Conserved mycobacterial sRNA B11 regulates lipooligosaccharide synthesis at posttranscriptional level in Mycobacterium marinum

Abstract

Extractable glycolipids of mycobacteria, such as lipooligosaccharides (LOSs), play crucial roles in responding to environmental stress and modulating the host immune response. Although the biosynthesis of LOS is likely regulated at multiple levels to ensure proper composition of the cell wall, the key regulators remain unknown. In this study, we investigated B11, a conserved mycobacterial small RNA (sRNA), and found that it post-transcriptionally regulates LOS synthesis in Mycobacterium marinum. Through a combination of RNA-seq and mass spectrometry screening, we identified specific genes within the LOS synthesis locus that are directly regulated by B11. We confirmed in vivo sRNA-mRNA interactions using MS2-tagged RNA affinity purification, and found that B11 utilizes the cytosine-rich loop of its Rho-independent transcriptional terminator to interact with guanine tracks adjacent to the ribosome binding sites of its target genes, thereby impeding translation and promoting mRNA degradation. Moreover, deletion of B11 altered the colony morphology associated with LOS composition. These comprehensive functional studies of the mycobacterial sRNA B11 reveal sRNA-based regulation of LOS synthesis, providing new insights into the regulatory mechanisms controlling the biosynthesis of the complex mycobacterial cell wall.

Keywords

lipooligosaccharides / MS2-RNA fishing / mycobacteria / posttranscriptional regulation / sRNA

References

[1] Dulberger CL, Rubin EJ, Boutte CC. The mycobacterial cell envelope—a moving target. Nat Rev Microbiol. 2020; 18: 47-59.

[2] Jackson M. The mycobacterial cell envelope-lipids. Cold Spring Harbor Perspect Med. 2014; 4: a021105.

[3] Bai B, Chu C, Lowary TL. Lipooligosaccharides from mycobacteria: structure, function, and synthesis. Isr J Chem. 2015; 55: 360-372.

[4] Camphausen RT, McNeil M, Jardine I, Brennan PJ. Location of acyl groups of trehalose-containing lipooligosaccharides of mycobacteria. J Bacteriol. 1987; 169: 5473-5480.

[5] Boritsch EC, Frigui W, Cascioferro A, Malaga W, Etienne G, Laval F, et al. pks5-recombination-mediated surface remodelling in Mycobacterium tuberculosis emergence. Nat Microbiol. 2016; 1: 15019.

[6] Pawlik A, Garnier G, Orgeur M, Tong P, Lohan A, Le Chevalier F, et al. Identification and characterization of the genetic changes responsible for the characteristic smooth-to-rough morphotype alterations of clinically persistent Mycobacterium abscessus. Mol Microbiol. 2013; 90: 612-629.

[7] Ortega C, Liao R, Anderson LN, Rustad T, Ollodart AR, Wright AT, et al. Mycobacterium tuberculosis Ser/Thr protein kinase B mediates an oxygen-dependent replication switch. PLoS Biol. 2014; 12: e1001746.

[8] Baez-Ramirez E, Querales L, Aranaga CA, Lopez G, Guerrero E, Kremer L, et al. Elimination of PknL and MSMEG_4242 in Mycobacterium smegmatis alters the character of the outer cell envelope and selects for mutations in Lsr2. Cell Surf. 2021; 7: 100060.

[9] Papenfort K, Melamed S. Small RNAs, large networks: posttranscriptional regulons in Gram-negative bacteria. Annu Rev Microbiol. 2023; 77: 23-43.

[10] Bouvier M, Sharma CM, Mika F, Nierhaus KH, Vogel J. Small RNA binding to 5′ mRNA coding region inhibits translational initiation. Mol Cell. 2008; 32: 827-837.

[11] Sharma CM, Darfeuille F, Plantinga TH, Vogel J. A small RNA regulates multiple ABC transporter mRNAs by targeting C/A-rich elements inside and upstream of ribosome-binding sites. Genes Dev. 2007; 21: 2804-2817.

[12] Storz G, Vogel J, Wassarman KM. Regulation by small RNAs in bacteria: expanding frontiers. Mol Cell. 2011; 43: 880-891.

[13] Fröhlich KS, Papenfort K, Fekete A, Vogel J. A small RNA activates CFA synthase by isoform-specific mRNA stabilization. EMBO J. 2013; 32: 2963-2979.

[14] Papenfort K, Sun Y, Miyakoshi M, Vanderpool CK, Vogel J. Small RNA-mediated activation of sugar phosphatase mRNA regulates glucose homeostasis. Cell. 2013; 153: 426-437.

[15] Sedlyarova N, Shamovsky I, Bharati BK, Epshtein V, Chen J, Gottesman S, et al. sRNA-mediated control of transcription termination in E. coli. Cell. 2016; 167: 111-121.e13.

[16] Vogel J, Luisi BF. Hfq and its constellation of RNA. Nat Rev Microbiol. 2011; 9: 578-589.

[17] Smirnov A, Wang C, Drewry LL, Vogel J. Molecular mechanism of mRNA repression in trans by a ProQ-dependent small RNA. EMBO J. 2017; 36: 1029-1045.

