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Modern molecular biology techniques, particularly new DNA sequencing methods, have opened our eyes to the fact that microorganisms live in an extraordinary range of natural environments, from desert soils and [...]

A microbial community (short-chain alkane-degrading culture, SCADC) enriched from an oil sands tailings pond was shown to degrade C6-C10 alkanes under methanogenic conditions. Total genomic DNA from SCADC was subjected to 454 pyrosequencing, Illumina paired-end sequencing, and 16S rRNA amplicon pyrotag sequencing; the latter revealed 320 operational taxonomic units at 5% distance. Metagenomic sequences were subjected to in-house quality control and co- assembly, yielding 984 086 contigs, and annotation using MG-Rast and IMG. Substantial nucleotide and protein recruitment to Methanosaeta concilii, Syntrophus aciditrophicus, and Desulfobulbus propionicus reference genomes suggested the presence of closely related strains in SCADC; other genomes were not well mapped, reflecting the paucity of suitable reference sequences for such communities. Nonetheless, we detected numerous homologues of putative hydrocarbon succinate synthase genes (e.g., assA, bssA, and nmsA) implicated in anaerobic hydrocarbon degradation, suggesting the ability of the SCADC microbial community to initiate methanogenic alkane degradation by addition to fumarate. Annotation of a large contig revealed analogues of the ass operon 1 in the alkane-degrading sulphate-reducing bacterium Desulfatibacillum alkenivorans AK-01. Despite being enriched under methanogenic-fermentative conditions, additional metabolic functions inferred by COG profiling indicated multiple C[O.sub.2] fixation pathways, organic acid utilization, hydrogenase activity, and sulphate reduction. Key words: oil sands tailings ponds, anaerobic hydrocarbon biodegradation, alkanes, metagenome, bioinformatics. Une communaute bacterienne capable de degrader les alcanes a courtes chaines (SDADC, << short-chain alkanedegrading culture >>), isolee a partir d'un bassin de decantation des residus d'exploitation des sables bitumineux, s'est averee capable de degrader des alcanes C6-C10 dans des conditions methanogenes. L'ADN genomique total extrait de la culture SCADC a ete assujetti a du pyrosequen^age 454, a du sequencage Illumina des deux extremites (<< paired-end >>), et a du sequencage 454 d'etiquettes de l'amplicon d'ADNr 16S. Cette derniere analyse a permis de distinguer 320 unites taxonomiques operationnelles a une distance de 5 %. Les sequences issues de cette analyse metagenomique ont ete examinees pour leur qualite et leur assemblage, ce qui a permis d'obtenir 984 086 contigs, lesquels ont ete annotes avec MG-Rast et IMG. Une portion substantielle des sequences nucleotidiques et proteiques s'alignait sur les genomes de reference de Methanosaeta concilii, Syntrophus aciditrophicus et Desulfobulbus propionicus, ce qui suggere que les souches presentes au sein du SCADC etaient tres apparentees a ces especes. D'autres genomes n'etaient pas bien couverts, ce qui refleterait le faible nombre de genomes de reference pour ce genre de communaute bacterienne. Neanmoins, les auteurs ont detecte de nombreux homologues possibles de genes codant pour des hydrocarbure succinate synthetases (e.g. assA, bssA et nmsA) impliquees dans la degradation des hydrocarbures en conditions anaerobiques. Cela suggere que cette communaute serait capable d'initier la degradation methanogene d'alcanes par addition au fumarate. L'annotation d'un grand contig a revele la presence d'analogues de l'operon 1 d'ass chez la souche AK-01 du Desulfatibacillum alkenivorans, laquelle est capable de degrader les alcanes et de reduire les sulfates. En depit d'avoir ete enrichie sous des conditions methanogenes et de fermentation, d'autres fonctions metaboliques deduites grace au profil COG suggerent des activites liees a plusieurs autres sentiers metaboliques dont la fixation du C[O.sub.2], l'utilisation des acides organiques, une activite hydrogenase et de reduction des sulfates. [Traduit par la Redaction] Mots-cles: bassins de decantation des residus d'exploitation de sables bitumineux, degradation biologique des hydrocarbures en conditions anaerobiques, alcanes, metagenome, bioinformatique., Introduction Anaerobic biodegradation of petroleum hydrocarbons can be an important process in anoxic environments (Head et al. 2003; Gray et al. 2010; Berdugo-Clavijo et al. 2012). For example, biodegradation occurring [...]

