Study on a two-component signal transduction system RimA1A2 that negatively regulates oxytetracycline biosynthesis in Streptomyces rimosus M4018

Abstract

Biosynthesis of secondary metabolites in actinobacteria is regulated by complex regulatory mechanisms on responding environmental changes. In this study, we have identified a two-component system (TCS) designated as RimA1A2 in the genome of Streptomyces rimosus M4018, with high homology to the TCS RapA1A2 from Streptomyces coelicolor, known for its positive regulatory function towards actinorhodin (ACT) biosynthesis. Using RT-PCR analysis, we demonstrate that rimA1 encodes response regulator (RR) and rimA2 encoding histidine kinase (HK) from S. rimosus that are co-transcribed as a single-polycistronic mRNA. When S. rimosus was cultivated on standard media, no significant difference in culture growth or morphological properties was observed between the rimA1-disrupted mutant and parent strain M4018. However, the rimA1-disrupted strain displayed significant increase in oxytetracycline (OTC) titer when cultivated in minimal medium (MM) containing glycine as sole nitrogen source, and the transcription of selected genes involved in OTC biosynthesis was increased, supporting the hypothesis that RimA1A2 has a negative regulatory role in OTC biosynthesis via global regulation. We observed an increased resistance of the rimA1-disrupted mutant strain to selected antibiotics. Interestingly, in the rimA1-disrupted strain, OTC biosynthesis was affected under different environmental stress conditions such as osmotic and oxidative stress. Accordingly, this phenotype was observed in a medium-dependent manner. Considering complexity of regulatory networks in antibiotic-producing organisms, this study demonstrates the importance of cultivation conditions, which is often neglected.

Keywords

RimA1A2 / Oxytetracycline biosynthesis / Two-component regulatory system / Environmental stress / Streptomyces rimosus / Negative regulation

References

[1] Adamidis T, Riggle P, Champness W. Mutations in a new Streptomyces coelicolor locus which globally block antibiotic biosynthesis but not sporulation. J Bacteriol, 1990, 172: 2962-2969.

[2] Ahn SJ, Qu MD, Roberts E, Burne RA, Rice KC. Identification of the Streptococcus mutans LytST two-component regulon reveals its contribution to oxidative stress tolerance. BMC Microbiol, 2012, 12: 187.

[3] Bang IS, Kim BH, Foster JW, Park YK. OmpR regulates the stationary-phase acid tolerance response of Salmonella enterica serovar typhimurium. J Bacteriol, 2000, 182: 2245-2252.

[4] Beites T, Pires SDS, Santos CL, Osório H, Moradas-Ferreira P, Mendes MV. Crosstalk between ROS homeostasis and secondary metabolism in S. natalensis ATCC 27448: modulation of pimaricin production by intracellular ROS. PLoS ONE, 2011, 6: e27472.

[5] Bentley SD, Chater KF, Cerdeno-Tarraga A, Challis GL, Thomson NR, James KD, Harris DE, Quail MA, Kieser H, Harper D. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature, 2002, 417: 141-147.

[6] Bierman M, Logan R, O’Brien K, Seno ET, Nagaraja Rao R, Schoner BE. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene, 1992, 116: 43-49.

[7] Bijlsma JJE, Groisman EA. Making informed decisions: regulatory interactions between two-component systems. Trends Microbiol, 2003, 11: 359-366.

[8] Bishop A, Fielding S, Dyson P, Herron P. Systematic insertional mutagenesis of a Streptomycete genome: a link between osmoadaptation and antibiotic production. Genome Res, 2003, 14: 893-900.

[9] Brian P, Riggle PJ, Santos RA, Champness WC. Global negative regulation of Streptomyces coelicolor antibiotic synthesis mediated by an absA-encoded putative signal transduction system. J Bacteriol, 1996, 178: 3221-3231.

[10] Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. The MIQE guidelines: minimum information for publication of quantitative Real-Time PCR experiments. Clin Chem, 2009, 55: 611-622.

[11] Djoric D, Kristich CJ. Oxidative stress enhances cephalosporin resistance of Enterococcus faecalis through activation of a two-component signaling system. Antimicrob Agents Chemother, 2015, 59: 159-169.

