AggA is required for aggregation and increased biofilm formation of a hyper-aggregating mutant of Shewanella oneidensis MR-1

1 Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Gent, Belgium 2 Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA 3 Eurogentec Proteomics GmbH, Warthestraße 21, D-14513 Teltow/Berlin, Germany 4 VAR Ukkel, Groeselenberg 99, B-1180 Brussel, Belgium Correspondence Willy Verstraete willy.verstraete{at}ugent.be Shewanella oneidensis COAG, a hyper-aggregating mutant of MR-1, was isolated from a rifampicin-challenged culture. Compared to the wild-type, COAG exhibited increased biofilm formation on glass carrier material. The role of surface-located proteins in the process of COAG auto-aggregation was confirmed by different proteolytic treatments of the aggregates. All of the tested proteolytic enzymes resulted in deflocculation within 3 h of incubation. In order to examine the altered expression of outer-membrane proteins in COAG, membrane-enriched cell preparations were analysed by proteomics and the protein pattern was compared to that of MR-1. From the proteomics results, it was hypothesized that the agglutination protein AggA, associated with the secretion of a putative RTX protein, was involved in the hyper-aggregating phenotype. These results were confirmed with a DNA microarray study of COAG versus MR-1. An insertional mutation in the S. oneidensis COAG aggA locus resulted in loss of the hyper-aggregating properties and the increased biofilm-forming capability. The insertional mutation resulted in strongly decreased attachment during the initial stage of biofilm formation. By complementing this mutation with the vector pCM62, expressing the aggA gene, this effect could be nullified and biofilm formation was restored to at least the level of the MR-1 wild-type. Abbreviations: AI, aggregation index; 2D, two-dimensional; Rif, rifampicin The results of gel electrophoresis of aggA PCR amplification products are shown in Supplementary Fig. S1, and an overview of the 11 identified protein spots and their relative spot intensities, and a complete list of all the genes that showed significant upregulation in the COAG strain in at least one sampling event, are shown in Supplementary Tables S1 and S2, respectively, with the online version of this paper.