The Bacterial and Fungal Diversity of an Aged PAH- and Heavy Metal-Contaminated Soil is Affected by Plant Cover and Edaphic Parameters.

Industrial wasteland soils with aged PAH and heavy metal contaminations are environments where pollutant toxicity has been maintained for decades. Although the communities may be well adapted to the presence of stressors, knowledge about microbial diversity in such soils is scarce. Soil microbial community dynamics can be driven by the presence of plants, but the impact of plant development on selection or diversification of microorganisms in these soils has not been established yet. To test these hypotheses, aged-contaminated soil samples from a field trial were collected. Plots planted with alfalfa were compared to bare soil plots, and bacterial and fungal diversity and abundance were assessed after 2 and 6 years. Using pyrosequencing of 16S rRNA gene and ITS amplicons, we showed that the bacterial community was dominated by Proteobacteria, Actinobacteria, and Bacteroidetes and was characterized by low Acidobacteria abundance, while the fungal community was mainly represented by members of the Ascomycota. The short-term toxic impact of pollutants usually reduces the microbial diversity, yet in our samples bacterial and fungal species richness and diversity was high suggesting that the community structure and diversity adapted to the contaminated soil over decades. The presence of plants induced higher bacterial and fungal diversity than in bare soil. It also increased the relative abundance of bacterial members of the Actinomycetales, Rhizobiales, and Xanthomonadales orders and of most fungal orders. Multivariate analysis showed correlations between microbial community structure and heavy metal and PAH concentrations over time, but also with edaphic parameters (C/N, pH, phosphorus, and nitrogen concentrations).

Leaf-associated fungal diversity in acidified streams: insights from combining traditional and molecular approaches.

We combined microscopic and molecular methods to investigate fungal assemblages on alder leaf litter exposed in the benthic and hyporheic zones of five streams across a gradient of increasing acidification for 4 weeks. The results showed that acidification and elevated Al concentrations strongly depressed sporulating aquatic hyphomycetes diversity in both zones of streams, while fungal diversity assessed by denaturing gradient gel electrophoresis (DGGE) appeared unaffected. Clone library analyses revealed that fungal communities on leaves were dominated by members of Ascomycetes and to a lesser extent by Basidiomycetes and Chytridiomycetes. An important contribution of terrestrial fungi was observed in both zones of the most acidified stream and in the hyporheic zone of the reference circumneutral stream. The highest leaf breakdown rate was observed in the circumneutral stream and occurred in the presence of both the highest diversity of sporulating aquatic hyphomycetes and the highest contribution to clone libraries of sequences affiliated with aquatic hyphomycetes. Both methods underline the major role played by aquatic hyphomycetes in leaf decomposition process. Our findings also bring out new highlights on the identity of leaf-associated fungal communities and their responses to anthropogenic alteration of running water ecosystems.

Impaired leaf litter processing in acidified streams : learning from microbial enzyme activities.

Anthropogenic acidification in headwater streams is known to affect microbial assemblages involved in leaf litter breakdown. Far less is known about its potential effects on microbial enzyme activities. To assess the effects of acidification on microbial activities associated with decaying leaves, a 70-day litter bag experiment was conducted in headwater streams at six sites across an acidification gradient. The results revealed that microbial leaf decomposition was strongly and negatively correlated with total Al concentrations (r = -0.99, p < 0.001) and positively correlated with Ca(2+) concentrations (r = 0.94, p = 0.005) and pH (r = 0.93, p = 0.008). Denaturing gradient gel electrophoresis analyses showed that microbial assemblages differed between non-impacted and impacted sites, whereas fungal biomass associated with decaying leaves was unaffected. The nutrient content of leaf detritus and ecoenzymatic activities of carbon (C), nitrogen (N) and phosphorus (P) acquisition revealed that N acquisition was unaltered, while P acquisition was significantly reduced across the acidification gradient. The P content of leaf litter was negatively correlated with total Al concentrations (r = -0.94, p < 0.01) and positively correlated with decomposition rates (r = 0.95, p < 0.01). This potential P limitation of microbial decomposers in impacted sites was confirmed by the particularly high turnover activity for phosphatase and imbalanced ratios between the ecoenzymatic activities of C and P acquisition. The toxic form of Al has well-known direct effects on aquatic biota under acidic conditions, but in this study, Al was found to also potentially affect microbially mediated leaf processing by interfering with the P cycle. These effects may in turn have repercussions on higher trophic levels and whole ecosystem functioning.

Biodegradation of Phenanthrene by Pseudomonas putida and a Bacterial Consortium in the Presence and in the Absence of a Surfactant.

