic Acids Res. 2013; 41:5679–5691. PubMed PMC

Hu Y., Morichaud Z., Chen S., Leonetti J.P., Brodolin K.. Mycobacterium tuberculosis RbpA protein is a new type of transcriptional activator that stabilizes the sigma A-containing RNA polymerase holoenzyme. Nucleic Acids Res. 2012; 40:6547–6557. PubMed PMC

Hu Y., Morichaud Z., Perumal A.S., Roquet-Baneres F., Brodolin K.. Mycobacterium RbpA cooperates with the stress-response sigmaB subunit of RNA polymerase in promoter DNA unwinding. Nucleic Acids Res. 2014; 42:10399–10408. PubMed PMC

Forti F., Mauri V., Dehò G., Ghisotti D.. Isolation of conditional expression mutants in Mycobacterium tuberculosis by transposon mutagenesis. Tuberculosis. 2011; 91:569–578. PubMed

Newell K.V., Thomas D.P., Brekasis D., Paget M.S.. The RNA polymerase-binding protein RbpA confers basal levels of rifampicin resistance on Streptomyces coelicolor. Mol. Microbiol. 2006; 60:687–696. PubMed

Dey A., Verma A.K., Chatterji D.. Molecular insights into the mechanism of phenotypic tolerance to rifampicin conferred on mycobacterial RNA polymerase by MsRbpA. Microbiology. 2011; 157:2056–2071. PubMed

Dey A., Verma A.K., Chatterji D.. Role of an RNA polymerase interacting protein, MsRbpA, from Mycobacterium smegmatis in phenotypic tolerance to rifampicin. Microbiology. 2010; 156:873–883. PubMed

Paget M.S., Molle V., Cohen G., Aharonowitz Y., Buttner M.J.. Defining the disulphide stress response in Streptomyces coelicolor A3(2): identification of the sigmaR regulon. Mol. Microbiol. 2001; 42:1007–1020. PubMed

Hu Y., Wang Z., Feng L., Chen Z., Mao C., Zhu Y., Chen S.. sigma(E) -dependent activation of RbpA controls transcription of the furA-katG operon in response to oxidative stress in mycobacteria. Mol. Microbiol. 2016; 102:107–120. PubMed

Datsenko K.A., Wanner B.L.. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 2000; 97:6640–6645. PubMed PMC

MacNeil D.J., Gewain K.M., Ruby C.L., Dezeny G., Gibbons P.H., MacNeil T.. Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene. 1992; 111:61–68. PubMed

Kieser T., Bibb M.J., Buttner M.J., Chater K.F., Hopwood D.A.. Practical Streptomyces Genetics. 2000; Norwich: John Innes Foundation.

Nieselt K., Battke F., Herbig A., Bruheim P., Wentzel A., Jakobsen O.M., Sletta H., Alam M.T., Merlo M.E., Moore J. et al. . The dynamic architecture of the metabolic switch in Streptomyces coelicolor. BMC Genomics. 2010; 11:10. PubMed PMC

Gust B., Chandra G., Jakimowicz D., Yuqing T., Bruton C.J., Chater K.F.. Lambda red-mediated genetic manipulation of antibiotic-producing Streptomyces. Adv. Appl. Microbiol. 2004; 54:107–128. PubMed

Flett F., Mersinias V., Smith C.P.. High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes. FEMS Microbiol. Lett. 1997; 155:223–229. PubMed

Spencer V.A., Sun J.M., Li L., Davie J.R.. Chromatin immunoprecipitation: a tool for studying histone acetylation and transcription factor binding. Methods. 2003; 31:67–75. PubMed

Castro-Melchor M., Charaniya S., Karypis G., Takano E., Hu W.S.. Genome-wide inference of regulatory networks in Streptomyces coelicolor. BMC Genomics. 2010; 11:578. PubMed PMC

