Functional Roles of arcA, etrA, Cyclic AMP (cAMP)-cAMP Receptor Protein, and cya in the Arsenate Respiration Pathway in Shewanella sp Strain ANA-3

Microbial arsenate respiration can enhance arsenic release from arsenic-bearing minerals-a process that can cause arsenic contamination of water. In Shewanella sp. strain ANA-3, the arsenate respiration genes (arrAB) are induced under anaerobic conditions with arsenate and arsenite. Here we report how genes that encode anaerobic regulator ( arcA and etrA [fnr homolog]) and carbon catabolite repression (crp and cya) proteins affect arsenate respiration in ANA-3. Transcription of arcA, etrA, and crp in ANA-3 was similar in cells grown on arsenate and cells grown under aerobic conditions. ANA-3 strains lacking arcA and etrA showed minor to moderate growth defects, respectively, with arsenate. However, crp was essential for growth on arsenate. In contrast to the wild-type strain, arrA was not induced in the crp mutant in cultures shifted from aerobic to anaerobic conditions containing arsenate. This indicated that cyclic AMP (cAMP)-cyclic AMP receptor (CRP) activates arr operon transcription. Computation analysis for genome-wide CRP binding motifs identified a putative binding motif within the arr promoter region. This was verified by electrophoretic mobility shift assays with cAMP-CRP and several DNA probes. Lastly, four putative adenylate cyclase ( cya) genes were identified in the genome. One particular cya-like gene was differentially expressed under aerobic versus arsenate respiration conditions. Moreover, a double mutant lacking two of the cya-like genes could not grow with arsenate as a terminal electron acceptor; exogenous cAMP could complement growth of the double cya mutant. It is concluded that the components of the carbon catabolite repression system are essential to regulating arsenate respiratory reduction in Shewanella sp. strain ANA-3.