Genome sequence and characterisation of a freshwater photoarsenotroph, Cereibacter azotoformans strain ORIO, isolated from sediments capable of cyclic light-dark arsenic oxidation and reduction.

A freshwater photosynthetic arsenite-oxidizing bacterium, Cereibacter azotoformans strain ORIO, was isolated from Owens River, CA, USA. The waters from Owens River are elevated in arsenic and serve as the headwaters to the Los Angeles Aqueduct. The complete genome sequence of strain ORIO is 4.8 Mb genome (68% G + C content) and comprises two chromosomes and six plasmids. Taxonomic analysis placed ORIO within the Cereibacter genus (formerly Rhodobacter). The ORIO genome contains arxB2 AB1 CD (encoding an arsenite oxidase), arxXSR (regulators) and several ars arsenic resistance genes all co-localised on a 136 kb plasmid, named pORIO3. Phylogenetic analysis of ArxA, the molybdenum-containing arsenite oxidase catalytic subunit, demonstrated photoarsenotrophy is likely to occur within members of the Alphaproteobacteria. ORIO is a mixotroph, oxidises arsenite to arsenate (As(V)) photoheterotrophically, and expresses arxA in cultures grown with arsenite. Further ecophysiology studies with Owens River sediment demonstrated the interconversion of arsenite and As(V) was dependent on light-dark cycling. arxA and arrA (As(V) respiratory reductase) genes were detected in the light-dark cycled sediment metagenomes suggesting syntrophic interactions among arsenotrophs. This work establishes C. azotoformans str. ORIO as a new model organism for studying photoarsenotrophy and light-dark arsenic biogeochemical cycling.

Linking nitrate removal, carbon cycling, and mobilization of geogenic trace metals during infiltration for managed recharge.

We present results from a series of laboratory column studies investigating the impacts of infiltration dynamics and the addition of a soil-carbon amendment (wood mulch or almond shells) on water quality during infiltration for flood-managed aquifer recharge (flood-MAR). Recent studies suggest that nitrate removal could be enhanced during infiltration for MAR through the application of a wood chip permeable reactive barrier (PRB). However, less is understood about how other readily available carbon sources, such as almond shells, could be used as a PRB material, and how carbon amendments could impact other solutes, such as trace metals. Here we show that the presence of a carbon amendment increases nitrate removal relative to native soil, and that there is greater nitrate removal in association with longer fluid retention times (slower infiltration rates). Almond shells promoted more efficient nitrate removal than wood mulch or native soil, but also promoted the mobilization of geogenic trace metals (Mn, Fe, and As) during experiments. Almond shells in a PRB likely enhanced nitrate removal and trace metal cycling by releasing labile carbon, promoting reducing conditions, and providing habitat for microbial communities, the composition of which shifted in response. These results suggest that limiting the amount of bioavailable carbon released by a carbon-rich PRB may be preferred where geogenic trace metals are common in soils. Given the dual threats to groundwater supplies and quality worldwide, incorporating a suitable carbon source into the soil for managed infiltration projects could help to generate co-benefits and avoid undesirable results.

Soil characteristics and redox properties of infiltrating water are determinants of microbial communities at managed aquifer recharge sites.

In this study, we conducted a meta-analysis of soil microbial communities at three, pilot-scale field sites simulating shallow infiltration for managed aquifer recharge (MAR). We evaluated shifts in microbial communities after infiltration across site location, through different soils, with and without carbon-rich amendments added to test plots. Our meta-analysis aims to enable more effective MAR basin design by identifying potentially important interactions between soil physical-geochemical parameters and microbial communities across several geographically separate MAR basins. We hypothesized infiltration and carbon amendments would lead to common changes in subsurface microbial communities at multiple field sites but instead found distinct differences. Sites with coarser (mainly sandy) soil had large changes in diversity and taxa abundance, while sites with finer soils had fewer significant changes in genera, despite having the greatest increase in nitrogen cycling. Below test plots amended with a carbon-rich permeable reactive barrier, we observed more nitrate removal and a decrease in genera capable of nitrification. Multivariate statistics determined that the soil texture (a proxy for numerous soil characteristics) was the main determinant of whether the microbial community composition changed because of infiltration. These results suggest that microbial communities in sandy soil with carbon-rich amendments are most impacted by infiltration. Soil composition is a critical parameter that links between microbial communities and nutrient cycling during infiltration and could influence the citing and operation of MAR to benefit water quality and supply.

