Applied Microbiology and Biotechnology

, Volume 88, Issue 6, pp 1343–1354 | Cite as

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

  • Rajkumar Pyla
  • Tae-Jo Kim
  • Juan L. Silva
  • Yean-Sung Jung
Applied Microbial and Cell Physiology

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.

Keywords

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

Notes

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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Copyright information

© Springer-Verlag 2010

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

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