Applied Microbiology and Biotechnology

, Volume 98, Issue 5, pp 2173–2182 | Cite as

Posttranscriptional regulation of PhbR, the transcriptional activator of polyhydroxybutyrate synthesis, by iron and the sRNA ArrF in Azotobacter vinelandii

  • Luis Felipe Muriel-Millán
  • Mildred Castellanos
  • Jose Alberto Hernandez-Eligio
  • Soledad Moreno
  • Guadalupe Espín
Applied genetics and molecular biotechnology


Azotobacter vinelandii is a Gram-negative bacterium able to synthesize poly-β-hydroxybutyrate (PHB), a biodegradable plastic of industrial interest. The phbBAC operon encodes the enzymes of PHB synthesis and is activated by the transcriptional regulator PhbR and the sigma factor RpoS. Iron limitation has been previously reported to increase PHB accumulation in A. vinelandii; however, the mechanism by which iron controls PHB synthesis is unknown. Under iron starvation in Escherichia coli, the RyhB sRNA modulates the translation of genes involved in iron homeostasis. ArrF is the RyhB analogue in A. vinelandii and similarly increases in quantity during Fe2+ depletion. In this study, we evaluate the effect of iron and ArrF on PHB accumulation, and on phbR and phbBAC expression in A. vinelandii strain UW136. Using transcriptional and translational fusions of phbR and phbB with gusA reporter gene, we found that iron limitation increased the expression of phbBAC at the transcriptional level and posttranscriptionally increased the expression of phbR. We also found that the ArrF sRNA is a positive regulator of phbR expression at the posttranscriptional level. Collectively, these data suggest that iron limitation increases the translation of phbR through ArrF.


