1887

Abstract

In the two-component GacS/GacA system is required for synthesis of polyhydroxybutyrate (PHB) and of the exopolysaccharide alginate. The RsmA protein was shown to interact with the alginate biosynthetic mRNA, acting as a translational repressor, and GacA was found to activate transcription of the and genes that encode small RNAs interacting with RsmA to counteract its repressor activity. The operon encodes the enzymes of PHB synthesis and is activated by the transcriptional regulator PhbR. This study shows that GacA is required for transcription of one and seven genes present in the genome, and that inactivation of results in increased PHB production. Transcriptional and translational gene fusions were used to show that the mutation negatively affected the expression of the gene at the translational level. We also demonstrated an interaction of RsmA with RNAs corresponding to and mRNA leaders, and showed that the stability of and mRNAs is increased in the mutant. Taken together these results indicate that in , RsmA post-transcriptionally represses the expression of PhbR.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.059329-0
2012-08-01
2020-01-25
Loading full text...

Full text loading...

/deliver/fulltext/micro/158/8/1953.html?itemId=/content/journal/micro/10.1099/mic.0.059329-0&mimeType=html&fmt=ahah

References

  1. Alexeyev M. F., Shokolenko I. N., Croughan T. P.. ( 1995;). Improved antibiotic-resistance gene cassettes and omega elements for Escherichia coli vector construction and in vitro deletion/insertion mutagenesis. Gene160:63–67 [CrossRef][PubMed]
    [Google Scholar]
  2. Babitzke P., Romeo T.. ( 2007;). CsrB sRNA family: sequestration of RNA-binding regulatory proteins. Curr Opin Microbiol10:156–163 [CrossRef][PubMed]
    [Google Scholar]
  3. Baker C. S., Morozov I., Suzuki K., Romeo T., Babitzke P.. ( 2002;). CsrA regulates glycogen biosynthesis by preventing translation of glgC in Escherichia coli. Mol Microbiol44:1599–1610 [CrossRef][PubMed]
    [Google Scholar]
  4. Bali A., Blanco G., Hill S., Kennedy C.. ( 1992;). Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen. Appl Environ Microbiol58:1711–1718[PubMed]
    [Google Scholar]
  5. Bush J. A., Wilson P. W.. ( 1959;). A non-gummy chromogenic strain of Azotobacter vinelandii. Nature184:381 [CrossRef][PubMed]
    [Google Scholar]
  6. Campos M. E., Martínez-Salazar J. M., Lloret L., Moreno S., Núñez C., Espín G., Soberón-Chávez G.. ( 1996;). Characterization of the gene coding for GDP-mannose dehydrogenase (algD) from Azotobacter vinelandii. J Bacteriol178:1793–1799[PubMed]
    [Google Scholar]
  7. Castañeda M., Guzmán J., Moreno S., Espín G.. ( 2000;). The GacS sensor kinase regulates alginate and poly-β-hydroxybutyrate production in Azotobacter vinelandii. J Bacteriol182:2624–2628 [CrossRef][PubMed]
    [Google Scholar]
  8. 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 Bacteriol183:6787–6793 [CrossRef][PubMed]
    [Google Scholar]
  9. Dubey A. K., Baker C. S., Romeo T., Babitzke P.. ( 2005;). RNA sequence and secondary structure participate in high-affinity CsrA–RNA interaction. RNA11:1579–1587 [CrossRef][PubMed]
    [Google Scholar]
  10. Galindo E., Peña C., Núñez C., Segura D., Espín G.. ( 2007;). Molecular and bioengineering strategies to improve alginate and polyhydroxyalkanoate production by Azotobacter vinelandii. Microb Cell Fact6:7 [CrossRef][PubMed]
    [Google Scholar]
  11. Hanahan D.. ( 1983;). Studies on transformation of Escherichia coli with plasmids. J Mol Biol166:557–580 [CrossRef][PubMed]
    [Google Scholar]
  12. Hernandez-Eligio A., Castellanos M., Moreno S., Espín G.. ( 2011;). Transcriptional activation of the Azotobacter vinelandii polyhydroxybutyrate biosynthetic genes phbBAC by PhbR and RpoS. Microbiology157:3014–3023 [CrossRef][PubMed]
    [Google Scholar]