[12] Elsayed EA, Omar HG, El-Enshasy HA. Development of fed-batch cultivation strategy for efficient oxytetracycline production by Streptomyces rimosus at semi-industrial scale. Braz Arch Biol Technol, 2015, 58: 676-685.

[13] Fabret C, Feher VA, Hoch JA. Two-component signal transduction in Bacillus subtilis: how one organism sees its world. J Bacteriol, 1999, 181: 1975-1983.

[14] Finlay AC, Hobby GL, P’an SY, Regna PP, Routien JB, Seeley DB, Shull GM, Sobin BA, Solomons IA, Vinson JW, Kane JH. Terramycin, a new antibiotic. Science, 1950, 111: 85.

[15] Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-Vegas A, Salazar GA, Tate J, Bateman A. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res, 2016, 44: D279-D285.

[16] Hakenbeck R, Stock JB. Analysis of two-component signal transduction systems involved in transcriptional regulation. Methods Enzymol, 1996, 273: 281-300.

[17] Hoch JA, Silhavy TJ. Two-component signal transduction, 1995, Washington, D.C: ASM Press

[18] Hong S, Roze LV, Linz JE. Oxidative stress-related transcription factors in the regulation of secondary metabolism. Toxins, 2013, 5: 683-702.

[19] Hopwood DA, Bibb MJ, Chater KF, Kieser T, Bruton CJ, Kieser HM, Lydiate DJ, Smith CP, Ward JM, Schrempf H. Genetic manipulation of Streptomyces: a laboratory manual, 1985, Norwich: John Innes Foundation.

[20] Hutchings MI, Hoskisson PA, Chandra G, Buttner MJ. Sensing and responding to diverse extracellular signals? Analysis of the sensor kinases and response regulators of Streptomyces coelicolor A3(2). Microbiology, 2004, 150: 2795-2806.

[21] Karnicar K, Drobnak I, Petek M, Magdevska V, Horvat J, Vidmar R, Baebler S, Rotter A, Jamnik P, Fujs S, Turk B, Fonovic M, Gruden K, Kosec G, Petkovic H. Integrated omics approaches provide strategies for rapid erythromycin yield increase in Saccharopolyspora erythraea. Microb Cell Fact, 2016, 15: 1-17.

[22] Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. Practical streptomyces genetics, 2000, Norwich: The John Innes Foundation.

[23] Kim D, Forst S. Genomic analysis of the histidine kinase family in bacteria and archaea. Microbiology, 2001, 147: 1197-1212.

[24] Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol, 2016, 33: 1870-1874.

[25] Lesnik U, Gormand A, Magdevska V, Fujs S, Raspor P, Hunter I, Petkovic H. Regulatory elements in tetracycline-encoding gene clusters: the otcG gene positively regulates the production of oxytetracycline in Streptomyces rimosus. Food Technol Biotechnol, 2009, 47: 323-330.

[26] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔC_T method. Methods, 2001, 25: 402-408.

[27] Lu YH, Wang WH, Shu D, Zhang WW, Chen L, Qin ZJ, Yang S, Jiang WH. Characterization of a novel two-component regulatory system involved in the regulation of both actinorhodin and a type I polyketide in Streptomyces coelicolor. Appl Microbiol Biotechnol, 2007, 77: 625-635.

[28] MacNeil DJ, Gewain KM, Ruby CL, Dezeny G, Gibbons PH, MacNeil T. Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene, 1992, 111: 61-68.

[29] Mascher T, Helmann JD, Unden G. Stimulus perception in bacterial signal-transducing histidine kinases. Microbiol Mol Biol Rev, 2006, 70: 910-938.

[30] McKenzie NL, Nodwell JR. Phosphorylated AbsA2 negatively regulates antibiotic production in Streptomyces coelicolor through interactions with pathway-specific regulatory gene promoters. J Bacteriol, 2007, 189: 5284-5292.

[31] McMurry LM, Levy SB. Revised sequence of OtrB (Tet347) tetracycline efflux protein from Streptomyces rimosus. Antimicrob Agents Chemother, 1998, 42: 3050.