Modrak M., Vohradsky J.. Genexpi: a toolset for identifying regulons and validating gene regulatory networks using time-course expression data. BMC Bioinformatics. 2018; 19:137. PubMed PMC

Jeong Y., Kim J.N., Kim M.W., Bucca G., Cho S., Yoon Y.J., Kim B.G., Roe J.H., Kim S.C., Smith C.P. et al. . The dynamic transcriptional and translational landscape of the model antibiotic producer Streptomyces coelicolor A3(2). Nat. Commun. 2016; 7:11605. PubMed PMC

Zhu Y., Mao C., Ge X., Wang Z., Lu P., Zhang Y., Chen S., Hu Y.. Characterization of a minimal type of promoter containing the −10 element and a guanine at the −14 or −13 Position in Mycobacteria. J. Bacteriol. 2017; 199:doi:10.1128/JB.00385-17. PubMed PMC

Lundgren J. SplineFit, fit a spline to noisy data. 2010; http://www.mathworks.com/matlabcentral/fileexchange/13812-splinefit.

Moody M.J., Young R.A., Jones S.E., Elliot M.A.. Comparative analysis of non-coding RNAs in the antibiotic-producing Streptomyces bacteria. BMC Genomics. 2013; 14:558. PubMed PMC

Panek J., Bobek J., Mikulik K., Basler M., Vohradsky J.. Biocomputational prediction of small non-coding RNAs in Streptomyces. BMC Genomics. 2008; 9:217. PubMed PMC

Cortes T., Schubert O.T., Rose G., Arnvig K.B., Comas I., Aebersold R., Young D.B.. Genome-wide mapping of transcriptional start sites defines an extensive leaderless transcriptome in Mycobacterium tuberculosis. Cell Rep. 2013; 5:1121–1131. PubMed PMC

Hubin E.A., Fay A., Xu C., Bean J.M., Saecker R.M., Glickman M.S., Darst S.A., Campbell E.A.. Structure and function of the mycobacterial transcription initiation complex with the essential regulator RbpA. Elife. 2017; 6:e22520. PubMed PMC

Buttner M.J., Chater K.F., Bibb M.J.. Cloning, disruption, and transcriptional analysis of three RNA polymerase sigma factor genes of Streptomyces coelicolor A3(2). J. Bacteriol. 1990; 172:3367–3378. PubMed PMC

Petrone B.L., Stringer A.M., Wade J.T.. Identification of HilD-regulated genes in Salmonella enterica serovar Typhimurium. J. Bacteriol. 2014; 196:1094–1101. PubMed PMC

Qian Z., Trostel A., Lewis D.E., Lee S.J., He X., Stringer A.M., Wade J.T., Schneider T.D., Durfee T., Adhya S.. Genome-wide transcriptional regulation and chromosome structural arrangement by GalR in E. coli. Front. Mol. Biosci. 2016; 3:74. PubMed PMC

Stringer A.M., Currenti S., Bonocora R.P., Baranowski C., Petrone B.L., Palumbo M.J., Reilly A.A., Zhang Z., Erill I., Wade J.T.. Genome-scale analyses of Escherichia coli and Salmonella enterica AraC reveal noncanonical targets and an expanded core regulon. J. Bacteriol. 2014; 196:660–671. PubMed PMC

Gust B., Challis G.L., Fowler K., Kieser T., Chater K.F.. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc. Natl. Acad. Sci. U.S.A. 2003; 100:1541–1546. PubMed PMC

Kim J.N., Yi J.S., Lee B.R., Kim E.J., Kim M.W., Song Y., Cho B.K., Kim B.G.. A versatile PCR-based tandem epitope tagging system for Streptomyces coelicolor genome. Biochem. Biophys. Res. Commun. 2012; 424:22–27. PubMed

Ren B., Robert F., Wyrick J.J., Aparicio O., Jennings E.G., Simon I., Zeitlinger J., Schreiber J., Hannett N., Kanin E. et al. . Genome-wide location and function of DNA binding proteins. Science. 2000; 290:2306–2309. PubMed

