σ factors are considered as positive regulators of gene expression. Here we reveal the opposite, inhibitory role of these proteins. We used a combination of molecular biology methods and computational modeling to analyze the regulatory activity of the extracytoplasmic σE factor from Streptomyces coelicolor. The direct activator/repressor function of σE was then explored by experimental analysis of selected promoter regions in vivo. Additionally, the σE interactome was defined. Taken together, the results characterize σE, its regulation, regulon, and suggest its direct inhibitory function (as a repressor) in gene expression, a phenomenon that may be common also to other σ factors and organisms.

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo nam 542 2 160 00 Prague 6 Czech Republic

Laboratory of Bioinformatics Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague 4 Czech Republic

Laboratory of Genomics and Bioinformatics Institute of Molecular Genetics of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague 4 Czech Republic

Laboratory of Microbial Genetics and Gene Expression Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 142 20 Prague 4 Czech Republic

Zobrazit více v PubMed

Bentley SD, et al. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2) Nature. 2002;417:141–147. doi: 10.1038/417141a. PubMed DOI

Burton AT, DeLoughery A, Li GW, Kearns DB. Transcriptional regulation and mechanism of SigN (ZpdN), a pBS32-encoded sigma factor in Bacillus subtilis. mBio. 2019;10:e01899–19. doi: 10.1128/mBio.01899-19. PubMed DOI PMC

Nicolas P, et al. Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis. Science. 2012;335:1103–1106. doi: 10.1126/science.1206848. PubMed DOI

Smidova K, et al. DNA mapping and kinetic modeling of the HrdB regulon in Streptomyces coelicolor. Nucl. Acids Res. 2019;47:621–633. doi: 10.1093/nar/gky1018. PubMed DOI PMC

Todor H, et al. Rewiring the specificity of extracytoplasmic function sigma factors. Proc. Natl Acad. Sci. USA. 2020;117:33496–33506. doi: 10.1073/pnas.2020204117. PubMed DOI PMC

Helmann JD. The extracytoplasmic function (ECF) sigma factors. Adv. Micro. Physiol. 2002;46:47–110. doi: 10.1016/S0065-2911(02)46002-X. PubMed DOI

Lopez-Garcia MT, Yague P, Gonzalez-Quinonez N, Rioseras B, Manteca A. The SCO4117 ECF sigma factor pleiotropically controls secondary metabolism and morphogenesis in Streptomyces coelicolor. Front. Microbiol. 2018;9:312. doi: 10.3389/fmicb.2018.00312. PubMed DOI PMC

Feng WH, Mao XM, Liu ZH, Li YQ. The ECF sigma factor SigT regulates actinorhodin production in response to nitrogen stress in Streptomyces coelicolor. Appl. Microbiol. Biotechnol. 2011;92:1009–1021. doi: 10.1007/s00253-011-3619-2. PubMed DOI

Kallifidas D, Thomas D, Doughty P, Paget MS. The sigmaR regulon of Streptomyces coelicolor A32 reveals a key role in protein quality control during disulphide stress. Microbiology. 2010;156:1661–1672. doi: 10.1099/mic.0.037804-0. PubMed DOI

Paget MS, Leibovitz E, Buttner MJ. A putative two-component signal transduction system regulates sigmaE, a sigma factor required for normal cell wall integrity in Streptomyces coelicolor A3(2) Mol. Microbiol. 1999;33:97–107. doi: 10.1046/j.1365-2958.1999.01452.x. PubMed DOI

Hong HJ, Paget MS, Buttner MJ. A signal transduction system in Streptomyces coelicolor that activates the expression of a putative cell wall glycan operon in response to vancomycin and other cell wall-specific antibiotics. Mol. Microbiol. 2002;44:1199–1211. doi: 10.1046/j.1365-2958.2002.02960.x. PubMed DOI

Hutchings MI, Hong HJ, Leibovitz E, Sutcliffe IC, Buttner MJ. The sigma(E) cell envelope stress response of Streptomyces coelicolor is influenced by a novel lipoprotein, CseA. J. Bacteriol. 2006;188:7222–7229. doi: 10.1128/JB.00818-06. PubMed DOI PMC

Tran NT, et al. Defining the regulon of genes controlled by σE, a key regulator of the cell envelope stress response in Streptomyces coelicolor. Mol. Microbiol. 2019;112:461–481. doi: 10.1111/mmi.14250. PubMed DOI PMC

