Characteristics of the iron uptake-related process of a pathogenic Vibrio splendidus strain associated with massive mortalities of the sea cucumber Apostichopus japonicus

Under low iron bioavailability environment, many bacteria acquire iron for growth and survival through siderophore-mediated iron acquisition systems. However, until now, little research on the growth, siderophore production and siderophore receptors of Vibrio splendidus Vs under iron limited conditions has been reported. In our present study, V. splendidus Vs could survive in media supplemented with 160 mu M 2,2'-dipyridyl (DIP), but 74.5% of the growth was suppressed at 48 h, while the siderophore production of V. splendidus Vs increased by 35.9%. As the OD600 of V. splendidus Vs decreased when the concentration of DIP was increased from 40 to 80 NI, the siderophore production of V. splendidus Vs increased from 34.0% to 43.4% at 24 h, and it was further determined to be a hydroxamate siderophore. To explore the potential siderophore receptors anchored on the outer membrane of V. splendidus Vs, outer membrane proteins from cells grown with and without 80 mu M DIP were extracted and the differentially expressed proteins were identified by SDS-PAGE and MALDI-TOF/TOF MS. Five proteins, aerobactin siderophore receptor IutA, enterobactin receptor protein FepA, ATP synthase subunit A, ATP synthase subunit B and the ATP synthase F0F1 subunit beta were identified. Real-time reverse transcriptase PCR showed that mRNA levels of iutA, fepA, capA, atpB and atp beta-F0F1 were upregulated 271.5-, 15.1-, 1.1-, 2.5- and 67.9-fold respectively, after 6 h in cells treated with 80 mu M DIP. In addition, the promoters of the siderophore receptor genes of iutA and fepA had apparent ferric uptake regulator (Fur) binding sites. Combined with the simultaneous production of both the hydroxamate siderophore and its corresponding aerobactin siderophore receptor IutA, these results suggested that there might be a hydroxamate siderophore-IutA mediated iron uptake pathway in V. splendidus Vs.