Azotobacter vinelandii Poly-β-hydroxybutyrate Iron ArrF sRNA Gene regulation 



We thank Paul Gaytan, Eugenio López, Santiago Becerra, and Jorge Yanez for oligonucleotide synthesis and DNA sequencing. L.F.M. thanks CONACyT for financial support during his M.Sc. studies.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Alexeyev MF, Shokolenko IN, Croughan TP (1995) Improved antibiotic-resistance gene cassettes and omega elements for Escherichia coli vector construction and in vitro deletion/insertion mutagenesis. Gene 160:63–67Google 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 58:1711–1718Google Scholar
  3. Bardill JP, Zhao X, Hammer BK (2011) The Vibrio cholerae quorum sensing response is mediated by Hfq-dependent sRNA/mRNA base pairing interactions. Mol Microbiol 80:1381–1394Google Scholar
  4. Barry T, Geary S, Hannify S, MacGearailt C, Shalloo M, Heery D, Gannon F, Powell R (1992) Rapid mini-preparations of total RNA from bacteria. Nucleic Acids Res 20:4940PubMedCentralPubMedCrossRefGoogle Scholar
  5. Bishop PE, Brill WJ (1977) Genetic analysis of Azotobacter vinelandii mutant strains unable to fix nitrogen. J Bacteriol 130:954–956PubMedCentralPubMedGoogle Scholar
  6. Castañeda M, Sanchez 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 183:6787–6793PubMedCentralPubMedCrossRefGoogle Scholar
  7. Cereda A, Carpen A, Picariello G, Iriti M, Faoro F, Ferranti P, Pagani S (2007) Effects of the deficiency of the rhodanese-like protein RhdA in Azotobacter vinelandii. FEBS Lett 581:1625–1630PubMedCrossRefGoogle Scholar
  8. Cornish A, Page WJ (1998) The catecholate siderophores of Azotobacter vinelandii: their affinity for iron and role in oxygen stress management. Microbiology 144:1747–1754CrossRefGoogle Scholar
  9. Demange P, Bateman A, Dell A, Abdallah M (1988) Structure of azotobactin D, a siderophore of Azotobacter vinelandii strain D (CCM 289). Biochemistry 27:2745–2752CrossRefGoogle Scholar
  10. Enos-Berlage JL, Downs DM (1997) Mutations in sdh (succinate dehydrogenase genes) alters the thiamine requirement of Salmonella typhimurium. J Bacteriol 179:3989–3996Google Scholar
  11. Escolar L, Perez-Martin J, De Lorenzo V (1999) Opening the iron box: transcriptional metalloregulation by the Fur protein. J Bacteriol 181:6223–6229PubMedCentralPubMedGoogle Scholar
  12. Fröhlich K, Vogel J (2009) Activation of gene expression by small RNA. Curr Opin Microbiol 12:674–682PubMedCrossRefGoogle Scholar
  13. 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 Factories 6:7CrossRefGoogle Scholar
  14. Hernandez-Eligio A, Castellanos M, Moreno S, Espín G (2011) Transcriptional activation of the Azotobacter vinelandii polyhydroxybutyrate biosynthetic genes phbBAC by PhbR and RpoS. Microbiology 157:3014–3023Google Scholar
  15. Hernandez-Eligio A, Moreno S, Castellanos M, Castañeda M, Núñez C, Muriel-Millán LF, Espín G (2012) RsmA post-transcriptionally controls PhbR expression and polyhydroxybutyrate biosynthesis in Azotobacter vinelandii. Microbiology 158:1953–1963Google Scholar
  16. Jung YS, Kwon YM (2008) Small RNA ArrF regulates the expression of sodB and feSII genes in Azotobacter vinelandii. Curr Microbiol 57:593–597Google Scholar
  17. Kennedy C, Gamal R, Hummprey 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 bank. Mol Gen Genet 205:318–325Google Scholar
  18. Law JH, Slepecky RA (1961) Assay of poly-β-hydroxybutyric acid. J Bacteriol 82:33–36PubMedCentralPubMedGoogle Scholar
  19. Livak K, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2\( {}^{-\Delta \Delta {\mathrm{C}}_{\mathrm{T}}} \) method. Methods 25:402–408Google Scholar
  20. Lloyd JR, Leang C, Hodges Myerson AL, Coppi MV, Cuifo S, Methé B, Sandler SJ, Lovley DR (2003) Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens. Biochem J 369:153–161Google Scholar
  21. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  22. Martínez-Salazar JM, Moreno S, Nájera R, Boucher JC, Espín G, Soberón-Chávez G, Deretic V (1996) Characterization of the genes coding for the putative sigma factor AlgU and its regulators MucA, MucB, MucC, and MucD in Azotobacter vinelandii and evaluation of their roles in alginate biosynthesis. J Bacteriol 178:1800–1808Google Scholar
  23. Massé E, Gottesman S (2002) A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci U S A 99:4620–4625Google Scholar
  24. Massé E, Vanderpool CK, Gottesman S (2005) Effect of RyhB small RNA on global iron use in Escherichia coli. J Bacteriol 187:6962–6971Google Scholar
  25. Noguez R, Segura D, Moreno S, Hernandez A, Juarez 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 15:244–254Google Scholar
  26. Page WJ (1982) Optimal conditions for induction of competence in nitrogen-fixing Azotobacter vinelandii. Can J Microbiol 28:389–397Google Scholar
  27. Page WJ, Huyer M (1984) Derepression of the Azotobacter vinelandii siderophore system, using iron-containing minerals to limit iron repletion. J Bacteriol 158:496–502Google Scholar
  28. Page WJ, von Tigerstrom M (1978) Induction of transformation competence in Azotobacter vinelandii iron-limited cultures. Can J Microbiol 24:1590–1594Google Scholar
  29. Page WJ, von Tigerstrom M (1982) Iron- and molybdenum-repressible outer membrane proteins in competent Azotobacter vinelandii. J Bacteriol 151:237–242Google Scholar
  30. Peña C, López S, García A, Espín G, Romo-Uribe A, Segura D (2013) Biosynthesis of poly-β-hydroxybutyrate with a high molecular mass by a mutant strain of Azotobacter vinelandii (OPN). Ann Microbiol. doi: 10.1007/s13213-013-0630-0
  31. Peralta-Gil M, Segura D, Guzmán J, Servín-Gonzalez 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 184:5672–5677Google Scholar
  32. Prévost K, Salvail H, Desnoyers G, Jacques JF, Phaneuf É, Massé 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 64:1260–1273Google Scholar
  33. Pyla R, Kim TK, Silva JL, Jung YS (2009) Overproduction of poly-β-hydroxybutyrate in the Azotobacter vinelandii mutant that does not express small RNA ArrF. Appl Microbiol Biotechnol 84:717–724PubMedCrossRefGoogle Scholar
  34. Pyla R, Kim TK, Silva JL, Jung YS (2010) Proteome analysis of Azotobacter vinelandiiarrF mutant that overproduces poly-β-hydroxybutyrate polymer. Appl Microbiol Biotechnol 88:1343–1354Google Scholar
  35. Rehm BH (2010) Bacterial polymers: biosynthesis, modifications and applications. Nat Rev Microbiol 8:578–592PubMedCrossRefGoogle Scholar
  36. Reusch RN, Sadoff HL (1983) D-(-)-poly-β-hydroxybutyrate in membranes of genetically competent bacteria. J Bacteriol 156:778–788PubMedCentralPubMedGoogle Scholar
  37. Salvail H, Massé E (2012) Regulating iron storage and metabolism with RNA: an overview of posttranscriptional controls of intracellular iron homeostasis. Wiley Interdiscip Rev RNA 3:26–36PubMedCrossRefGoogle Scholar
  38. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  39. 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: 4790–4798Google Scholar
  40. Segura D, Cruz T, Espín G (2003) Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-β-hydroxybutyrate synthesis. Arch Microbiol 179:437–443PubMedGoogle Scholar
  41. Segura D, Espín G (2004) Inactivation of pycA, encoding pyruvate carboxylase activity, increases poly-β-hydroxybutyrate accumulation in Azotobacter vinelandii on solid medium. Appl Microbiol Biotechnol 65:414–418Google Scholar
  42. Senior PJ, Beech GA, Ritchie GA, Dawes EA (1972) The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii. Biochem J 128:1193–1201PubMedGoogle Scholar
  43. Senior PJ, Dawes EA (1973) The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter beijerinckii. Biochem J 134:225–238PubMedGoogle Scholar
  44. Setubal JC, dos Santos P, Goldman BS, Ertesvåg H, Espín 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, Mærk 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 CV, 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 191:4534–4545PubMedCentralPubMedCrossRefGoogle Scholar
  45. 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 U S A 101:9792–9797PubMedCentralPubMedCrossRefGoogle Scholar
  46. Wilson KJ, Sessitsch A, Corbo JC, Giller KE, Akkermans AD, Jefferson RA (1995) β-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other gram-negative bacteria. Microbiology 141:1691–1705PubMedCrossRefGoogle Scholar
  47. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Luis Felipe Muriel-Millán
    • 1
  • Mildred Castellanos
    • 1
  • Jose Alberto Hernandez-Eligio
    • 1
  • Soledad Moreno
    • 1
  • Guadalupe Espín
    • 1
  1. 1.Departamento de Microbiología Molecular, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMéxico

Personalised recommendations