Characterization of a small non-coding RNA S612 in Bacillus subtilis

Abstract

Small regulatory RNAs (sRNAs) are non-coding RNA molecules that fine-tune various cellular processes and respond to various environmental stimuli. In Bacillus subtilis, the regulatory mechanisms and specific targets of several sRNAs remain largely unknown. In this study, we identified and characterized S612 as a self-terminating sRNA in B. subtilis. The expression of S612 is regulated by external signals, including nutrient availability and salt concentration. Overexpression of S612 induced filamentous cells with extensive cellular elongation and complete inhibition of sporulation, indicating its potential to control cell morphology and spore formation. S612 directly targets and downregulates genes through post-transcriptional base pairing with mRNAs, including ylmD, trpE, ycxC, yycS, rapH, and amyE, some of which are involved in cell membrane integrity, cell wall synthesis, and sporulation initiation. Therefore, we propose that S612 is an important post-transcriptional regulator of cell morphology and sporulation.

Keywords

Bacillus subtilis / Small RNA / Non-coding RNA / Sporulation / Filamentation / Post-transcription regulation

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its supplementary information files or are available upon request.

Declaration of Competing Interest

This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. We have read and understood your journal's policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare.

CRediT authorship contribution statement

Anqi Peng: Writing - review & editing, Writing - original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Weijiao Zhang: Writing - review & editing, Validation, Supervision. Haibo Xiong: Investigation. Luyao Zhang: Investigation. Jian Cheng: Investigation. Yang Wang: Writing - review & editing, Supervision, Data curation, Conceptualization. Zhen Kang: Writing - review & editing, Supervision, Funding acquisition, Conceptualization.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (32370066).

Supplementary Materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.engmic.2024.100186.

References

[1] L.S. Waters, G. Storz, Regulatory RNAs in Bacteria, Cell 136(4) (2009) 615-628.

[2] B. Felden, Y. Augagneur, Diversity and Versatility in Small RNA-Mediated Regulation in Bacterial Pathogens, Front. Microbiol. 12(2021) 719977.

[3] A. Peng, G. Yin, W. Zuo, L. Zhang, G. Du, J. Chen, Y. Wang, Z. Kang, Regulatory RNAs in Bacillus subtilis : A review on regulatory mechanism and applications in synthetic biology, Synth. Syst. Biotechnol. 9(2) (2024) 223-233.

[4] T. Dutta, S. Srivastava, Small RNA-mediated regulation in bacteria: A growing palette of diverse mechanisms, Gene 656(2018) 60-72.

[5] M.L. Pinel-Marie, R. Brielle, C. Riffaud, N. Germain-Amiot, N. Polacek, B. Felden, RNA antitoxin SprF1 binds ribosomes to attenuate translation and promote persister cell formation in Staphylococcus aureus, Nat. Microbiol. 6(2) (2021) 209-220.

[6] F.L. Short, C. Akusobi, W.R. Broadhurst, G.P.C. Salmond, The bacterial Type III toxin- antitoxin system, ToxIN, is a dynamic protein-RNA complex with stability-dependent antiviral abortive infection activity, Sci. Rep. 8(1) (2018) 1013.

[7] S.M. Kang, D.H. Kim, C. Jin, B.J. Lee, A Systematic Overview of Type II and III Toxin- Antitoxin Systems with a Focus on Druggability, Toxins. ( Basel) 10 (12) (2018).

[8] J. Lin, Y. Guo, J. Yao, K. Tang, X. Wang, Applications of toxin-antitoxin systems in synthetic biology, Engineering Microbiology 3(2) (2023).

[9] M.G. Jørgensen, J.S. Pettersen, B.H. Kallipolitis, sRNA-mediated control in bacteria: An increasing diversity of regulatory mechanisms, Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1863 (5) (2020).

[10] M. Lejars, E. Hajnsdorf, The world of asRNAs in Gram-negative and Gram-positive bacteria, Biochim. Biophys. Acta Gene Regul. Mech. 1863(2) (2020) 194489.

[11] R.A.T. Mars, P. Nicolas, E.L. Denham, J.M. van Dijl, Regulatory RNAs in Bacillus subtilis : a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression, Microbiology and Molecular Biology Reviews 80(4) (2016) 1029-1057.