  13. Humair B., Wackwitz B., Haas D.. ( 2010;). GacA-controlled activation of promoters for small RNA genes in Pseudomonas fluorescens. Appl Environ Microbiol76:1497–1506 [CrossRef][PubMed]
    [Google Scholar]
  14. 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 Genet205:318–325 [CrossRef]
    [Google Scholar]
  15. Lapouge K., Schubert M., Allain F. H., Haas D.. ( 2008;). Gac/Rsm signal transduction pathway of gamma-proteobacteria: from RNA recognition to regulation of social behaviour. Mol Microbiol67:241–253 [CrossRef][PubMed]
    [Google Scholar]
  16. Liu M. Y., Yang H., Romeo T.. ( 1995;). The product of the pleiotropic Escherichia coli gene csrA modulates glycogen biosynthesis via effects on mRNA stability. J Bacteriol177:2663–2672[PubMed]
    [Google Scholar]
  17. Livak K. J., Schmittgen T. D.. ( 2001;). Analysis of relative gene expression data using real-time quantitative PCR and the 2ΔΔCT method. Methods25:402–408 [CrossRef][PubMed]
    [Google Scholar]
  18. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J.. ( 1951;). Protein measurement with the Folin phenol reagent. J Biol Chem193:265–275[PubMed]
    [Google Scholar]
  19. Manzo J., Cocotl-Yañez M., Tzontecomani T., Martínez V. M., Bustillos R., Velásquez C., Goiz Y., Solís Y., López L.. & other authors ( 2011;). Post-transcriptional regulation of the alginate biosynthetic gene algD by the Gac/Rsm system in Azotobacter vinelandii. J Mol Microbiol Biotechnol21:147–159 [CrossRef][PubMed]
    [Google Scholar]
  20. Noguez R., Segura D., Moreno S., Hernandez A., Juárez K., Espín G.. ( 2008;). Enzyme I NPr, NPr and IIA Ntr are involved in regulation of the poly-β-hydroxybutyrate biosynthetic genes in Azotobacter vinelandii. J Mol Microbiol Biotechnol15:244–254 [CrossRef][PubMed]
    [Google Scholar]
  21. Peralta-Gil M., Segura D., Guzmán J., Servín-González L., Espín G.. ( 2002;). Expression of the Azotobacter vinelandii poly-β-hydroxybutyrate biosynthetic phbBAC operon is driven by two overlapping promoters and is dependent on the transcriptional activator PhbR. J Bacteriol184:5672–5677 [CrossRef][PubMed]
    [Google Scholar]
  22. Romeo T.. ( 1998;). Global regulation by the small RNA-binding protein CsrA and the non-coding RNA molecule CsrB. Mol Microbiol29:1321–1330 [CrossRef][PubMed]
    [Google Scholar]
  23. Sambrook J., Fritsch E. F., Maniatis T.. ( 1989;). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  24. Segura D., Espín G.. ( 1998;). Mutational inactivation of a gene homologous to Escherichia coli ptsP affects poly-β-hydroxybutyrate accumulation and nitrogen fixation in Azotobacter vinelandii. J Bacteriol180:4790–4798[PubMed]
    [Google Scholar]
  25. Segura D., Cruz T., Espín G.. ( 2003;). Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-β-hydroxybutyrate synthesis. Arch Microbiol179:437–443[PubMed]
    [Google Scholar]
  26. Setubal J. C., dos Santos P., Goldman B. S., Ertesvåg H., Espín G., Rubio L. M., Valla S., Almeida N. F., Balasubramanian D.. & other authors ( 2009;). Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes. J Bacteriol191:4534–4545 [CrossRef][PubMed]
    [Google Scholar]
  27. Sonnleitner E., Haas D.. ( 2011;). Small RNAs as regulators of primary and secondary metabolism in Pseudomonas species. Appl Microbiol Biotechnol91:63–79 [CrossRef][PubMed]
    [Google Scholar]
  28. Wang X., Dubey A. K., Suzuki K., Baker C. S., Babitzke P., Romeo T.. ( 2005;). CsrA post-transcriptionally represses pgaABCD, responsible for synthesis of a biofilm polysaccharide adhesin of Escherichia coli. Mol Microbiol56:1648–1663 [CrossRef][PubMed]
    [Google Scholar]
  29. Wilson K. J., Sessitsch A., Corbo J. C., Giller K. E., Akkermans A. D., Jefferson R. A.. ( 1995;). β-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other Gram-negative bacteria. Microbiology141:1691–1705 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.059329-0
Loading
/content/journal/micro/10.1099/mic.0.059329-0
Loading

Data & Media loading...

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