Enhanced cycling of nitrogen and metals during rapid infiltration: Implications for managed recharge.

We present results from a series of plot-scale field experiments to quantify physical infiltration dynamics and the influence of adding a carbon-rich, permeable reactive barrier (PRB) for the cycling of nitrogen and associated trace metals during rapid infiltration for managed aquifer recharge (MAR). Recent studies suggest that adding a bio-available carbon source to soils can enhance denitrification rates and associated N load reduction during moderate-to-rapid infiltration (≤1 m/day). We examined the potential for N removal during faster infiltration (>1 m/day), through coarse and carbon-poor soils, and how adding a carbon-rich PRB (wood chips) affects subsurface redox conditions and trace metal mobilization. During rapid infiltration, plots amended with a carbon-rich PRB generally demonstrated modest increases in subsurface loads of dissolved organic carbon, nitrite, manganese and iron, decreases in loads of nitrate and ammonium, and variable changes in arsenic. These trends differed considerably from those seen during infiltration through native soil without a carbon-rich PRB. Use of a carbon-rich soil amendment increased the fraction of dissolved N species that was removed at equivalent inflowing N loads. There is evidence that N removal took place primarily via denitrification. Shifts in microbial ecology following infiltration in all of the plots included increases in the relative abundances of microbes in the families Comamonadaceae, Pseudomonadaceae, Methylophilaceae, Rhodocyclaceae and Sphingomonadaceae, all of which contain genera capable of carrying out denitrification. These results, in combination with studies that have tested other soil types, flow rates, and system scales, show how water quality can be improved during infiltration for managed recharge, even during rapid infiltration, with a carbon-rich soil amendment.

Photodynamic Activity of Graphene Oxide/Polyaniline/Manganese Oxide Ternary Composites toward Both Gram-Positive and Gram-Negative Bacteria.

Graphene derivatives have been attracting extensive interest as effective antimicrobial agents. In the present study, ternary nanocomposites are prepared based on graphene oxide quantum dots (GOQD), polyaniline (PANI), and manganese oxides. Because of the hydrophilic GOQD and PANI, the resulting GPM nanocomposites are readily dispersible in water and upon photoirradiation at 365 nm exhibit antimicrobial activity toward both Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus epidermidis (S. epidermidis). Notably, the nanocomposite with a high Mn2+ and Mn4+ content is found to be far more active than that with a predominant Mn3+ component, although both samples feature a similar elemental composition and average Mn valence state. The bactericidal activity is largely ascribed to the photocatalytic production of hydroxy radicals and photogenerated holes; both are known to exert oxidative stress on bacterial cells. Further antimicrobial contributions may arise from the strong affinity of the nanocomposites to the cell surfaces. These results suggest that the metal valence state may be a critical parameter in the design and engineering of high-performance antimicrobial agents based on metal oxide nanocomposites.

Antibacterial Activity of Nitrogen-Doped Carbon Dots Enhanced by Atomic Dispersion of Copper.