Abstract: In 1982 diesel fuel migrated from a well lease site into underlying fractured bedrock contaminating the groundwater-bearing zone approximately 30 m below ground surface. Historical contaminant concentrations, geochemical indicators, [...]

The EmhABC efflux system in Pseudomonas fluorescens cLP6a is homologous to the muitidrug and solvent efflux systems belonging to the resistance-nodulation-division (RND) family and is responsible for polycyclic aromatic hydrocarbon transport, antibiotic resistance, and toluene efflux. To gain a better understanding of substrate transport in RND efflux pumps, the EmhB pump was subjected to mutational analysis. Mutagenesis of amino acids within the central cavity of the predicted three-dimensional structure of EmhB showed selective activity towards antibiotic substrates. An A384P/A385Y double mutant showed increased susceptibility toward rhodamine 6G compared to the wild type, and F386A and N99A single mutants showed increased susceptibility to dequalinium compared to the wild type. As well, the carboxylic acid side chain of D101, located in the central cavity region, was found to be essential for polycyclic aromatic hydrocarbon transport and resistance to all antibiotic substrates of EmhB. Phenylalanine residues located within the periplasmic pore domain were also targeted for mutagenesis, and the F325A and F281A mutations significantly impaired etflux activity for all EmhB substrates. One mutation (A206S) in the outer membrane protein docking domain increased antibiotic resistance and toluene tolerance, demonstrating the important role of this domain in transport activity. These data demonstrate the roles of the central cavity and periplasmic domains in the function of the RND efflux pump EmhB.

The hydrocarbon-degrading environmental isolate Pseudomonas fluorescens LP6a possesses an active efflux mechanism for the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene but not for naphthalene or toluene. PCR was used to detect efflux pump genes belonging to the resistance-nodulation-cell division (RND) superfamily in a plasmid-cured derivative, P. fluorescens cLP6a, which is unable to metabolize hydrocarbons. One RND pump, whose gene was identified in P. fluorescens cLP6a and was designated emhB, showed homology to the multidrug and solvent efflux pumps in Pseudomonas aeruginosa and Pseudomonas putida. The emhB gene is located in a gene cluster with the emhA and emhC genes, which encode the membrane fusion protein and outer membrane protein components of the efflux system, respectively. Disruption of emhB by insertion of an antibiotic resistance cassette demonstrated that the corresponding gene product was responsible for the efflux of polycyclic aromatic hydrocarbons. The emhB gene disruption did not affect the resistance of P. fluorescens cLP6a to tetracycline, erythromycin, trimethoprim, or streptomycin, but it did decrease resistance to chloramphenicol and nalidixic acid, indicating that the EmhABC system also functions in the efflux of these compounds and has an unusual selectivity. Phenanthrene efflux was observed in P. aeruginosa, P. putida, and Burkholderia cepacia but not in Azotobacter vinelandii. Polycyclic aromatic hydrocarbons represent a new class of nontoxic, highly hydrophobic compounds that are substrates of RND efflux systems, and the EmbABC system in P. fluorescens cLP6a has a narrow substrate range for these hydrocarbons and certain antibiotics.

Research reveals that five bacterial isolates from fuel-contaminated Antarctic soils are capable of fixing nitrogen heterotrophically and nonsymbiotically. Data show that two isolates grow using jet fuel vapors and volatile hydrocarbons in nitrogen medium.

Pseudomonas fluorescens LP6a uses passive diffusion and an energy-driven efflux system to transport polycyclic aromatic hydrocarbons (PAHs) out of the cell. The efflux system appears to be selective because some PAHs are transported out of the cell whereas others are not.

Researchers discovered several different types of microbes underneath the John Evans Glacier on eastern Ellesmere Island, Nunavut, Canada. The organisms included aerobic chemoheterotrophs, anaerobic nitrate reducers, sulfate reducers, and methanogens. Similar conditions may exist under Martian polar ice caps, creating a viable habitat for life on Mars.

A modified form of reverse sample genome probe (RSGP) method was developed for the isolation of heterotrophic bacteria from oil fields. The RSGP method can be utilized to monitor the response of microbial communities to environmental changes such as high-potential electron acceptor nitrate availability. The quantitation of hybridization signals was also enhanced by utilizing phosphorimaging plate technology. Furthermore, RSGP analysis indicated the inability of the heterotropic bacteria from the master filter to become dominant community components after the injection of nitrate.