The ars detoxification system is advantageous but not required for As(V) respiration by the genetically tractable Shewanella species strain ANA-3

Abstract

Arsenate [As(V); HAsO(4)(2-)] respiration by bacteria is poorly understood at the molecular level largely due to a paucity of genetically tractable organisms with this metabolic capability. We report here the isolation of a new As(V)-respiring strain (ANA-3) that is phylogenetically related to members of the genus Shewanella and that also provides a useful model system with which to explore the molecular basis of As(V) respiration. This gram-negative strain stoichiometrically couples the oxidation of lactate to acetate with the reduction of As(V) to arsenite [As(III); HAsO(2)]. The generation time and lactate molar growth yield (Y(lactate)) are 2.8 h and 10.0 g of cells mol of lactate(-1), respectively, when it is grown anaerobically on lactate and As(V). ANA-3 uses a wide variety of terminal electron acceptors, including oxygen, soluble ferric iron, oxides of iron and manganese, nitrate, fumarate, the humic acid functional analog 2,6-anthraquinone disulfonate, and thiosulfate. ANA-3 also reduces As(V) to As(III) in the presence of oxygen and resists high concentrations of As(III) (up to 10 mM) when grown under either aerobic or anaerobic conditions. ANA-3 possesses an ars operon (arsDABC) that allows it to resist high levels of As(III); this operon also confers resistance to the As-sensitive strains Shewanella oneidensis MR-1 and Escherichia coli AW3110. When the gene encoding the As(III) efflux pump, arsB, is inactivated in ANA-3 by a polar mutation that also eliminates the expression of arsC, which encodes an As(V) reductase, the resulting As(III)-sensitive strain still respires As(V); however, the generation time and the Y(lactate) value are two- and threefold lower, respectively, than those of the wild type. These results suggest that ArsB and ArsC may be useful for As(V)-respiring bacteria in environments where As concentrations are high, but that neither is required for respiration.

FIG. 1.

Phylogenetic relationships among 16S rDNA sequences from Shewanella strains. Shewanella sp. strain ANA-3 is boxed and in boldface type. The phylogenetic tree was constructed according to the distance criterion. The scale represents the number of substitutions per site. The percentage of 1,000 bootstrap replicates that supported the branching order is shown near the relevant nodes. Nodes without bootstrap values occurred <50%. Outgroups included Alteromonas macleodii (X82145) and Pseudoalteromonas haloplanktis (X67024). GenBank accession numbers for Shewanella species are given parenthetically as follows: S. figidimarina (U85903), S. japonica (Af145921), S. algae (U91546), S. amazonensis (AF005248), Shewanella sp. strain ANA-3 (AF136392), S. baltica (AJ000214), S. putrefaciens (U91550), S. oneidensis (AF005251), Shewanella sp. strain MR-8 (AF005254), Shewanella sp. strain MR-7 (AF005253), Shewanella sp. strain MR-4 (AF005252), S. gelidimarina (U85907), S. pealeana (AF011335), S. benthica (X82131), S. violacea (D21225), S. hanedai (U91590), S. woodyi (AF003549), and S. colwelliana (AF170794).

FIG. 2.

(A) Respiratory arsenate reduction and growth of Shewanella sp. strain ANA-3 on lactate as the electron donor. (B) Oxidation of lactate and accumulation of acetate during respiration on arsenate. Data are representative of triplicate cultures.

FIG. 3.

The As^r cosmid pSALT1 confers As(III) resistance to E. coli AW3110 (A) and S. oneidensis MR-1 (B). Strains were grown aerobically in LB medium with the specified As(III) concentrations. Tetracycline was added at 15 μg/ml to strains harboring pSALT1 or the cosmid vector pLAFR5. The initial OD₆₃₀ was <0.05 on average in all experiments. Values and error bars represent the averages and standard deviations of quadruplicate samples, respectively, after 24 h of incubation.

FIG. 4.

(A) Map of the ANA-3 ars operon and location of the arsB mutation in ARSB1. The mutation in arsB with the EZ::TN<KAN-2> transposon is indicated by the black 1.2-kb size box. Numbers under the genes indicate the percent identity (similarity) to the corresponding R773 ars operon homolog. (B) Gel picture showing the results of PCR with primers TNARSBF and TNARSBR with genomic DNA of the ARSB1 strain (lane 2), pSALT1-B10 cosmid (lane 3), wild-type ANA-3 (lane 4), and a reagent negative control (lane 5). Lane 1 contains a 1-kb ladder. (C) RT-PCR analysis for the expression of arsC, arsA, and 16S rDNA genes after ANA-3 and ARSB1 were grown for 4 h in the presence of 1 mM As(V). Lanes 1 to 3 and lanes 4 to 6 correspond to ANA-3 and ARSB1, respectively. Lanes also correspond to RT with primer (lanes 1 and 4), no RT added (lanes 2 and 5), RT without primer (lanes 3 and 6), ANA-3 genomic DNA (PCR-positive control) (lane 7), water (PCR-negative control) (lane 8), and DNA ladders in kilobases (lane 9).

FIG. 5.

Anaerobic As(V) respiration with lactate as the sole carbon source and electron donor (A), resistance profile to increasing As(III) concentrations (B), and time course for growth in 5 mM As(III) (C) for ANA-3 and ARSB1. In panels B and C, both strains were grown anaerobically on lactate and fumarate with As(III) added at the specified concentrations, and the initial OD₆₀₀ values were similar to that of the blank medium (i.e., 0.005). The values and error bars in all three panels represent the averages and ranges of duplicate samples, respectively.