Nieselt K, et al. The dynamic architecture of the metabolic switch in Streptomyces coelicolor. BMC Genom. 2010;11:10. doi: 10.1186/1471-2164-11-10. PubMed DOI PMC

Paget MS, Chamberlin L, Atrih A, Foster SJ, Buttner MJ. 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. doi: 10.1128/JB.181.1.204-211.1999. PubMed DOI PMC

Jeong Y, et al. The dynamic transcriptional and translational landscape of the model antibiotic producer Streptomyces coelicolor A3(2) Nat. Commun. 2016;7:11605. doi: 10.1038/ncomms11605. PubMed DOI PMC

Lane WJ, Darst SA. The structural basis for promoter -35 element recognition by the group IV sigma factors. PLoS Biol. 2006;4:e269. doi: 10.1371/journal.pbio.0040269. PubMed DOI PMC

Sievers F, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 2011;7:539. doi: 10.1038/msb.2011.75. PubMed DOI PMC

Touzain F, et al. SIGffRid: a tool to search for sigma factor binding sites in bacterial genomes using comparative approach and biologically driven statistics. BMC Bioinformat. 2008;9:73. doi: 10.1186/1471-2105-9-73. PubMed DOI PMC

Kwon HJ, Tirumalai R, Landy A, Ellenberger T. Flexibility in DNA recombination: structure of the lambda integrase catalytic core. Science. 1997;276:126–131. doi: 10.1126/science.276.5309.126. PubMed DOI PMC

Schaerlaekens K, Mellaert Van, Lammertyn L, Geukens E, Anne N. J. The importance of the Tat-dependent protein secretion pathway in Streptomyces as revealed by phenotypic changes in tat deletion mutants and genome analysis. Microbiology. 2004;150:21–31. doi: 10.1099/mic.0.26684-0. PubMed DOI

Koebsch I, Overbeck J, Piepmeyer S, Meschke H, Schrempf H. A molecular key for building hyphae aggregates: the role of the newly identified Streptomyces protein HyaS. Microb. Biotechnol. 2009;2:343–360. doi: 10.1111/j.1751-7915.2009.00093.x. PubMed DOI PMC

Cao H, et al. Systems-level understanding of ethanol-induced stresses and adaptation in E. coli. Sci. Rep. 2017;7:44150. doi: 10.1038/srep44150. PubMed DOI PMC

Hesketh A, et al. Genome-wide dynamics of a bacterial response to antibiotics that target the cell envelope. BMC Genom. 2011;12:226. doi: 10.1186/1471-2164-12-226. PubMed DOI PMC

Noens EE, et al. SsgA-like proteins determine the fate of peptidoglycan during sporulation of Streptomyces coelicolor. Mol. Microbiol. 2005;58:929–944. doi: 10.1111/j.1365-2958.2005.04883.x. PubMed DOI

Lu Y, et al. An orphan histidine kinase, OhkA, regulates both secondary metabolism and morphological differentiation in Streptomyces coelicolor. J. Bacteriol. 2011;193:3020–3032. doi: 10.1128/JB.00017-11. PubMed DOI PMC

Jayapal KP, Lian W, Glod F, Sherman DH, Hu WS. Comparative genomic hybridizations reveal absence of large Streptomyces coelicolor genomic islands in Streptomyces lividans. BMC Genom. 2007;8:229. doi: 10.1186/1471-2164-8-229. PubMed DOI PMC

Du C, et al. System-wide analysis of the GATC-binding nucleoid-associated protein Gbn and its impact on streptomyces development. mSystems. 2022;7:e0006122. doi: 10.1128/msystems.00061-22. PubMed DOI PMC

Falke D, et al. Co-purification of nitrate reductase 1 with components of the cytochrome bcc-aa(3) oxidase supercomplex from spores of Streptomyces coelicolor A3(2) FEBS Open Bio. 2021;11:652–669. doi: 10.1002/2211-5463.13086. PubMed DOI PMC

Fischer M, Alderson J, van Keulen G, White J, Sawers RG. The obligate aerobe Streptomyces coelicolor A3(2) synthesizes three active respiratory nitrate reductases. Microbiology. 2010;156:3166–3179. doi: 10.1099/mic.0.042572-0. PubMed DOI