Antibiotic resistance is an imminent threat to human health, requiring the development of effective alternate antibacterial agents. One such alternative includes nanoparticle (photo)catalysts that are good at producing reactive oxygen species (ROS). Herein, we report the design and preparation of nitrogen-doped carbon dots functionalized with atomically dispersed copper centers by Cu-N coordination (Cu/NCD) that exhibit apparent antibacterial activity toward Gram-negative Escherichia coli (E. coli) under photoirradiation. The growth of E. coli cells is found to be markedly inhibited by Cu/NCD under 365 nm photoirradiation, whereas no apparent inhibition is observed in the dark or with the copper-free carbon dots alone. This is ascribed to the prolonged photoluminescence lifetime of Cu/NCD that facilitates the separation of photogenerated electron-hole pairs and ROS formation. The addition of tert-butyl alcohol is found to completely diminish the antimicrobial activity, suggesting that hydroxyl radicals are responsible for microbial death. Consistent results are obtained from fluorescence microscopic studies using CellROX green as the probe. Similar bactericidal behaviors are observed with Gram-positive Staphylococcus epidermidis (S. epidermidis). The copper content within the carbon material is optimized at a low loading of 1.09 wt %, reducing the possibility of toxic copper-ion leaching. Results from this study highlight the significance of carbon-based nanocomposites with isolated metal species as potent antimicrobial reagents.

Denitrification during infiltration for managed aquifer recharge: Infiltration rate controls and microbial response.

Managed aquifer recharge (MAR) systems can be designed and operated to improve water supply and quality simultaneously by creating favorable conditions for contaminant removal during infiltration through shallow soils. We present results from laboratory flow-through column experiments, using intact soil cores from two MAR sites, elucidating conditions that are favorable to nitrate (NO3) removal via microbial denitrification during infiltration. Experiments focused on quantitative relations between infiltration rate and the presence or absence of a carbon-rich permeable reactive barrier (PRB) on both amounts and rates of nitrate removal during infiltration and associated shifts in microbial ecology. Experiments were conducted using a range of infiltration rates relevant to MAR (0.3-1.4 m/day), with PRBs made of native soil (NS), woodchips (WC) and a 50:50 mixture of woodchips and native soil (MIX). The latter two (carbon-rich) PRB treatments led to statistically significant increases in the amount of nitrate removed by increasing zero-order denitrification rates, both within the PRB materials and in the underlying soil. The highest fraction of nitrate removal occurred at the lowest infiltration rates for all treatments. However, the highest nitrogen mass removal (∆NL) was observed at 0.4-0.7 m/day for both the WC and MIX treatments. In contrast, the maximum ∆NL for the NS treatment was observed at the lowest infiltration rates measured (~0.3 m/day). Further, both carbon-rich PRBs had a substantial impact on the soil microbial ecology in the underlying soil, with lower overall diversity and a greater relative abundance of groups known to degrade carbon and metabolize nitrogen. These results demonstrate that infiltration rates and carbon availability can combine to create favorable conditions for denitrification during infiltration for MAR and show how these factors shape and sustain the microbial community structures responsible for nutrient cycling in associated soils.

Antimicrobial activity of graphene oxide quantum dots: impacts of chemical reduction.

Design and engineering of graphene-based functional nanomaterials for effective antimicrobial applications has been attracting extensive interest. In the present study, graphene oxide quantum dots (GOQDs) were prepared by chemical exfoliation of carbon fibers and exhibited apparent antimicrobial activity. Transmission electron microscopic measurements showed that the lateral length ranged from a few tens to a few hundred nanometers. Upon reduction by sodium borohydride, whereas the UV-vis absorption profile remained largely unchanged, steady-state photoluminescence measurements exhibited a marked blue-shift and increase in intensity of the emission, due to (partial) removal of phenanthroline-like structural defects within the carbon skeletons. Consistent results were obtained in Raman and time-resolved photoluminescence measurements. Interestingly, the samples exhibited apparent, but clearly different, antimicrobial activity against Staphylococcus epidermidis cells. In the dark and under photoirradiation (400 nm), the as-produced GOQDs exhibited markedly higher cytotoxicity than the chemically reduced counterparts, likely because of (i) effective removal by NaBH4 reduction of redox-active phenanthroline-like moieties that interacted with the electron-transport chain of the bacterial cells, and (ii) diminished production of hydroxyl radicals that were potent bactericidal agents after chemical reduction as a result of increased conjugation within the carbon skeletons.

