Applied Microbiology and Biotechnology

, Volume 84, Issue 4, pp 717–724 | Cite as

Overproduction of poly-β-hydroxybutyrate in the Azotobacter vinelandii mutant that does not express small RNA ArrF

  • Rajkumar Pyla
  • Tae-Jo Kim
  • Juan L. Silva
  • Yean-Sung Jung
Applied Genetics and Molecular Biotechnology


Azotobacter vinelandii contains an iron-regulatory small RNA ArrF whose expression is dependent upon the levels of iron and ferric uptake regulator. The deletion of this ArrF-encoding gene resulted in a 300-fold increase in the production of poly-β-hydroxybutyrate (PHB), a polymer of industrial importance. This ∆arrF mutant exhibited wild-type growth and growth-associated PHB production. Limited iron and aeration elevated the PHB production in the mutant as well as wild type. Real-time RT-PCR revealed that phbB, phbA, and phbC were upregulated ∼61-, 18-, and eightfold, respectively, in the mutant. The phbR transcript of the activator PhbR for this operon was also ∼11 times more abundant. The analysis of phbR transcript predicted a region of complementarity near its Shine–Dalgarno sequence that could potentially basepair with the conserved region of ArrF. These results suggest that ArrF represses the expression of PhbR in an antisense manner and derepression of this activator in the mutant elevates the expression of phbB, phbA, and phbC, resulting in the PHB overproduction.


Polyhydroxybutyrate Azotobacter vinelandii Small RNA ArrF Iron Aeration 



The authors would like to thank Professors Scott Willard, Din-Pow Ma, and Ken Willeford for reviewing the paper. The authors thank Professor Jeff Wilkinson for allowing us to use LightcyclerR 2.0. This work was supported in part by a grant from Robert M Hearing Foundation, by the Mississippi Agricultural and Forestry Experiment Station (MAFES) Project Number MIS-401030, and by a grant of MAFES SRI. This paper was approved for publication as Journal Article No. J-11447 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University.


