On the 230-kilobase-pair (kb) virulence plasmid of Shigella flexneri 2a strain YSH6000, at least seven separate genetic determinants have been identified. One of them, an approximately 4-kb region, virG, that is required for the Sereny reaction, was extensively studied to examine the role of the virG region. The phenotype of a VirG-mutant (M94) of YSH6000 in the cytoplasm of cultured MK cels was characterized by a kinetic study of the invading shigellae. The observed phenotype of M94 in the cytoplasm indicated that the virG locus is not required for multiplication of the invading shigellae, but is essential for their spread to adjacent cells. The DNA region necessary for the VirG function was localized to a 3.6-kb DNA sequence on the 230-kb plasmid. A 130-kilodalton polypeptide was confirmed to be the virG product. External labeling of bacteria with 1251 indicated that the 130-kilodalton virG protein is exposed on the bacterial surface. The nucleotide sequence of 4,472 bp, which contains the functional virG gene and its own regulatory sequence, was determined, and a large open reading frame encoding 1,102 amino acid residues was identified.

The ␤-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.Arsenic is present in various environments, is released either by natural weathering of rocks or by anthropogenic sources (e.g., mining industries and agricultural practices), and is found in the oxidation states ϩ5 (arsenate), ϩ3 (arsenite), 0 (elemental arsenic), and Ϫ3 (arsine). Contamination of drinkingwater supplies with the inorganic soluble forms arsenite and arsenate has often been reported, and arsenic has been identified as a major risk for human health in different parts of the world (northeast India, Bangladesh, northwest United States) (31). The biogeochemical cycle of this element strongly depends on microbial transformation, which affects the mobility and the distribution of arsenic species in the environment (33, 41). Several bacteria involved in transformation processes comprising reduction, oxidation, and methylation of arsenic species have been described (8,11,26,36,40).The toxicological effects of arsenic are related to its chemical form and oxidation state; the organic forms are the less toxic. Among inorganic forms, As(III) is reported to be on average 100 times more toxic than the less mobile As(V) (25). Several remediation processes have been described for arsenic removal (19) based on chemical oxidation of arsenite to arsenate followed by alkaline precipitation (5, 15-17, 24). The major drawbacks of these processes are that they generate additional pollution and are expensive. This has led to the exploration of alternative methods for arsenic remediation based on its biological oxidation. Several arsenite-oxidizing bacteria have been isolated, starting with an Achromobacter strain in 1918 (14). Since then, different arsenite-oxidizing bacteria, including several Pseudomonas strains (18, 42-44), A...

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.

The ability of Shigella to spread within and between epithelial cells is a prerequisite for causing bacillary dysentery and requires the function encoded by the virG gene on the large plasmid. The outer membrane VirG (IcsA) protein is essential for bacterial spreading by eliciting polar deposition of filamentous actin (F-actin) in the cytoplasm of epithelial cells. Recent studies have indicated that an N-terminal 80-kDa VirG portion is exposed on the bacterial cell surface and released into the external medium, while the following 37-kDa C-terminal portion is embedded in the outer membrane, although little is known about the extracellular transport of the VirG protein. In this study, we attempted to elucidate the export pathway of VirG protein across the outer membrane and found that the C-terminal 37-kDa portion, termed VirG ␤-core, serves as the self-transporter for the secretion of the preceding 80-kDa portion from the periplasmic side of the outer membrane to the external side. Indeed, foreign polypeptides such as MalE or PhoA covalently linked to the N terminus of VirG ␤-core were transported to the external side of the outer membrane, and it was further shown that the folding structure of the passenger polypeptide at the periplasmic side of the outer membrane interferes with its translocation. Analysis of the secondary structure of VirG ␤-core predicted that the critical structural property was a ␤-barrel channel consisting of amphipathic antiparallel transmembrane ␤-strands, interspersed by hairpin turns and loops. These results thus strongly suggest that the secretion of VirG protein from Shigella is similar to the export system utilized by the IgA protease of Neisseria.

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An autotrophic bacterium able to gain energy from the oxidation of arsenite was isolated from arsenite-containing acid mine drainage waters. It belongs to the genus Thiomonas as shown by DNA-DNA hybridization experiments, 16S rRNA gene sequence, quinone and fatty acid content analyses. Carboxysomes were observed and the cbbSL genes encoding the ribulose 1,5-bisphosphate carboxylase/oxygenase were detected, confirming that this bacterium is able to fix CO(2). Arsenite oxidation was catalysed by a membrane-bound enzyme, and this activity was detected essentially in cells grown in the presence of arsenite. The genes encoding the two subunits of the arsenite oxidase of the Thiomonas isolate have been sequenced. The small subunit has a characteristic Tat signal sequence and contains the residues binding the [2Fe-2S] Rieske-type cluster. The large subunit has the [3Fe-4S] cluster-binding motif as well as the residues proposed to bind arsenite. In addition, most of the residues interacting with the molybdenum cofactor are conserved. The genes encoding both subunits belong to an operon, likely with a gene encoding a cytochrome c. The expression of this operon is greater in cells grown in the presence than in the absence of arsenite, in agreement with a transcriptional regulation in the presence of this metalloid.

e This study investigates the mechanisms of UV-A (315 to 400 nm) photocatalysis with titanium dioxide (TiO 2 ) applied to the degradation of Escherichia coli and their effects on two key cellular components: lipids and proteins. The impact of TiO 2 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 (O 2 · ؊ ), inhibited this effect by half, showing us that O 2 · ؊ radicals participate in the photocatalytic antimicrobial effect. Qualitative analysis using twodimensional 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 TiO 2 , respectively, which is accounted for by the cytotoxic effect of TiO 2 . Exposure to 30 min of UV-A radiation in the presence of 0.1 g/liter and 0.4 g/liter TiO 2 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 O 2 · ؊ on lipid peroxidation and on the proteome during photocatalytic treatment and therefore contributes to a better understanding of molecular mechanisms in antibacterial photocatalytic treatment.

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