[32] Montibus M, Pinson-Gadais L, Richard-Forget F, Barreau C, Ponts N. Coupling of transcriptional response to oxidative stress and secondary metabolism regulation in filamentous fungi. Crit Rev Microbiol, 2015, 41: 295-308.

[33] Ogura M, Tanaka T. Recent progress in Bacillus subtilis two-component regulation. Front Biosci, 2002, 7: D1815-D1824.

[34] Ohnuki T, Katoh T, Imanaka T, Aiba S. Molecular cloning of tetracycline resistance genes from Streptomyces rimosus in Streptomyces griseus and characterization of the cloned genes. J Bacteriol, 1985, 161: 1010-1016.

[35] Paget M, Leibovitz E, Buttner MJ. A putative two-component signal transduction system regulates sigma(E), a sigma factor required for normal cell wall integrity in Streptomyces coelicolor A3(2). Mol Microbiol, 1999, 33: 97-107.

[36] Pethick FE, MacFadyen AC, Tang Z, Sangal V, Liu T, Chu J, Kosec G, Petkovic H, Guo M, Kirby R, Hoskisson PA, Herron PR, Hunter IS. Draft genome sequence of the oxytetracycline-producing bacterium Streptomyces rimosus ATCC 10970. Genome Announc, 2013, 1: 1-2.

[37] Petkovic H, Cullum J, Hranueli D, Hunter IS, Peric-Concha N, Pigac J, Thamchaipenet A, Vujaklija D, Long PF. Genetics of Streptomyces rimosus, the oxytetracycline producer. Microbiol Mol Biol Rev, 2006, 70: 704-728.

[38] Petkovic H, Lukezic T, Suskovic J. Biosynthesis of oxytetracycline by Streptomyces rimosus: past, present and future directions in the development of tetracycline antibiotics. Food Technol Biotechnol, 2017, 55: 3-13.

[39] Pickens LB, Tang Y. Oxytetracycline biosynthesis. J Biol Chem, 2010, 285: 27509-27515.

[40] Rao YM, Sureshkumar GK. Improvement in bioreactor productivities using free radicals: HOCl-induced overproduction of xanthan gum from Xanthomonas campestris and its mechanism. Biotechnol Bioeng, 2001, 72: 62-68.

[41] Rhodes PM, Hunter IS, Friend EJ, Warren M. Recombinant DNA methods for the oxytetracycline producer Streptomyces rimosus. Biochem Soc T, 1984, 12: 586-587.

[42] Rodriguez H, Rico S, Diaz M, Santamaria RI. Two-component systems in Streptomyces: key regulators of antibiotic complex pathways. Microb Cell Fact, 2013, 12: 1-10.

[43] Rodriguez-Garcia A, Barreiro C, Santos-Beneit F, Sola-Landa A, Martin JF. Genome-wide transcriptomic and proteomic analysis of the primary response to phosphate limitation in Streptomyces coelicolor M145 and in a ΔphoP mutant. Proteomics, 2007, 7: 2410-2429.

[44] Rozas D, Gullon S, Mellado RP. A novel two-component system involved in the transition to secondary metabolism in Streptomyces coelicolor. PLoS ONE, 2012, 7: 1-10.

[45] Ryding NJ, Anderson TB, Champness WC. Regulation of the Streptomyces coelicolor calcium-dependent antibiotic by absA, encoding a cluster-linked two-component system. J Bacteriol, 2002, 184: 794-805.

[46] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, 1989, New York: Cold spring harbor laboratory press.

[47] Santos-Beneit F, Rodriguez-Garcia A, Sola-Landa A, Martin JF. Cross-talk between two global regulators in Streptomyces: PhoP and AfsR interact in the control of afsS, pstS and phoRP transcription. Mol Microbiol, 2009, 72: 53-68.

[48] Shu D, Chen L, Wang WH, Yu ZY, Ren C, Zhang WW, Yang S, Lu YH, Jiang WH. afsQ1-Q2-sigQ is a pleiotropic but conditionally required signal transduction system for both secondary metabolism and morphological development in Streptomyces coelicolor. Appl Microbiol Biotechnol, 2009, 81: 1149-1160.

