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Proteome analysis of Azotobacter vinelandiiarrF mutant that overproduces poly-β-hydroxybutyrate polymer

Abstract

Azotobacter vinelandii ArrF is an iron-responsive small RNA that is under negative control of Ferric uptake regulator protein. A. vinelandiiarrF mutant that had a deletion of the entire arrF gene was known to overproduce poly-β-hydroxybutyrate (PHB). Proteins differentially expressed in the mutant were identified by gel-based proteomics and confirmed by real-time RT-PCR. 6-Phosphogluconolactonase and E1 component of pyruvate dehydrogenase complex, which leads to the production of NADPH and acetyl-CoA, were upregulated, while proteins in the tricarboxylic acid cycle that consumes acetyl-CoA were downregulated. Heat-shock proteins such as HSP20 and GroEL were highly overexpressed in the mutant. Antioxidant proteins such as Fe-containing superoxide dismutase (FeSOD), a putative oxidoreductase, alkyl hydroperoxide reductase, flavorprotein WrbA, and cysteine synthase were also overexpressed in the ∆arrF mutant, indicating that the PHB accumulation is stressful to the cells. Upregulated in the ∆arrF mutant were acetyl-CoA carboxylase, flagellin, and adenylate kinase, though the reasons for their overexpression are unclear. Among genes upregulated in the mutant, sodB coding for FeSOD and phbF encoding PHB synthesis regulator PhbF were negatively regulated by small RNA ArrF probably in an antisense mechanism. The deletion of arrF gene, therefore, would increase PhbF and FeSOD levels, which favors PHB synthesis in the mutant. On the other hand, glutamate synthetase, elongation factor-Tu, iron ABC transporter, and major outer membrane porin OprF were downregulated in the ∆arrF mutant. Based on the results, it is concluded that multiple factors including the direct effect of small RNA ArrF might be responsible for the PHB overproduction in the mutant.

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References

  • 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 33:1678–1698

    CAS  Article  Google Scholar 

  • Aldor IS, Keasling JD (2003) Process design for microbial plastic factories: metabolic engineering of polyhydroxyalkanoates. Curr Opin Biotechnol 14:475–483

    CAS  Article  Google Scholar 

  • Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472

    CAS  Google Scholar 

  • Andrade SLL, Patridge EV, Ferry JG, Einsle O (2007) Crystal structure of the NADH:quinone oxidoreductase WrbA from Escherichia coli. J Bacteriol 189:9101–9107

    CAS  Article  Google Scholar 

  • Becker MA, Kredich NM, Tomkins GM (1969) The purification and characterization of O-acetylserine sulfhydrylase-A from Salmonella typhimurium. J Biol Chem 244:2418–2427

    CAS  Google Scholar 

  • Berlett BS, Stadtman ER (1997) Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 272:20313–20316

    CAS  Article  Google Scholar 

  • 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 182:2624–2628

    CAS  Article  Google Scholar 

  • 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 183:6787–6793

    CAS  Article  Google Scholar 

  • Chung JW, Speert DP (2007) Proteomic identification and characterization of bacterial factors associated with Burkholderia cenocepacia survival in a murine host. Microbiology 153:206–214

    CAS  Article  Google Scholar 

  • 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 187:4005–4014

    CAS  Article  Google Scholar 

  • Escolar L, Perez-Martin J, De Lorenzo V (1999) Opening the iron box: transcriptional metalloregulation by the Fur protein. J Bacteriol 181:6223–6229

    CAS  Google Scholar 

  • Galindo E, Pena C, Nunez C, Segura D, Epsin G (2007) Molecular and bioengineering strategies to improve alginate and polyhydroxyalkanoate production by Azotobacter vinelandii. Microb Cell Factories 6:7

    Article  Google Scholar 

  • Han MJ, Yoon SS, Lee SY (2001) Proteome analysis of metabolically engineered Escherichia coli producing poly (3-hydroxybutyrate). J Bacteriol 183:301–308

    CAS  Article  Google Scholar 

  • Hansford RG (1980) Control of mitochondrial substrate oxidation. In: Sanadi DR (ed) Current topics in bioenergetics, vol 10. Academic, New York, pp 217–278

    Google Scholar 

  • Hubbard JS, Stadtman ER (1967) Regulation of glutamine synthetase. II. Patterns of feedback inhibition in microorganisms. J Bacteriol 93:1045–1055

    CAS  Google Scholar 

  • Isas J, Yannone SM, Burgess BK (1995) Azotobacter vinelandii NADPH: ferredoxin reductase cloning, sequencing, and overexpression. J Biol Chem 270:21258–21263

    CAS  Article  Google Scholar 

  • 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 97:303–312

    CAS  Google Scholar 

  • Jung YS, Kwon YM (2008) Small RNA ArrF regulates the expression of sodB and fesII genes in Azotobacter vinelandii. Curr Microbiol 57:593–597

    CAS  Article  Google Scholar 

  • Kabir M, Shimizu K (2001) Proteome analysis of a temperature-inducible recombinant Escherichia coli for poly-β-hydroxybutyrate production. J Biosci Bioeng 92:277–284

