Theses and Dissertations

Rajkumar Pyla

Issuing Body

Mississippi State University

Advisor

Jung, Yean-Sung

Committee Member

Peterson, Daniel

Committee Member

Li, Jiaxu

Committee Member

Silva, L. Juan

Committee Member

Willard, T. Scott

Date of Degree

8-7-2010

Original embargo terms

MSU Only Indefinitely

Document Type

Dissertation - Campus Access Only

Major

Molecular Biology

Degree Name

Doctor of Philosophy

College

College of Agriculture and Life Sciences

Department

Department of Biochemistry and Molecular Biology

Abstract

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 ArrF-encoding gene resulted in a 300old increase in the production of polyhydroxybutyrate (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. SDS-PAGE and MALDI-MS/MS revealed the overexpression of acetyl-CoA reductase, a phbBAC operon enzyme and the proteins that would alleviate the stress due to PHB accumulation in the ∆arrF mutant. Real-time RT-PCR revealed that phbR, phbB, phbA and phbC were upregulated in the mutant. Increased levels of activator PhbR in the mutant elevates the expression of phbB, phbA and phbC, resulting in the PHB overproduction. The proteins differentially expressed in the ∆arrF mutant were determined by gel-based proteomics and confirmed by real time RT-PCR. 6-phosphogluconolactonase that involve in the production of NADPH and acetyl-CoA, was upregulated, while the proteins involved in the TCA cycle that consumes acetyl-CoA were downregulated. Heat-shock proteins such as HSP20 and GroEL were overexpressed in the mutant. In addition, antioxidant proteins such as Fe-containing supeoxide dismutase (FeSOD), a putative oxidoreductase with unknown function, alkyl hydroperoxide reductase, flavorprotein WrbA and cysteine synthase were also upregulated, indicating that the PHB accumulation is highly stressful to the cells. Upregulated in the ∆arrF mutant were acetyl-CoA carboxylase, flagellin, and adenylate kinase. Among the genes upregulated in the ∆arrF mutant, sodB gene coding for Fe-superoxide dismutase and phbF gene encoding PHB synthesis regulator appears to be negatively regulated by small RNA ArrF in an antisense mechanism. However, all the TCA cycle genes were downregulated in the ∆arrF mutant. In addition to the TCA cycles enzyme, 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 several factors are responsible for the overproduction of PHB polymer in the ∆arrF mutant and one of which is the direct effect of small RNA ArrF on the expression of PhbF .

URI

https://hdl.handle.net/11668/16302

Comments

ArrF||Proteome analysis||Small RNA||Polyhydroxybutyrate||Azotobacter vinelandii

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