This study describes the biodegradation of phenanthrene in aqueous media in the presence and in the absence of a surfactant, Brij 30. Biodegradations were performed using either Pseudomonas putida DSMZ 8368 or a bacterial consortium Pyr01 isolated from one PAHs-polluted site. P. putida degraded phenanthrene to form 1-hydroxy-2-naphthoic acid (1H2Na) as the major metabolite. LC-MS analysis revealed the production of complementary intermediates in the presence of Brij 30, showing intense ions at mass-to-charge ratios (m/z) 97 and 195. Higher phenanthrene biodegradation rate was obtained in the presence of Brij 30. Conversely, in the case of Pyr01consortium, the addition of Brij 30 (0.5 g L(-1)) had a negative effect on biodegradation: no phenanthrene biodegradation products were detected in the medium, whereas a production of several intermediates (m/z 97, 195 and 293) was obtained without surfactant. New results on phenanthrene metabolism by P. putida DSMZ 8368 and Pyr01 consortium in the presence and in the absence of Brij 30 we obtained. They confirm that the knowledge of the effect of a surfactant on bacterial cultures is crucial for the optimization of surfactant-enhanced PAHs biodegradation.

Influence of microorganisms on uranium release from mining-impacted lake sediments under various oxygenation conditions.

Microbial processes can be involved in the remobilization of uranium (U) from reduced sediments under O2 reoxidation events such as water table fluctuations. Such reactions could be typically encountered after U-bearing sediment dredging operations. Solid U(IV) species may thus reoxidize into U(VI) that can be released in pore waters in the form of aqueous complexes with organic and inorganic ligands. Non-uraninite U(IV) species may be especially sensitive to reoxidation and remobilization processes. Nevertheless, little is known regarding the effect of microbially mediated processes on the behaviour of U under these conditions.

Transcriptomic effects of di-(2-ethylhexyl)-phthalate in Syrian hamster embryo cells: an important role of early cytoskeleton disturbances in carcinogenesis?

BACKGROUND: Di-(2-ethylhexyl)-phthalate (DEHP) is a commonly used plasticizer in polyvinylchloride (PVC) formulations and a potentially non-genotoxic carcinogen. The aim of this study was to identify genes whose level of expression is altered by DEHP by using a global wide-genome approach in Syrian hamster embryo (SHE) cells, a model similar to human cells regarding their responses to this type of carcinogen. With mRNA Differential Display (DD), we analysed the transcriptional regulation of SHE cells exposed to 0, 12.5, 25 and 50 µM of DEHP for 24 hrs, conditions which induced neoplastic transformation of these cells. A real-time quantitative polymerase chain reaction (qPCR) was used to confirm differential expression of genes identified by DD. RESULTS: Gene expression profiling showed 178 differentially-expressed fragments corresponding to 122 genes after tblastx comparisons, 79 up-regulated and 43 down-regulated. The genes of interest were involved in many biological pathways, including signal transduction, regulation of the cytoskeleton, xenobiotic metabolism, apoptosis, lipidogenesis, protein conformation, transport and cell cycle. We then focused particularly on genes involved in the regulation of the cytoskeleton, one of the processes occurring during carcinogenesis and in the early steps of neoplastic transformation. Twenty one cytoskeleton-related genes were studied by qPCR. The down-regulated genes were involved in focal adhesion or cell junction. The up-regulated genes were involved in the regulation of the actin cytoskeleton and this would suggest a role of cellular plasticity in the mechanism of chemical carcinogenesis. The gene expression changes identified in the present study were PPAR-independent. CONCLUSION: This study identified a set of genes whose expression is altered by DEHP exposure in mammalian embryo cells. This is the first study that elucidates the genomic changes of DEHP involved in the organization of the cytoskeleton. The latter genes may be candidates as biomarkers predictive of early events in the multistep carcinogenic process.

Characterization of the ars gene cluster from extremely arsenic-resistant Microbacterium sp. strain A33.

The arsenic resistance gene cluster of Microbacterium sp. A33 contains a novel pair of genes (arsTX) encoding a thioredoxin system that are cotranscribed with an unusual arsRC2 fusion gene, ACR3, and arsC1 in an operon divergent from arsC3. The whole ars gene cluster is required to complement an Escherichia coli ars mutant. ArsRC2 negatively regulates the expression of the pentacistronic operon. ArsC1 and ArsC3 are related to thioredoxin-dependent arsenate reductases; however, ArsC3 lacks the two distal catalytic cysteine residues of this class of enzymes.

Environmental transcriptomes of invasive dreissena, a model species in ecotoxicology and invasion biology.

Dreissenids are established model species for ecological and ecotoxicological studies, since they are sessile and filter feeder organisms and reflect in situ freshwater quality. Despite this strong interest for hydrosystem biomonitoring, omics data are still scarce. In the present study, we achieved full de novo assembly transcriptomes of digestive glands to gain insight into Dreissena polymorpha and D. rostriformis bugensis molecular knowledge. Transcriptomes were obtained by Illumina RNA sequencing of seventy-nine organisms issued from fifteen populations inhabiting sites that exhibits multiple freshwater contamination levels and different hydrosystem topographies (open or closed systems). Based on a recent de novo assembly algorithm, we carried out a complete, quality-checked and annotated transcriptomes. The power of the present study lies in the completeness of transcriptomes gathering multipopulational organisms sequencing and its full availability through an open access interface that gives a friendly and ready-to-use access to data. The use of such data for proteogenomic and targeted biological pathway investigations purpose is promising as they are first full transcriptomes for this two Dreissena species.

