AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways.

The regulatory programme of multicellular behaviour in Salmonella typhimurium is determined by mutations in the agfD promoter. AgfD has already been identified to regulate the extracellular matrix associated with the multicellular morphotype composed of thin aggregative fimbriae (agf). To detect additional components contributing to the multicellular morphotype in S. typhimurium, we constructed a mutant in agfD, the positive transcriptional regulator of the agfBA(C) operon encoding for fimbrial subunit proteins. The agfD mutant lacked any form of multicellular behaviour as shown by analysis at the macroscopic and microscopic level. In contrast, the agfBA mutant unable to form thin aggregative fimbriae still maintained long-range intercellular adhesion. Promoter and expression analysis revealed that the genes downstream of agfD agfEFG most likely did not contribute to the remaining aggregative behaviour. Screening of transcriptional fusions for agfD dependency uncovered adrA, a homologue of yaiC in Escherichia coli. Environmental factors regulating adrA correspond to the regulation of thin aggregative fimbriae. AdrA is a putative transmembrane protein with a C-terminal GGDEF domain of unknown function although it is present in over 50 bacterial proteins. AdrA mutant cells, which still formed thin aggregative fimbriae with all binding characteristics, exhibited community behaviour but, unlike the wild type, lacked long-range intercellular adhesion. An agfBA adrA double mutant behaved like the agfD mutant. Therefore, it was concluded that agfD regulates at least two independent pathways contributing to the multicellular morphotype in S. typhimurium.