1887

Abstract

Small non-coding RNAs (sRNAs) are well-established post-transcriptional regulators of gene expression in bacteria that respond to a variety of environmental stimuli. They usually act by base-pairing with their target mRNAs, which is commonly facilitated by the RNA chaperone Hfq. In this study we initiated the analysis of the sRNA FnrS of Neisseria gonorrhoeae, which is induced under anaerobic conditions. We identified four putative FnrS target genes using bioinformatics approaches and validated these target genes using translational reporter gene fusions in both Escherichia coli and N. gonorrhoeae, thereby demonstrating their downregulation by direct base-pairing between the respective mRNA and FnrS. We demonstrate deregulation of target mRNAs upon deletion of fnrS and provide evidence that the isc gene cluster required for iron–sulfur cluster biosynthesis, which harbours iscS, which is a direct target of FnrS, is coordinately downregulated by the sRNA. By mutational analysis we show that, surprisingly, three distinct regions of FnrS are employed for interaction with different target genes.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000484
2017-07-10
2019-10-17
Loading full text...

Full text loading...

/deliver/fulltext/micro/163/7/1081.html?itemId=/content/journal/micro/10.1099/mic.0.000484&mimeType=html&fmt=ahah

References

  1. Lewis DA. Global resistance of Neisseria gonorrhoeae: when theory becomes reality. Curr Opin Infect Dis 2014;27:62–67 [CrossRef][PubMed]
    [Google Scholar]
  2. Hook EW, Hansfield HH. Gonococcal infection in the adult. In Holmes KK. (editor) Sexually Transmitted Diseases New York, NY: McGraw-Hill; 2008; pp.627–645
    [Google Scholar]
  3. Knapp JS, Clark VL. Anaerobic growth of Neisseria gonorrhoeae coupled to nitrite reduction. Infect Immun 1984;46:176–181[PubMed]
    [Google Scholar]
  4. Barth KR, Isabella VM, Clark VL. Biochemical and genomic analysis of the denitrification pathway within the genus Neisseria. Microbiology 2009;155:4093–4103 [CrossRef][PubMed]
    [Google Scholar]
  5. Clark VL, Knapp JS, Thompson S, Klimpel KW. Presence of antibodies to the major anaerobically induced gonococcal outer membrane protein in sera from patients with gonococcal infections. Microb Pathog 1988;5:381–390 [CrossRef][PubMed]
    [Google Scholar]
  6. Falsetta ML, Bair TB, Ku SC, Vanden Hoven RN, Steichen CT et al. Transcriptional profiling identifies the metabolic phenotype of gonococcal biofilms. Infect Immun 2009;77:3522–3532 [CrossRef][PubMed]
    [Google Scholar]
  7. Philipps NJ, Steichen CT, Schilling B, Post DMB, Niles RK et al. Proteomic analysis of Neisseria gonorrhoeae biofilms shows shift to anaerobic respiration and changes in nutrient transport and outer membrane proteins. PLoS ONE 2012;6:e38303
    [Google Scholar]
  8. Steichen CT, Shao JQ, Ketterer MR, Apicella MA. Gonococcal cervicitis: a role for biofilm in pathogenesis. J Infect Dis 2008;198:1856–1861 [CrossRef][PubMed]
    [Google Scholar]
  9. Isabella VM, Clark VL. Deep sequencing-based analysis of the anaerobic stimulon in Neisseria gonorrhoeae. BMC Genomics 2011;12:51 [CrossRef][PubMed]
    [Google Scholar]
  10. Whitehead RN, Overton TW, Snyder LA, Mcgowan SJ, Smith H et al. The small FNR regulon of Neisseria gonorrhoeae: comparison with the larger Escherichia coli FNR regulon and interaction with the NarQ-NarP regulon. BMC Genomics 2007;8:35 [CrossRef][PubMed]
    [Google Scholar]