A new approach to biomining: Bioengineering surfaces for metal recovery from aqueous solutions.

Electronics waste production has been fueled by economic growth and the demand for faster, more efficient consumer electronics. The glass and metals in end-of-life electronics components can be reused or recycled; however, conventional extraction methods rely on energy-intensive processes that are inefficient when applied to recycling e-waste that contains mixed materials and small amounts of metals. To make e-waste recycling economically viable and competitive with obtaining raw materials, recovery methods that lower the cost of metal reclamation and minimize environmental impact need to be developed. Microbial surface adsorption can aid in metal recovery with lower costs and energy requirements than traditional metal-extraction approaches. We introduce a novel method for metal recovery by utilizing metal-binding peptides to functionalize fungal mycelia and enhance metal recovery from aqueous solutions such as those found in bioremediation or biomining processes. Using copper-binding as a proof-of-concept, we compared binding parameters between natural motifs and those derived in silico, and found comparable binding affinity and specificity for Cu. We then combined metal-binding peptides with chitin-binding domains to functionalize a mycelium-based filter to enhance metal recovery from a Cu-rich solution. This finding suggests that engineered peptides could be used to functionalize biological surfaces to recover metals of economic interest and allow for metal recovery from metal-rich effluent with a low environmental footprint, at ambient temperatures, and under circumneutral pH.

Field and Laboratory Studies Linking Hydrologic, Geochemical, and Microbiological Processes and Enhanced Denitrification during Infiltration for Managed Recharge.

We present linked field and laboratory studies investigating controls on enhanced nitrate processing during infiltration for managed aquifer recharge. We examine how carbon-rich permeable reactive barriers (PRBs) made of woodchips or biochar, placed in the path of infiltrating water, stimulate microbial denitrification. In field studies with infiltration of 0.2-0.3 m/day and initial nitrate concentrations of [NO3-N] = 20-28 mg/L, we observed that woodchips promoted 37 ± 6.6% nitrate removal (primarily via denitrification), and biochar promoted 33 ± 12% nitrate removal (likely via denitrification and physical absorption effects). In contrast, unamended soil at the same site generated <5% denitrification. We find that the presence of a carbon-rich PRB has a modest effect on the underlying soil microbial community structure in these experiments, indicating that existing consortia have the capability to carry out denitrification given favorable conditions. In laboratory studies using intact cores from the same site, we extend the results to quantify how infiltration rate influences denitrification, with and without a carbon-rich PRB. We find that the influence of both PRB materials is diminished at higher infiltration rates (>0.7 m/day) but can still result in denitrification. These results demonstrate a quantitative relationship between infiltration rate and denitrification that depends on the presence and nature of a PRB. Combined results from these field and laboratory experiments, with complementary studies of denitrification during infiltration through other soils, suggest a framework for understanding linked hydrologic and chemical controls on microbial denitrification (and potentially other redox-sensitive processes) that could improve water quality during managed recharge.

Arsenate-dependent growth is independent of an ArrA mechanism of arsenate respiration in the termite hindgut isolate Citrobacter sp. strain TSA-1.

