Genome Sequence of the Photoarsenotrophic Bacterium Ectothiorhodospira sp. Strain BSL-9, Isolated from a Hypersaline Alkaline Arsenic-Rich Extreme Environment.

The full genome sequence of Ectothiorhodospira sp. strain BSL-9 is reported here. This purple sulfur bacterium encodes an arxA-type arsenite oxidase within the arxB2AB1CD gene island and is capable of carrying out "photoarsenotrophy" anoxygenic photosynthetic arsenite oxidation. Its genome is composed of 3.5 Mb and has approximately 63% G+C content

Lysine-91 of the tetraheme c-type cytochrome CymA is essential for quinone interaction and arsenate respiration in Shewanella sp. strain ANA-3

The tetraheme c-type cytochrome, CymA, is essential for arsenate respiratory reduction in Shewanella sp. ANA-3, a model arsenate reducer. CymA is predicted to mediate electron transfer from quinols to the arsenate respiratory reductase (ArrAB). Here, we present biochemical and physiological evidence that CymA interacts with menaquinol (MQH2) substrates. Fluorescence quench titration with the MQH2 analog, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), was used to demonstrate quinol binding of E. coli cytoplasmic membranes enriched with various forms of CymA. Wild-type CymA bound HOQNO with a Kd of 0.1–1 μM. It was also shown that the redox active MQH2 analog, 2,3-dimethoxy-1,4-naphthoquinone (DMNH2), could reduce CymA in cytoplasmic membrane preparations. Based on a CymA homology model made from the NrfH tetraheme cytochrome structure, it was predicted that Lys91 would be involved in CymA-quinol interactions. CymA with a K91Q substitution showed little interaction with HOQNO. In addition, DMNH2-dependent reduction of CymA-K91Q was diminished by 45% compared to wild-type CymA. A ΔcymA ANA-3 strain containing a plasmid copy of cymA-K91Q failed to grow with arsenate as an electron acceptor. These results suggest that Lys91 is physiologically important for arsenate respiration and support the hypothesis that CymA interacts with menaquinol resulting in the reduction of the cytochrome

Homology of Escherichia coli R773 arsA, arsB, and arsC Genes in Arsenic-Resistant Bacteria Isolated from Raw Sewage and Arsenic-Enriched Creek Waters

The occurrence and diversity of the Escherichia coli R773 ars operon were investigated among arsenic-resistant enteric and nonenteric bacteria isolated from raw sewage and arsenic-enriched creek waters. Selected isolates from each creek location were screened for ars genes by colony hybridization and PCR. The occurrence of arsA, arsB, and arsC determined by low-stringency colony hybridization (31 to 53% estimated mismatch) was 81, 87, and 86%, respectively, for 84 bacteria isolated on arsenate- and arsenite-amended media from three locations. At moderate stringency (21 to 36% estimated mismatch), the occurrence decreased to 42, 56, and 63% for arsA, arsB, and arsC, respectively. PCR results showed that the ars operon is conserved in some enteric bacteria isolated from creek waters and raw sewage. The occurrence of the arsBC genotype was about 50% in raw sewage enteric bacteria, while arsA was detected in only 9.4% of the isolates (n = 32). The arsABC and arsBC genotypes occurred more frequently in enteric bacteria isolated from creek samples: 71.4 and 85.7% (n = 7), respectively. Average sequence divergence within arsB for six creek enteric bacteria was 20% compared to that of the E. coli R773 ars operon. Only 1 of 11 pseudomonads screened by PCR was positive for arsB. The results from this study suggest that significant divergence has occurred in the ars operon among As-resistant E. coli strains and in Pseudomonas spp

The cymA Gene, Encoding a Tetraheme c-Type Cytochrome, Is Required for Arsenate Respiration in Shewanella Species▿

In Shewanella sp. strain ANA-3, utilization of arsenate as a terminal electron acceptor is conferred by a two-gene operon, arrAB, which lacks a gene encoding a membrane-anchoring subunit for the soluble ArrAB protein complex. Analysis of the genome sequence of Shewanella putrefaciens strain CN-32 showed that it also contained the same arrAB operon with 100% nucleotide identity. Here, we report that CN-32 respires arsenate and that this metabolism is dependent on arrA and an additional gene encoding a membrane-associated tetraheme c-type cytochrome, cymA. Deletion of cymA in ANA-3 also eliminated growth on and reduction of arsenate. The ΔcymA strains of CN-32 and ANA-3 negatively affected the reduction of Fe(III) and Mn(IV) but not growth on nitrate. Unlike the CN-32 ΔcymA strain, growth on fumarate was absent in the ΔcymA strain of ANA-3. Both homologous and heterologous complementation of cymA in trans restored growth on arsenate in ΔcymA strains of both CN-32 and ANA-3. Transcription patterns of cymA showed that it was induced under anaerobic conditions in the presence of fumarate and arsenate. Nitrate-grown cells exhibited the greatest level of cymA expression in both wild-type strains. Lastly, site-directed mutagenesis of the first Cys to Ser in each of the four CXXCH c-heme binding motifs of the CN-32 CymA nearly eliminated growth on and reduction of arsenate. Together, these results indicate that the biochemical mechanism of arsenate respiration and reduction requires the interactions of ArrAB with a membrane-associated tetraheme cytochrome, which in the non-arsenate-respiring Shewanella species Shewanella oneidensis strain MR-1, has pleiotropic effects on Fe(III), Mn(IV), dimethyl sulfoxide, nitrate, nitrite, and fumarate respiration