Isolation and characterization of a gene cluster involved in PAH degradation in Mycobacterium sp. strain SNP11: expression in Mycobacterium smegmatis mc(2)155.

Mycobacterium sp. strain SNP11 is able to grow with pyrene, fluoranthene, phenanthrene and fluorene the sole carbon and energy sources. A probe based on the previously described gene pdoA2, which encodes the alpha subunit of a PAH ring-hydroxylating dioxygenase in Mycobacterium sp. strain 6PY1 [S. Krivobok et al., Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases, J. Bacteriol. 185(13) (2003) 3828-3841], was used to isolate a 14kb DNA fragment from strain SNP11. Twelve putative open reading frames (ORFs), divided into two groups by a promoter intergenic region, were detected in this DNA sequence. The first gene cluster, located upstream of the promoter region, showed low but significant deduced amino acid sequence homologies with enzymes involved in aromatic degradation. The second gene cluster, under control of the promoter, contained pdoA2 (designated phdA in this study) and several other ORFs with deduced amino acid sequences closely related to enzymes involved in the phenanthrene-degrading pathway of Nocardioides sp. strain KP7. Gene expression analysis in Mycobacterium smegmatis mc(2)155 revealed broad substrate specificity of the ring-hydroxylating dioxygenase, since transformant cells containing phdAB strongly oxidized fluoranthene, phenanthrene, anthracene, fluorine and dibenzofuran. Laser desorption/ionization time-of-flight mass spectrometry (LDI-ToF MS) analyses of culture media after PAH degradation by M. smegmatis transformants also revealed that the second gene cluster, located downstream of the promoter, takes an active share in initial phenanthrene and anthracene degradation by allowing transformation of these two PAHs in aromatic ring-cleaved metabolites.

Characterization of IS1110-like sequences found in Mycobacterium species other than Mycobacterium avium.

During study of a polycyclic aromatic hydrocarbon-degrading gene cluster in Mycobacterium sp. SNP11, which is a fast-growing strain related to Mycobacterium gilvum, a DNA segment with very high similarities to the IS1110 element of Mycobacterium avium was identified. Insertion sequence IS1110 was discovered for the first time in 1994 during a study of plasmid incidence in AIDS-derived M. avium strains. This element had thus far been detected in most human, veterinary, and environmental M. avium isolates, but not in other Mycobacterium species such as M. bovis, M. tuberculosis, M. xenopi, M. kansasii or M. gordonae. In the present paper, we describe the isolation and characterization of ISMysp1, an IS1110-like element present in several copies in the genome of Mycobacterium sp. strain SNP11. PCR and hybridization experiments revealed that this element is commonly found in fast-growing Mycobacterium strains. Moreover, Blast searches against the recently sequenced genome of M. smegmatis mc(2)155 revealed that this strain contains four IS1110-like elements. Analysis and sequence comparison of the whole of the IS1110-like elements revealed several common features not found in the most closely related mycobacterial members, IS900, IS901, IS902, IS1626 and IS1547.

Molecular cloning, nucleotide sequencing and expression of genes encoding a cytochrome P450 system involved in secondary amine utilization in Mycobacterium sp. strain RP1.

Mycobacterium sp. strain RP1 degrades morpholine, piperidine, and pyrrolidine and is able to use these compounds as the sole source of carbon, nitrogen, and energy. Cytochrome P450 (MorA) is involved in the biodegradation of these secondary amines. A 3.9-PstI genomic DNA fragment, containing the gene encoding MorA, was cloned and sequenced. Four open reading frames were detected on this DNA fragment. The first encoded a cytochrome P450 designated as MorA which was the second member of the CYP151 family and was named CYP151A2. The second open reading frame (morB) featured a [3Fe-4S] type of ferredoxin. A third gene (morC), exhibiting sequence identity to known reductases, and a fourth truncated gene encoding a putative glutamine reductase (orf1' ), were found downstream of morB. Recombinant MorA cytochrome P450 was purified to homogeneity from Escherichia coli. The purified enzyme was a monomeric soluble protein with an apparent Mr of about 45,000. CYP151A2 catalyzed the ring cleavage of the secondary amines and the Vmax/KMapp values indicated that pyrrolidine is the preferred substrate for this monooxygenase.

Ecotoxicological assessment of PAHs and their dead-end metabolites after degradation by Mycobacterium sp. strain SNP11.

Mycobacterium sp. SNP11 has a high PAH biodegradation potential. In this paper, the toxicity of pyrene, fluoranthene, phenanthrene, and their dead-end metabolites, accumulated in the media after biodegradation by Mycobacterium sp. SNP11, were evaluated by a screening battery of acute, chronic, and genotoxic tests. According to the bioassays, performed on bacteria (Vibrio fischeri, Salmonella typhimurium strains TA1535/pSK1002, TA97a, TA98, TA100), algae (Pseudokirchneriella subcapitata), and crustaceans (Daphnia magna, Ceriodaphnia dubia), total disappearance or a very significant reduction of the (geno)toxic potential was observed after PAH degradation by Mycobacterium sp. SNP11.