[12] I. Irnov, C.M. Sharma, J. Vogel, W.C. Winkler, Identification of regulatory RNAs in Bacillus subtilis, Nucleic. Acids. Res. 38(19) (2010) 6637-6651.

[13] S. Durand, F. Braun, A.C. Helfer, P. Romby, C. Condon, sRNA-mediated activation of gene expression by inhibition of 5 ′ -3 ′ exonucleolytic mRNA degradation, Elife, 6(2017) e23602.

[14] G.T. Smaldone, O. Revelles, A. Gaballa, U. Sauer, H. Antelmann, J.D. Helmann, A global investigation of the Bacillus subtilis iron-sparing response identifies major changes in metabolism, J. Bacteriol. 194(10) (2012) 2594-2605.

[15] S. Durand, A. Callan-Sidat, J. McKeown, S. Li, G. Kostova, J.R. Hernandez-Fernaud, M.T. Alam, A. Millard, D. Allouche, C. Constantinidou, C. Condon, E.L. Denham, Identification of an RNA sponge that controls the RoxS riboregulator of central metabolism in Bacillus subtilis, Nucleic. Acids. Res. 49(11) (2021) 6399-6419.

[16] S. Brantl, P. Muller, Toxin(-)Antitoxin Systems in Bacillus subtilis, Toxins. ( Basel) 11 (5) (2019).

[17] S. Durand, N. Jahn, C. Condon, S. Brantl, Type I toxin-antitoxin systems in Bacillus subtilis, RNa Biol. 9(12) (2012) 1491-1497.

[18] C. L. a. S. B. Celine Reif †, Bacillus subtilis Type I antitoxin SR6 Promotes Degradation of Toxin yonT mRNA and Is Required to Prevent Toxic yoyJ Overexpression, Toxins. (Basel) 10 (2) (2018) 74.

[19] D.H. Bechhofer, M.P. Deutscher, Bacterial ribonucleases and their roles in RNA metabolism, Crit. Rev. Biochem. Mol. Biol. 54(3) (2019) 242-300.

[20] N. Christopoulou, S. Granneman, The role of RNA-binding proteins in mediating adaptive responses in Gram-positive bacteria, FEBS. J. 289(7) (2022) 1746-1764.

[21] I. Ul Haq, P. Muller, S. Brantl, Intermolecular Communication in Bacillus subtilis : RNA-RNA, RNA-Protein and Small Protein-Protein Interactions, Front. Mol. Biosci. 7(2020) 178.

[22] L. Djapgne, A.G. Oglesby, Impacts of Small RNAs and Their Chaperones on Bacterial Pathogenicity, Front. Cell Infect. Microbiol. 11(2021) 604511.

[23] S. Durand, L. Gilet, P. Bessieres, P. Nicolas, C. Condon, Three essential ribonucleases- RNase Y, J1, and III-control the abundance of a majority of Bacillus subtilis mRNAs, PLoS. Genet. 8(3) (2012) e1002520.

[24] M. Lehnik-Habrink, R.J. Lewis, U. Mader, J. Stulke, RNA degradation in Bacillus subtilis : an interplay of essential endo- and exoribonucleases, Mol. Microbiol. 84(6) (2012) 1005-1017.

[25] S. Durand, A. Tomasini, F. Braun, C. Condon, P. Romby, sRNA and mRNA turnover in Gram-positive bacteria, FEMS Microbiol. Rev. 39(3) (2015) 316-330.

[26] J. Le Derout, I.V. Boni, P. Régnier, E Hajnsdorf, Hfq affects mRNA levels independently of degradation, BMC. Mol. Biol. 11(17) (2010).

[27] T.B. Updegrove, A. Zhang, G. Storz, Hfq: the flexible RNA matchmaker, Curr. Opin. Microbiol. 30(2016) 133-138.

[28] J. Vogel, B.F. Luisi, Hfq and its constellation of RNA, Nature Reviews Microbiology 9(8) (2011) 578-589.

[29] H. Hammerle, F. Amman, B. Vecerek, J. Stulke, I. Hofacker, U. Blasi, Impact of Hfq on the Bacillus subtilis transcriptome, PLoS. One 9(6) (2014) e98661.

[30] M. Dambach, I. Irnov, W.C. Winkler, Association of RNAs with Bacillus subtilis Hfq, PLoS. One 8(2) (2013) e55156.

