Azotobacter vinelandii Small RNAs: Their Roles in the Formation of Cysts and Other Processes

  • Miguel Castañeda
  • Liliana López-Pliego
  • Guadalupe Espín


Azotobacter vinelandii is a soil bacterium that undergoes differentiation to form cysts resistant to desiccation. Alginate, polyhydroxybutyrate (PHB) and alkylresorcinols (AR) are structural components of the cysts. The synthesis of these compounds has been shown to be under control of the Gac/Rsm signal transduction pathway. This pathway includes eight small non-coding RNAs (RsmZ1-7 and RsmY ), and the translational repressor protein RsmA. Binding of RsmA to Rsm-RNAs inhibits its repressor activity. Here, we review and discuss the roles of sRNAs in the formation of cysts and other processes in A. vinelandii. We also report a search for genes encoding small RNAs in the A. vinelandii genome . Two new sRNAs potentially related to the control of cyst components synthesis were identified in this search.


Carbohydrate Superoxide Respiration Polysaccharide Polypeptide 


  1. Babitzke P, Romeo T (2007) CsrB sRNA family: sequestration of RNA-binding regulatory proteins. Curr Opin Microbiol 10(2):156–163. doi: 10.1016/j.mib.2007.03.007 PubMedCrossRefGoogle Scholar
  2. Beisel CL, Storz G (2011) The base-pairing RNA spot 42 participates in a multioutput feedforward loop to help enact catabolite repression in Escherichia coli. Mol Cell 41(3):286–297. doi: 10.1016/j.molcel.2010.12.027 PubMedPubMedCentralCrossRefGoogle Scholar
  3. Brantl S (2002) Antisense-RNA regulation and RNA interference. Biochim Biophys Acta 1575(1–3):15–25PubMedCrossRefGoogle Scholar
  4. Brencic A, McFarland KA, McManus HR, Castang S, Mogno I, Dove SL, Lory S (2009) The GacS/GacA signal transduction system of Pseudomonas aeruginosa acts exclusively through its control over the transcription of the RsmY and RsmZ regulatory small RNAs. Mol Microbiol 73(3):434–445. doi: 10.1111/j.1365-2958.2009.06782.x PubMedPubMedCentralCrossRefGoogle Scholar
  5. Campos M, Martinez-Salazar JM, Lloret L, Moreno S, Nunez C, Espin G, Soberon-Chavez G (1996) Characterization of the gene coding for GDP-mannose dehydrogenase (algD) from Azotobacter vinelandii. J Bacteriol 178(7):1793–1799PubMedPubMedCentralGoogle Scholar
  6. Casida LE (1983) Interaction of Agromyces ramosus with Other Bacteria in Soil. Appl Environ Microbiol 46(4):881–888PubMedPubMedCentralGoogle Scholar
  7. Castañeda M, Guzman J, Moreno S, Espin G (2000) The GacS sensor kinase regulates alginate and poly-beta-hydroxybutyrate production in Azotobacter vinelandii. J Bacteriol 182(9):2624–2628PubMedPubMedCentralCrossRefGoogle Scholar
  8. Castañeda M, Sanchez J, Moreno S, Nunez C, Espin G (2001) The global regulators GacA and sigma(S) form part of a cascade that controls alginate production in Azotobacter vinelandii. J Bacteriol 183(23):6787–6793. doi: 10.1128/JB.183.23.6787-6793.2001 PubMedPubMedCentralCrossRefGoogle Scholar
  9. Citron M, Schuster H (1990) The c4 repressors of bacteriophages P1 and P7 are antisense RNAs. Cell 62(3):591–598PubMedCrossRefGoogle Scholar
  10. Cocotl-Yañez M, Moreno S, Encarnacion S, Lopez-Pliego L, Castañeda M, Espin G (2014) A small heat-shock protein (Hsp20) regulated by RpoS is essential for cyst desiccation resistance in Azotobacter vinelandii. Microbiology 160(Pt 3):479–487. doi: 10.1099/mic.0.073353-0 PubMedCrossRefGoogle Scholar
