1887

Abstract

In the N-fixing symbiont of alfalfa root nodules, 2011, the gene encodes a 77 nt small untranslated RNA (sRNA) that negatively regulates the accumulation of polyhydroxybutyrate (PHB) when the bacterium is grown under conditions of surplus carbon (C) in relation to nitrogen (N). We previously showed that the expression of is primarily controlled at the transcriptional level and that it depends on the cellular N status, although the regulatory mechanism and the factors involved were unknown. In this study, we provide experimental data supporting that: (a) is induced upon N limitation with the maximum expression found at the highest tested C/N molar ratio in the growth medium; (b) a conserved heptamer TTGTGCA located between the −35 and −10 promoter elements is necessary and sufficient for induction by N limitation; (c) induction of requires the N-status regulator NtrC; (d) under C limitation, transcription is repressed by AniA, a global regulator of C flow; (e) the promoter contains a conserved dyadic motif (TGC[N]GCA) partially overlapping the heptamer TTGTGCA, which was also found in the promoters of the PHB-related genes , , and () of and other alpha-proteobacteria. Taken together, these results suggest that the promoter would integrate signals from the metabolism of C and N through – at least – the global regulators NtrC and AniA, to provide an optimal level of the MmgR sRNA to fine-tune gene expression post-transcriptionally according to varying C and N availability.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000586
2018-01-01
2020-01-27
Loading full text...

Full text loading...

/deliver/fulltext/micro/164/1/88.html?itemId=/content/journal/micro/10.1099/mic.0.000586&mimeType=html&fmt=ahah

