Proteome analysis of Azotobacter vinelandii a†arrF mutant that overproduces poly-β-hydroxybutyrate polymer

Azotobacter vinelandii ArrF is an iron-responsive small RNA that is under negative control of Ferric uptake regulator protein. A. vinelandii a dagger arrF mutant that had a deletion of the entire arrF gene was known to overproduce poly-beta-hydroxybutyrate (PHB). Proteins differentially expressed in the mutant were identified by gel-based proteomics and confirmed by real-time RT-PCR. 6-Phosphogluconolactonase and E-1 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 a dagger arrF mutant, indicating that the PHB accumulation is stressful to the cells. Upregulated in the a dagger 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 a dagger 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.