Arsenite oxidation regulator AioR regulates bacterial chemotaxis towards arsenite in Agrobacterium tumefaciens GW4

Abstract

Some arsenite [As(III)]-oxidizing bacteria exhibit positive chemotaxis towards As(III), however, the related As(III) chemoreceptor and regulatory mechanism remain unknown. The As(III)-oxidizing bacterium Agrobacterium tumefaciens GW4 displays positive chemotaxis towards 0.5–2 mM As(III). Genomic analyses revealed a putative chemoreceptor-encoding gene, mcp, located in the arsenic gene island and having a predicted promoter binding site for the As(III) oxidation regulator AioR. Expression of mcp and other chemotaxis related genes (cheA, cheY2 and fliG) was inducible by As(III), but not in the aioR mutant. Using capillary assays and intrinsic tryptophan fluorescence spectra analysis, Mcp was confirmed to be responsible for chemotaxis towards As(III) and to bind As(III) (but not As(V) nor phosphate) as part of the sensing mechanism. A bacterial one-hybrid system technique and electrophoretic mobility shift assays showed that AioR interacts with the mcp regulatory region in vivo and in vitro, and the precise AioR binding site was confirmed using DNase I foot-printing. Taken together, these results indicate that this Mcp is responsible for the chemotactic response towards As(III) and is regulated by AioR. Additionally, disrupting the mcp gene affected bacterial As(III) oxidation and growth, inferring that Mcp may exert some sort of functional connection between As(III) oxidation and As(III) chemotaxis.

Transcriptomics analysis defines global cellular response ofAgrobacterium tumefaciens5A to arsenite exposure regulated through the histidine kinases PhoR and AioS

“…Of the 148 genes influenced in the Δ aioS mutant, only slightly over half (78 genes) were associated with one or the other putative AioR‐type binding sites (Supporting Information Table 2). The Corsini and colleagues () motif was associated with only 18 of these genes, whereas the motif described by Shi and colleagues () was found associated with 67 genes; seven genes were found to be associated with both motifs. As would be predicted, the degenerate sequence of Shi and colleagues (), allowed for greater flexibility and thus it was identified more frequently.…”

“…The Corsini and colleagues () motif was associated with only 18 of these genes, whereas the motif described by Shi and colleagues () was found associated with 67 genes; seven genes were found to be associated with both motifs. As would be predicted, the degenerate sequence of Shi and colleagues (), allowed for greater flexibility and thus it was identified more frequently. We did not observe any relationship between As III ‐related gene expression and occurrence or abundance of either type of AioR binding site (Supporting Information Table 2).…”

“…The predicted and experimentally demonstrated degenerate AioR binding site motif (Li et al ., ; Shi et al ., ) and that predicted by Corsini and colleagues () were used to search for potential AioR binding sites (see Supporting Information Table 2). Of the 148 genes influenced in the Δ aioS mutant, only slightly over half (78 genes) were associated with one or the other putative AioR‐type binding sites (Supporting Information Table 2).…”

“…Consistent with this view, the great majority of the transcriptional changes registered for the ΔaioS mutant only occurred in As III -exposed cells, clearly linking this sensor kinase with primarily As IIIspecific responses, which conforms to current expectations. Based on our recent report , positive control of aioS by PhoR-PhoB (Table 2) was anticipated and in turn AioS positively controls aioB, aioA, aioC and aioD (Table 2) through its interaction with its cognate response regulator AioR (Kashyap et al, 2006;Shi et al, 2017). Note that aioX (encodes the required periplasmic As III binding protein) is controlled by PhoR (Table 2), consistent with the evidence of a Pho box associated with its promoter region , and again linking As III oxidation with the PSR.…”

“…The RpoN search motif is shown on the left and the AioR search motifs are as shown on the right. ‘S’ indicates AioR sequence from Shi and colleagues (); ‘C’ indicates AioR sequence from Corsini and colleagues (2017). Lack of fold change indicates expression changes did not meet the base criteria of (±) twofold change and p value < 0.05.…”