  11. Escolar L, Perez-Martin J, de Lorenzo V (1999) Opening the iron box: transcriptional metalloregulation by the Fur protein. J Bacteriol 181(20):6223–6229PubMedPubMedCentralGoogle Scholar
  12. Ferrara S, Carloni S, Fulco R, Falcone M, Macchi R, Bertoni G (2015) Post-transcriptional regulation of the virulence-associated enzyme AlgC by the sigma(22)-dependent small RNA ErsA of Pseudomonas aeruginosa. Environ Microbiol 17(1):199–214. doi: 10.1111/1462-2920.12590 PubMedCrossRefGoogle Scholar
  13. Filiatrault MJ, Stodghill PV, Wilson J, Butcher BG, Chen H, Myers CR, Cartinhour SW (2013) CrcZ and CrcX regulate carbon source utilization in Pseudomonas syringae pathovar tomato strain DC3000. RNA Biol 10(2):245–255. doi: 10.4161/rna.23019 PubMedPubMedCentralCrossRefGoogle Scholar
  14. Gaona G, Nunez C, Goldberg JB, Linford AS, Najera R, Castañeda M, Guzman J, Espin G, Soberon-Chavez G (2004) Characterization of the Azotobacter vinelandii algC gene involved in alginate and lipopolysaccharide production. FEMS Microbiol Lett 238(1):199–206. doi: 10.1016/j.femsle.2004.07.044 PubMedGoogle Scholar
  15. Gonzalez-Casanova A, Aguirre-von-Wobeser E, Espin G, Servin-Gonzalez L, Kurt N, Spano D, Blath J, Soberon-Chavez G (2014) Strong seed-bank effects in bacterial evolution. J Theor Biol 356:62–70. doi: 10.1016/j.jtbi.2014.04.009 PubMedCrossRefGoogle Scholar
  16. Hernandez-Eligio A, Moreno S, Castellanos M, Castañeda M, Nunez C, Muriel-Millan LF, Espin G (2012) RsmA post-transcriptionally controls PhbR expression and polyhydroxybutyrate biosynthesis in Azotobacter vinelandii. Microbiology 158(Pt 8):1953–1963. doi: 10.1099/mic.0.059329-0 PubMedCrossRefGoogle Scholar
  17. Heroven AK, Bohme K, Dersch P (2012) Regulation of virulence gene expression by regulatory RNA elements in Yersinia pseudotuberculosis. Adv Exp Med Biol 954:315–323. doi: 10.1007/978-1-4614-3561-7_39 PubMedCrossRefGoogle Scholar
  18. Huerta JM, Aguilar I, Lopez-Pliego L, Fuentes-Ramirez LE, Castañeda M (2016) The Role of the ncRNA RgsA in the Oxidative Stress Response and Biofilm Formation in Azotobacter vinelandii. Curr Microbiol. doi: 10.1007/s00284-016-1003-2 PubMedGoogle Scholar
  19. Humair B, Wackwitz B, Haas D (2010) GacA-controlled activation of promoters for small RNA genes in Pseudomonas fluorescens. Appl Environ Microbiol 76(5):1497–1506. doi: 10.1128/AEM.02014-09 PubMedPubMedCentralCrossRefGoogle Scholar
  20. Jung YS, Kwon YM (2008) Small RNA ArrF regulates the expression of sodB and feSII genes in Azotobacter vinelandii. Curr Microbiol 57(6):593–597. doi: 10.1007/s00284-008-9248-z PubMedCrossRefGoogle Scholar
  21. Keiler KC (2015) Mechanisms of ribosome rescue in bacteria. Nat Rev Microbiol 13(5):285–297. doi: 10.1038/nrmicro3438 PubMedCrossRefGoogle Scholar
  22. Kirsebom LA (2007) RNase P RNA mediated cleavage: substrate recognition and catalysis. Biochimie 89(10):1183–1194. doi: 10.1016/j.biochi.2007.05.009 PubMedCrossRefGoogle Scholar
  23. Lapouge K, Schubert M, Allain FH, Haas D (2008) Gac/Rsm signal transduction pathway of gamma-proteobacteria: from RNA recognition to regulation of social behaviour. Mol Microbiol 67(2):241–253. doi: 10.1111/j.1365-2958.2007.06042.x PubMedCrossRefGoogle Scholar