References

  1. Wagner EG, Romby P. Small RNAs in bacteria and archaea: who they are, what they do, and how they do it. Adv Genet 2015;90:133–208 [CrossRef][PubMed]
    [Google Scholar]
  2. 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 [CrossRef][PubMed]
    [Google Scholar]
  3. Attaiech L, Boughammoura A, Brochier-Armanet C, Allatif O, Peillard-Fiorente F et al. Silencing of natural transformation by an RNA chaperone and a multitarget small RNA. Proc Natl Acad Sci USA 2016;113:8813–8818 [CrossRef][PubMed]
    [Google Scholar]
  4. Smirnov A, Förstner KU, Holmqvist E, Otto A, Günster R et al. Grad-seq guides the discovery of ProQ as a major small RNA-binding protein. Proc Natl Acad Sci USA 2016;113:11591–11596 [CrossRef][PubMed]
    [Google Scholar]
  5. Vogel J, Luisi BF. Hfq and its constellation of RNA. Nat Rev Microbiol 2011;9:578–589 [CrossRef][PubMed]
    [Google Scholar]
  6. Levine E, Zhang Z, Kuhlman T, Hwa T. Quantitative characteristics of gene regulation by small RNA. PLoS Biol 2007;5:e229 [CrossRef][PubMed]
    [Google Scholar]
  7. Viegas SC, Arraiano CM. Regulating the regulators: how ribonucleases dictate the rules in the control of small non-coding RNAs. RNA Biol 2008;5:230–243 [CrossRef][PubMed]
    [Google Scholar]
  8. Göpel Y, Görke B. Rewiring two-component signal transduction with small RNAs. Curr Opin Microbiol 2012;15:132–139 [CrossRef][PubMed]
    [Google Scholar]
  9. Valverde C, Haas D. Small RNAs controlled by two-component systems. Adv Exp Med Biol 2008;631:54–79 [CrossRef][PubMed]
    [Google Scholar]
  10. Beisel CL, Storz G. Base pairing small RNAs and their roles in global regulatory networks. FEMS Microbiol Rev 2010;34:866–882 [CrossRef][PubMed]
    [Google Scholar]
  11. Richards GR, Vanderpool CK. Molecular call and response: the physiology of bacterial small RNAs. Biochim Biophys Acta 2011;1809:525–531 [CrossRef][PubMed]
    [Google Scholar]
  12. Massé E, Salvail H, Desnoyers G, Arguin M. Small RNAs controlling iron metabolism. Curr Opin Microbiol 2007;10:140–145 [CrossRef][PubMed]
    [Google Scholar]
  13. Becker A, Overlöper A, Schlüter JP, Reinkensmeier J, Robledo M et al. Riboregulation in plant-associated α-proteobacteria. RNA Biol 2014;11:550–562 [CrossRef][PubMed]
    [Google Scholar]
  14. Roux B, Rodde N, Jardinaud MF, Timmers T, Sauviac L et al. An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing. Plant J 2014;77:817–837 [CrossRef][PubMed]
    [Google Scholar]
  15. Robledo M, Frage B, Wright PR, Becker A. A stress-induced small RNA modulates alpha-rhizobial cell cycle progression. PLoS Genet 2015;11:e1005153 [CrossRef][PubMed]
    [Google Scholar]
  16. Robledo M, Peregrina A, Millán V, García-Tomsig NI, Torres-Quesada O et al. A conserved α-proteobacterial small RNA contributes to osmoadaptation and symbiotic efficiency of rhizobia on legume roots. Environ Microbiol 2017;19:2661–2680 [CrossRef][PubMed]
    [Google Scholar]
  17. Baumgardt K, Šmídová K, Rahn H, Lochnit G, Robledo M et al. The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti. RNA Biol 2016;13:486–499 [CrossRef][PubMed]
    [Google Scholar]
  18. Torres-Quesada O, Millán V, Nisa-Martínez R, Bardou F, Crespi M et al. Independent activity of the homologous small regulatory RNAs AbcR1 and AbcR2 in the legume symbiont Sinorhizobium meliloti. PLoS One 2013;8:e68147 [CrossRef][PubMed]
    [Google Scholar]
  19. Torres-Quesada O, Reinkensmeier J, Schlüter JP, Robledo M, Peregrina A et al. Genome-wide profiling of Hfq-binding RNAs uncovers extensive post-transcriptional rewiring of major stress response and symbiotic regulons in Sinorhizobium meliloti. RNA Biol 2014;11:563–579 [CrossRef][PubMed]
    [Google Scholar]
  20. Sobrero P, Valverde C. Evidences of autoregulation of hfq expression in Sinorhizobium meliloti strain 2011. Arch Microbiol 2011;193:629–639 [CrossRef][PubMed]
    [Google Scholar]
  21. Lagares Jr A, Roux I, Valverde C. Phylogenetic distribution and evolutionary pattern of an α-proteobacterial small RNA gene that controls polyhydroxybutyrate accumulation in Sinorhizobium meliloti. Mol Phylogenet Evol 2016;99:182–193 [CrossRef][PubMed]
    [Google Scholar]
  22. Lagares Jr A, Ceizel Borella G, Linne U, Becker A, Valverde C. Regulation of polyhydroxybutyrate accumulation in Sinorhizobium meliloti by the trans-encoded small RNA MmgR. J Bacteriol 2017;199:e00776-16 [CrossRef][PubMed]
    [Google Scholar]
  23. Ceizel Borella G, Lagares Jr A, Valverde C. Expression of the Sinorhizobium meliloti small RNA gene mmgR is controlled by the nitrogen source. FEMS Microbiol Lett 2016;363:fnw069 [CrossRef][PubMed]
    [Google Scholar]
  24. Vincent JM. A Manual for the Practical Study of Root Nodule Bacteria Oxford: Blackwell Scientific Publications; 1970
    [Google Scholar]
  25. Sambrook J, Fritsch E, Maniatis T. Molecular Cloning: A Laboratory Manual New York: Cold Spring Harbor Laboratory; 1989
    [Google Scholar]
