Skip to main content
Log in

The Role of the ncRNA RgsA in the Oxidative Stress Response and Biofilm Formation in Azotobacter vinelandii

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

Azotobacter vinelandii is a soil bacterium that forms desiccation-resistant cysts, and the exopolysaccharide alginate is essential for this process. A. vinelandii also produces alginate under vegetative growth conditions, and this production has biotechnological significance. Poly-β-hydroxybutyrate (PHB) is another polymer synthetized by A. vinelandii that is of biotechnological interest. The GacS/A two-component signal transduction system plays an important role in regulating alginate production, PHB synthesis, and encystment. GacS/A in turn controls other important regulators such as RpoS and the ncRNAs that belong to the Rsm family. In A. vinelandii, RpoS is necessary for resisting oxidative stress as a result of its control over the expression of the catalase Kat1. In this work, we characterized a new ncRNA in A. vinelandii that is homologous to the P16/RsgA reported in Pseudomonas. We found that the expression of rgsA is regulated by GacA and RpoS and that it was essential for oxidative stress resistance. However, the activity of the catalase Kat1 is unaffected in rgsA mutants. Unlike those reported in Pseudomonas, RgsA in A. vinelandii regulates biofilm formation but not polymer synthesis or the encystment process.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Alexeyev MF, Shokolenko IN, Croughan TP (1995) Improved antibiotic-resistance gene cassette and omega elements for Escherichia coli vector construction and in vitro deletion/insertion mutagenesis. Gene 160:63–67

    Article  CAS  PubMed  Google Scholar 

  2. Bali A, Blanco G, Hill S, Kennedy C (1992) Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen. Appl Environ Microbiol 58:1711–1718

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Blumenkrantz N, Asboe-Hansen G (1973) New method for quantitative determination of uronic acids. Anal Biochem 54:484–489

    Article  CAS  PubMed  Google Scholar 

  4. Boucher JC, Martinez-Salazar J, Schurr MJ, Mudd MH, Yu H, Deretic V (1996) Two distinct loci affecting conversion to mucoidy in Pseudomonas aeruginosa in cystic fibrosis encode homologs of the serine protease HtrA. J Bacteriol 178:511–523

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Busch A, Richter AS, Backofen R (2008) IntaRNA: efficient prediction of bacterial sRNA targets incorporating target site accessibility and seed regions. Bioinformatics 24:2849–2856

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Castañeda M, Guzmán J, Moreno S, Espín G (2000) The GacS sensor kinase regulates alginate and poly-β-hydroxybutyrate production in Azotobacter vinelandii. J Bacteriol 182:2624–2628

    Article  PubMed  PubMed Central  Google Scholar 

  7. Castañeda M, Sánchez J, Moreno S, Núñez C, Espín G (2001) The global regulators GacA and σs form part of a cascade that controls alginate production in Azotobacter vinelandii. J Bacteriol 183:6787–6793

    Article  PubMed  PubMed Central  Google Scholar 

  8. Chatterjee A, Cui Y, Yang H, Collmer A, Alfano JR, Chatterjee AK (2003) GacA, the response regulator of a two-component system, acts as a master regulator in Pseudomonas syringae pv. tomato DC3000 by controlling regulatory RNA, transcriptional activators, and alternate sigma factors. Mol Plant Microbe Interact 12:1106–1117

    Article  Google Scholar 

  9. Clementi F (1998) Alginate production by Azotobacter vinelandii. Crit Rev Biotechnol 17:327–361

    Article  Google Scholar 

  10. Cocotl-Yañez M, Moreno S, Encarnación S, López-Pliego L, Castañeda M, Espín G (2014) A small heat-shock protein (Hsp20) regulated by RpoS is essential for cyst desiccation resistance in Azotobacter vinelandii. Microbiology 160:479–487

    Article  PubMed  Google Scholar 

  11. Cocotl-Yañez M, Sampieri A, Moreno S, Núñez C, Castañeda M, Segura D, Espín G (2011) Roles of RpoS and PsrA in cyst formation and alkylresorcinol synthesis in Azotobacter vinelandii. Microbiology 157:1685–1693

    Article  PubMed  Google Scholar 

  12. Galindo E, Peña C, Núñez C, Segura D, Espín G (2007) Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by Azotobacter vinelandii. Microb Cell Fact 6:7. doi:10.1186/1475-2859-6-7

    Article  PubMed  PubMed Central  Google Scholar 

  13. Gómez-Lozano M, Marvig RL, Molina-Santiago C, Tribelli PM, Ramos JL, Molin S (2015) Diversity of small RNAs expressed in Pseudomonas species. Environ Microbiol Rep 2:227–236

    Article  Google Scholar 

  14. González N, Heeb S, Valverde C, Kay E, Reimmann C, Junier T, Hass D (2008) Genome-wide search reveals a novel GacA-regulated small RNA in Pseudomonas species. BMC Genom 9:167

    Article  Google Scholar 

  15. Hashimoto W, Miyamoto Y, Yamamoto M, Yoneyama F, Murata K (2013) A novel bleb-dependent polysaccharide export system in nitrogen-fixing Azotobacter vinelandii subjected to low nitrogen gas levels. Int Microbiol 16:35–44