  11. Crack JC, Green J, Hutchings MI, Thomson AJ, Le Brun NE. Bacterial iron-sulfur regulatory proteins as biological sensor-switches. Antioxid Redox Signal 2012;17:1215–1231 [CrossRef][PubMed]
    [Google Scholar]
  12. Fantappiè L, Oriente F, Muzzi A, Serruto D, Scarlato V et al. A novel Hfq-dependent sRNA that is under FNR control and is synthesized in oxygen limitation in Neisseria meningitidis. Mol Microbiol 2011;80:507–523 [CrossRef][PubMed]
    [Google Scholar]
  13. Storz G, Vogel J, Wassarman KM. Regulation by small RNAs in bacteria: expanding frontiers. Mol Cell 2011;43:880–891 [CrossRef][PubMed]
    [Google Scholar]
  14. de Lay N, Schu DJ, Gottesman S. Bacterial small RNA-based negative regulation: Hfq and its accomplices. J Biol Chem 2013;288:7996–8003 [CrossRef]
    [Google Scholar]
  15. Papenfort K, Vanderpool CK. Target activation by regulatory RNAs in bacteria. FEMS Microbiol Rev 2015;39:286–300 [CrossRef][PubMed]
    [Google Scholar]
  16. Georg J, Hess WR. cis-antisense RNA, another level of gene regulation in bacteria. Microbiol Mol Biol Rev 2011;75:286–300 [CrossRef]
    [Google Scholar]
  17. Mellin JR, Goswami S, Grogan S, Tjaden B, Genco CA. A novel Fur- and iron-regulated small RNA, NrrF, is required for indirect Fur-mediated regulation of the sdhA and sdhC genes in Neisseria meningitidis. J Bacteriol 2007;189:3686–3694 [CrossRef][PubMed]
    [Google Scholar]
  18. Ducey TF, Jackson L, Orvis J, Dyer DW. Transcript analysis of nrrF, a Fur repressed sRNA of Neisseria gonorrhoeae. Microb Pathog 2009;46:166–170 [CrossRef][PubMed]
    [Google Scholar]
  19. Metruccio MM, Fantappiè L, Serruto D, Muzzi A, Roncarati D et al. The Hfq-dependent small noncoding RNA NrrF directly mediates Fur-dependent positive regulation of succinate dehydrogenase in Neisseria meningitidis. J Bacteriol 2009;191:1330–1342 [CrossRef][PubMed]
    [Google Scholar]
  20. Jackson LA, Pan JC, Day MW, Dyer DW. Control of RNA stability by NrrF, an iron-regulated small RNA in Neisseria gonorrhoeae. J Bacteriol 2013;195:5166–5173 [CrossRef][PubMed]
    [Google Scholar]
  21. Del Tordello E, Bottini S, Muzzi A, Serruto D. Analysis of the regulated transcriptome of Neisseria meningitidis in human blood using a tiling array. J Bacteriol 2012;194:6217–6232 [CrossRef][PubMed]
    [Google Scholar]
  22. Fagnocchi L, Bottini S, Golfieri G, Fantappiè L, Ferlicca F et al. Global transcriptome analysis reveals small RNAs affecting Neisseria meningitidis bacteremia. PLoS One 2015;10:e0126325 [CrossRef][PubMed]
    [Google Scholar]
  23. Cahoon LA, Seifert HS. Transcription of a cis-acting, noncoding, small RNA is required for pilin antigenic variation in Neisseria gonorrhoeae. PLoS Pathog 2013;9:e1003074 [CrossRef][PubMed]
    [Google Scholar]
  24. Tan FY, Wörmann ME, Loh E, Tang CM, Exley RM. Characterization of a novel antisense RNA in the major pilin locus of Neisseria meningitidis influencing antigenic variation. J Bacteriol 2015;197:1757–1768 [CrossRef][PubMed]
    [Google Scholar]
  25. Urban JH, Vogel J. Translational control and target recognition by Escherichia coli small RNAs in vivo. Nucleic Acids Res 2007;35:1018–1037 [CrossRef][PubMed]
    [Google Scholar]
  26. Corcoran CP, Podkaminski D, Papenfort K, Urban JH, Hinton JC et al. Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA. Mol Microbiol 2012;84:428–445 [CrossRef][PubMed]
    [Google Scholar]