    CAS  Article  Google Scholar 

  • Keyer K, Imlay JA (1996) Superoxide accelerates DNA damage by elevating free-iron levels. Proc Natl Acad Sci USA 93:13635–13640

    CAS  Article  Google Scholar 

  • 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 99:4620–4625

    CAS  Article  Google Scholar 

  • Masse E, Escorcia FE, Gottesman S (2003) Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli. Genes Dev 17:2374–2383

    CAS  Article  Google Scholar 

  • Masse E, Vanderpool CK, Gottesman S (2005) Effect of RyhB small RNA on global iron use in Escherichia coli. J Bacteriol 187:6962–6971

    CAS  Article  Google Scholar 

  • Mey AR, Craig SA, Payne SM (2005) Characterization of Vibrio cholerae RyhB: the RyhB regulon and role of RyhB in biofilm formation. Infect Immun 73:5706–5719

    CAS  Article  Google Scholar 

  • Meyer JM (1992) Exogenous siderophore-mediated iron uptake in Pseudomonas aeruginosa: possible involvement of porin OprF in iron translocation. J Gen Microbiol 138:951–958

    CAS  Google Scholar 

  • Noda LH (1973) Adenylate kinases. In: Boyer PD (ed) The enzyme, vol 8. Academic, New York, pp 279–305

    Google Scholar 

  • Page WJ, Knosp O (1989) Hyperproduction of poly-β-hydroxybutyrate during exponential growth of Azotobacter vinelandii UWD. Appl Environ Microbiol 55:1334–1339

    CAS  Google Scholar 

  • 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 147:483–490

    CAS  Google Scholar 

  • Park S, Imlay JA (2003) High levels of intracellular cysteine promote oxidative DNA damage by driving the Fenton reaction. J Bacteriol 185:1942–1950

    CAS  Article  Google Scholar 

  • 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 273:244–252

    CAS  Article  Google Scholar 

  • Patridge EV, Ferry JG (2006) WrbA from Escherichia coli and Archaeoglobus fulgidus is an NAD(P)H:quinone oxidoreductase. J Bacteriol 188:3498–3506

    CAS  Article  Google Scholar 

  • 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 184:5672–5677

    CAS  Article  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007

    Article  Google Scholar 

  • 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 84:717–724

    CAS  Article  Google Scholar 

  • Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Meth Mol Biol 132:365–386

    CAS  Google Scholar 

  • 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 65:414–418

    CAS  Article  Google Scholar 

  • Segura D, Guzmán J, Espín G (2003) Azotobacter vinelandii mutants that overproduce poly-β-hydroxybutyrate or alginate. Appl Microbiol Biotechnol 63:159–163

    CAS  Article  Google Scholar 

  • Senior PJ, Dawes EA (1973) The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter beijerinckii. Biochem J 134:225–238

    CAS  Google Scholar 

  • Tong L (2005) Acetyl-coenzyme A carboxylase: crucial metabolic enzyme and attractive target for drug discovery. Cell Mol Life Sci 62:1784–1803

    CAS  Article  Google Scholar 

  • Tzeng CM, Kornberg A (1998) Polyphosphate kinase is highly conserved in many bacterial pathogens. Mol Microbiol 29:381–382

    CAS  Article  Google Scholar 

  • Vasil ML (2007) How we learnt about iron acquisition in Pseudomonas aeruginosa: a series of very fortunate events. Biometals 20:587–601

    CAS  Article  Google Scholar 

  • 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 101:9792–9297

    CAS  Article  Google Scholar 

  • Wu G, Moir AJG, Sawers G, Hill S, Poole RK (2001) Biosynthesis of poly-β-hydroxybutyrate (PHB) is controlled by CydR (Fnr) in the obligate aerobe Azotobacter vinelandii. FEMS Microbiol Lett 194:215–220

    CAS  Google Scholar 

  • York GM, Stubbe JA, Sinskey AJ (2002) The Ralstonia eutropha PhaR protein couples synthesis of the PhaP phasin to the presence of polyhydroxybutyrate in cells and promotes polyhydroxybutyrate production. J Bacteriol 184:59–66

    CAS  Article  Google Scholar 

Download references

Acknowledgments

We would like to thank Dr. Pechan Tibor in Life Science & Biotechnology Institute for running mass spectrometer for our protein samples and Pradeep Dumpala and Babi Ramesh Nallamilli for their extended help in the project. This paper was approved for publication as Journal Article No. J-11818 of the Mississippi Agricultural and Forestry Experiment Station (MAFES), Mississippi State University. This work was supported in part by the MAFES Project Number MIS-401090 and by a MAFES SRI grant.

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Correspondence to Yean-Sung Jung.

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Pyla, R., Kim, TJ., Silva, J.L. et al. Proteome analysis of Azotobacter vinelandiiarrF mutant that overproduces poly-β-hydroxybutyrate polymer. Appl Microbiol Biotechnol 88, 1343–1354 (2010). https://doi.org/10.1007/s00253-010-2852-4

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  • DOI: https://doi.org/10.1007/s00253-010-2852-4

Keywords

  • Poly-β-hydroxybutyrate
  • Azotobacter vinelandii
  • Small RNA ArrF
  • Proteomics
  • Real-time reverse transcription PCR