The ArsR Repressor Mediates Arsenite-Dependent Regulation of Arsenate Respiration and Detoxification Operons of Shewanella sp. Strain ANA-3▿ †

Microbial arsenate reduction affects the fate and transport of arsenic in the environment. Arsenate respiratory (arr) and detoxifying (ars) reduction pathways in Shewanella sp. strain ANA-3 are induced by arsenite and under anaerobic conditions. Here it is shown that an ArsR family protein, called ArsR2, regulates the arsenate respiratory reduction pathway in response to elevated arsenite under anaerobic conditions. Strains lacking arsR2 grew faster in the presence of high levels of arsenite (3 mM). Moreover, expression of arrA and arsC (arsenate reductase-encoding genes) in the ΔarsR2 mutant of ANA-3 were increased in cells grown under anaerobic conditions and in the absence of arsenic. Mutations in putative arsenic binding amino acid residues in ArsR2 (substitutions of Cys-30 and Cys-32 with Ser) resulted in ANA-3 strains that exhibited anaerobic growth deficiencies with high levels of arsenite and arsenate. DNA binding studies with purified ArsR2 showed that ArsR2 binding to the arr promoter region was impaired by trivalent arsenicals such as arsenite and phenylarsine oxide. However, ArsR2 binding occurred in the presence of arsenate. A second known regulator of the arr operon, cyclic AMP (cAMP)-cAMP receptor protein (CRP), could bind simultaneously with ArsR2 within the arr promoter region. It is concluded that ArsR2 is most likely the major arsenite-dependent regulator of arr and ars operons in Shewanella sp. strain ANA-3. However, anaerobic growth on arsenate will require coregulation with global regulators such as cAMP-CRP

Genetic identification of a respiratory arsenate reductase

For more than a decade, it has been recognized that arsenate [H(2)AsO(4)(1-); As(V)] can be used by microorganisms as a terminal electron acceptor in anaerobic respiration. Given the toxicity of arsenic, the mechanistic basis of this process is intriguing, as is its evolutionary origin. Here we show that a two-gene cluster (arrAB; arsenate respiratory reduction) in the bacterium Shewanella sp. strain ANA-3 specifically confers respiratory As(V) reductase activity. Mutants with in-frame deletions of either arrA or arrB are incapable of growing on As(V), yet both are able to grow on a wide variety of other electron acceptors as efficiently as the wild-type. Complementation by the wild-type sequence rescues the mutants' ability to respire As(V). arrA is predicted to encode a 95.2-kDa protein with sequence motifs similar to the molybdenum containing enzymes of the dimethyl sulfoxide reductase family. arrB is predicted to encode a 25.7-kDa iron–sulfur protein. arrA and arrB comprise an operon that contains a twin arginine translocation (Tat) motif in ArrA (but not in ArrB) as well as a putative anaerobic transcription factor binding site upstream of arrA, suggesting that the respiratory As(V) reductase is exported to the periplasm via the Tat pathway and under anaerobic transcriptional control. These genes appear to define a new class of reductases that are specific for respiratory As(V) reduction

Expression Dynamics of Arsenic Respiration and Detoxification in Shewanella sp. Strain ANA-3

Because arsenate [As(V)] reduction by bacteria can significantly enhance arsenic mobility in the environment, it is important to be able to predict when this activity will occur. Currently, two bacterial systems are known that specifically reduce As(V), namely, a respiratory system (encoded by the arr genes) and a detoxification system (encoded by the ars genes). Here we analyze the conditions under which these two systems are expressed in Shewanella sp. strain ANA-3. The ars system is expressed under both aerobic and anaerobic conditions, whereas the arr system is only expressed anaerobically and is repressed by oxygen and nitrate. When cells are grown on As(V), the arr system is maximally induced during exponential growth, with peak expression of the ars system occurring at the beginning of stationary phase. Both the arr and ars systems are specifically induced by arsenite [As(III)], but the arr system is activated by a concentration of As(III) that is 1,000 times lower than that required for the arsC system (≤100 nM versus ≤100 μM, respectively). A double mutant was constructed that does not reduce As(V) under any growth conditions. In this strain background, As(V) is capable of inducing the arr system at low micromolar concentrations, but it does not induce the ars system. Collectively, these results demonstrate that the two As(V) reductase systems in ANA-3 respond to different amounts and types of inorganic arsenic

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

The ars Detoxification System Is Advantageous but Not Required for As(V) Respiration by the Genetically Tractable Shewanella Species Strain ANA-3

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