Rhodococcus erythropolis CCM2595 is a bacterial strain, which has been studied for its capability to degrade phenol and other toxic aromatic compounds. Its cell wall contains mycolic acids, which are also an attribute of other bacteria of the Mycolata group, such as Corynebacterium and Mycobacterium species. We suppose that many genes upregulated by phenol stress in R. erythropolis are controlled by the alternative sigma factors of RNA polymerase, which are active in response to the cell envelope or oxidative stress. We developed in vitro and in vivo assays to examine the connection between the stress sigma factors and genes activated by various extreme conditions, e.g., heat, cell surface, and oxidative stress. These assays are based on the procedures of such tests carried out in the related species, Corynebacterium glutamicum. We showed that the R. erythropolis CCM2595 genes frmB1 and frmB2, which encode S-formylglutathione hydrolases (named corynomycolyl transferases in C. glutamicum), are controlled by SigD, just like the homologous genes cmt1 and cmt2 in C. glutamicum. The new protocol of the in vivo and in vitro assays will enable us to classify R. erythropolis promoters according to their connection to sigma factors and to assign the genes to the corresponding sigma regulons. The complex stress responses, such as that induced by phenol, could, thus, be analyzed with respect to the gene regulation by sigma factors.

Department of Genetics and Microbiology Faculty of Science Charles University Prague Czech Republic

Institute of Microbiology of the CAS v v i Prague Czech Republic

See more in PubMed

Martínková L, Uhnákova B, Pátek M, Nešvera J, Křen V (2009) Biodegradation potential of the genus Rhodococcus. Environ Int 35:162–177. https://doi.org/10.1016/j.envint.2008.07.018 PubMed DOI

Donini E, Firrincieli A, Cappelletti M (2021) Systems biology and metabolic engineering of Rhodococcus for bioconversion and biosynthesis processes. Folia Microbiol 66:701–713. https://doi.org/10.1007/s12223-021-00892-y DOI

Cappelletti M, Presentato A, Piacenza E, Firrincieli A, Turner RJ, Zannoni D (2020) Biotechnology of Rhodococcus for the production of valuable compounds. Appl Microbiol Biotechnol 104:8567–8594. https://doi.org/10.1007/s00253-020-10861-z PubMed DOI PMC

Li X, He Y, Zhang L, Xu Z, Ben H, Gaffrey MJ, Yang Y, Yang S, Yuan JS, Qian WJ, Yang B (2019) Discovery of potential pathways for biological conversion of poplar wood into lipids by co-fermentation of Rhodococci strains. Biotechnol Biofuels 12:60–75. https://doi.org/10.1186/s13068-019-1395-x PubMed DOI PMC

Wang M, Chen J, Yu H, Shen Z (2018) Improving stress tolerance and cell integrity of Rhodococcus ruber by overexpressing small-shock-protein Hsp16 of Rhodococcus. J Ind Microbiol Biotechnol 45:929–938. https://doi.org/10.1007/s10295-018-2066-9 PubMed DOI

Bukliarevich HA, Charniauskaya MI, Akhremchuk AE, Valentovich LN, Titok MA (2019) Effect of the structural and regulatory heat shock proteins on hydrocarbon degradation by Rhodococcus pyridinivorans 5Ap. Microbiology 88:573–579. https://doi.org/10.1134/s0026261719050023 DOI

Kobayashi M, Yanaka N, Nagasawa T, Yamada H (1992) Primary structure of an aliphatic nitrile-degrading enzyme, aliphatic nitrilase, from Rhodococcus rhodochrous K22 and expression of its gene and identification of its active site residue. Biochemistry 31:9000–9007. https://doi.org/10.1021/bi00152a042 PubMed DOI

Kuyukina MS, Krivoruchko A, Ivshina IB (2018) Hydrocarbon- and metal-polluted soil bioremediation: progress and challenges. Microbiol Aust 39:133–136. https://doi.org/10.1071/MA18041 DOI

Zheng YT, Toyofuku M, Nomura N, Shigeto S (2013) Correlation of carotenoid accumulation with aggregation and biofilm development in Rhodococcus sp. SD-74. Anal Chem 85:7295–7301. https://doi.org/10.1021/ac401188f PubMed DOI

Pátek M, Grulich M, Nešvera J (2021) Stress response in Rhodococcus strains. Biotechnol Adv. https://doi.org/10.1016/j.biotechadv.2021.107698 PubMed DOI

Gruber TM, Gross CA (2003) Multiple sigma subunits and the partitioning of bacterial transcription space. Annu Rev Microbiol 57:441–466. https://doi.org/10.1146/annurev.micro.57.030502.090913 PubMed DOI

