RESUMO
We determined the ability of Vibrio parahaemolyticus to utilize enterobactin (Ent) as a xenosiderophore. Homology searches of the V. parahaemolyticus genomic sequence revealed the presence of genes that are homologous to the V. cholerae ferric Ent utilization genes, which consist of the iron-repressible outer-membrane protein genes irgA and vctA, and the ATP-binding cassette transport system operon vctPDGC. Moreover, the irgB and vctR genes, which encode transcriptional regulators, were also found immediately upstream of irgA and vctA, respectively. Growth assays of V. parahaemolyticus indicated that both irgA and vctA mutants grew well in the presence of Ent under iron-limiting conditions, whereas both the irgA/vctA double mutant and the vctPDGC mutant barely grew under the same conditions. In addition, growth assays of three isogenic tonB mutants demonstrated that the TonB2 system, and to a lesser extent the TonB1 system, can provide energy for both IrgA and VctA to transport ferric Ent. SDS-PAGE analysis showed that expression of both IrgA and VctA was enhanced by the presence of Ent. Complementation of the irgB and vctR mutants with their respective genes resulted in the increased expression of IrgA and VctA, respectively. Finally, reverse transcriptase-quantitative PCR revealed that transcription of the Ent utilization system genes is iron-regulated, and that transcription of irgA and vctA under iron-limiting conditions is further activated by proteins encoded by irgB and vctR, respectively, together with Ent.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas da Membrana Bacteriana Externa/genética , Enterobactina/metabolismo , Regulação Bacteriana da Expressão Gênica , Sideróforos/metabolismo , Vibrio parahaemolyticus/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , Dados de Sequência Molecular , Vibrio parahaemolyticus/genéticaRESUMO
We previously reported that the Vibrio parahaemolyticus pvsABCDE and psuA-pvuABCDE operons are involved in the biosynthesis and transport of its own siderophore, vibrioferrin (VF). Of these, psuA and pvuA encode TonB-dependent outer-membrane proteins (OMPs). Although pvuA was characterized as the ferric vibrioferrin receptor gene, the role of the psuA product remains unknown. In this study, a growth assay of isogenic psuA, pvuA, and psuA-pvuA double-deletion mutants followed by complementation of the double-deletion mutant with psuA or pvuA was used to identify psuA as a gene encoding an OMP involved in the uptake of ferric VF. Thus, psuA and pvuA were renamed pvuA1 and pvuA2, respectively. Moreover, we clarified the TonB specificities of PvuA1 and PvuA2, because V. parahaemolyticus has three sets of the TonB systems. The triple deletion of pvuA1, tonB1, and tonB2, and the double deletion of pvuA2 and tonB2 resulted in the complete loss of growth promotion by VF. This finding indicates that the energy required for PvuA1 and PvuA2 to transport ferric VF across the outer membrane is provided by the TonB2 system and by both the TonB1 and TonB2 systems, respectively.