  26. del Sal G, Manfioletti G, Schneider C. A one-tube plasmid DNA mini-preparation suitable for sequencing. Nucleic Acids Res 1988;16:9878 [CrossRef][PubMed]
    [Google Scholar]
  27. Bahlawane C, Baumgarth B, Serrania J, Rüberg S, Becker A. Fine-tuning of galactoglucan biosynthesis in Sinorhizobium meliloti by differential WggR (ExpG)-, PhoB-, and MucR-dependent regulation of two promoters. J Bacteriol 2008;190:3456–3466 [CrossRef][PubMed]
    [Google Scholar]
  28. Simon R, Quandt J, Klipp W. New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria. Gene 1989;80:161–169 [CrossRef][PubMed]
    [Google Scholar]
  29. Valverde C, Livny J, Schlüter JP, Reinkensmeier J, Becker A et al. Prediction of Sinorhizobium meliloti sRNA genes and experimental detection in strain 2011. BMC Genomics 2008;9:416 [CrossRef][PubMed]
    [Google Scholar]
  30. Abramoff MD, Magelhaes PJ, Ram SJ. Image processing with ImageJ. Biophotonics International 2004;11:36–42
    [Google Scholar]
  31. Yurgel SN, Rice J, Kahn ML. Transcriptome analysis of the role of GlnD/GlnBK in nitrogen stress adaptation by Sinorhizobium meliloti Rm1021. PLoS One 2013;8:e58028 [CrossRef][PubMed]
    [Google Scholar]
  32. Ow DW, Sundaresan V, Rothstein DM, Brown SE, Ausubel FM. Promoters regulated by the glnG (ntrC) and nifA gene products share a heptameric consensus sequence in the -15 region. Proc Natl Acad Sci USA 1983;80:2524–2528[PubMed]
    [Google Scholar]
  33. Szeto WW, Nixon BT, Ronson CW, Ausubel FM. Identification and characterization of the Rhizobium meliloti ntrC gene: R. meliloti has separate regulatory pathways for activation of nitrogen fixation genes in free-living and symbiotic cells. J Bacteriol 1987;169:1423–1432 [CrossRef][PubMed]
    [Google Scholar]
  34. Povolo S, Casella S. A critical role for aniA in energy-carbon flux and symbiotic nitrogen fixation in Sinorhizobium meliloti. Arch Microbiol 2000;174:42–49 [CrossRef][PubMed]
    [Google Scholar]
  35. Encarnación S, del Carmen Vargas M, Dunn MF, Dávalos A, Mendoza G et al. AniA regulates reserve polymer accumulation and global protein expression in Rhizobium etli. J Bacteriol 2002;184:2287–2295 [CrossRef][PubMed]
    [Google Scholar]
  36. Medina-Rivera A, Defrance M, Sand O, Herrmann C, Castro-Mondragon JA et al. RSAT 2015: regulatory sequence analysis tools. Nucleic Acids Res 2015;43:W50–W56 [CrossRef][PubMed]
    [Google Scholar]
  37. Yamada M, Yamashita K, Wakuda A, Ichimura K, Maehara A et al. Autoregulator protein PhaR for biosynthesis of polyhydroxybutyrate [P(3HB)] possibly has two separate domains that bind to the target DNA and P(3HB): functional mapping of amino acid residues responsible for DNA binding. J Bacteriol 2007;189:1118–1127 [CrossRef][PubMed]
    [Google Scholar]
  38. Chou ME, Yang MK. Analyses of binding sequences of the PhaR protein of Rhodobacter sphaeroides FJ1. FEMS Microbiol Lett 2010;302:138–143 [CrossRef][PubMed]
    [Google Scholar]
  39. D'Alessio M, Nordeste R, Doxey AC, Charles TC. Transcriptome analysis of polyhydroxybutyrate cycle mutants reveals discrete loci connecting nitrogen utilization and carbon storage in Sinorhizobium meliloti. mSystems 2017;2:e00035-17 [CrossRef][PubMed]
    [Google Scholar]
  40. Brown NL, Stoyanov JV, Kidd SP, Hobman JL. The MerR family of transcriptional regulators. FEMS Microbiol Rev 2003;27:145–163 [CrossRef][PubMed]
    [Google Scholar]
  41. Prell J, White JP, Bourdes A, Bunnewell S, Bongaerts RJ et al. Legumes regulate Rhizobium bacteroid development and persistence by the supply of branched-chain amino acids. Proc Natl Acad Sci USA 2009;106:12477–12482 [CrossRef][PubMed]
    [Google Scholar]
  42. Nitzan M, Rehani R, Margalit H. Integration of bacterial small RNAs in regulatory networks. Annu Rev Biophys 2017;46:131–148 [CrossRef][PubMed]
    [Google Scholar]
  43. Shimoni Y, Friedlander G, Hetzroni G, Niv G, Altuvia S et al. Regulation of gene expression by small non-coding RNAs: a quantitative view. Mol Syst Biol 2007;3:138 [CrossRef][PubMed]
    [Google Scholar]
  44. Sun J, Peng X, van Impe J, Vanderleyden J. The ntrB and ntrC genes are involved in the regulation of poly-3-hydroxybutyrate biosynthesis by ammonia in Azospirillum brasilense Sp7. Appl Environ Microbiol 2000;66:113–117 [CrossRef][PubMed]
    [Google Scholar]
  45. Finan TM, Kunkel B, de Vos GF, Signer ER. Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol 1986;167:66–72 [CrossRef][PubMed]
    [Google Scholar]
  46. Simon R, Priefer U, Pühler A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Biotechnology 1983;1:784–791 [CrossRef]
    [Google Scholar]
  47. Meade HM, Signer ER. Genetic mapping of Rhizobium meliloti. Proc Natl Acad Sci USA 1977;74:2076–2078 [CrossRef][PubMed]
    [Google Scholar]
  48. Li W, Cowley A, Uludag M, Gur T, McWilliam H et al. The EMBL-EBI bioinformatics web and programmatic tools framework. Nucleic Acids Res 2015;43:W580–W584 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000586
Loading
/content/journal/micro/10.1099/mic.0.000586
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