Primary Characterization of Small RNAs in Symbiotic Nitrogen-Fixing Bacteria

  • Marta Robledo
  • Natalia I. García-Tomsig
  • José I. Jiménez-ZurdoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1734)


High-throughput transcriptome profiling (RNAseq) has uncovered large and heterogeneous populations of small noncoding RNA species (sRNAs) with potential regulatory roles in bacteria. A large fraction of sRNAs are differentially regulated and rely on protein-assisted antisense interactions to trans-encoded target mRNAs to fine-tune posttranscriptional reprogramming of gene expression in response to external cues. However, annotation and function of sRNAs are still largely overlooked in nonmodel bacteria with complex lifestyles. Here, we describe experimental protocols successfully applied for the accurate annotation, expression profiling and target mRNA identification of trans-acting sRNAs in the nitrogen-fixing α-rhizobium Sinorhizobium meliloti. The protocols presented here can be similarly applied for the characterization of trans-sRNAs in genetically tractable α-proteobacteria of agronomical or clinical relevance interacting with eukaryotic hosts.

Key words

RACE Northern hybridization mRNA target Genetic reporter assay GFP Riboregulation 


  1. 1.
    Gibson KE, Kobayashi H, Walker GC (2008) Molecular determinants of a symbiotic chronic infection. Annu Rev Genet 42:413–441CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Jones KM, Kobayashi H, Davies BW et al (2007) How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model. Nat Rev Microbiol 5:619–633CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Sorek R, Cossart P (2010) Prokaryotic transcriptomics: a new view on regulation, physiology and pathogenicity. Nat Rev Genet 11:9–16CrossRefPubMedGoogle Scholar
  4. 4.
    Storz G, Opdyke JA, Zhang A (2004) Controlling mRNA stability and translation with small, noncoding RNAs. Curr Opin Microbiol 7:140–144CrossRefPubMedGoogle Scholar
  5. 5.
    Jiménez-Zurdo JI, Valverde C, Becker A (2013) Insights into the noncoding RNome of nitrogen-fixing endosymbiotic α-proteobacteria. Mol Plant-Microbe Interact 26:160–167CrossRefPubMedGoogle Scholar
  6. 6.
    Jiménez-Zurdo JI, Robledo M (2015) Unraveling the universe of small RNA regulators in the legume symbiont Sinorhizobium meliloti. Symbiosis 67:43–54CrossRefGoogle Scholar
  7. 7.
    Schlüter JP, Reinkensmeier J, Daschkey S et al (2010) A genome-wide survey of sRNAs in the symbiotic nitrogen-fixing alpha-proteobacterium Sinorhizobium meliloti. BMC Genomics 11:245CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Schlüter JP, Reinkensmeier J, Barnett MJ et al (2013) Global mapping of transcription start sites and promoter motifs in the symbiotic α-proteobacterium Sinorhizobium meliloti 1021. BMC Genomics 14:156CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    del Val C, Romero-Zaliz R, Torres-Quesada O et al (2012) A survey of sRNA families in alpha-proteobacteria. RNA Biol 9:119–129CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    del Val C, Rivas E, Torres-Quesada O et al (2007) Identification of differentially expressed small non-coding RNAs in the legume endosymbiont Sinorhizobium meliloti by comparative genomics. Mol Microbiol 66:1080–1091CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Reinkensmeier J, Schlüter J-P, Giegerich R et al (2011) Conservation and occurrence of trans-encoded sRNAs in the Rhizobiales. Genes 2:925–956CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198PubMedGoogle Scholar
  13. 13.
    Robertsen BK, Aman P, Darvill AG et al (1981) Host-symbiont interactions: V. The structure of acidic extracellular polysaccharides secreted by Rhizobium leguminosarum and Rhizobium trifolii. Plant Physiol 67:389–400CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Rigaud J, Puppo A (1977) Effect of nitrite upon leghemoglobin and interaction with nitrogen fixation. Biochim Biophys Acta 497:702–706CrossRefPubMedGoogle Scholar
  15. 15.
    Torres-Quesada O, Millán V, Nisa-Martínez R et al (2013) Independent activity of the homologous small regulatory RNAs AbcR1 and AbcR2 in the legume symbiont Sinorhizobium meliloti. PLoS One 8:e68147CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Khan SR, Gaines J, Roop RM et al (2008) Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing. Appl Environ Microbiol 74:5053–5062CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Schafer A, Tauch A, Jager W et al (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73CrossRefPubMedGoogle Scholar
  18. 18.
    Torres-Quesada O, Reinkensmeier J, Schlueter J-P et al (2014) Genome-wide profiling of Hfq-binding RNAs uncovers extensive post-transcriptional rewiring of major stress response and symbiotic regulons in Sinorhizobium meliloti. RNA Biol 11:563–579CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Waters LS, Storz G (2009) Regulatory RNAs in bacteria. Cell 136:615–628CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Baumgardt K, Šmídová K, Rahn H et al (2015) The stress-related, rhizobial small RNA RcsR1 destabilizes the autoinducer synthase encoding mRNA sinI in Sinorhizobium meliloti. RNA Biol 13:486–499CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Robledo M, Frage B, Wright PR et al (2015) A stress-induced small RNA modulates alpha-rhizobial cell cycle progression. PLoS Genet 11:e1005153CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Robledo M, Jiménez-Zurdo JI, Becker A (2015) Antisense transcription of symbiotic genes in Sinorhizobium meliloti. Symbiosis 67:55–67CrossRefGoogle Scholar
  23. 23.
    McIntosh M, Meyer S, Becker A (2009) Novel Sinorhizobium meliloti quorum sensing positive and negative regulatory feedback mechanisms respond to phosphate availability. Mol Microbiol 74:1238–1256CrossRefPubMedGoogle Scholar
  24. 24.
    Wright PR, Richter AS, Papenfort K et al (2013) Comparative genomics boosts target prediction for bacterial small RNAs. Proc Natl Acad Sci U S A 110:E3487–E3496CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Wright PR, Georg J, Mann M et al (2014) CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains. Nucleic Acids Res 42(Web Server issue):W119–W123CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Will S, Joshi T, Hofacker IL et al (2012) LocARNA-P: accurate boundary prediction and improved detection of structural RNAs. RNA 18:900–914CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Charoenpanich P, Meyer S, Becker A et al (2013) Temporal expression program of quorum sensing-based transcription regulation in Sinorhizobium meliloti. J Bacteriol 195:3224–3236CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Marta Robledo
    • 1
  • Natalia I. García-Tomsig
    • 1
  • José I. Jiménez-Zurdo
    • 1
    Email author
  1. 1.Grupo de Ecología Genética de la RizosferaEstación Experimental del Zaidín (CSIC)GranadaSpain

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