Characterization of the Arsenate Respiratory Reductase from Shewanella sp. Strain ANA-3

Microbial arsenate respiration contributes to the mobilization of arsenic from the solid to the soluble phase in various locales worldwide. To begin to predict the extent to which As(V) respiration impacts arsenic geochemical cycling, we characterized the expression and activity of the Shewanella sp. strain ANA-3 arsenate respiratory reductase (ARR), the key enzyme involved in this metabolism. ARR is expressed at the beginning of the exponential phase and persists throughout the stationary phase, at which point it is released from the cell. In intact cells, the enzyme localizes to the periplasm. To purify ARR, a heterologous expression system was developed in Escherichia coli. ARR requires anaerobic conditions and molybdenum for activity. ARR is a heterodimer of ~131 kDa, composed of one ArrA subunit (~95 kDa) and one ArrB subunit (~27 kDa). For ARR to be functional, the two subunits must be expressed together. Elemental analysis of pure protein indicates that one Mo atom, four S atoms associated with a bis-molybdopterin guanine dinucleotide cofactor, and four to five [4Fe-4S] are present per ARR. ARR has an apparent melting temperature of 41°C, a Km of 5 µM, and a Vmax of 11,111 µmol of As(V) reduced min–1 mg of protein–1 and shows no activity in the presence of alternative electron acceptors such as antimonite, nitrate, selenate, and sulfate. The development of a heterologous overexpression system for ARR will facilitate future structural and/or functional studies of this protein family

Molecular and Environmental Studies of Bacterial Arsenate Respiration

Arsenate [As(V)]-respiring bacteria that reduce As(V) to arsenite, As(III), for energy production have been implicated as possible catalysts for arsenic mobilization into drinking water supplies. To understand how this metabolism contributes to arsenic geochemistry, this thesis explores the dynamics of As(V)-respiratory gene expression, the impact of As(V) respiration on microbial ferric [Fe(III)] reduction, and biochemical properties of the arsenate respiratory reductase, ARR. Using sequences for arrA, a gene encoding the terminal reductase involved in As(V) respiration, degenerate PCR primers were designed to amplify a diagnostic region of the gene in multiple As(V)-respiring isolates. These primers were used to track arrA transcription in microcosm studies involving synthetic sediments. arrA was required for As(V) reduction in this context, and the gene was expressed in contaminated sediments at Haiwee Reservoir in Olancha, CA. To understand the impact of As(V) respiration on Fe(III) reduction, native microbial consortia from Haiwee Reservoir and pure cultures of the genetically tractable Shewanella sp. strain ANA-3 were incubated with As-sorbed hydrous ferric oxide (HFO), and rates of As(V) and Fe(III) reduction were determined. As(V) reduction occurred simultaneously with or prior to Fe(III) reduction, consistent with the idea that electron acceptor utilization is determined by thermodynamic favorability. Furthermore, the presence of sorbed As(III) increased rates of Fe(III) reduction, potentially by increasing HFO surface area. Lastly, the expression, assembly, and kinetic properties of ARR from ANA-3 were characterized. ARR is a soluble periplasmic heterodimer that is expressed during early exponential growth and persists into late stationary phase. The enzyme contains molybdenum, Fe, and sulfur cofactors. It has a Km of 5 µM, a Vmax of 11,111 µmol As(V) reduced . min-1 . mg protein-1, and reduces only As(V). Mutational analysis of the residues corresponding to the diagnostic region of arrA mentioned above resulted in loss of enzyme activity. This work brings us closer to being able to quantify and predict the contribution of As(V) respiration to the solubilization of arsenic from sediments. Structural studies, the development of probes to detect ARR, and comparisons of ARR from different bacterial species are now possible.</p

A subset of the diverse COG0523 family of putative metal chaperones is linked to zinc homeostasis in all kingdoms of life

