1887

Abstract

We previously showed that in , accumulation of polyhydroxybutyrate (PHB) occurs mainly during the stationary phase, and that a mutation in , encoding a transcriptional regulator of the AraC family, reduces PHB accumulation. In this study, we characterized the roles of PhbR and RpoS, a central regulator during stationary phase in bacteria, in the regulation of expression of the PHB biosynthetic operon and We showed that inactivation of reduced PHB accumulation, similar to the mutation, and inactivation of both and resulted in an inability to produce PHB. We carried out expression studies with the wild-type, and the , and double - mutant strains, using quantitative RT-PCR, as well as  : :  and  : :  gene fusions. These studies showed that both PhbR and RpoS act as activators of and , and revealed a role for PhbR as an autoactivator. We also demonstrated that PhbR binds specifically to two almost identical 18 bp sites, TGTCACCAA-N-CACTA and TGTCACCAA-N-CAGTA, present in the promoter region. The activation of and transcription by RpoS reported here is in agreement with the observation that accumulation of PHB in occurs mainly during the stationary phase.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.051649-0
2011-11-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/157/11/3014.html?itemId=/content/journal/micro/10.1099/mic.0.051649-0&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. Gene 160:63–67 [View Article][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[PubMed]
    [Google Scholar]
  3. Belyaeva T. A., Wade J. T., Webster C. L., Howard V. J., Thomas M. S., Hyde E. I., Busby S. J. ( 2000). Transcription activation at the Escherichia coli melAB promoter: the role of MelR and the cyclic AMP receptor protein. Mol Microbiol 36:211–222 [View Article][PubMed]
    [Google Scholar]
  4. Browning D. F., Busby S. J. ( 2004). The regulation of bacterial transcription initiation. Nat Rev Microbiol 2:57–65 [View Article][PubMed]
    [Google Scholar]
  5. 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 [View Article][PubMed]
    [Google Scholar]
  6. 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 [View Article][PubMed]
    [Google Scholar]
  7. Hanahan D. ( 1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580 [View Article][PubMed]
    [Google Scholar]
  8. Johnson D. C., Unciuleac M. C., Dean D. R. ( 2006). Controlled expression and functional analysis of iron-sulfur cluster biosynthetic components within Azotobacter vinelandii. J Bacteriol 188:7551–7561 [View Article][PubMed]
    [Google Scholar]
  9. 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 [View Article]
    [Google Scholar]
  10. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. ( 1951). Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275[PubMed]
    [Google Scholar]
  11. Martínez P., Guzman J., Espín G. ( 1997). A mutation impairing alginate production increased accumulation of poly-β-hydroxybutyrate in Azotobacter vinelandii. Biotechnol Lett 19:909–912 [View Article]
    [Google Scholar]
  12. Matsusaki H., Manji S., Taguchi K., Kato M., Fukui T., Doi Y. ( 1998). Cloning and molecular analysis of the poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) biosynthesis genes in Pseudomonas sp. strain 61-3. J Bacteriol 180:6459–6467[PubMed]
    [Google Scholar]
  13. Navarro Llorens J. M., Tormo A., Martínez-García E. ( 2010). Stationary phase in Gram-negative bacteria. FEMS Microbiol Rev 34:476–495 [View Article][PubMed]
    [Google Scholar]
  14. Noguez R., Segura D., Moreno S., Hernandez A., Juárez K., Espín G. ( 2008). Enzyme INtr, NPr and IIANtr are involved in regulation of the poly-β-hydroxybutyrate biosynthetic genes in Azotobacter vinelandii. J Mol Microbiol Biotechnol 15:244–254 [View Article][PubMed]
    [Google Scholar]
  15. Page W. J., von Tigerstrom M. ( 1978). Induction of transformation competence in Azotobacter vinelandii iron-limited cultures. Can J Microbiol 24:1590–1594 [View Article][PubMed]
    [Google Scholar]
  16. 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 Bacteriol 184:5672–5677 [View Article][PubMed]
    [Google Scholar]
  17. Pettinari M. J., Vázquez G. J., Silberschmidt D., Rehm B., Steinbüchel A., Méndez B. S. ( 2001). Poly(3-hydroxybutyrate) synthesis genes in Azotobacter sp. strain FA8. Appl Environ Microbiol 67:5331–5334 [View Article][PubMed]
    [Google Scholar]
  18. Samarasinghe S., El-Robh M. S., Grainger D. C., Zhang W., Soultanas P., Busby S. J. ( 2008). Autoregulation of the Escherichia coli melR promoter: repression involves four molecules of MelR. Nucleic Acids Res 36:2667–2676 [View Article][PubMed]
    [Google Scholar]
  19. Sambrook J., Fritsch E. E., Maniatis T. ( 1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  20. Sandercock J. R., Page W. J. ( 2008). RpoS expression and the general stress response in Azotobacter vinelandii during carbon and nitrogen diauxic shifts. J Bacteriol 190:946–953 [View Article][PubMed]
    [Google Scholar]
  21. 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 Bacteriol 180:4790–4798[PubMed]
    [Google Scholar]
  22. Segura D., Cruz T., Espín G. ( 2003). Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-β-hydroxybutyrate synthesis. Arch Microbiol 179:437–443[PubMed]
    [Google Scholar]
  23. Tamai E., Belyaeva T. A., Busby S. J. W., Tsuchiya T. ( 2000). Mutations that increase the activity of the promoter of the Escherichia coli melibiose operon improve the binding of MelR, a transcription activator triggered by melibiose. J Biol Chem 275:17058–17063 [View Article][PubMed]
    [Google Scholar]
  24. Wade J. T., Belyaeva T. A., Hyde E. I., Busby S. J. W. ( 2000). Repression of the Escherichia coli melR promoter by MelR: evidence that efficient repression requires the formation of a repression loop. Mol Microbiol 36:223–229 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.051649-0
Loading
/content/journal/micro/10.1099/mic.0.051649-0
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