Brown AS, Calcott MJ, Collins VM, Owen JG, Ackerley DF. The indigoidine synthetase BpsA provides a colorimetric ATP assay that can be adapted to quantify the substrate preferences of other NRPS enzymes. Biotechnol. Lett. 2020;42:2665–2671. doi: 10.1007/s10529-020-02972-4. PubMed DOI

Salerno P, et al. One of the two genes encoding nucleoid-associated HU proteins in Streptomyces coelicolor is developmentally regulated and specifically involved in spore maturation. J. Bacteriol. 2009;191:6489–6500. doi: 10.1128/JB.00709-09. PubMed DOI PMC

Hesketh A, Deery MJ, Hong HJ. High-resolution mass spectrometry based proteomic analysis of the Response to vancomycin-induced cell wall stress in Streptomyces coelicolor A3(2) J. Proteome Res. 2015;14:2915–2928. doi: 10.1021/acs.jproteome.5b00242. PubMed DOI

Sekurova ON, et al. Targeted metabolomics and high-throughput RNA sequencing-based transcriptomics reveal massive changes in the Streptomyces venezuelae NRRL B-65442 metabolism caused by ethanol shock. Microbiol. Spectr. 2022;10:e0367222. doi: 10.1128/spectrum.03672-22. PubMed DOI PMC

Wang D, Xu P, Sun J, Yuan J, Zhao J. Effects of ethanol stress on epsilon-poly-l-lysine (epsilon-PL) biosynthesis in Streptomyces albulus X-18. Enzym. Micro. Technol. 2022;153:109907. doi: 10.1016/j.enzmictec.2021.109907. PubMed DOI

Dong G, Tian XL, Gomez ZA, Li YH. Regulated proteolysis of the alternative sigma factor SigX in Streptococcus mutans: implication in the escape from competence. BMC Microbiol. 2014;14:183. doi: 10.1186/1471-2180-14-183. PubMed DOI PMC

Chen YF, Helmann JD. The Bacillus subtilis flagellar regulatory protein sigma D: overproduction, domain analysis and DNA-binding properties. J. Mol. Biol. 1995;249:743–753. doi: 10.1006/jmbi.1995.0333. PubMed DOI

Goedhart J, Luijsterburg MS. VolcaNoseR is a web app for creating, exploring, labeling and sharing volcano plots. Sci. Rep. 2020;10:20560. doi: 10.1038/s41598-020-76603-3. PubMed DOI PMC

Mahmud A, et al. Genome-scale mapping reveals complex regulatory activities of RpoN in Yersinia pseudotuberculosis. mSystems. 2020;5:e01006–e01020. doi: 10.1128/mSystems.01006-20. PubMed DOI PMC

Samuels DJ, et al. Use of a promiscuous, constitutively-active bacterial enhancer-binding protein to define the sigma(5)(4) (RpoN) regulon of Salmonella Typhimurium LT2. BMC Genom. 2013;14:602. doi: 10.1186/1471-2164-14-602. PubMed DOI PMC

Fitzgerald DM, Bonocora RP, Wade JT. Comprehensive mapping of the Escherichia coli flagellar regulatory network. PLoS Genet. 2014;10:e1004649. doi: 10.1371/journal.pgen.1004649. PubMed DOI PMC

Bonocora RP, Smith C, Lapierre P, Wade JT. Genome-scale mapping of Escherichia coli sigma54 reveals widespread, conserved intragenic binding. PLoS Genet. 2015;11:e1005552. doi: 10.1371/journal.pgen.1005552. PubMed DOI PMC

Singh SS, et al. Widespread suppression of intragenic transcription initiation by H-NS. Genes Dev. 2014;28:214–219. doi: 10.1101/gad.234336.113. PubMed DOI PMC

Pukhrambam C, et al. Structural and mechanistic basis of sigma-dependent transcriptional pausing. Proc. Natl Acad. Sci. USA. 2022;119:e2201301119. doi: 10.1073/pnas.2201301119. PubMed DOI PMC

Campagne S, Marsh ME, Capitani G, Vorholt JA, Allain FH. Structural basis for -10 promoter element melting by environmentally induced sigma factors. Nat. Struct. Mol. Biol. 2014;21:269–276. doi: 10.1038/nsmb.2777. PubMed DOI

