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Iron-regulated outer membrane protein OM2 of Vibrio anguillarum is encoded by virulence plasmid pJM1
Abstract:
Vibrio anguillarum 775 harboring the virulence plasmid pJM1 synthesized an outer membrane protein of 86 kilodaltons, OM2, that was inducible under conditions of iron limitation. pJM1 DNA fragments obtained by digestion with restriction endonucleases were cloned into cosmid vectors and transferred into Escherichia coli. The OM2 protein was synthesized in E. coli, demonstrating that it is actually encoded by the pJM1 plasmid. Mobilization of the recombinant plasmids to V. anguillarum was accomplished by using the transfer factor pRK2013. A V. anguillarum exconjugant harboring the recombinant derivative pJHC-T7 and synthesizing the OM2 protein took up 55Fe3+ and grew under iron-limiting conditions, only in presence of the pJM1-mediated siderophore. Exconjugants harboring recombinant plasmids, such as pJHC-T2 which did not encode the OM2 protein, were transport negative. Membrane protein iodination experiments, together with protease treatment of whole cells, indicated that the OM2 protein is exposed to the outside environment of the V. anguillarum cells.
Insights
The OM2 outer membrane protein, encoded by the virulence plasmid pJM1 in Vibrio anguillarum, is crucial for iron uptake under iron-limiting conditions. This protein facilitates the transport of iron, enabling bacterial growth.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Pathogenesis
Background:
- Vibrio anguillarum is a fish pathogen that harbors the virulence plasmid pJM1.
- Iron is an essential nutrient for bacterial growth and a key factor in virulence.
- Virulence factors, including outer membrane proteins, are often regulated by iron availability.
Purpose of the Study:
- To identify and characterize the gene encoding the 86-kilodalton outer membrane protein (OM2) synthesized by Vibrio anguillarum 775 under iron limitation.
- To determine if the OM2 protein is involved in iron uptake mediated by the pJM1 plasmid.
- To confirm the localization of the OM2 protein on the bacterial cell surface.
Main Methods:
- Cloning of pJM1 DNA fragments into cosmid vectors and transfer into Escherichia coli.
- Expression of the OM2 protein in E. coli to confirm its genetic origin.
- Mobilization of recombinant plasmids to V. anguillarum using a transfer factor.
- Iron uptake assays (55Fe3+) in V. anguillarum exconjugants under iron-limiting conditions.
- Membrane protein iodination and protease treatment of whole cells to determine protein localization.
Main Results:
- The OM2 protein was synthesized in E. coli, confirming it is encoded by the pJM1 plasmid.
- V. anguillarum exconjugants expressing OM2 showed iron transport activity and grew under iron limitation when supplemented with the pJM1-mediated siderophore.
- Exconjugants lacking OM2 expression were transport negative.
- OM2 was identified as an outer membrane protein exposed to the external environment.
Conclusions:
- The OM2 protein is a pJM1-encoded outer membrane protein essential for iron acquisition in Vibrio anguillarum.
- OM2 plays a critical role in the utilization of siderophores for iron uptake under iron-limiting conditions.
- The localization of OM2 on the bacterial surface facilitates its function in iron transport.