Wilbanks E.G., Larsen D.J., Neches R.Y., Yao A.I., Wu C.Y., Kjolby R.A., Facciotti M.T.. A workflow for genome-wide mapping of archaeal transcription factors with ChIP-seq. Nucleic Acids Res. 2012; 40:e74. PubMed PMC

Ring B.Z., Yarnell W.S., Roberts J.W.. Function of E. coli RNA polymerase sigma factor sigma 70 in promoter-proximal pausing. Cell. 1996; 86:485–493. PubMed

Petushkov I., Esyunina D., Kulbachinskiy A.. Possible roles of sigma-dependent RNA polymerase pausing in transcription regulation. RNA Biol. 2017; 14:1678–1682. PubMed PMC

Delic I., Robbins P., Westpheling J.. Direct repeat sequences are implicated in the regulation of two Streptomyces chitinase promoters that are subject to carbon catabolite control. Proc. Natl. Acad. Sci. U.S.A. 1992; 89:1885–1889. PubMed PMC

Schumacher M.A., den Hengst C.D., Bush M.J., Le T.B.K., Tran N.T., Chandra G., Zeng W., Travis B., Brennan R.G., Buttner M.J.. The MerR-like protein BldC binds DNA direct repeats as cooperative multimers to regulate Streptomyces development. Nat. Commun. 2018; 9:1139. PubMed PMC

Pope M.K., Green B., Westpheling J.. The bldB gene encodes a small protein required for morphogenesis, antibiotic production, and catabolite control in Streptomyces coelicolor. J. Bacteriol. 1998; 180:1556–1562. PubMed PMC

den Hengst C.D., Tran N.T., Bibb M.J., Chandra G., Leskiw B.K., Buttner M.J.. Genes essential for morphological development and antibiotic production in Streptomyces coelicolor are targets of BldD during vegetative growth. Mol. Microbiol. 2010; 78:361–379. PubMed

Chater K.F., Bruton C.J., Plaskitt K.A., Buttner M.J., Mendez C., Helmann J.D.. The developmental fate of S. coelicolor hyphae depends upon a gene product homologous with the motility sigma factor of B. subtilis. Cell. 1989; 59:133–143. PubMed

Vujaklija D., Ueda K., Hong S.K., Beppu T., Horinouchi S.. Identification of an A-factor-dependent promoter in the streptomycin biosynthetic gene cluster of Streptomyces griseus. Mol. Gen. Genet. 1991; 229:119–128. PubMed

Vujaklija D., Horinouchi S., Beppu T.. Detection of an A-factor-responsive protein that binds to the upstream activation sequence of strR, a regulatory gene for streptomycin biosynthesis in Streptomyces griseus. J. Bacteriol. 1993; 175:2652–2661. PubMed PMC

Nguyen K.T., Tenor J., Stettler H., Nguyen L.T., Nguyen L.D., Thompson C.J.. Colonial differentiation in Streptomyces coelicolor depends on translation of a specific codon within the adpA gene. J. Bacteriol. 2003; 185:7291–7296. PubMed PMC

Setinova D., Smidova K., Pohl P., Music I., Bobek J.. RNase III-Binding-mRNAs revealed novel complementary transcripts in Streptomyces. Front. Microbiol. 2017; 8:2693. PubMed PMC

Rigali S., Titgemeyer F., Barends S., Mulder S., Thomae A.W., Hopwood D.A., van Wezel G.P.. Feast or famine: the global regulator DasR links nutrient stress to antibiotic production by Streptomyces. EMBO Rep. 2008; 9:670–675. PubMed PMC

Rigali S., Nothaft H., Noens E.E., Schlicht M., Colson S., Muller M., Joris B., Koerten H.K., Hopwood D.A., Titgemeyer F. et al. . The sugar phosphotransferase system of Streptomyces coelicolor is regulated by the GntR-family regulator DasR and links N-acetylglucosamine metabolism to the control of development. Mol. Microbiol. 2006; 61:1237–1251. PubMed