<p>Abstract</p> <p>Background</p> <p>COG0523 proteins are, like the nickel chaperones of the UreG family, part of the G3E family of GTPases linking them to metallocenter biosynthesis. Even though the first COG0523-encoding gene, <it>cobW</it>, was identified almost 20 years ago, little is known concerning the function of other members belonging to this ubiquitous family.</p> <p>Results</p> <p>Based on a combination of comparative genomics, literature and phylogenetic analyses and experimental validations, the COG0523 family can be separated into at least fifteen subgroups. The CobW subgroup involved in cobalamin synthesis represents only one small sub-fraction of the family. Another, larger subgroup, is suggested to play a predominant role in the response to zinc limitation based on the presence of the corresponding COG0523-encoding genes downstream from putative Zur binding sites in many bacterial genomes. Zur binding sites in these genomes are also associated with candidate zinc-independent paralogs of zinc-dependent enzymes. Finally, the potential role of COG0523 in zinc homeostasis is not limited to Bacteria. We have predicted a link between COG0523 and regulation by zinc in Archaea and show that two COG0523 genes are induced upon zinc depletion in a eukaryotic reference organism, <it>Chlamydomonas reinhardtii</it>.</p> <p>Conclusion</p> <p>This work lays the foundation for the pursuit by experimental methods of the specific role of COG0523 members in metal trafficking. Based on phylogeny and comparative genomics, both the metal specificity and the protein target(s) might vary from one COG0523 subgroup to another. Additionally, Zur-dependent expression of <it>COG0523 </it>and putative paralogs of zinc-dependent proteins may represent a mechanism for hierarchal zinc distribution and zinc sparing in the face of inadequate zinc nutrition.</p

The genetics of geochemistry

Bacteria are remarkable in their metabolic diversity due to their ability to harvest energy from myriad oxidation and reduction reactions. In some cases, their metabolisms involve redox transformations of metal(loid)s, which lead to the precipitation, transformation, or dissolution of minerals. Microorganism/mineral interactions not only affect the geochemistry of modern environments, but may also have contributed to shaping the near-surface environment of the early Earth. For example, bacterial anaerobic respiration of ferric iron or the toxic metalloid arsenic is well known to affect water quality in many parts of the world today, whereas the utilization of ferrous iron as an electron donor in anoxygenic photosynthesis may help explain the origin of Banded Iron Formations, a class of ancient sedimentary deposits. Bacterial genetics holds the key to understanding how these metabolisms work. Once the genes and gene products that catalyze geochemically relevant reactions are understood, as well as the conditions that trigger their expression, we may begin to predict when and to what extent these metabolisms influence modern geochemical cycles, as well as develop a basis for deciphering their origins and how organisms that utilized them may have altered the chemical and physical features of our planet

Microbial ecology of arsenic-mobilizing Cambodian sediments: lithological controls uncovered by stable-isotope probing

Microbially mediated arsenic release from Holocene and Pleistocene Cambodian aquifer sediments was investigated using microcosm experiments and substrate amendments. In the Holocene sediment, the metabolically active bacteria, including arsenate-respiring bacteria, were determined by DNA stable-isotope probing. After incubation with 13C-acetate and 13C-lactate, active bacterial community in the Holocene sediment was dominated by different Geobacter spp.-related 16S rRNA sequences. Substrate addition also resulted in the enrichment of sequences related to the arsenate-respiring Sulfurospirillum spp. 13C-acetate selected for ArrA related to Geobacter spp. whereas 13C-lactate selected for ArrA which were not closely related to any cultivated organism. Incubation of the Pleistocene sediment with lactate favoured a 16S rRNA-phylotype related to the sulphate-reducing Desulfovibrio oxamicus DSM1925, whereas the ArrA sequences clustered with environmental sequences distinct from those identified in the Holocene sediment. Whereas limited As(III) release was observed in Pleistocene sediment after lactate addition, no arsenic mobilization occurred from Holocene sediments, probably because of the initial reduced state of As, as determined by X-ray Absorption Near Edge Structure. Our findings demonstrate that in the presence of reactive organic carbon, As(III) mobilization can occur in Pleistocene sediments, having implications for future strategies that aim to reduce arsenic contamination in drinking waters by using aquifers containing Pleistocene sediments

