Marie-Claire Lett

Arsenite Oxidase aox Genes from a Metal-Resistant β-Proteobacterium

The beta-proteobacterial strain ULPAs1, isolated from an arsenic-contaminated environment, is able to efficiently oxidize arsenite [As(III)] to arsenate [As(V)]. Mutagenesis with a lacZ-based reporter transposon yielded two knockout derivatives deficient in arsenite oxidation. Sequence analysis of the DNA flanking the transposon insertions in the two mutants identified two adjacent open reading frames, named aoxA and aoxB, as well as a putative promoter upstream of the aoxA gene. Reverse transcription-PCR data indicated that these genes are organized in an operonic structure. The proteins encoded by aoxA and aoxB share 64 and 72% identity with the small Rieske subunit and the large subunit of the purified and crystallized arsenite oxidase of Alcaligenes faecalis, respectively (P. J. Ellis, T. Conrads, R. Hille, and P. Kuhn, Structure [Cambridge] 9:125-132, 2001). Importantly, almost all amino acids involved in cofactor interactions in both subunits of the A. faecalis enzyme were conserved in the corresponding sequences of strain ULPAs1. An additional Tat (twin-arginine translocation) signal peptide sequence was detected at the N terminus of the protein encoded by aoxA, strongly suggesting that the Tat pathway is involved in the translocation of the arsenite oxidase to its known periplasmic location.

Oxidation of arsenite to arsenate by a bacterium isolated from an aquatic environment.

Arsenic is ubiquitous in the biosphere and frequently reported to be an environmental pollutant. Global cycling of arsenic is affected by microorganisms. This paper describes a new bacterial strain which is able to efficiently oxidize arsenite (As[III]) into arsenate (As[V]) in liquid medium. The rate of the transformation depends on the cell density. Arsenic species were separated by high performance liquid chromatography (HPLC) and quantified by inductively coupled plasma-atomic emission spectrometry (ICP-AES). The strain also exhibits high minimum inhibitory concentrations (MICs) for As[III] (6.65 mM (500 mg L-1)) and other heavy metals, such as cadmium (1.42 mM (160 mg L-1)) or lead (1.20 mM (250 mg L-1)). Partial identification of the strain revealed a chemoorganotrophic, Gram-negative and motile rod. The results presented here demonstrate that this strain could represent a good candidate for arsenic remediation in heavily polluted sites.

A tale of two oxidation states: bacterial colonization of arsenic-rich environments.

Microbial biotransformations have a major impact on contamination by toxic elements, which threatens public health in developing and industrial countries. Finding a means of preserving natural environments—including ground and surface waters—from arsenic constitutes a major challenge facing modern society. Although this metalloid is ubiquitous on Earth, thus far no bacterium thriving in arsenic-contaminated environments has been fully characterized. In-depth exploration of the genome of the β-proteobacterium Herminiimonas arsenicoxydans with regard to physiology, genetics, and proteomics, revealed that it possesses heretofore unsuspected mechanisms for coping with arsenic. Aside from multiple biochemical processes such as arsenic oxidation, reduction, and efflux, H. arsenicoxydans also exhibits positive chemotaxis and motility towards arsenic and metalloid scavenging by exopolysaccharides. These observations demonstrate the existence of a novel strategy to efficiently colonize arsenic-rich environments, which extends beyond oxidoreduction reactions. Such a microbial mechanism of detoxification, which is possibly exploitable for bioremediation applications of contaminated sites, may have played a crucial role in the occupation of ancient ecological niches on earth.

Arsenic in contaminated waters: biogeochemical cycle, microbial metabolism and biotreatment processes.

Arsenic is responsible for the contamination of water supplies in various parts of the world and poses a major risk to human health. Its toxicity and bioavailability depend on its speciation, which in turn, depends on microbial transformations, including reduction, oxidation and methylation. This review describes the development of bioprocesses for the treatment of arsenic-contaminated waters based on bacterial metabolism and biogeochemical cycling of arsenic.

Unified Nomenclature for Genes Involved in Prokaryotic Aerobic Arsenite Oxidation

Published ahead of print 4 November 2011 The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.

TiO2 photocatalysis damages lipids and proteins in Escherichia coli

ABSTRACT This study investigates the mechanisms of UV-A (315 to 400 nm) photocatalysis with titanium dioxide (TiO2) applied to the degradation of Escherichia coli and their effects on two key cellular components: lipids and proteins. The impact of TiO2 photocatalysis on E. coli survival was monitored by counting on agar plate and by assessing lipid peroxidation and performing proteomic analysis. We observed through malondialdehyde quantification that lipid peroxidation occurred during the photocatalytic process, and the addition of superoxide dismutase, which acts as a scavenger of the superoxide anion radical (O2·−), inhibited this effect by half, showing us that O2·− radicals participate in the photocatalytic antimicrobial effect. Qualitative analysis using two-dimensional electrophoresis allowed selection of proteins for which spot modifications were observed during the applied treatments. Two-dimensional electrophoresis highlighted that among the selected protein spots, 7 and 19 spots had already disappeared in the dark in the presence of 0.1 g/liter and 0.4 g/liter TiO2, respectively, which is accounted for by the cytotoxic effect of TiO2. Exposure to 30 min of UV-A radiation in the presence of 0.1 g/liter and 0.4 g/liter TiO2 increased the numbers of missing spots to 14 and 22, respectively. The proteins affected by photocatalytic oxidation were strongly heterogeneous in terms of location and functional category. We identified several porins, proteins implicated in stress response, in transport, and in bacterial metabolism. This study reveals the simultaneous effects of O2·− on lipid peroxidation and on the proteome during photocatalytic treatment and therefore contributes to a better understanding of molecular mechanisms in antibacterial photocatalytic treatment.

