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2.  TiO2 Photocatalysis Damages Lipids and Proteins in Escherichia coli 
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.
PMCID: PMC3993174  PMID: 24532071
3.  Novel and unexpected bacterial diversity in an arsenic-rich ecosystem revealed by culture-dependent approaches 
Biology Direct  2012;7:28.
Acid 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.
Despite 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.
This 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.
This paper was reviewed by Sándor Pongor, Eugene V. Koonin and Brett Baker (nominated by Purificacion Lopez-Garcia).
PMCID: PMC3443666  PMID: 22963335
Acid mine drainage (AMD); Alkaliphilic bacteria; Neutrophilic bacteria; Functional redundancy; Rare biosphere; Uncultured bacteria; Molecular biases; Culture-dependent approaches; Actinobacteria; Bacterial diversity
4.  Amylases without known homologues discovered in an acid mine drainage: significance and impact 
Scientific Reports  2012;2:354.
Acid Mine Drainages (AMDs) are extreme environments characterized by acidic and oligotrophic conditions and by metal contaminations. A function-based screening of an AMD-derived metagenomic library led to the discovery and partial characterization of two non-homologous endo-acting amylases sharing no sequence similarity with any known amylase nor glycosidase. None carried known amylolytic domains, nor could be assigned to any GH-family. One amylase displayed no similarity with any known protein, whereas the second one was similar to TraC proteins involved in the bacterial type IV secretion system. According to the scarce similarities with known proteins, 3D-structure modelling using I-TASSER was unsuccessful. This study underlined the utility of a function-driven metagenomic approach to obtain a clearer image of the bacterial community enzymatic landscape. More generally, this work points out that screening for microorganisms or biomolecules in a priori incongruous environments could provide unconventional and new exciting ways for bioprospecting.
PMCID: PMC3319935  PMID: 22482035
5.  Deciphering the role of Paenibacillus strain Q8 in the organic matter recycling in the acid mine drainage of Carnoulès 
The recycling of the organic matter is a crucial function in any environment, especially in oligotrophic environments such as Acid Mine Drainages (AMDs). Polymer-degrading bacteria might play an important role in such ecosystem, at least by releasing by-products useful for the rest of the community. In this study, physiological, molecular and biochemical experiments were performed to decipher the role of a Paenibacillus strain isolated from the sediment of Carnoulès AMD.
Even though Paenibacillus sp. strain Q8 was isolated from an oligotrophic AMD showing an acidic pH, it developed under both acidic and alkaline conditions and showed a heterotrophic metabolism based on the utilization of a broad range of organic compounds. It resisted to numerous metallic stresses, particularly high arsenite (As(III)) concentrations (> 1,800 mg/L). Q8 was also able to efficiently degrade polymers such as cellulose, xylan and starch. Function-based screening of a Q8 DNA-library allowed the detection of 15 clones with starch-degrading activity and 3 clones with xylan-degrading activity. One clone positive for starch degradation carried a single gene encoding a "protein of unknown function". Amylolytic and xylanolytic activities were measured both in growing cells and with acellular extracts of Q8. The results showed the ability of Q8 to degrade both polymers under a broad pH range and high As(III) and As(V) concentrations. Activity measurements allowed to point out the constitutive expression of the amylase genes and the mainly inducible expression of the xylanase genes. PACE demonstrated the endo-acting activity of the amylases and the exo-acting activity of the xylanases.
AMDs have been studied for years especially with regard to interactions between bacteria and the inorganic compartment hosting them. To date, no study reported the role of microorganisms in the recycling of the organic matter. The present work suggests that the strain Q8 might play an important role in the community by recycling the scarce organic matter (cellulose, hemicellulose, starch...), especially when the conditions change. Furthermore, function-based screening of a Q8 DNA library allowed to assign an amylolytic function to a gene previously unknown. AMDs could be considered as a reservoir of genes with potential biotechnological properties.
PMCID: PMC3287962  PMID: 22305268
Paenibacillus; Functional redundancy; Acid Mine Drainage (AMD); Amylase; Xylanase; Polymer degradation; Organic matter; Function-based screening; Community function
6.  Subinhibitory Arsenite Concentrations Lead to Population Dispersal in Thiomonas sp. 
PLoS ONE  2011;6(8):e23181.
Biofilms represent the most common microbial lifestyle, allowing the survival of microbial populations exposed to harsh environmental conditions. Here, we show that the biofilm development of a bacterial species belonging to the Thiomonas genus, frequently found in arsenic polluted sites and playing a key role in arsenic natural remediation, is markedly modified when exposed to subinhibitory doses of this toxic element. Indeed, arsenite [As(III)] exposure led to a considerable impact on biofilm maturation by strongly increasing the extracellular matrix synthesis and by promoting significant cell death and lysis within microcolonies. These events were followed by the development of complex 3D-biofilm structures and subsequently by the dispersal of remobilized cells observed inside the previously formed hollow voids. Our results demonstrate that this biofilm community responds to arsenite stress in a multimodal way, enhancing both survival and dispersal. Addressing this complex bacterial response to As(III) stress, which might be used by other microorganisms under various adverse conditions, may be essential to understand how Thiomonas strains persist in extreme environments.
PMCID: PMC3158062  PMID: 21876737
7.  A Tale of Two Oxidation States: Bacterial Colonization of Arsenic-Rich Environments 
PLoS Genetics  2007;3(4):e53.
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.
Author Summary
Microorganisms play a crucial role in nutrient biogeochemical cycles. Arsenic is found throughout the environment from both natural and anthropogenic sources. Its inorganic forms are highly toxic and impair the physiology of most higher organisms. Arsenic contamination of groundwater supplies is giving rise to increasingly severe human health problems in both developing and industrial countries. In the present work, we investigated the metabolism of this metalloid in Herminiimonas arsenicoxydans, a representative organism of a novel bacterial genus widespread in aquatic environments. Examination of the genome sequence and experimental evidence revealed that it is remarkably capable of coping with arsenic. Our observations support the existence of multiple strategies allowing arsenic-metabolizing microbes to efficiently colonize toxic environments. In particular, arsenic oxidation and scavenging may have played a crucial role in the development of early stages of life on Earth. Such mechanisms may one day be exploited as part of a potential bioremediation strategy in toxic environments.
PMCID: PMC1851979  PMID: 17432936
8.  Arsenite Oxidase aox Genes from a Metal-Resistant β-Proteobacterium 
Journal of Bacteriology  2003;185(1):135-141.
The β-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.
PMCID: PMC141815  PMID: 12486049

Results 1-8 (8)