Desulfuribacillus alkaliarsenatis gen. nov. sp. nov., a deep-lineage, obligately anaerobic, dissimilatory sulfur and arsenate-reducing, haloalkaliphilic representative of the order Bacillales from soda lakes

An anaerobic enrichment culture inoculated with a sample of sediments from soda lakes of the Kulunda Steppe with elemental sulfur as electron acceptor and formate as electron donor at pH 10 and moderate salinity inoculated with sediments from soda lakes in Kulunda Steppe (Altai, Russia) resulted in the domination of a Gram-positive, spore-forming bacterium strain AHT28. The isolate is an obligate anaerobe capable of respiratory growth using elemental sulfur, thiosulfate (incomplete reduction) and arsenate as electron acceptor with H2, formate, pyruvate and lactate as electron donor. Growth was possible within a pH range from 9 to 10.5 (optimum at pH 10) and a salt concentration at pH 10 from 0.2 to 2 M total Na+ (optimum at 0.6 M). According to the phylogenetic analysis, strain AHT28 represents a deep independent lineage within the order Bacillales with a maximum of 90 % 16S rRNA gene similarity to its closest cultured representatives. On the basis of its distinct phenotype and phylogeny, the novel haloalkaliphilic anaerobe is suggested as a new genus and species, Desulfuribacillus alkaliarsenatis (type strain AHT28T = DSM24608T = UNIQEM U855T)

A new family of periplasmic-binding proteins that sense arsenic oxyanions

Arsenic contamination of drinking water affects more than 140 million people worldwide. While toxic to humans, inorganic forms of arsenic (arsenite and arsenate), can be used as energy sources for microbial respiration. AioX and its orthologues (ArxX and ArrX) represent the first members of a new sub-family of periplasmic-binding proteins that serve as the first component of a signal transduction system, that's role is to positively regulate expression of arsenic metabolism enzymes. As determined by X-ray crystallography for AioX, arsenite binding only requires subtle conformational changes in protein structure, providing insights into protein-ligand interactions. The binding pocket of all orthologues is conserved but this alone is not sufficient for oxyanion selectivity, with proteins selectively binding either arsenite or arsenate. Phylogenetic evidence, clearly demonstrates that the regulatory proteins evolved together early in prokaryotic evolution and had a separate origin from the metabolic enzymes whose expression they regulate

Metal bioremediation by CrMTP4 over-expressing Chlamydomonas reinhardtii in comparison to natural wastewater-tolerant microalgae strains

Metal pollution in freshwater bodies is a long-standing challenge with large expense required to clean-up pollutants such as Cd. There is widespread interest in the potentially low-cost and sustainable use of biological material to perform bioremediation, such as the use of microalgae. Efficient metal bioremediation capacity requires both the ability to tolerate metal stress and metal accumulation. Here, the role of a Chlamydomonas reinhardtii metal tolerance protein (MTP) was examined for enhanced Cd tolerance and uptake. The CrMTP4 gene is a member of the Mn-CDF clade of the cation diffusion facilitator family of metal transporters but is able to provide tolerance and sequestration for Mn and Cd, but not other metals, when expressed in yeast. Over-expression of CrMTP4 in C. reinhardtii yielded a significant increase in tolerance to Cd toxicity and increased Cd accumulation although tolerance to Mn was not increased. In comparison, the metal tolerance of three chlorophyte microalgae strains (Chlorella luteoviridis, Parachlorella hussii, and Parachlorella kessleri) that had previously been adapted to wastewater growth was examined. In comparison to wild type C. reinhardtii, all three natural strains showed significantly increased tolerance to Cd, Cu, Al and Zn, and furthermore their Cd tolerance and uptake was greater than that of the CrMTP4 over-expression strains. Despite CrMTP4 gene over-expression being a successful strategy to enhance the Cd bioremediation potential of a metal-sensitive microalga, a single gene manipulation cannot compete with naturally adapted strain mechanisms that are likely to be multigenic and due in part to oxidative stress tolerance