Citrobacter sp. strain TSA-1 is an enteric bacterium isolated from the hindgut of the termite. Strain TSA-1 displays anaerobic growth with selenite, fumarate, tetrathionate, nitrate, or arsenate serving as electron acceptors, and it also grows aerobically. In regards to arsenate, genome sequencing revealed that strain TSA-1 lacks a homolog for respiratory arsenate reductase, arrAB, and we were unable to obtain amplicons of arrA. This raises the question as to how strain TSA-1 achieves As(V)-dependent growth. We show that growth of strain TSA-1 on glycerol, which it cannot ferment, is linked to the electron acceptor arsenate. A series of transcriptomic experiments were conducted to discern which genes were upregulated during growth on arsenate, as opposed to those on fumarate or oxygen. For As(V), upregulation was noted for 1 of the 2 annotated arsC genes, while there was no clear upregulation for tetrathionate reductase (ttr), suggesting that this enzyme is not an alternative to arrAB as occurs in certain hyperthermophilic archaea. A gene-deletion mutant strain of TSA-1 deficient in arsC could not achieve anaerobic respiratory growth on As(V). Our results suggest that Citrobacter sp. strain TSA-1 has an unusual and as yet undefined means of achieving arsenate respiration, perhaps involving its ArsC as a respiratory reductase as well as a detoxifying agent.

A horizontal permeable reactive barrier stimulates nitrate removal and shifts microbial ecology during rapid infiltration for managed recharge.

We present results from field experiments linking hydrology, geochemistry, and microbiology during infiltration at a field site that is used for managed aquifer recharge (MAR). These experiments measured how a horizontal permeable reactive barrier (PRB) made of woodchips impacted subsurface nitrate removal and microbial ecology. Concentrations of dissolved organic carbon consistently increased in infiltrating water below the PRB, but not in un-amended native soil. The average nitrate removal rate in soils below the PRB was 1.5 g/m2/day NO3-N, despite rapid infiltration (up to 1.9 m/d) and a short fluid residence time within the woodchips (≤6 h). In contrast, 0.09 g/m2/day NO3-N was removed on average in native soil. Residual nitrate in infiltrating water below the PRB was enriched in δ15N and δ18O, with low and variable isotopic enrichment factors that are consistent with denitrification during rapid infiltration. Many putative denitrifying bacteria were significantly enhanced in the soil below a PRB; Methylotenera mobilis and genera Microbacterium, Polaromonas, and Novosphingobium had log2 fold-changes of +4.9, +5.6, +7.2, and +11.8, respectively. These bacteria were present before infiltration and were not enhanced in native soil. It appears that the woodchip PRB contributed to favorable conditions in the underlying soil for enhanced nitrate removal, quantitatively shifting soil microbial ecology. These results suggest that using a horizontal PRB could improve water quality during rapid infiltration for MAR.

Photo-enhanced antibacterial activity of ZnO/graphene quantum dot nanocomposites.

Synthesis of new, highly active antibacterial agents has become increasingly important in light of emerging antibiotic resistance. In the present study, ZnO/graphene quantum dot (GQD) nanocomposites were produced by a facile hydrothermal method and characterized by an array of microscopic and spectroscopic measurements, including transmission electron microscopy, X-ray photoelectron spectroscopy, UV-vis and photoluminescence spectroscopy. Antibacterial activity of the ZnO/GQD nanocomposites was evaluated with Escherichia coli within the context of minimum inhibitory concentration and the reduction of the number of bacterial colonies in a standard plate count method, in comparison to those with ZnO and GQD separately. It was found that the activity was markedly enhanced under UV photoirradiation as compared to that in ambient light. This was ascribed to the enhanced generation of reactive oxygen species under UV photoirradiation, with minor contributions from membrane damage, as manifested in electron paramagnetic resonance and fluorescence microscopic measurements. The results highlight the significance of functional nanocomposites based on semiconductor nanoparticles and graphene derivatives in the development of effective bactericidal agents.

Autotrophic microbial arsenotrophy in arsenic-rich soda lakes.