  24. Lengeler JW, Jahreis K (2009) Bacterial PEP-dependent carbohydrate: phosphotransferase systems couple sensing and global control mechanisms. Contrib Microbiol 16:65–87. doi: 10.1159/000219373 PubMedCrossRefGoogle Scholar
  25. Livny J, Brencic A, Lory S, Waldor MK (2006) Identification of 17 Pseudomonas aeruginosa sRNAs and prediction of sRNA-encoding genes in 10 diverse pathogens using the bioinformatic tool sRNAPredict2. Nucleic Acids Res 34(12):3484–3493. doi: 10.1093/nar/gkl453 PubMedPubMedCentralCrossRefGoogle Scholar
  26. Manzo J, Cocotl-Yañez M, Tzontecomani T, Martinez VM, Bustillos R, Velasquez C, Goiz Y, Solis Y, Lopez L, Fuentes LE, Nunez C, Segura D, Espin G, Castañeda M (2011) Post-transcriptional regulation of the alginate biosynthetic gene algD by the Gac/Rsm system in Azotobacter vinelandii. J Mol Microbiol Biotechnol 21(3–4):147–159. doi: 10.1159/000334244 PubMedCrossRefGoogle Scholar
  27. Masse E, Gottesman S (2002) A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci USA 99(7):4620–4625. doi: 10.1073/pnas.032066599 PubMedPubMedCentralCrossRefGoogle Scholar
  28. Matz C, Bergfeld T, Rice SA, Kjelleberg S (2004) Microcolonies, quorum sensing and cytotoxicity determine the survival of Pseudomonas aeruginosa biofilms exposed to protozoan grazing. Environ Microbiol 6(3):218–226PubMedCrossRefGoogle Scholar
  29. Mejia-Ruiz H, Moreno S, Guzman J, Najera R, Leon R, Soberon-Chavez G, Espin G (1997) Isolation and characterization of an Azotobacter vinelandii algK mutant. FEMS Microbiol Lett 156(1):101–106PubMedCrossRefGoogle Scholar
  30. Moll S, Schneider DJ, Stodghill P, Myers CR, Cartinhour SW, Filiatrault MJ (2010) Construction of an rsmX co-variance model and identification of five rsmX non-coding RNAs in Pseudomonas syringae pv. tomato DC3000. RNA Biol 7(5):508–516PubMedPubMedCentralCrossRefGoogle Scholar
  31. Moreno R, Hernandez-Arranz S, La Rosa R, Yuste L, Madhushani A, Shingler V, Rojo F (2015) The Crc and Hfq proteins of Pseudomonas putida cooperate in catabolite repression and formation of ribonucleic acid complexes with specific target motifs. Environ Microbiol 17(1):105–118. doi: 10.1111/1462-2920.12499 PubMedCrossRefGoogle Scholar
  32. Moreno S, Najera R, Guzman J, Soberon-Chavez G, Espin G (1998) Role of alteRNAtive sigma factor algU in encystment of Azotobacter vinelandii. J Bacteriol 180(10):2766–2769PubMedPubMedCentralGoogle Scholar
  33. Muriel-Millan LF, Castellanos M, Hernandez-Eligio JA, Moreno S, Espin G (2014) Posttranscriptional regulation of PhbR, the transcriptional activator of polyhydroxybutyrate synthesis, by iron and the sRNA ArrF in Azotobacter vinelandii. Appl Microbiol Biotechnol 98(5):2173–2182. doi: 10.1007/s00253-013-5407-7 PubMedCrossRefGoogle Scholar
  34. Ozen AI, Ussery DW (2012) Defining the Pseudomonas genus: where do we draw the line with Azotobacter? Microb Ecol 63(2):239–248. doi: 10.1007/s00248-011-9914-8 PubMedCrossRefGoogle Scholar
  35. Padalon-Brauch G, Hershberg R, Elgrably-Weiss M, Baruch K, Rosenshine I, Margalit H, Altuvia S (2008) Small RNAs encoded within genetic islands of Salmonella typhimurium show host-induced expression and role in virulence. Nucleic Acids Res 36(6):1913–1927. doi: 10.1093/nar/gkn050 PubMedPubMedCentralCrossRefGoogle Scholar
