Microbial arsenate respiration can enhance arsenic release from arsenic-bearing mineralsa process

Microbial arsenate respiration can enhance arsenic release from arsenic-bearing mineralsa process that can cause arsenic contamination of water. Computation analysis for genome-wide CRP binding motifs identified a putative binding motif within the promoter region. This was verified by electrophoretic mobility shift assays with cAMP-CRP and several DNA probes. Lastly, four putative adenylate cyclase (mutant. It is concluded that the components of the carbon catabolite repression system are essential to regulating arsenate respiratory reduction in sp. strain ANA-3. Chronic consumption of well water contaminated with arsenic has become a global public health crisis of epidemic proportions (3). In most cases, arsenic contamination originates GW 9662 from natural geological sources (36). Elevated levels of dissolved arsenic in contaminated sediments and aquifers are generally associated with low oxygen tensions (2, 13, 26, 28). Under these conditions, bacteria are able to utilize arsenate as a terminal electron acceptor, reducing it to arsenite, which has greater toxicity and hydrological mobility than arsenate. Microbial respiratory reduction of arsenate is likely a major pathway that leads to the accumulation of toxic arsenite in groundwater (6). The biochemical mechanism of reduction of arsenate as a terminal electron acceptor involves a molybdenum-containing terminal reductase, ArrA, and a Fe-S subunit, ArrB (1, 18, GW 9662 21). In sp. strain ANA-3, ArrA and ArrB are encoded by a two-gene operon, (32). Moreover, a membrane-associated tetraheme operons are regulated in arsenate-respiring bacteria. In sp. strain ANA-3, the expression of the operon is usually greatest in arsenate-grown cells (33). Arsenite is also a strong inducer of expression, but only in anaerobically grown cells. Oxygen and nitrate repress expression. A small arsenite-binding repressor, ArsR, mediates the GW 9662 arsenite-dependent regulation of the sp. strain ANA-3 operon (J. N. Murphy and C. W. Saltikov, unpublished results). The oxygen-dependent regulation of follows expression patterns similar to those of pathways regulated by redox-sensing regulators like FNR and the two-component sensor/response regulator proteins ArcB and ArcA, respectively (22, 37). Previous work with non-arsenate-respiring MR-1 has shown that regulation of anaerobic respiration relies mostly around the global regulator cyclic AMP (cAMP) receptor protein (CRP) (30). FNR (referred to as EtrA in (4, 9, 12, 20). The molecular details of EtrA-, ArcA-, and CRP-dependent regulation of anaerobic respiratory pathways in are not well understood. In this study, we investigated the functional roles of sp. strain ANA-3 as a model system. MATERIALS AND METHODS Strains and plasmids. All of the and strains and plasmids used in this study are described in Table ?Table1.1. strain BL21 and pGEX-6P-2 were kindly provided by Karen Ottemann, University of California, Santa Cruz. TABLE 1. Bacterial strains and plasmids used in this study Growth conditions. strains were grown in Luria-Bertani (LB) medium or 2X YT medium (described in reference 34). sp. strain ANA-3 was grown at 30C in LB or minimal salts medium (referred to as TME medium) consisting of 1.5 g liter?1 NH4Cl, 0.6 g liter?1 NaHPO4, 0.1 g liter?1 KCl, 0.5 g liter?1 yeast extract, 10 mM HEPES, 20 mM lactate, and 10 ml liter?1 each trace mineral and vitamin solutions, pH 7 (described in reference 25). ANA-3 aerobic liquid cultures were shaken at 250 rpm. Preparation of anaerobic GW 9662 medium, electron acceptors, medium additions, and anaerobic culturing were done as previously described (25). Growth experiments. Aerobic cultures were grown overnight in TME medium. The optical densities at 600 nm (OD600) of the cultures were adjusted to below 0.6 and standardized to each other by the addition of medium to ensure Mouse monoclonal to BNP that the inoculation levels of all of the strains were equal. Cells were then inoculated at a 1/100 dilution into anaerobic Balch tubes containing 10 ml medium or, for aerobic growth curves, into 250-ml flasks with 20 ml medium (shaken at 250 rpm). Growth was monitored with a Spectronic 20D+. Control cultures were also GW 9662 grown and monitored in anaerobic medium without electron acceptors. Mutagenesis. In-frame, nonpolar deletions of (Shewana3_0650), (Shewana3_0617), (Shewana3_2115), (Shewana3_0387), (Shewana3_0814), (Shewana3_0823), and (Shewana3_3045) were generated by previously developed methods (25, 31, 32); for the primers used, see Table S1 in the supplemental material. Because an insertion element (Shewana3_0815) overlapped the 3 end of sp. strain ANA-3 null mutants. Complementation plasmids pArcA, pCRP, and pEtrA were generated by cloning PCR products of the genes into the SpeI site of pBBR1-MCS2; for the primers used, see Table S1 in the supplemental material. Conjugation of the complementation vectors into sp. strain ANA-3 was performed as previously described (31, 32). All plasmid constructs were verified by PCR, restriction mapping, and sequencing. Quantification of gene transcription. The methods for culturing, preparing cells, RNA extraction, and quantitative.

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