A number of prokaryotes are capable of employing arsenic oxy-anions as either electron acceptors [arsenate; As(V)] or electron donors [arsenite; As(III)] to sustain arsenic-dependent growth ('arsenotrophy'). A subset of these microorganisms function as either chemoautotrophs or photoautotrophs, whereby they gain sufficient energy from their redox metabolism of arsenic to completely satisfy their carbon needs for growth by autotrophy, that is the fixation of inorganic carbon (e.g. HCO3-) into their biomass. Here we review what has been learned of these processes by investigations we have undertaken in three soda lakes of the western USA and from the physiological characterizations of the relevant bacteria, which include the critical genes involved, such as respiratory arsenate reductase (arrA) and the discovery of its arsenite-oxidizing counterpart (arxA). When possible, we refer to instances of similar process occurring in other, less extreme ecosystems and by microbes other than haloalkaliphiles.

Antibacterial mechanisms of graphene-based composite nanomaterials.

Pathogenic bacteria are gaining resistance to conventional antibiotics at an alarming rate due to overuse and rapid transfer of resistance genes between bacterial populations. As bacterial resistance to antibiotics causes millions of fatalities worldwide, it is of urgent importance to develop a new class of antibiotic materials with both broad-spectrum bactericidal activity and suitable biocompatibility. Graphene derivatives are rapidly emerging as an extremely promising class of antimicrobial materials due to their diverse bactericidal mechanisms and relatively low cytotoxicity towards mammalian cells. By combining graphene derivatives with currently utilized antibacterial metal and metal-oxide nanostructures, composite materials with exceptional bactericidal activity can be achieved. In this review, the antibacterial activities of graphene derivatives as well as their metal and metal-oxide composite nanostructures will be presented. The synthetic methodology for these various materials will be briefly mentioned, and emphasis will be placed on the evaluation of their mechanisms of action. This information will provide a valuable insight into the current understanding of the interactions governing the microbial toxicity of graphene-based composite nanostructures.

The genetic basis of anoxygenic photosynthetic arsenite oxidation.

'Photoarsenotrophy', the use of arsenite as an electron donor for anoxygenic photosynthesis, is thought to be an ancient form of phototrophy along with the photosynthetic oxidation of Fe(II), H2 S, H2 and NO2-. Photoarsenotrophy was recently identified from Paoha Island's (Mono Lake, CA) arsenic-rich hot springs. The genomes of several photoarsenotrophs revealed a gene cluster, arxB2AB1CD, where arxA is predicted to encode for the sole arsenite oxidase. The role of arxA in photosynthetic arsenite oxidation was confirmed by disrupting the gene in a representative photoarsenotrophic bacterium, resulting in the loss of light-dependent arsenite oxidation. In situ evidence of active photoarsenotrophic microbes was supported by arxA mRNA detection for the first time, in red-pigmented microbial mats within the hot springs of Paoha Island. This work expands on the genetics for photosynthesis coupled to new electron donors and elaborates on known mechanisms for arsenic metabolism, thereby highlighting the complexities of arsenic biogeochemical cycling.

Arsenite as an Electron Donor for Anoxygenic Photosynthesis: Description of Three Strains of Ectothiorhodospira from Mono Lake, California and Big Soda Lake, Nevada.

Three novel strains of photosynthetic bacteria from the family Ectothiorhodospiraceae were isolated from soda lakes of the Great Basin Desert, USA by employing arsenite (As(III)) as the sole electron donor in the enrichment/isolation process. Strain PHS-1 was previously isolated from a hot spring in Mono Lake, while strain MLW-1 was obtained from Mono Lake sediment, and strain BSL-9 was isolated from Big Soda Lake. Strains PHS-1, MLW-1, and BSL-9 were all capable of As(III)-dependent growth via anoxygenic photosynthesis and contained homologs of arxA, but displayed different phenotypes. Comparisons were made with three related species: Ectothiorhodospira shaposhnikovii DSM 2111, Ectothiorhodospira shaposhnikovii DSM 243T, and Halorhodospira halophila DSM 244. All three type cultures oxidized arsenite to arsenate but did not grow with As(III) as the sole electron donor. DNA-DNA hybridization indicated that strain PHS-1 belongs to the same species as Ect. shaposhnikovii DSM 2111 (81.1% sequence similarity), distinct from Ect. shaposhnikovii DSM 243T (58.1% sequence similarity). These results suggest that the capacity for light-driven As(III) oxidation is a common phenomenon among purple photosynthetic bacteria in soda lakes. However, the use of As(III) as a sole electron donor to sustain growth via anoxygenic photosynthesis is confined to novel isolates that were screened for by this selective cultivation criterion.

