Bundle-forming pilus locus of Aeromonas veronii bv. Sobria

Little is known about the colonizationmechanisms of Aeromonas spp. Previouswork has suggested that the type IVbundle-forming pilus (Bfp) is an aeromonad intestinal colonization factor. This study provides the first genetic characterization of this structure. To define the role of Bfp in Aeromonas veronii bv. Sobria adherence, a 22-kb locus encoding the bundle-forming piluswas isolated; this contained 17 pilus-related genes similar to themannose-sensitive hemagglutinin (MSHA) of Vibrio cholerae. Reverse transcriptase PCR (RT-PCR) demonstrated that the locus had twomajor transcriptional units,mshI tomshF andmshB tomshQ. Transcriptional fusion experiments demonstrated the presence of two strong promoters upstreamofmshI andmshB. The locus encoded four putative prepilin proteins, one ofwhich (MshA) corresponded to theN-terminal sequence of the previously isolatedmajor pilin protein.All the pilin geneswere inactivated,mutation of eachminor ormajor pilin gene greatly reduced the bacterium’s ability to adhere and form biofilms, and complementation of eachmutant in trans rescued this phenotype.Mutation of themajor pilinMshAandMshB, aminor pilin, resulted in their loss. The position of themshHgene is conservedwithin a number of bacteria, andwe have shown it is not tran- scriptionally linked to the othermsh genes;moreover, itsmutation did not have a dramatic effect on either adhesion or biofilmforma- tion.We conclude that the bundle-forming pilus is required for A. veronii bv. Sobria adherence and biofilmformation; furthermore, both themajor andminor pilin proteins are essential for this process.