[31] C. Pourciau, Y.J. Lai, M. Gorelik, P. Babitzke, T. Romeo, Diverse Mechanisms and Circuitry for Global Regulation by the RNA-Binding Protein CsrA, Front. Microbiol. 11(2020).

[32] P. Muller, M. Gimpel, T. Wildenhain, S. Brantl, A new role for CsrA: promotion of complex formation between an sRNA and its mRNA target in Bacillus subtilis, RNa Biol. 16(7) (2019) 972-987.

[33] S. Yang, Y. Wang, C. Wei, Q. Liu, X. Jin, G. Du, J. Chen, Z. Kang, A new sRNA-mediated posttranscriptional regulation system for Bacillus subtilis, Biotechnol. Bioeng. 115(12) (2018) 2986-2995.

[34] G. Yin, A. Peng, L. Zhang, Y. Wang, G. Du, J. Chen, Z. Kang, Design of artificial small regulatory trans -RNA for gene knockdown in Bacillus subtilis, Synth. Syst. Biotechnol. 8(1) (2023) 61-68.

[35] M. Lins, L. Amorim, G.G. Correa, B.W. Picao, M. Mack, M.O. Cerri, D.B. Pedrolli, Targeting riboswitches with synthetic small RNAs for metabolic engineering, Metab. Eng. 68(2021) 59-67.

[36] Y. Wu, Y. Liu, X. Lv, J. Li, G. Du, L. Liu, CAMERS-B: CRISPR/Cpf1 assisted multiple- genes editing and regulation system for Bacillus subtilis, Biotechnol. Bioeng. 117(6) (2020) 1817-1825.

[37] P.Z. Wang, R.H. Doi, Overlapping promoters transcribed by Bacillus subtilis sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary phases, Journal of Biological Chemistry 259(13) (1984) 8619-8625.

[38] X.Z. Zhang, Z.L. Cui, Q. Hong, S.P. Li, High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800, Appl. Environ. Microbiol. 71(7) (2005) 4101-4103.

[39] J.H. Kim, B.Y. Hwang, J. Roh, J.K. Lee, K. Kim, S.L. Wong, H. Yun, S.G. Lee, B.G. Kim, Comparison of P aprE, P amyE, and P P43 promoter strength for β-galactosidase and staphylokinase expression in Bacillus subtilis, Biotechnology and Bioprocess Engineering 13(3) (2008) 313-318.

[40] B.A. Traag, A. Ramirez-Peralta, A.F. Wang Erickson, P. Setlow, R. Losick, A novel RNA polymerase-binding protein controlling genes involved in spore germination in Bacillus subtilis, Mol. Microbiol. 89(1) (2013) 113-122.

[41] S.T. Wang, B. Setlow, E.M. Conlon, J.L. Lyon, D. Imamura, T. Sato, P. Setlow, R. Losick, P. Eichenberger, The forespore line of gene expression in Bacillus subtilis, J. Mol. Biol. 358(1) (2006) 16-37.

[42] S.M. Monteiro, A Procedure for High-Yield Spore Production by Bacillus subtilis, Biotechnol. Prog (21) (2005) 1026-1031.

[43] N.C. Shaner, P.A. Steinbach, R.Y. Tsien, A guide to choosing fluorescent proteins, Nat. Methods 2(12) (2005) 905-909.

[44] W.W. Su, Fluorescent proteins as tools to aid protein production, Microb. Cell Fact. 4(1) (2005) 12.

[45] S. Li, P. He, H. Fan, L. Liu, K. Yin, B. Yang, Y. Li, S.M. Huang, X. Li, S.J. Zheng, A Real-Time Fluorescent Reverse Transcription Quantitative PCR Assay for Rapid Detection of Genetic Markers’ Expression Associated with Fusarium Wilt of Banana Biocontrol Activities in Bacillus, J. Fungi. ( Basel) 7 (5) (2021).

[46] A.R. Gruber, R. Lorenz, S.H. Bernhart, R. Neubock, I.L. Hofacker, The Vienna RNA websuite, Nucleic. Acids. Res. 36 (Web Server issue) (2008) W70-W74.

[47] I.L. Hofacker, Vienna RNA secondary structure server, Nucleic. Acids. Res. 31(13) (2003) 3429-3431.

[48] R. Lorenz, S.H. Bernhart, C. Höner Zu Siederdissen, H. Tafer, C. Flamm, P.F. Stadler, I.L. Hofacker, ViennaRNA Package 2.0, Algorithms. Mol. Biol. 6(2011) 26.

