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 Saracatinib 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 ABT-263 ic50 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. this website 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 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, Benzatropine 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 6). This central σ54 interaction domain has been already used to identify new σ54 EBPs [30–37].