Jensen-Cain DM, Quinn FD. Differential expression of sigE by Mycobacterium tuberculosis during intracellular growth. Micro. Pathog. 2001;30:271–278. doi: 10.1006/mpat.2001.0431. PubMed DOI

Mauri M, Klumpp S. A model for sigma factor competition in bacterial cells. PLoS Comput. Biol. 2014;10:e1003845. doi: 10.1371/journal.pcbi.1003845. PubMed DOI PMC

Ryu YG, Butler MJ, Chater KF, Lee KJ. Engineering of primary carbohydrate metabolism for increased production of actinorhodin in Streptomyces coelicolor. Appl. Environ. Microbiol. 2006;72:7132–7139. doi: 10.1128/AEM.01308-06. PubMed DOI PMC

Stankovic N, Senerovic L, Ilic-Tomic T, Vasiljevic B, Nikodinovic-Runic J. Properties and applications of undecylprodigiosin and other bacterial prodigiosins. Appl. Microbiol. Biotechnol. 2014;98:3841–3858. doi: 10.1007/s00253-014-5590-1. PubMed DOI

Millan-Oropeza A, Henry C, Lejeune C, David M, Virolle MJ. Expression of genes of the Pho regulon is altered in Streptomyces coelicolor. Sci. Rep. 2020;10:8492. doi: 10.1038/s41598-020-65087-w. PubMed DOI PMC

Tenconi E, Traxler MF, Hoebreck C, van Wezel GP, Rigali S. Production of prodiginines is part of a programmed cell death process in Streptomyces coelicolor. Front. Microbiol. 2018;9:1742. doi: 10.3389/fmicb.2018.01742. PubMed DOI PMC

Ramaniuk O, Cerny M, Krasny L, Vohradsky J. Kinetic modelling and meta-analysis of the B. subtilis SigA regulatory network during spore germination and outgrowth. Biochim. Biophys. Acta Gene Regul. Mech. 2017;1860:894–904. doi: 10.1016/j.bbagrm.2017.06.003. PubMed DOI

Vohradsky J, et al. Kinetic modeling and meta-analysis of the bacillus subtilis SigB regulon during spore germination and outgrowth. Microorganisms. 2021;9:112. doi: 10.3390/microorganisms9010112. PubMed DOI PMC

Ferooz J, Lemaire J, Delory M, De Bolle X, Letesson JJ. RpoE1, an extracytoplasmic function sigma factor, is a repressor of the flagellar system in Brucella melitensis. Microbiology. 2011;157:1263–1268. doi: 10.1099/mic.0.044875-0. PubMed DOI

Bowers CW, Dombroski AJ. A mutation in region 1.1 of sigma70 affects promoter DNA binding by Escherichia coli RNA polymerase holoenzyme. EMBO J. 1999;18:709–716. doi: 10.1093/emboj/18.3.709. PubMed DOI PMC

Bibb MJ, Molle V, Buttner MJ. sigma(BldN), an extracytoplasmic function RNA polymerase sigma factor required for aerial mycelium formation in Streptomyces coelicolor A3(2) J. Bacteriol. 2000;182:4606–4616. doi: 10.1128/JB.182.16.4606-4616.2000. PubMed DOI PMC

Paget MS, Molle V, Cohen G, Aharonowitz Y, Buttner MJ. Defining the disulphide stress response in Streptomyces coelicolor A3(2): identification of the sigmaR regulon. Mol. Microbiol. 2001;42:1007–1020. doi: 10.1046/j.1365-2958.2001.02675.x. PubMed DOI

Shimada T, Furuhata S, Ishihama A. Whole set of constitutive promoters for RpoN sigma factor and the regulatory role of its enhancer protein NtrC in Escherichia coli K-12. Micro. Genom. 2021;7:000653. PubMed PMC

Schumacher MA, et al. The MerR-like protein BldC binds DNA direct repeats as cooperative multimers to regulate Streptomyces development. Nat. Commun. 2018;9:1139. doi: 10.1038/s41467-018-03576-3. PubMed DOI PMC

Srivastava DB, et al. Structure and function of CarD, an essential mycobacterial transcription factor. Proc. Natl Acad. Sci. USA. 2013;110:12619–12624. doi: 10.1073/pnas.1308270110. PubMed DOI PMC

Sevcikova B, Rezuchova B, Homerova D, Kormanec J. The anti-anti-sigma factor BldG is involved in activation of the stress response sigma factor sigma(H) in Streptomyces coelicolor A3(2) J. Bacteriol. 2010;192:5674–5681. doi: 10.1128/JB.00828-10. PubMed DOI PMC

Parashar A, Colvin KR, Bignell DR, Leskiw BK. BldG and SCO3548 interact antagonistically to control key developmental processes in Streptomyces coelicolor. J. Bacteriol. 2009;191:2541–2550. doi: 10.1128/JB.01695-08. PubMed DOI PMC

Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. Practical Streptomyces Genetics (John Innes Foundation, 2000).