  36. Page WJ, Huyer M (1984) Derepression of the Azotobacter vinelandii siderophore system, using iron-containing minerals to limit iron repletion. J Bacteriol 158(2):496–502PubMedPubMedCentralGoogle Scholar
  37. Page WJ, von Tigerstrom M (1982) Iron- and molybdenum-repressible outer membrane proteins in competent Azotobacter vinelandii. J Bacteriol 151(1):237–242PubMedPubMedCentralGoogle Scholar
  38. Park SH, Butcher BG, Anderson Z, Pellegrini N, Bao Z, D’Amico K, Filiatrault MJ (2013) Analysis of the small RNA P16/RgsA in the plant pathogen Pseudomonas syringae pv. tomato strain DC3000. Microbiology 159 (Pt 2):296–306. doi: 10.1099/mic.0.063826-0
  39. Prevost K, Salvail H, Desnoyers G, Jacques JF, Phaneuf E, Masse E (2007) The small RNA RyhB activates the translation of shiA mRNA encoding a permease of shikimate, a compound involved in siderophore synthesis. Mol Microbiol 64(5):1260–1273. doi: 10.1111/j.1365-2958.2007.05733.x PubMedCrossRefGoogle Scholar
  40. Pyla R, Kim TJ, Silva JL, Jung YS (2009) Overproduction of poly-beta-hydroxybutyrate in the Azotobacter vinelandii mutant that does not express small RNA ArrF. Appl Microbiol Biotechnol 84(4):717–724. doi: 10.1007/s00253-009-2002-z PubMedCrossRefGoogle Scholar
  41. Rediers H, Vanderleyden J, De Mot R (2004) Azotobacter vinelandii: a Pseudomonas in disguise? Microbiology 150(Pt 5):1117–1119. doi: 10.1099/mic.0.27096-0 PubMedCrossRefGoogle Scholar
  42. Romero Y, Guzman J, Moreno S, Cocotl M, Espin G, Castañeda M, Vences M, Segura D (2016) The GacS/A-RsmA signal transduction pathway controls the synthesis of alkylresorcinol lipids that replace membrane phospholipids during encystment of Azotobacter vinelandii SW136. PLoS ONE  11, e0153266. doi: 10.1371/journal.pone.0153266
  43. Romero Y, Moreno S, Guzman J, Espin G, Segura D (2013) Sigma factor RpoS controls alkylresorcinol synthesis through ArpR, a LysR-type regulatory protein, during encystment of Azotobacter vinelandii. J Bacteriol 195(8):1834–1844. doi: 10.1128/JB.01946-12 PubMedPubMedCentralCrossRefGoogle Scholar
  44. Salvail H, Masse E (2012) Regulating iron storage and metabolism with RNA: an overview of posttranscriptional controls of intracellular iron homeostasis. Wiley Interdiscip Rev RNA 3(1):26–36. doi: 10.1002/wrna.102 PubMedCrossRefGoogle Scholar
  45. Segura D, Cruz T, Espin G (2003) Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-beta-hydroxybutyrate synthesis. Arch Microbiol 179(6):437–443. doi: 10.1007/s00203-003-0553-4 PubMedGoogle Scholar
  46. Segura D, Nunez C, Espín G (2014) Azotobacter Cysts. In: Ltd JWS (ed) Encyclopedia of life sciences. Chichester, West Sussex, UK. doi: 10.1002/9780470015902.a0000295.pub2
  47. Segura D, Vite O, Romero Y, Moreno S, Castañeda M, Espin G (2009) Isolation and characterization of Azotobacter vinelandii mutants impaired in alkylresorcinol synthesis: alkylresorcinols are not essential for cyst desiccation resistance. J Bacteriol 191(9):3142–3148. doi: 10.1128/JB.01575-08 PubMedPubMedCentralCrossRefGoogle Scholar