Gehring A.M., Wang S.T., Kearns D.B., Storer N.Y., Losick R.. Novel genes that influence development in Streptomyces coelicolor. J. Bacteriol. 2004; 186:3570–3577. PubMed PMC

Flardh K. Growth polarity and cell division in Streptomyces. Curr. Opin. Microbiol. 2003; 6:564–571. PubMed

McCormick J.R. Cell division is dispensable but not irrelevant in Streptomyces. Curr. Opin. Microbiol. 2009; 12:689–698. PubMed

Strohl W.R. Compilation and analysis of DNA sequences associated with apparent streptomycete promoters. Nucleic Acids Res. 1992; 20:961–974. PubMed PMC

Buttner M.J., Lewis C.G.. Construction and characterization of Streptomyces coelicolor A3(2) mutants that are multiply deficient in the nonessential hrd-encoded RNA polymerase sigma factors. J. Bacteriol. 1992; 174:5165–5167. PubMed PMC

Fujii T., Gramajo H.C., Takano E., Bibb M.J.. redD and actII-ORF4, pathway-specific regulatory genes for antibiotic production in Streptomyces coelicolor A3(2), are transcribed in vitro by an RNA polymerase holoenzyme containing sigma hrdD. J. Bacteriol. 1996; 178:3402–3405. PubMed PMC

Paget M.S., Chamberlin L., Atrih A., Foster S.J., Buttner M.J.. Evidence that the extracytoplasmic function sigma factor sigmaE is required for normal cell wall structure in Streptomyces coelicolor A3(2). J. Bacteriol. 1999; 181:204–211. PubMed PMC

Helmann J.D., Chamberlin M.J.. Structure and function of bacterial sigma factors. Annu. Rev. Biochem. 1988; 57:839–872. PubMed

Karoonuthaisiri N., Weaver D., Huang J., Cohen S.N., Kao C.M.. Regional organization of gene expression in Streptomyces coelicolor. Gene. 2005; 353:53–66. PubMed

Bobek J., Strakova E., Zikova A., Vohradsky J.. Changes in activity of metabolic and regulatory pathways during germination of S. coelicolor. BMC Genomics. 2014; 15:1173. PubMed PMC

Viollier P.H., Minas W., Dale G.E., Folcher M., Thompson C.J.. Role of acid metabolism in Streptomyces coelicolor morphological differentiation and antibiotic biosynthesis. J. Bacteriol. 2001; 183:3184–3192. PubMed PMC

Ward B. Sussman M, Liu D, Poxton I, Schwartzman J. Bacterial Energy Metabolism. Molecular Medical Microbiology. 2015; 2nd ednBoston: Academic Press; 201–233.

Brekasis D., Paget M.S.. A novel sensor of NADH/NAD+ redox poise in Streptomyces coelicolor A3(2). EMBO J. 2003; 22:4856–4865. PubMed PMC

Liu X., Cheng Y., Lyu M., Wen Y., Song Y., Chen Z., Li J.. Redox-sensing regulator Rex regulates aerobic metabolism, morphological differentiation, and avermectin production in Streptomyces avermitilis. Sci. Rep. 2017; 7:44567. PubMed PMC

van Keulen G., Jonkers H.M., Claessen D., Dijkhuizen L., Wosten H.A.. Differentiation and anaerobiosis in standing liquid cultures of Streptomyces coelicolor. J. Bacteriol. 2003; 185:1455–1458. PubMed PMC

RIP-seq reveals RNAs that interact with RNA polymerase and primary sigma factors in bacteria

σE of Streptomyces coelicolor can function both as a direct activator or repressor of transcription

Ms1 RNA Interacts With the RNA Polymerase Core in Streptomyces coelicolor and Was Identified in Majority of Actinobacteria Using a Linguistic Gene Synteny Search