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.

Archaeal and bacterial diversity in an arsenic-rich shallow-sea hydrothermal system undergoing phase separation.

Phase separation is a ubiquitous process in seafloor hydrothermal vents, creating a large range of salinities. Toxic elements (e.g., arsenic) partition into the vapor phase, and thus can be enriched in both high and low salinity fluids. However, investigations of microbial diversity at sites associated with phase separation are rare. We evaluated prokaryotic diversity in arsenic-rich shallow-sea vents off Milos Island (Greece) by comparative analysis of 16S rRNA clone sequences from two vent sites with similar pH and temperature but marked differences in salinity. Clone sequences were also obtained for aioA-like functional genes (AFGs). Bacteria in the surface sediments (0-1.5 cm) at the high salinity site consisted of mainly Epsilonproteobacteria (Arcobacter sp.), which transitioned to almost exclusively Firmicutes (Bacillus sp.) at ~10 cm depth. However, the low salinity site consisted of Bacteroidetes (Flavobacteria) in the surface and Epsilonproteobacteria (Arcobacter sp.) at ~10 cm depth. Archaea in the high salinity surface sediments were dominated by the orders Archaeoglobales and Thermococcales, transitioning to Thermoproteales and Desulfurococcales (Staphylothermus sp.) in the deeper sediments. In contrast, the low salinity site was dominated by Thermoplasmatales in the surface and Thermoproteales at depth. Similarities in gas and redox chemistry suggest that salinity and/or arsenic concentrations may select for microbial communities that can tolerate these parameters. Many of the archaeal 16S rRNA sequences contained inserts, possibly introns, including members of the Euryarchaeota. Clones containing AFGs affiliated with either Alpha- or Betaproteobacteria, although most were only distantly related to published representatives. Most clones (89%) originated from the deeper layer of the low salinity, highest arsenic site. This is the only sample with overlap in 16S rRNA data, suggesting arsenotrophy as an important metabolism in similar environments.

Characterization of axial and proximal histidine mutations of the decaheme cytochrome MtrA from Shewanella sp. strain ANA-3 and implications for the electron transport system.

Extracellular respiration of solid-phase electron acceptors in some microorganisms requires a complex chain of multiheme c-type cytochromes that span the inner and outer membranes. In Shewanella species, MtrA, an ~35-kDa periplasmic decaheme c-type cytochrome, is an essential component for extracellular respiration of iron(III). The exact mechanism of electron transport has not yet been resolved, but the arrangement of the polypeptide chain may have a strong influence on the capability of the MtrA cytochrome to transport electrons. The iron hemes of MtrA are bound to its polypeptide chain via proximal (CXXCH) and distal histidine residues. In this study, we show the effects of mutating histidine residues of MtrA to arginine on protein expression and extracellular respiration using Shewanella sp. strain ANA-3 as a model organism. Individual mutations to six out of nine proximal histidines in CXXCH of MtrA led to decreased protein expression. However, distal histidine mutations resulted in various degrees of protein expression. In addition, the effects of histidine mutations on extracellular respiration were tested using ferrihydrite and current production in microbial fuel cells. These results show that proximal histidine mutants were unable to reduce ferrihydrite. Mutations to the distal histidine residues resulted in various degrees of ferrihydrite reduction. These findings indicate that mutations to the proximal histidine residues affect MtrA expression, leading to loss of extracellular respiration ability. In contrast, mutations to the distal histidine residues are less detrimental to protein expression, and extracellular respiration can proceed.