  1. Afonyushkin T, Vecerek B, Moll I, Blasi U, Kaberdin VR (2005) Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB. Nucleic Acids Res 33:1678–1698CrossRefGoogle Scholar
  2. Aldor IS, Keasling JD (2003) Process design for microbial plastic factories: metabolic engineering of polyhydroxyalkanoates. Curr Opin Biotechnol 14:475–483CrossRefGoogle Scholar
  3. Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472Google Scholar
  4. Castaneda M, Guzman J, Moreno S, Espin G (2000) The GacS sensor kinase regulates alginate and poly-β-hydroxybutyrate production in Azotobacter vinelandii. J Bacteriol 182:2624–2628CrossRefGoogle Scholar
  5. Castaneda M, Sanchez J, Moreno S, Nunez C, Espin G (2001) The global regulators GacA and σS form part of a cascade that controls alginate production in Azotobacter vinelandii. J Bacteriol 183:6787–6793CrossRefGoogle Scholar
  6. 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 581:1625–1630CrossRefGoogle Scholar
  7. Davis BM, Quinones M, Pratt J, Ding Y, Waldor MK (2005) Characterization of the small untranslated RNA RyhB and its regulon in Vibrio cholerae. J Bacteriol 187:4005–4014CrossRefGoogle Scholar
  8. Escolar L, Perez-Martin J, De Lorenzo V (1999) Opening the iron box: transcriptional metalloregulation by the Fur protein. J Bacteriol 181:6223–6229Google Scholar
  9. Gardner PR, Fridovich I (1991) Superoxide sensitivity of the Escherichia coli aconitase. J Biol Chem 266:19328–19333Google Scholar
  10. Galindo E, Pena C, Nunez C, Segura D, Epsin G (2007) Molecular and bioengineering strategies to improve alginate and polyhydroxyalkanoate production by Azotobacter vinelandii. Microbial Cell Factories 6:7CrossRefGoogle Scholar
  11. Hantke K (2001) Iron and metal regulation in bacteria. Curr Opin Microbiol. 4:172–177CrossRefGoogle Scholar
  12. Hengge-Aronis R (2002) Signal transduction and regulatory mechanism involved in control of sigma (S) (RpoS) subunit of RNA polymerase. Microbiol Mol Biol Rev 66:373–395CrossRefGoogle Scholar
  13. Huang R, Reusch RN (1996) Poly (3-hydroxybutyrate) is associated with specific proteins in the cytoplasm and membranes of Escherichia coli. J Biol Chem 271:22196–22202CrossRefGoogle Scholar
  14. Isas J, Yannone SM, Burgess BK (1995) Azotobacter vinelandii NADPH: ferredoxin reductase cloning, sequencing, and overexpression. J Biol Chem 270:21258–21263CrossRefGoogle Scholar
  15. Jackson FA, Dawes EA (1976) Regulation of the tricarboxylic acid cycle and poly-β-hydroxybutyrate metabolism in Azotobacter beijeinckii grown under nitrogen or oxygen limitation. J Gen Microbiol 97:303–312Google Scholar
  16. Jung YS, Kwon YM (2008) Small RNA ArrF regulates the expression of sodB and fesII genes in Azotobacter vinelandii. Curr Microbiol 57:593–597CrossRefGoogle Scholar
  17. Kadouri D, Jurkevitch E, Okon Y, Castro-Sowinski S (2005) Ecological and agricultural significance of bacterial polyhydroxyalkanoates. Crit Rev Microbiol 31:55–67CrossRefGoogle Scholar
  18. 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 99:4620–4625CrossRefGoogle Scholar
  19. Masse E, Escorcia FE, Gottesman S (2003) Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli. Genes Dev 17:2374–2383CrossRefGoogle Scholar
  20. Masse E, Vanderpool CK, Gottesman S (2005) Effect of RyhB small RNA on global iron use in Escherichia coli. J Bacteriol 187:6962–6971CrossRefGoogle Scholar
  21. Mey AR, Craig SA, Payne SM (2005) Characterization of Vibrio cholerae RyhB: the RyhB regulon and role of RyhB in biofilm formation. Infect Immun 73:5706–5719CrossRefGoogle Scholar
  22. Page WJ, Tindale A, Chandra M, Kwon E (2001) Alginate formation in Azotobacter vinelandii UWD during stationary phase and the turnover of poly-β-hydroxybutyrate. Microbiology 147:483–490Google Scholar
  23. Park BS, Kwon YM, Pyla R, Boyle JA, Jung YS (2007) E1 component of pyruvate dehydrogenase complex does not regulate the expression of NADPH-ferredoxin reductase in Azotobacter vinelandii. FEMS Microbiol Lett 273:244–252CrossRefGoogle Scholar
  24. Peralta-Gil M, Segura D, Guzman J, Servin-Gonzalez L, Espin 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 184:5672–5677CrossRefGoogle Scholar
  25. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007CrossRefGoogle Scholar
  26. Reusch RN, Sadoff HL (1983) D-(-)-Poly-β-hydroxybutyrate in membranes of genetically competent bacteria. J Bacterol 156:778–788Google Scholar
  27. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386Google Scholar
  28. Senior PJ, Dawes EA (1973) The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter beijerinckii. Biochem J 134:225–238Google Scholar
  29. Suh MH, Pulakat L, Gavini N (2002) Functional expression of the FeMo-cofactor-specific biosynthetic gene nifEN as a NifE-N fusion protein synthesizing unit in Azotobacter vinelandii. Biochem Biophys Res Commun 299:233–240CrossRefGoogle Scholar
  30. Tindale AE, Mehrotra M, Ottem D, Page WJ (2000) Dual regulation of catecholate siderophore biosynthesis in Azotobacter vinelandii by iron and oxidative stress. Microbiology 146:1617–1626Google Scholar
  31. Vasil ML (2007) How we learnt about iron acquisition in Pseudomonas aeruginosa: a series of very fortunate events. Biometals 20:587–601CrossRefGoogle Scholar
  32. 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 101:9792–9297CrossRefGoogle Scholar
  33. Wu G, Moir AJG, Sawers G, Hill S, Poole RK (2001) Biosynthesis of poly-β-hydroxybutyrate (PHB) is controlled by CydR (Fnr) is the obligate aerobe Azotobacter vinelandii. FEMS Microbiol Lett 194:215–220Google Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Rajkumar Pyla
    • 1
  • Tae-Jo Kim
    • 2
  • Juan L. Silva
    • 2
  • Yean-Sung Jung
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyMississippi State UniversityMississippi StateUSA
  2. 2.Department of Food Science, Nutrition and Health PromotionMississippi State UniversityMississippi StateUSA

Personalised recommendations