  27. Sittka A, Pfeiffer V, Tedin K, Vogel J. The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium. Mol Microbiol 2007;63:193–217 [CrossRef][PubMed]
    [Google Scholar]
  28. Remmele CW, Xian Y, Albrecht M, Faulstich M, Fraunholz M et al. Transcriptional landscape and essential genes of Neisseria gonorrhoeae. Nucleic Acids Res 2014;42:10579–10595 [CrossRef][PubMed]
    [Google Scholar]
  29. Ramsey ME, Hackett KT, Kotha C, Dillard JP. New complementation constructs for inducible and constitutive gene expression in Neisseria gonorrhoeae and Neisseria meningitidis. Appl Environ Microbiol 2012;78:3068–3078 [CrossRef][PubMed]
    [Google Scholar]
  30. Brosius J. Superpolylinkers in cloning and expression vectors. DNA 1989;8:759–777 [CrossRef][PubMed]
    [Google Scholar]
  31. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001;25:402–408 [CrossRef]
    [Google Scholar]
  32. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012;9:671–675 [CrossRef][PubMed]
    [Google Scholar]
  33. Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 2003;31:3406–3415 [CrossRef][PubMed]
    [Google Scholar]
  34. Kery MB, Feldman M, Livny J, Tjaden B. TargetRNA2: identifying targets of small regulatory RNAs in bacteria. Nucleic Acids Res 2014;42:W124–W129 [CrossRef][PubMed]
    [Google Scholar]
  35. Wright PR, Richter AS, Papenfort K, Mann M, Vogel J et al. Comparative genomics boosts target prediction for bacterial small RNAs. Proc Natl Acad Sci USA 2013;110:E3487E3496 [CrossRef][PubMed]
    [Google Scholar]
  36. Wright PR, Georg J, Mann M, Sorescu DA, Richter AS et al. CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains. Nucleic Acids Res 2014;42:W119–W123 [CrossRef][PubMed]
    [Google Scholar]
  37. McClure R, Tjaden B, Genco C. Identification of sRNAs expressed by the human pathogen Neisseria gonorrhoeae under disparate growth conditions. Front Microbiol 2014;5:456 [CrossRef][PubMed]
    [Google Scholar]
  38. Wachter J, Hill SA. Small transcriptome analysis indicates that the enzyme RppH influences both the quality and quantity of sRNAs in Neisseria gonorrhoeae. FEMS Microbiol Lett 2015;362:1–7 [CrossRef]
    [Google Scholar]
  39. Capel E, Zomer AL, Nussbaumer T, Bole C, Izac B et al. Comprehensive identification of meningococcal genes and small noncoding RNAs required for host cell colonization. MBio 2016;7:e01173-16 [CrossRef]
    [Google Scholar]
  40. Boysen A, Møller-Jensen J, Kallipolitis B, Valentin-Hansen P, Overgaard M. Translational regulation of gene expression by an anaerobically induced small non-coding RNA in Escherichia coli. J Biol Chem 2010;285:10690–10702 [CrossRef][PubMed]
    [Google Scholar]
  41. Durand S, Storz G. Reprogramming of anaerobic metabolism by the FnrS small RNA. Mol Microbiol 2010;75:1215–1231 [CrossRef][PubMed]
    [Google Scholar]
  42. Vogel J, Luisi BF. Hfq and its constellation of RNA. Nat Rev Microbiol 2011;9:578–589 [CrossRef][PubMed]
    [Google Scholar]
  43. Dietrich M, Munke R, Gottschald M, Ziska E, Boettcher JP et al. The effect of hfq on global gene expression and virulence in Neisseria gonorrhoeae. FEBS 2009;276:5507–5520 [CrossRef][PubMed]
    [Google Scholar]
  44. Fröhlich KS, Haneke K, Papenfort K, Vogel J. The target spectrum of SdsR small RNA in Salmonella. Nucleic Acids Res 2016;44:10406–10422 [CrossRef][PubMed]
    [Google Scholar]
  45. Sergiev PV, Bogdanov AA, Dontsova OA. Ribosomal RNA guanine-(N2)-methyltransferases and their targets. Nucleic Acids Res 2007;35:2295–2301 [CrossRef][PubMed]
    [Google Scholar]
