Determination and Analysis of the Putative AcaCD-Responsive Promoters of Salmonella Genomic Island 1

The integrative genomic island SGI1 and its variants confer multidrug resistance in numerous Salmonella enterica serovariants and several Proteus mirabilis and Acinetobacter strains. SGI1 is mobilized by the IncA/C family plasmids. The island exploits not only the conjugation apparatus of the plasmid, but also utilizes the plasmid-encoded master regulator AcaCD to induce the excision and formation of its transfer-competent form, which is a key step in the horizontal transfer of SGI1. Triggering of SGI1 excision occurs via the AcaCD-dependent activation of xis gene expression. AcaCD binds in P-xis to an unusually long recognition sequence. Beside the Pxis promoter, upstream regions of four additional SGI1 genes, S004, S005, S012 and S018, also contain putative AcaCD-binding sites. Furthermore, SGI1 also encodes an AcaCD-related activator, FlhDC(SGI1), which has no known function. Here, we have analysed the functionality of the putative AcaCD-dependent promoter regions and proved their activation by either AcaCD or FlhDC(SGI1). Moreover, we provide evidence that both activators act on the same binding site in P-xis and that FlhDC(SGI1) is able to complement the acaCD deletion of the IncA/C family plasmid R16a. We determined the transcription start sites for the AcaCD-responsive promoters and showed that orf S004 is expressed probably from a different start codon than predicted earlier. Additionally, expression of S003 from promoter P-S004 was ruled out. Pxis and the four SGI1 promoters examined here also lack obvious -35 promoter box and their promoter profile is consistent with the class II-type activation pathway. Although the role of the four additionally analysed AcaCD/FlhDC(SGI1)-controlled genes in transfer and/or maintenance of SGI1 is not yet clear, the conservation of the whole region suggests the existence of some selection for their functionality.