[18] Holmqvist E, Vogel J. RNA-binding proteins in bacteria. Nat Rev Microbiol. 2018; 16: 601-615.

[19] Arnvig KB, Young DB. Identification of small RNAs in Mycobacterium tuberculosis. Mol Microbiol. 2009; 73: 397-408.

[20] DiChiara JM, Contreras-Martinez LM, Livny J, Smith D, McDonough KA, Belfort M. Multiple small RNAs identified in Mycobacterium bovis BCG are also expressed in Mycobacterium tuberculosis and Mycobacterium smegmatis. Nucleic Acids Res. 2010; 38: 4067-4078.

[21] Cortes T, Schubert OT, Rose G, Arnvig KB, Comas I, Aebersold R, et al. Genome-wide mapping of transcriptional start sites defines an extensive leaderless transcriptome in Mycobacterium tuberculosis. Cell Rep. 2013; 5: 1121-1131.

[22] Dinan AM, Tong P, Lohan AJ, Conlon KM, Miranda-CasoLuengo AA, Malone KM, et al. Relaxed selection drives a noisy noncoding transcriptome in members of the Mycobacterium tuberculosis complex. mBio. 2014; 5: e01169-14.

[23] Arnvig KB, Comas I, Thomson NR, Houghton J, Boshoff HI, Croucher NJ, et al. Sequence-based analysis uncovers an abundance of non-coding RNA in the total transcriptome of Mycobacterium tuberculosis. PLoS Pathog. 2011; 7: e1002342.

[24] Li SK, Ng PKS, Qin H, Lau JKY, Lau JPY, Tsui SKW, et al. Identification of small RNAs in Mycobacterium smegmatis using heterologous Hfq. RNA. 2013; 19: 74-84.

[25] Gerrick ER, Barbier T, Chase MR, Xu R, François J, Lin VH, et al. Small RNA profiling in Mycobacterium tuberculosis identifies MrsI as necessary for an anticipatory iron sparing response. Proc Natl Acad Sci USA. 2018; 115: 6464-6469.

[26] Mai J, Rao C, Watt J, Sun X, Lin C, Zhang L, et al. Mycobacterium tuberculosis 6C sRNA binds multiple mRNA targets via C-rich loops independent of RNA chaperones. Nucleic Acids Res. 2019; 47: 4292-4307.

[27] Bar-Oz M, Martini MC, Alonso MN, Meir M, Lore NI, Miotto P, et al. The small non-coding RNA B11 regulates multiple facets of Mycobacterium abscessus virulence. PLoS Pathog. 2023; 19: e1011575.

[28] Shell SS, Wang J, Lapierre P, Mir M, Chase MR, Pyle MM, et al. Leaderless transcripts and small proteins are common features of the mycobacterial translational landscape. PLoS Genet. 2015; 11: e1005641.

[29] Walter ND, Born SEM, Robertson GT, Reichlen M, Dide-Agossou C, Ektnitphong VA, et al. Mycobacterium tuberculosis precursor rRNA as a measure of treatment-shortening activity of drugs and regimens. Nat Commun. 2021; 12: 2899.

[30] Fröhlich KS, Papenfort K, Berger AA, Vogel J. A conserved RpoS-dependent small RNA controls the synthesis of major porin OmpD. Nucleic Acids Res. 2012; 40: 3623-3640.

[31] Rustad TR, Minch KJ, Brabant W, Winkler JK, Reiss DJ, Baliga NS, et al. Global analysis of mRNA stability in Mycobacterium tuberculosis. Nucleic Acids Res. 2013; 41: 509-517.

[32] Pelicic V, Jackson M, Reyrat JM, Jacobs, WR, Gicquel B, et al. Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci USA. 1997; 94: 10955-10960.

[33] van der Woude AD, Sarkar D, Bhatt A, Sparrius M, Raadsen SA, Boon L, et al. Unexpected link between lipooligosaccharide biosynthesis and surface protein release in Mycobacterium marinum. J Biol Chem. 2012; 287: 20417-20429.

[34] Gorski SA, Vogel J, Doudna JA. RNA-based recognition and targeting: sowing the seeds of specificity. Nat Rev Mol Cell Biol. 2017; 18: 215-228.

[35] Waters LS, Storz G. Regulatory RNAs in bacteria. Cell. 2009; 136: 615-628.

[36] Lalaouna D, Carrier MC, Semsey S, Brouard JS, Wang J, Wade JT, et al. A 3′ external transcribed spacer in a tRNA transcript acts as a sponge for small RNAs to prevent transcriptional noise. Mol Cell. 2015; 58: 393-405.

[37] Carrier MC, Laliberte G, Masse E. Identification of new bacterial small RNA targets using MS2 affinity purification coupled to RNA sequencing. Methods Mol Biol. 2018; 1737: 77-88.

[38] Bolstad BM, Irizarry RA, Åstrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics. 2003; 19: 185-193.

[39] Ray-Soni A, Bellecourt MJ, Landick R. Mechanisms of bacterial transcription termination: all good things must end. Annu Rev Biochem. 2016; 85: 319-347.