  46. Giel JL, Nesbit AD, Mettert EL, Fleischhacker AS, Wanta BT et al. Regulation of iron–sulphur cluster homeostasis through transcriptional control of the Isc pathway by [2Fe–2S]–IscR in Escherichia coli. Mol Microbiol 2013;87:478–492 [CrossRef][PubMed]
    [Google Scholar]
  47. Schwartz CJ, Djaman O, Imlay JA, Kiley PJ. The cysteine desulfurase, IscS, has a major role in in vivo Fe-S cluster formation in Escherichia coli. Proc Natl Acad Sci USA 2000;97:9009–9014 [CrossRef][PubMed]
    [Google Scholar]
  48. Rice JB, Vanderpool CK. The small RNA SgrS controls sugar–phosphate accumulation by regulating multiple PTS genes. Nucleic Acids Res 2011;39:3806–3819 [CrossRef][PubMed]
    [Google Scholar]
  49. Rice JB, Balasubramanian D, Vanderpool CK. Small RNA binding-site multiplicity involved in translational regulation of a polycistronic mRNA. Proc Natl Acad Sci USA 2012;109:E2691E2698 [CrossRef][PubMed]
    [Google Scholar]
  50. Lu P, Zhang Y, Li L, Hu Y, Huang L et al. Small non-coding RNA SraG regulates the operon YPK_1206-1205 in Yersinia pseudotuberculosis. FEMS Microbiol Lett 2012;331:37–43 [CrossRef][PubMed]
    [Google Scholar]
  51. Masse 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 [CrossRef]
    [Google Scholar]
  52. Massé E, Vanderpool CK, Gottesman S. Effect of RyhB small RNA on global iron use in Escherichia coli. J Bacteriol 2005;187:6962–6971 [CrossRef][PubMed]
    [Google Scholar]
  53. Desnoyers G, Morissette A, Prévost K, Massé E. Small RNA-induced differential degradation of the polycistronic mRNA iscRSUA. EMBO J 2009;28:1551–1561 [CrossRef][PubMed]
    [Google Scholar]
  54. Giel JL, Rodionov D, Liu M, Blattner FR, Kiley PJ. IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O2-regulated genes in Escherichia coli. Mol Microbiol 2006;60:1058–1075 [CrossRef][PubMed]
    [Google Scholar]
  55. Yeo WS, Lee JH, Lee KC, Roe JH. IscR acts as an activator in response to oxidative stress for the suf operon encoding Fe-S assembly proteins. Mol Microbiol 2006;61:206–218 [CrossRef][PubMed]
    [Google Scholar]
  56. Papenfort K, Bouvier M, Mika F, Sharma CM, Vogel J. Evidence for an autonomous 5' target recognition domain in an Hfq-associated small RNA. Proc Natl Acad Sci USA 2010;107:20435–20440 [CrossRef][PubMed]
    [Google Scholar]
  57. 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 [CrossRef][PubMed]
    [Google Scholar]
  58. Beisel CL, Updegrove TB, Janson BJ, Storz G. Multiple factors dictate target selection by Hfq-binding small RNAs. EMBO J 2012;31:1961–1974 [CrossRef][PubMed]
    [Google Scholar]
  59. Sharma CM, Papenfort K, Pernitzsch SR, Mollenkopf HJ, Hinton JC et al. Pervasive post-transcriptional control of genes involved in amino acid metabolism by the Hfq-dependent GcvB small RNA. Mol Microbiol 2011;81:1144–1165 [CrossRef][PubMed]
    [Google Scholar]
  60. Coornaert A, Chiaruttini C, Springer M, Guillier M. Post-transcriptional control of the Escherichia coli PhoQ-PhoP two-component system by multiple sRNAs involves a novel pairing region of GcvB. PLoS Genet 2013;9:e1003156 [CrossRef][PubMed]
    [Google Scholar]
  61. Papenfort K, Förstner KU, Cong JP, Sharma CM, Bassler BL. Differential RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm formation. Proc Natl Acad Sci USA 2015;112:E766E775 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000484
Loading
/content/journal/micro/10.1099/mic.0.000484
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error