Pátek M, Dostálová H, Nešvera J (2020) Sigma factors of RNA polymerase in Corynebacterium glutamicum. In: Corynebacterium glutamicum, biology and biotechnology. pp 89–112. https://doi.org/10.1007/978-3-030-39267-3_4

Strnad H, Pátek M, Fousek J, Szokol J, Ulbrich P, Nešvera J, Pačes V, Vlček Č (2014) Genome sequence of Rhodococcus erythropolis strain CCM2595, a phenol derivatives degrading bacterium. Genome Announc. https://doi.org/10.1128/genomeA.00208-14 PubMed DOI PMC

McLeod MP, Warren RL, Hsiao WWK, Araki N, Myhre M, Fernandes C, Miyazawa D, Wong W, Lillquist AL, Wang D, Dosanjh M, Hara H, Petrescu A, Morin RD, Yang G, Stott JM, Schein JE, Shin H, Smailus D, Siddiqui AS, Marra MA, Jones SJM, Holt R, Brinkman FSL, Miyauchi K, Fukuda M, Davies JE, Mohn WW, Eltis LD (2006) The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse. Proc Natl Acad Sci USA 103:15582–15587. https://doi.org/10.1073/pnas.0607048103 PubMed DOI PMC

Ekpanyaskun P (2006) Transcriptomic analysis of Rhodococcus sp. RHA1 responses to heat shock and osmotic stress. Master Thesis, University of British Columbia, Vancouver

Toyoda K, Inui M (2016) The extracytoplasmic function σ factor σ PubMed DOI

Taniguchi H, Busche T, Patschkowski T, Niehaus K, Pátek M, Kalinowski J, Wendisch VF (2017) Physiological roles of sigma factor SigD in Corynebacterium glutamicum. BMC Microbiol 17:158–168. https://doi.org/10.1186/s12866-017-1067-6 PubMed DOI PMC

Toyoda K, Inui M (2018) Extracytoplasmic function sigma factor σ PubMed DOI

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. https://doi.org/10.1186/1471-2164-13-445 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. https://doi.org/10.1128/JB.00112-09 PubMed DOI PMC

Holátko J, Šilar R, Rabatinová A, Šanderová H, Halada P, Nešvera J, Krásny L, Pátek M (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. https://doi.org/10.1007/s00253-012-4336-1 PubMed DOI

Dostálová H, Holátko J, Busche T, Rucká L, Rapoport A, Halada P, Nešvera J, Kalinowski J, Pátek M (2017) Assignment of sigma factors of RNA polymerase to promoters in Corynebacterium glutamicum. AMB Express 7:133–146. https://doi.org/10.1186/s13568-017-0436-8 PubMed DOI PMC

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. https://doi.org/10.1007/s00284-007-0106-1 PubMed DOI

Dostálová H, Busche T, Holátko J, Rucká L, Štěpánek V, Barvík I, Nešvera J, Kalinowski J, Pátek M (2019) Overlap of promoter recognition specificity of stress response sigma factors SigD and SigH in Corynebacterium glutamicum ATCC 13032. Front Microbiol 9:3287–3304. https://doi.org/10.3389/fmicb.2018.03287 PubMed DOI PMC

Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

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

Ross W, Gourse RL (2009) Analysis of RNA polymerase-promoter complex formation. Methods 47:13–24. https://doi.org/10.1016/j.ymeth.2008.10.018 PubMed DOI

Concordet JP, Haeussler M (2018) CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res 2:242–245. https://doi.org/10.1093/nar/gky354 DOI

Zhang B, Zhou N, Liu YM, Liu C, Lou CB, Jiang CY, Liu SJ (2015) Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum. Microb Cell Factories 14:1–14. https://doi.org/10.1186/s12934-015-0254-0 DOI

Gibson DG, Young L, Chuang RY, Venter JC, Hutchinson CA III, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345. https://doi.org/10.1038/nmeth.1318 PubMed DOI

van der Rest ME, 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. https://doi.org/10.1007/s002530051557 PubMed DOI

Qi Y, Hulett FM (1998) PhoP~P and RNA polymerase σ PubMed DOI

Homerova D, Surdova K, Mikusova K, Kormanec J (2007) Identification of promoters recognized by RNA polymerase containing Mycobacterium tuberculosis stress-response sigma factor σ PubMed DOI

Homerova D, Bischoff M, Dumolin A, Kormanec J (2004) Optimization of a two-plasmid system for the identification of promoters recognized by RNA polymerase containing Staphylococcus aureus alternative sigma factor σ PubMed DOI

Calamita H, Ko C, Tyagi S, Yoshimatsu T, Morrison NE, Bishai WR (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. https://doi.org/10.1111/j.1462-5822.2004.00454.x PubMed DOI

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

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. https://doi.org/10.1016/j.jbiotec.2017.04.008 PubMed DOI

Overlapping SigH and SigE sigma factor regulons in Corynebacterium glutamicum