The long term objective of the proposed research is the detailed analysis of a virulence associated property, plasmid-mediated iron uptake, which plays an important role in the pathogenesis of many invasive septicemic diseases of man and domestic animals. Our model system is the fish pathogen Vibrio anguillarum which causes a terminal hemorrhagic septicemia. This bacterium possesses a plasmid-mediated iron uptake system that is an important component of its virulence repertoire. The features of this plasmid-mediated virulence determinant makes it a very attractive model to study host-bacterial interactions. In the past granting period we have characterized the essential components of this system: the siderophore anguibactin; the transport proteins OM2 and P40, and regulatory elements controlling their biosynthesis. The specific mechanisms by which these components regulate the process of iron uptake, and thus virulence, in V. anguillarum will be further elucidated by the proposed investigation. The specific aims to achieve these objectives are: 1) To identify the genetic determinants and their encoded polypeptides involved in the biosynthesis of the siderophore anguibactin. We will use a combination of genetic and physical methods to dissect the biosynthetic pathway of anguibactin. 2) To characterize the mechanism of transport of iron into the cell cytosol and investigation of the role played by P40, OM2 and the products of ORFs 1 and 2 also encoded in the iron transport region. 3) To define the nature of the positive regulation of the expression of iron-uptake genes in V. anguillarum by the transcriptional regulators AngR and TAF. 4) to define the nature of the negative regulation by iron of the expression of iron transport genes. We have identified an antisense RNA encoded in the P40-OM2 intergenic region that is induced under iron-rich conditions, concomitant with a decrease in the synthesis of the P40 and OM2 mRNAs. Our evidence indicates that this antisense RNA may play a role in the iron regulation of the expression of the P40 and OM2 iron transport genes in V.anguillarum. We will determine whether this is the central mechanism of repression of iron transport in this bacterium or whether it is part of a more complex regulatory circuit that may also include Fur-like elements. In summary, the molecular studies of this plasmid-mediated iron-uptake system should provide useful information in both the basic aspects of plasmid-mediated iron uptake and its role as a bacterial virulence factor.