Arsenite Oxidase from Ralstonia sp. 22: CHARACTERIZATION OF THE ENZYME AND ITS INTERACTION WITH SOLUBLE CYTOCHROMES

We characterized the aro arsenite oxidation system in the novel strain Ralstonia sp. 22, a β-proteobacterium isolated from soil samples of the Salsigne mine in southern France. The inducible aro system consists of a heterodimeric membrane-associated enzyme reacting with a dedicated soluble cytochrome c554. Our biochemical results suggest that the weak association of the enzyme to the membrane probably arises from a still unknown interaction partner. Analysis of the phylogeny of the aro gene cluster revealed that it results from a lateral gene transfer from a species closely related to Achromobacter sp. SY8. This constitutes the first clear cut case of such a transfer in the Aro phylogeny. The biochemical study of the enzyme demonstrates that it can accommodate in vitro various cytochromes, two of which, c552 and c554, are from the parent species. Cytochrome c552 belongs to the sox and not the aro system. Kinetic studies furthermore established that sulfite and sulfide, substrates of the sox system, are both inhibitors of Aro activity. These results reinforce the idea that sulfur and arsenic metabolism are linked.

Novel and unexpected bacterial diversity in an arsenic-rich ecosystem revealed by culture-dependent approaches

BackgroundAcid Mine Drainages (AMDs) are extreme environments characterized by very acid conditions and heavy metal contaminations. In these ecosystems, the bacterial diversity is considered to be low. Previous culture-independent approaches performed in the AMD of Carnoulès (France) confirmed this low species richness. However, very little is known about the cultured bacteria in this ecosystem. The aims of the study were firstly to apply novel culture methods in order to access to the largest cultured bacterial diversity, and secondly to better define the robustness of the community for 3 important functions: As(III) oxidation, cellulose degradation and cobalamine biosynthesis.ResultsDespite the oligotrophic and acidic conditions found in AMDs, the newly designed media covered a large range of nutrient concentrations and a pH range from 3.5 to 9.8, in order to target also non-acidophilic bacteria. These approaches generated 49 isolates representing 19 genera belonging to 4 different phyla. Importantly, overall diversity gained 16 extra genera never detected in Carnoulès. Among the 19 genera, 3 were previously uncultured, one of them being novel in databases. This strategy increased the overall diversity in the Carnoulès sediment by 70% when compared with previous culture-independent approaches, as specific phylogenetic groups (e.g. the subclass Actinobacteridae or the order Rhizobiales) were only detected by culture. Cobalamin auxotrophy, cellulose degradation and As(III)-oxidation are 3 crucial functions in this ecosystem, and a previous meta- and proteo-genomic work attributed each function to only one taxon. Here, we demonstrate that other members of this community can also assume these functions, thus increasing the overall community robustness.ConclusionsThis work highlights that bacterial diversity in AMDs is much higher than previously envisaged, thus pointing out that the AMD system is functionally more robust than expected. The isolated bacteria may be part of the rare biosphere which remained previously undetected due to molecular biases. No matter their current ecological relevance, the exploration of the full diversity remains crucial to decipher the function and dynamic of any community. This work also underlines the importance to associate culture-dependent and -independent approaches to gain an integrative view of the community function.ReviewersThis paper was reviewed by Sándor Pongor, Eugene V. Koonin and Brett Baker (nominated by Purificacion Lopez-Garcia).

UV-A photocatalytic treatment of Legionella pneumophila bacteria contaminated airflows through three-dimensional solid foam structured photocatalytic reactors

A 3D-structured photocatalytic media was designed for allowing a tubular reactor to work in a traversing-flow mode at low pressure drops with a strong increase in the surface area-to-volume ratio inside the reactor. A protective polysiloxane coating was performed for protecting a structured polyurethane foam and anchoring the active TiO(2) particles. Filled with the 3D-structured solid foam supporting TiO(2) photocatalyst, the reactor could thus take advantages from the static mixer effect and from the low pressure drop resulting from the reticulated foam support. Very efficient decontamination levels towards airborne Legionella pneumophila bacteria were reached in a single-pass test mode.

Heat-treated Saccharomyces cerevisiae for antimony speciation and antimony(III) preconcentration in water samples.

An analytical method was developed for antimony speciation and antimony(III) preconcentration in water samples. The method is based on the selective retention of Sb(III) by modified Saccharomyces cerevisiae in the presence of Sb(V). Heat, caustic and solvent pretreatments of the biomass were investigated to improve the kinetics and thermodynamics of Sb(III) uptake process at room temperature. Heating for 30 min at 80 degrees C was defined as the optimal treatment. Antimony accumulation by the cells was independent of pH (5-10) and ionic strength (0.01-0.1 mol L(-1)). 140 mg of yeast and 2h of contact were necessary to ensure quantitative sequestration of Sb(III) up to 750 microg L(-1). In these conditions, Sb(V) was not retained. Sb(V) was quantified in sorption supernatant by inductively coupled plasma mass spectrometry (ICP-MS) or inductively coupled plasma optical emission spectrometry (ICP-OES). Sb(III) was determined after elution with 40 mmol L(-1) thioglycolic acid at pH 10. A preconcentration factor close to nine was achieved for Sb(III) when 100mL of sample was processed. After preconcentration, the detection limits for Sb(III) and Sb(V) were 2 and 5 ng L(-1), respectively, using ICP-MS, 7 and 0.9 microg L(-1) using ICP-OES. The proposed method was successfully applied to the determination of Sb(III) and Sb(V) in spiked river and mineral water samples. The relative standard deviations (n=3) were in the 2-5% range at the tenth microg L(-1) level and less than 10% at the lowest Sb(III) and Sb(V) tested concentration (0.1 microg L(-1)). Corrected recoveries were in all cases close to 100%.