[49] Singh N, Rai V, Tripathi C. Production and optimization of oxytetracycline by a new isolate Streptomyces rimosus using response surface methodology. Med Chem Res, 2012, 21: 3140-3145.

[50] Sola-Landa A, Moura RS, Martin JF. The two-component PhoR-PhoP system controls both primary metabolism and secondary metabolite biosynthesis in Streptomyces lividans. Proc Natl Acad Sci USA, 2003, 100: 6133-6138.

[51] Sola-Landa A, Rodriguez-Garcia A, Franco-Dominguez E, Martin JF. Binding of PhoP to promoters of phosphate-regulated genes in Streptomyces coelicolor: identification of PHO boxes. Mol Microbiol, 2005, 56: 1373-1385.

[52] Sola-Landa A, Rodriguez-Garci A, Apel AK, Martin JF. Target genes and structure of the direct repeats in the DNA-binding sequences of the response regulator PhoP in Streptomyces coelicolor. Nucleic Acids Res, 2008, 36: 1358-1368.

[53] Stock JB, Ninfa AJ, Stock AM. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev, 1989, 53: 450-490.

[54] Stock AM, Robinson VL, Goudreau PN. Two-component signal transduction. Annu Rev Biochem, 2000, 69: 183-215.

[55] Sugiura A, Hirokawa K, Nakashima K, Mizurto T. Signal-sensing mechanisms of the putative osmosensor KdpD in Escherichia coli. Mol Microbiol, 1994, 14: 929-938.

[56] Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M. A practical approach to RT-qPCR—publishing data that conform to the MIQE guidelines. Methods, 2010, 50: S1-S5.

[57] Wang JY, Yang SS. Morphogenesis, biomass and oxytetracycline production of Streptomyces rimosus in submerged cultivation. Chin J Microbiol Immunol, 1995, 28: 21-31.

[58] Wang R, Mast Y, Wang J, Zhang WW, Zhao GP, Wohlleben W, Lu YH, Jiang WH. Identification of two-component system AfsQ1/Q2 regulon and its cross-regulation with GlnR in Streptomyces coelicolor. Mol Microbiol, 2013, 87: 30-48.

[59] Wei Z, Bai L, Deng Z, Zhong J. Enhanced production of validamycin A by H₂O₂-induced reactive oxygen species in fermentation of Streptomyces hygroscopicus 5008. Bioresour Technol, 2011, 102: 1783-1787.

[60] Wietzorrek A, Bibb M. A novel family of proteins that regulates antibiotic production in streptomycetes appears to contain an OmpR-like DNA-binding fold. Mol Microbiol, 1997, 25: 1181-1184.

[61] Wilkinson CJ, Hughes-Thomas ZA, Martin CJ, Bohm I, Mironenko T, Deacon M, Wheatcroft M, Wirtz G, Staunton J, Leadlay PF. Increasing the efficiency of heterologous promoters in actinomycetes. J Mol Microbiol Biotechnol, 2002, 4: 417-426.

[62] Wood JM. Osmosensing by bacteria: signals and membrane-based sensors. Microbiol Mol Biol Rev, 1999, 63: 230-262.

[63] Yin SL, Wang WS, Wang XF, Zhu YX, Jia XL, Li SS, Yuan F, Zhang YX, Yang KQ. Identification of a cluster-situated activator of oxytetracycline biosynthesis and manipulation of its expression for improved oxytetracycline production in Streptomyces rimosus. Microb Cell Fact, 2015, 14: 1-12.

[64] Yu L, Yan X, Wang L, Chu J, Zhuang Y, Zhang S, Guo M. Molecular cloning and functional characterization of an ATP-binding cassette transporter OtrC from Streptomyces rimosus. BMC Biotechnol, 2012, 12: 52.

[65] Yu ZY, Zhu H, Dang FJ, Zhang WW, Qin ZJ, Yang S, Tan HR, Lu YH, Jiang WH. Differential regulation of antibiotic biosynthesis by DraR-K, a novel two-component system in Streptomyces coelicolor. Mol Microbiol, 2012, 85: 535-556.

Funding

Fundamental Research Funds for the Central Universities(22221818014)

Slovenian Research Agency(J4-7637)