Several bacteria and prokaryotes have developed adaptation, resistance and colonization mechanisms, which allow them to live in hostile arsenic contaminated environments. H. arsenicoxydans is a Gram-negative β-proteobacterium isolated from an industrial activated sludge plant and exhibiting a remarkable set of arsenic resistance determinants [4]. The H. arsenicoxydans adaptive response to arsenic is organized in a complex and sophisticated network. In particular, differential proteome studies have recently demonstrated the synthesis of several proteins encoded by the three ars resistance operons, e.g. arsenate reductase ArsC, flavoprotein ArsH and regulator ArsR [5,6] and the induction of oxidative stress protein encoding genes, e.g. catalase (katA), superoxide dismutase (sodB) and alkyl hydroperoxide reductase (ahpC) [7].

One of the most noticeable response to arsenic in H. arsenicoxydans is the ability of this bacterium to oxidize As(III) to As(V), a less toxic and less mobile form, via an arsenite oxidase activity. The two genes coding for this heterodimeric enzyme are organized in an operonic structure, and have been named aoxA and aoxB for the small and the large subunit, respectively [6,8,9]. Homologous genes have been since identified in various microorganisms [6,10-13]. In Agrobacterium tumefaciens, a complex transcriptional regulation has been recently suggested, involving As(III) sensing, two-component signal transduction by an AoxS sensor kinase and an AoxR regulator, and quorum sensing [14]. Nevertheless, the molecular mechanisms involved in the control of arsenite oxidase expression remain largely unknown.

More importantly, our results suggest that AoxR and RpoN constitute a transcriptional complex that play a major role in the initiation of aoxAB operon transcription. Three mutants, i.e. Ha482 (aoxS), Ha483 (aoxR) and Ha3109 (rpoN), were affected in this process. The amino acids sequence analysis of H. arsenicoxydans AoxR and AoxS revealed the existence in these proteins of structural features common to partners of two-component signal transduction systems, which are composed of a sensor kinase and a response regulator [24]. Moreover, the comparison of AoxS and AoxR protein sequences with those of A. tumefaciens revealed similarities. Indeed, the AoxS protein sequence contains short blocks of conserved motifs that are consistent with a role of sensor histidine kinase, e.g. the "H" (amino acids 279 to 287: LAHEVNNPL), the "G2" (amino acids 435 to 441: GRIGLGL) and the "N" (amino acids 380 to 391: VRQIVLNLVLNA) domains. In addition, four highly invariant residues playing a central role in phosphorylation correspond to Asp9, Asp10, Asp57 and Lys107 in the H. arsenicoxydans AoxR protein. The three asparagine residues are known to interact together to form an "acid pocket", characteristic of orthodox receivers, into which the side chain of Lys109 protrudes [24]. Finally, a transmembrane region and a 17 amino acids residue cluster possibly exposed to the periplasm are present in AoxS and could serve as a signal receptor in the presence of As(III) in the medium. The detection of As(III) would then lead to AoxS autophosphorylation at a histidine residue via ATP hydrolysis and phosphotransfer to an aspartate residue in the response regulator AoxR, as recently proposed in A. tumefaciens [14].

Remarkably, our results demonstrated for the first time that the alternative N sigma factor (σ⁵⁴) is essential for the initiation of arsenite oxidase transcription. Indeed, a mutation in the corresponding gene led to a complete loss of As(III) oxidation and aoxB transcription in Ha3109 (rpoN). σ⁵⁴ is one of the alternative sigma subunits of RNA polymerase responsible for specific binding to DNA. The core RNA polymerase complexed with σ⁵⁴ is usually associated with nitrogen assimilation and fixation, but is also known to play a role in various physiological processes, e.g. flagellar synthesis, carbon source utilization or bacterial virulence [25]. To date, only one report has shown that σ⁵⁴ participates in the transcription of genes possibly involved in metal tolerance, i.e. the zraR/S genes that code for a zinc and lead responsive two-component regulatory system in E. coli [26]. RNA polymerase together with σ⁵⁴ binds to a specific promoter site, with the consensus DNA sequence YTGGCACGNNNNTTGCWNNw [27], forming a transcriptionally inactive closed complex. Such a characteristic -12/-24 σ⁵⁴-dependent promoter motif, i.e. TGGCACGCAGTTTGC, was identified 26 pb upstream of the transcriptional initiation codon of aoxAB with respect to the +1 transcriptional start site (Figure ​(Figure5),5), which confirmed the need for RpoN in the initiation of aoxAB transcription. Changes in the conformation of σ⁵⁴-RNA polymerase are nucleotide dependent. Indeed, the DNA melting step absolutely requires the interaction with a transcriptional activator protein. Most of these σ⁵⁴-dependent activators share three domains found in AoxR, i.e. a C-terminal DNA binding domain that binds to upstream activation sequences, a conserved central domain belonging to the AAA+ (ATPases associated with various cellular activities) protein family to proceed with initiation of transcription and a N-terminal receiver domain that regulates its own AAA+ domain [20,28,29]. A multiple alignment of the central domain revealed a conservation of a common architecture between AoxR and σ⁵⁴ EBPs. Indeed, seven highly conserved sequence motifs corresponding to a σ⁵⁴ interaction domain of AoxR further support the direct interaction of AoxR with RpoN to stimulate the transcription of aoxAB operon in H. arsenicoxydans (Figure ​(Figure6).6). This central σ⁵⁴ interaction domain has been already used to identify new σ⁵⁴ EBPs [30-37]. It contains the highly conserved signature sequence GAFTGA loop (conserved region CIII, Figure ​Figure6)6) proved to be required for direct interaction with σ⁵⁴ [21,38,39], the Walker A (conserved region I, Figure ​Figure6)6) and B (conserved region CIV, Figure ​Figure6)6) ATPase motifs necessary for the open complex formation.

Both multiple sequences alignment and genetic environment analysis of aox promoters suggest the existence of a wide diversity in the transcriptional control of the aox operon. Indeed, as in H. arsenicoxydans, a σ⁵⁴-dependent promoter signature was identified in bacteria possessing a two-component transduction system AoxRS operon downstream of the aoxAB operon, e.g. A. tumefaciens and O. tritici (Figure ​(Figure5A).5A). In contrast, no σ⁵⁴-dependent promoter motif and no aoxR homologous gene were found in other bacteria, e.g. C. aurantiacus or C. aggregans (Figure ​(Figure5B).5B). These observations suggest that the transcription of the aox operon in these bacteria may involve other regulatory proteins and that AoxR may represent a specific co-activator of RpoN in the initiation of the aox operon transcription. Finally, our results provide evidence that the DnaJ co-chaperone is required for As(III) oxidation. DnaJ is part of the DnaK-DnaJ-GrpE Hsp70 machinery. Hsp70 chaperones represent one of the most potent defence cellular mechanism against environmental insults as DnaK-DnaJ-GrpE are known to assist protein folding [40,41] or to be involved in mRNA stability [42]. In the present study we showed that there is no induction of aoxAB transcription in the dnaJ mutant, resulting in a loss of AoxAB synthesis.