  48. Setubal JC, dos Santos P, Goldman BS, Ertesvag H, Espin G, Rubio LM, Valla S, Almeida NF, Balasubramanian D, Cromes L, Curatti L, Du Z, Godsy E, Goodner B, Hellner-Burris K, HeRNAndez JA, Houmiel K, Imperial J, Kennedy C, Larson TJ, Latreille P, Ligon LS, Lu J, Maerk M, Miller NM, Norton S, O’Carroll IP, Paulsen I, Raulfs EC, Roemer R, Rosser J, Segura D, Slater S, Stricklin SL, Studholme DJ, Sun J, Viana CJ, Wallin E, Wang B, Wheeler C, Zhu H, Dean DR, Dixon R, Wood D (2009) Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes. J Bacteriol 191(14):4534–4545. doi: 10.1128/JB.00504-09 PubMedPubMedCentralCrossRefGoogle Scholar
  49. Siu FY, Spanggord RJ, Doudna JA (2007) SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting. RNA 13(2):240-250. doi: 10.1261/rna.13540 Google Scholar
  50. Seyll E, Van Melderen L (2013) The ribonucleoprotein Csr network. Int J Mol Sci 14(11):22117–22131. doi: 10.3390/ijms141122117 PubMedPubMedCentralCrossRefGoogle Scholar
  51. Sonnleitner E, Blasi U (2014) Regulation of Hfq by the RNA CrcZ in Pseudomonas aeruginosa carbon catabolite repression. PLoS Genet 10(6):e1004440. doi: 10.1371/journal.pgen.1004440 PubMedPubMedCentralCrossRefGoogle Scholar
  52. Sonnleitner E, Haas D (2011) Small RNAs as regulators of primary and secondary metabolism in Pseudomonas species. Appl Microbiol Biotechnol 91(1):63–79. doi: 10.1007/s00253-011-3332-1 PubMedCrossRefGoogle Scholar
  53. Storz G, Vogel J, Wassarman KM (2011) Regulation by small RNAs in bacteria: expanding frontiers. Mol Cell 43(6):880–891. doi: 10.1016/j.molcel.2011.08.022 PubMedPubMedCentralCrossRefGoogle Scholar
  54. Tjaden B, Saxena RM, Stolyar S, Haynor DR, Kolker E, Rosenow C (2002) Transcriptome analysis of Escherichia coli using high-density oligonucleotide probe arrays. Nucleic Acids Res 30(17):3732–3738PubMedPubMedCentralCrossRefGoogle Scholar
  55. Ulbrandt ND, Newitt JA, Bernstein HD (1997) The E. coli signal recognition particle is required for the insertion of a subset of inner membrane proteins. Cell 88(2):187–196PubMedCrossRefGoogle Scholar
  56. Wassarman KM (2007) 6S RNA: a regulator of transcription. Mol Microbiol 65(6):1425–1431. doi: 10.1111/j.1365-2958.2007.05894.x PubMedCrossRefGoogle Scholar
  57. Waters LS, Storz G (2009) Regulatory RNAs in bacteria. Cell 136(4):615–628. doi: 10.1016/j.cell.2009.01.043 PubMedPubMedCentralCrossRefGoogle Scholar
  58. Wilderman PJ, Sowa NA, FitzGerald DJ, FitzGerald PC, Gottesman S, Ochsner UA, Vasil ML (2004) Identification of tandem duplicate regulatory small RNAs in Pseudomonas aeruginosa involved in iron homeostasis. Proc Natl Acad Sci USA 101(26):9792–9797. doi: 10.1073/pnas.0403423101 PubMedPubMedCentralCrossRefGoogle Scholar
  59. Zhang A, Wassarman KM, Rosenow C, Tjaden BC, Storz G, Gottesman S (2003) Global analysis of small RNA and mRNA targets of Hfq. Mol Microbiol 50(4):1111–1124PubMedCrossRefGoogle Scholar
  60. Zielinski NA, Chakrabarty AM, Berry A (1991) Characterization and regulation of the Pseudomonas aeruginosa algC gene encoding phosphomannomutase. J Biol Chem 266(15):9754–9763PubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Miguel Castañeda
    • 1
  • Liliana López-Pliego
    • 1
  • Guadalupe Espín
    • 2
  1. 1.Centro de Investigaciones en Ciencias Microbiologicas, Instituto de CienciasBenemerita Universidad Autónoma de PueblaPueblaMexico
  2. 2.Department of Molecular Microbiology, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MexicoCuernavacaMexico

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