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Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification
Published on: November 16, 2016
Outer membrane protein OmpU is related to iron balance in Vibrio alginolyticus
Tengteng Lv1, Fa Dai1, Qiuting Zhuang1
1State Key Laboratory for Quality and Safety of Agro-products, Ningbo University, Ningbo, 315211, PR China.
Abstract:
Outer membrane protein U (OmpU) is a major porin from Vibrio alginolyticus and has been considered a vaccine candidate against infection by V. alginolyticus. After pre-incubated with polyclonal antibody against rOmpU, V. alginolyticus showed a 78% decrease in extracellular iron level, suggesting that interruption of OmpU could increase intracellular iron level. The mRNA expression of ompU under iron-limited conditions was determined using real-time reverse transcriptase PCR. The mRNA level of ompU was downregulated to 0.27-, 0.036- and 0.019-fold after the addition of the iron chelator 2,2'-bipyridyl for 10, 30 and 60 min, respectively. In addition, the promoter of ompU contained a ferric uptake regulator (Fur) binding site, which revealed the potential regulation of ompU by Fur and iron. Fur from V. alginolyticus was purified and used for electrophoretic mobility shift assay. The result showed that in the absence of Fe2+, purified recombinant Fur could specifically bind to the promoter DNA of ompU, while in the presence of Fe2+, the binding of Fur and the promoter DNA was suppressed. Our study preliminarily explored the function of OmpU in iron balance in V. alginolyticus, and these findings were helpful in understanding iron metabolism in V. alginolyticus.
Insights
Outer membrane protein U (OmpU) in Vibrio alginolyticus is crucial for iron uptake. Blocking OmpU increases intracellular iron, revealing its role in bacterial iron metabolism.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Outer membrane protein U (OmpU) is a key porin in Vibrio alginolyticus, a significant pathogen.
- OmpU has been investigated as a potential vaccine target against V. alginolyticus infections.
Purpose of the Study:
- To investigate the role of OmpU in regulating iron balance within V. alginolyticus.
- To explore the mechanism of OmpU regulation by iron and the ferric uptake regulator (Fur) protein.
Main Methods:
- Real-time reverse transcriptase PCR (RT-PCR) to quantify ompU mRNA expression under iron-limited conditions.
- Electrophoretic mobility shift assay (EMSA) to assess the binding of purified Fur protein to the ompU promoter DNA in the presence and absence of Fe2+.
Main Results:
- Antibody-mediated interruption of OmpU led to a 78% decrease in extracellular iron, suggesting increased intracellular iron levels.
- Iron-limited conditions and the iron chelator 2,2'-bipyridyl significantly downregulated ompU mRNA expression.
- Purified Fur protein bound specifically to the ompU promoter DNA in the absence of Fe2+, but this binding was suppressed by Fe2+.
Conclusions:
- OmpU plays a critical role in maintaining iron homeostasis in V. alginolyticus.
- The expression of ompU is regulated by iron availability, likely through the Fur protein, which binds to the ompU promoter in an iron-dependent manner.
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