[49] J. W.R.W.S. Yarnell, Mechanism of Intrinsic Transcription Termination and Antitermination, Science (1979) 284(1999) 611-615.

[50] T. Nojima, A.C. Lin, T. Fujii, I. Endo, Determination of the termination efficiency of the transcription terminator using different fluorescent profiles in green fluorescent protein mutants, Anal. Sci. 21(12) (2005) 1479-1481.

[51] R. Borriss, A. Danchin, C.R. Harwood, C. Medigue, E.P.C. Rocha, A. Sekowska, D. Vallenet, Bacillus subtilis, the model Gram-positive bacterium: 20 years of annotation refinement, Microb. Biotechnol. 11(1) (2018) 3-17.

[52] P.R. Wright, J. Georg, M. Mann, D.A. Sorescu, A.S. Richter, S. Lott, R. Kleinkauf, W.R. Hess, R. Backofen, CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains, Nucleic. Acids. Res. 42 (Web Server issue) (2014) W119-W123.

[53] M. Raden, S.M. Ali, O.S. Alkhnbashi, A. Busch, F. Costa, J.A. Davis, F. Eggenhofer, R. Gelhausen, J. Georg, S. Heyne, M. Hiller, K. Kundu, R. Kleinkauf, S.C. Lott, M.M. Mohamed, A. Mattheis, M. Miladi, A.S. Richter, S. Will, J. Wolff, P.R. Wright, R. Backofen, Freiburg RNA tools: a central online resource for RNA-focused research and teaching, Nucleic. Acids. Res. 46(W1) (2018) W25-W29.

[54] H. Hall, The Sporulation-Specific Small Regulatory RNAs of Bacillus subtilis, University of Warwick, 2017.

[55] M. Mann, P.R. Wright, R. Backofen, IntaRNA 2.0: enhanced and customizable prediction of RNA-RNA interactions, Nucleic. Acids. Res. 45(W1) (2017) W435-W439.

[56] N. Peschek, R. Herzog, P.K. Singh, M. Sprenger, F. Meyer, K.S. Frohlich, L. Schroger, M. Bramkamp, K. Drescher, K. Papenfort, RNA-mediated control of cell shape modulates antibiotic resistance in Vibrio cholerae, Nat. Commun. 11(1) (2020) 6067.

[57] C.P. Corcoran, D. Podkaminski, K. Papenfort, J.H. Urban, J.C. Hinton, J. Vogel, Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA, Mol. Microbiol. 84(3) (2012) 428-445.

[58] K. Papenfort, K.U. Forstner, J.P. Cong, C.M. Sharma, B.L. Bassler, Differential RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm formation, Proc. Natl. Acad. Sci. u S. a 112(7) (2015) E766-E775.

[59] A. Krogh, B. Larsson, G. von Heijne, E.L.L. Sonnhammer, Predicting transmembrane protein topology with a hidden markov model: application to complete genomes, J. Mol. Biol. 305(3) (2001) 567-580.

[60] V. Molle, M. Fujita, S.T. Jensen, P. Eichenberger, J.E. Gonzalez-Pastor, J.S. Liu, R. Losick, The Spo0A regulon of Bacillus subtilis, Mol Microbiol, 50(5) (2003) 1683-1701.

[61] S. Parveen, M. Reddy, Identification of YfiH (PgeF) as a factor contributing to the maintenance of bacterial peptidoglycan composition, Mol. Microbiol. 105(5) (2017) 705-720.

[62] L. Zhang, J. Willemse, P.A. Hoskisson, G.P. van Wezel, Sporulation-specific cell division defects in ylmE mutants of Streptomyces coelicolor are rescued by additional deletion of ylmD, Sci. Rep. 8(1) (2018) 7328.

[63] E.P. White, ML. ylm Has More than a (Z Anchor) Ring to It!, J. Bacteriol. 203(3) (2021) e00460-00420.

[64] A.R. Diaz, L.J. Core, M. Jiang, M. Morelli, C.H. Chiang, H. Szurmant, M. Perego, Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide, J. Bacteriol. 194(6) (2012) 1378-1388.

[65] V. Parashar, N. Mirouze, D.A. Dubnau, M.B. Neiditch, Structural basis of response regulator dephosphorylation by Rap phosphatases, PLoS. Biol. 9(2) (2011) e1000589.