[40] Rock JM, Hopkins FF, Chavez A, Diallo M, Chase MR, Gerrick ER, et al. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nat Microbiol. 2017; 2: 16274.

[41] Klein G, Raina S. Regulated control of the assembly and diversity of LPS by noncoding sRNAs. BioMed Res Int. 2015; 2015: 153561.

[42] Moon K, Gottesman S. A PhoQ/P-regulated small RNA regulates sensitivity of Escherichia coli to antimicrobial peptides. Mol Microbiol. 2009; 74: 1314-1330.

[43] Corcoran CP, Podkaminski D, Papenfort K, Urban JH, Hinton JCD, Vogel J. Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA. Mol Microbiol. 2012; 84: 428-445.

[44] Peschek N, Herzog R, Singh PK, Sprenger M, Meyer F, Fröhlich KS, et al. RNA-mediated control of cell shape modulates antibiotic resistance in Vibrio cholerae. Nat Commun. 2020; 11: 6067.

[45] Alexander DC, Jones JRW, Tan T, Chen JM, Liu J. PimF, a mannosyltransferase of mycobacteria, is involved in the biosynthesis of phosphatidylinositol mannosides and lipoarabinomannan. J Biol Chem. 2004; 279: 18824-18833.

[46] Ren H, Dover LG, Islam ST, Alexander DC, Chen JM, Besra GS, et al. Identification of the lipooligosaccharide biosynthetic gene cluster from Mycobacterium marinum. Mol Microbiol. 2007; 63: 1345-1359.

[47] Alibaud L, Pawelczyk J, Gannoun-Zaki L, Singh VK, Rombouts Y, Drancourt M, et al. Increased phagocytosis of Mycobacterium marinum mutants defective in lipooligosaccharide production. J Biol Chem. 2014; 289: 215-228.

[48] Budell WC, Germain GA, Janisch N, McKie-Krisberg Z, Jayaprakash AD, Resnick AE, et al. Transposon mutagenesis in Mycobacterium kansasii links a small RNA gene to colony morphology and biofilm formation and identifies 9,885 intragenic insertions that do not compromise colony outgrowth. MicrobiologyOpen. 2020; 9: e988.

[49] Massé E, Gottesman S. A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci USA. 2002; 99: 4620-4625.

[50] Massé E, Vanderpool CK, Gottesman S. Effect of RyhB small RNA on global iron use in Escherichia coli. J Bacteriol. 2005; 187: 6962-6971.

[51] Updegrove TB, Shabalina SA, Storz G. How do base-pairing small RNAs evolve? FEMS Microbiol Rev. 2015; 39: 379-391.

[52] Mey AR, Craig SA, Payne SM. Characterization of Vibrio cholerae RyhB: the RyhB regulon and role of ryhB in biofilm formation. Infect Immun. 2005; 73: 5706-5719.

[53] Pernitzsch SR, Tirier SM, Beier D, Sharma CM. A variable homopolymeric G-repeat defines small RNA-mediated posttranscriptional regulation of a chemotaxis receptor in Helicobacter pylori. Proc Natl Acad Sci USA. 2014; 111: E501-E510.

[54] Chen Z, Yang Y, Chen X, Bei C, Gao Q, Chao Y, et al. An RNase III-processed sRNA coordinates sialic acid metabolism of Salmonella enterica during gut colonization. Proc Natl Acad Sci USA. 2025; 122: e2414563122.

[55] Liu F, Chen Z, Zhang S, Wu K, Bei C, Wang C, et al. In vivo RNA interactome profiling reveals 3'UTR-processed small RNA targeting a central regulatory hub. Nat Commun. 2023; 14: 8106.

[56] Huber M, Lippegaus A, Melamed S, Siemers M, Wucher BR, Hoyos M, et al. An RNA sponge controls quorum sensing dynamics and biofilm formation in Vibrio cholerae. Nat Commun. 2022; 13: 7585.

[57] Ruhland E, Siemers M, Gerst R, Späth F, Vogt LN, Figge MT, et al. The global RNA-RNA interactome of Klebsiella pneumoniae unveils a small RNA regulator of cell division. Proc Natl Acad Sci USA. 2024; 121: e2317322121.

[58] Cai Z, Cao C, Ji L, Ye R, Wang D, Xia C, et al. RIC-seq for global in situ profiling of RNA-RNA spatial interactions. Nature. 2020; 582: 432-437.

[59] Ju X, Li S, Froom R, Wang L, Lilic M, Delbeau M, et al. Incomplete transcripts dominate the Mycobacterium tuberculosis transcriptome. Nature. 2024; 627: 424-430.

[60] Li R, Li Y, Kristiansen K, Wang J. SOAP: short oligonucleotide alignment program. Bioinformatics. 2008; 24: 713-714.

[61] Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015; 12: 357-360.

[62] Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9: 357-359.

[63] Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011; 12: 323.

[64] Schwanhäusser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, et al. Global quantification of mammalian gene expression control. Nature. 2011; 473: 337-342.

RIGHTS & PERMISSIONS

2025 The Author(s). mLife published by John Wiley & Sons Australia, Ltd on behalf of Institute of Microbiology, Chinese Academy of Sciences.