Pospisil J, Strunin D, Zikova A, Hubalek M, Vohradsky J. A comparison of protein and mRNA expression during development of the soil dwelling prokaryote (S. coelicolor) Proteomics. 2020;20:e2000032. doi: 10.1002/pmic.202000032. PubMed DOI

Gust B, et al. Lambda red-mediated genetic manipulation of antibiotic-producing Streptomyces. Adv. Appl. Microbiol. 2004;54:107–128. doi: 10.1016/S0065-2164(04)54004-2. PubMed DOI

Flett F, Mersinias V, Smith CP. High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes. FEMS Microbiol. Lett. 1997;155:223–229. doi: 10.1111/j.1574-6968.1997.tb13882.x. PubMed DOI

Knirschova R, et al. Utilization of a reporter system based on the blue pigment indigoidine biosynthetic gene bpsA for detection of promoter activity and deletion of genes in Streptomyces. J. Microbiol. Methods. 2015;113:1–3. doi: 10.1016/j.mimet.2015.03.017. PubMed DOI

Erde J, Loo RR, Loo JA. Enhanced FASP (eFASP) to increase proteome coverage and sample recovery for quantitative proteomic experiments. J. Proteome Res. 2014;13:1885–1895. doi: 10.1021/pr4010019. PubMed DOI PMC

Keilhauer EC, Hein MY, Mann M. Accurate protein complex retrieval by affinity enrichment mass spectrometry (AE-MS) rather than affinity purification mass spectrometry (AP-MS) Mol. Cell Proteom. 2015;14:120–135. doi: 10.1074/mcp.M114.041012. PubMed DOI PMC

Langerova H, et al. Hepatitis B core protein is post-translationally modified through K29-linked ubiquitination. Cells. 2020;9:2547. doi: 10.3390/cells9122547. PubMed DOI PMC

Kallio MA, et al. Chipster: user-friendly analysis software for microarray and other high-throughput data. BMC Genom. 2011;12:507. doi: 10.1186/1471-2164-12-507. PubMed DOI PMC

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat. Methods. 2012;9:357–359. doi: 10.1038/nmeth.1923. PubMed DOI PMC

Zhang Y, et al. Model-based analysis of ChIP-Seq (MACS) Genome Biol. 2008;9:R137. doi: 10.1186/gb-2008-9-9-r137. PubMed DOI PMC

Milne I, et al. Using tablet for visual exploration of second-generation sequencing data. Brief. Bioinform. 2013;14:193–202. doi: 10.1093/bib/bbs012. PubMed DOI

Bailey TL, Elkan C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc. Int Conf. Intell. Syst. Mol. Biol. 1994;2:28–36. PubMed

Grant CE, Bailey TL, Noble WS. FIMO: scanning for occurrences of a given motif. Bioinformatics. 2011;27:1017–1018. doi: 10.1093/bioinformatics/btr064. PubMed DOI PMC

Kim MS, et al. Conservation of thiol-oxidative stress responses regulated by SigR orthologues in actinomycetes. Mol. Microbiol. 2012;85:326–344. doi: 10.1111/j.1365-2958.2012.08115.x. PubMed DOI PMC

Vohradsky J. Neural network model of gene expression. FASEB J. 2001;15:846–854. doi: 10.1096/fj.00-0361com. PubMed DOI

Modrak M, Vohradsky J. Genexpi: a toolset for identifying regulons and validating gene regulatory networks using time-course expression data. BMC Bioinform. 2018;19:137. doi: 10.1186/s12859-018-2138-x. PubMed DOI PMC

Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucl. Acids Res. 2001;29:e45. doi: 10.1093/nar/29.9.e45. PubMed DOI PMC