    CAS  PubMed  Google Scholar 

  16. Heeb S, Valverde C, Gigot-Bonnefoy C, Haas D (2005) Role of the stress sigma factor RpoS in GacA/RsmA-controlled secondary metabolism and resistance to oxidative stress in Pseudomonas fluorescens CHA0. FEMS Microbiol Lett 243:251–258

    Article  CAS  PubMed  Google Scholar 

  17. Hernandez-Eligio A, Moreno S, Castellanos M, Castañeda M, Nuñez C, Muriel-Millan LF, Espin G (2012) RsmA post-transcriptionally controls PhbR expression and polyhydroxybutyrate biosynthesis in Azotobacter vinelandii. Microbiology 158:1956–1963

    Article  Google Scholar 

  18. Kennedy C, Gamal R, Humphrey R, Ramos J, Brigle K, Dean D (1986) The nifH, nifM, and nifN genes of Azotobacter vinelandii: characterization by Tn5 mutagenesis and isolation from pLARF1 gene banks. Mol Gen Genet 205:318–325

    Article  CAS  Google Scholar 

  19. Kim J, Park W (2014) Oxidative stress response in Pseudomonas putida. Appl Microbiol Biotechnol 98:6933–6946

    Article  CAS  PubMed  Google Scholar 

  20. 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 67:241–253

    Article  CAS  PubMed  Google Scholar 

  21. Larsen B, Haug A (1971) Biosynthesis of alginate. 1. Composition and structure of alginate produced by Azotobacter vinelandii (Lipman). Carbohydr Res 17:287–296

    Article  CAS  PubMed  Google Scholar 

  22. Law J, Slepcky A (1961) Assay of poly-β-hydroxybutyric acid. J Bacteriol 82:33–36

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  PubMed  Google Scholar 

  24. Manzo J, Cocotl-Yañez M, Tzontecomani T et al (2011) Post-transcriptional regulation of the alginate biosynthetic gene algD by the Gac/Rsm system in Azotobacter vinelandii. J Mol Microbiol Biotechnol 21:147–159

    Article  CAS  PubMed  Google Scholar 

  25. Moreno S, Guzmán J, Nájera R, Soberón-Chávez G, Espín G (1998) Role of the alternative σ factor AlgU in encystment of Azotobacter vinelandii. J Bacteriol 180:2766–2769

    CAS  PubMed  PubMed Central  Google Scholar 

  26. O’Toole GA, Kolter R (1998) Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30:295–304

    Article  PubMed  Google Scholar 

  27. Papenfort K, Vanderpool CK (2015) Target activation by regulatory RNAs in bacteria. FEMS Microbiol Rev 39:362–378

    Article  PubMed  Google Scholar 

  28. 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 159:296–306

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Robson RL, Jones R, Robson RM, Schwartz A, Richardson TH (2015) Azotobacter genomes: The genome of Azotobacter chroococcum NCIMB 8003 (ATCC 4412). PLoS One 10(6):e0127997. doi:10.1371/journal.pone.0127997

    Article  PubMed  PubMed Central  Google Scholar 

  30. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  31. Sandercock JR, Page WJ (2008) Identification of two catalases in Azotobacter vinelandii: a KatG homologue and a novel bacterial cytochrome c catalase, CCCAv. J Bacteriol 190:954–962

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sandercock JR, Page WJ (2008) RpoS expression and the general stress response in Azotobacter vinelandii during carbon and nitrogen diauxic shifts. J Bacteriol 190:946–953

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Segura D, Cruz T, Espín G (2003) Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-beta-hydroxybutyrate synthesis. Arch Microbiol 179:437–443

    CAS  PubMed  Google Scholar 

  34. Segura D, Núñez C, Espín G (2014) Azotobacter Cysts. In: eLS. Wiley, Chichester. doi:10.1002/9780470015902.a0000295.pub2

  35. Setubal JC, dos Santos P, Goldman BS et al (2009) Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes. J Bacteriol 191:4534–4545

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Villa F, Remelli W, Forlani F, Gambino M, Landini P, Cappitelli F (2012) Effects of chronic sub-lethal oxidative stress on biofilm formation by Azotobacter vinelandii. Biofouling. 28:823–833

    Article  CAS  PubMed  Google Scholar 

  37. Wagner EG, Romby P (2015) Small RNAs in bacteria and archaea: who they are, what they do, and how they do it. Adv Genet 90:133–208

    Article  PubMed  Google Scholar 

  38. Woodbury W, Spencer AK, Stahman MA (1971) An improved procedure using ferricyanide for detecting catalase isozymes. Anal Biochem 44:301–305

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by CONACyT Grant CB-2009-01-129525. We thank Cinthia Núñez for helpful comments regarding the manuscript. We also thank Daniel Segura for helpful discussions regarding the results. J. M. Huerta thanks CONACyT for M.Sc. scholarships.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Miguel Castañeda.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Huerta, J.M., Aguilar, I., López-Pliego, L. et al. The Role of the ncRNA RgsA in the Oxidative Stress Response and Biofilm Formation in Azotobacter vinelandii . Curr Microbiol 72, 671–679 (2016). https://doi.org/10.1007/s00284-016-1003-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-016-1003-2

Keywords

Navigation