Publications by authors named "Denis Le Paslier"

39 Publications

Genetic Analysis of sp.86 Reveals Involvement of Corrinoids in Chlordecone and Lindane Biotransformations.

Front Microbiol 2020 9;11:590061. Epub 2020 Nov 9.

Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, Evry, France.

Chlordecone (Kepone®) and γ-hexachlorocyclohexane (γ-HCH or lindane) have been used for decades in the French West Indies (FWI) resulting in long-term soil and water pollution. In a previous work, we have identified a new species (sp.86) that is able to transform chlordecone into numerous products under anaerobic conditions. No homologs to known reductive dehalogenases or other candidate genes were found in the genome sequence of sp.86. However, a complete anaerobic pathway for cobalamin biosynthesis was identified. In this study, we investigated whether cobalamin or intermediates of cobalamin biosynthesis was required for chlordecone microbiological transformation. For this purpose, we constructed a set of four sp.86 mutant strains defective in several genes belonging to the anaerobic cobalamin biosynthesis pathway. We monitored chlordecone and its transformation products (TPs) during long-term incubation in liquid cultures under anaerobic conditions. Chlordecone TPs were detected in the case of cobalamin-producing sp.86 wild-type strain but also in the case of mutants able to produce corrinoids devoid of lower ligand. In contrast, mutants unable to insert the cobalt atom in precorrin-2 did not induce any transformation of chlordecone. In addition, it was found that lindane, previously shown to be anaerobically transformed by without evidence of a mechanism, was also degraded in the presence of the wild-type strain of sp.86. The lindane degradation abilities of the various sp.86 mutant strains paralleled chlordecone transformation. The present study shows the involvement of cobalt-containing corrinoids in the microbial degradation of chlorinated compounds with different chemical structures. Their increased production in contaminated environments could accelerate the decontamination processes.
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http://dx.doi.org/10.3389/fmicb.2020.590061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7680753PMC
November 2020

Transformation of the recalcitrant pesticide chlordecone by Desulfovibrio sp.86 with a switch from ring-opening dechlorination to reductive sulfidation activity.

Sci Rep 2020 08 11;10(1):13545. Epub 2020 Aug 11.

Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057, Evry, France.

The insecticide chlordecone has been used in the French West Indies for decades, resulting in long term pollution, human health problems and social crisis. In addition to bacterial consortia and Citrobacter sp.86 previously described to transform chlordecone into three families of transformation products (A: hydrochlordecones, B: polychloroindenes and C: polychloroindenecarboxylic acids), another bacterium Desulfovibrio sp.86, showing the same abilities has been isolated and its genome was sequenced. Ring-opening dechlorination, leading to A, B and C families, was observed as previously described. Changing operating conditions in the presence of chlordecone gave rise to the formation of an unknown sulfur-containing transformation product instead of the aforementioned ones. Its structural elucidation enabled to conclude to a thiol derivative, which corresponds to an undocumented bacterial reductive sulfidation. Microbial experiments pointed out that the chlordecone thiol derivative was observed in anaerobiosis, and required the presence of an electron acceptor containing sulfur or hydrogen sulfide, in a confined atmosphere. It seems that this new reaction is also active on hydrochlordecones, as the 10-monohydrochlordecone A1 was transformed the same way. Moreover, the chlordecone thiol derivative called F1 was detected in several chlordecone contaminated mangrove bed sediments from Martinique Island, highlighting the environmental relevance of these results.
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http://dx.doi.org/10.1038/s41598-020-70124-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419502PMC
August 2020

In situ metabolic activities of uncultivated Ferrovum sp. CARN8 evidenced by metatranscriptomic analysis.

Res Microbiol 2020 Jan - Feb;171(1):37-43. Epub 2019 Oct 10.

Génétique Moléculaire, Génomique et Microbiologie, UMR7156 CNRS Université de Strasbourg, Strasbourg, France.

Amongst iron-oxidizing bacteria playing a key role in the natural attenuation of arsenic in acid mine drainages (AMDs), members of the Ferrovum genus were identified in mine effluent or water treatment plants, and were shown to dominate biogenic precipitates in field pilot experiments. In order to address the question of the in situ activity of the uncultivated Ferrovum sp. CARN8 strain in the Carnoulès AMD, we assembled its genome using metagenomic and metatranscriptomic sequences and we determined standardized expression values for protein-encoding genes. Our results showed that this microorganism was indeed metabolically active and allowed us to sketch out its metabolic activity in its natural environment. Expression of genes related to the respiratory chain and carbon fixation suggests aerobic energy production coupled to ferrous iron oxidation and chemolithoautotrophic growth. Notwithstanding the presence of nitrogenase genes in its genome, expression data also indicated that Ferrovum sp. CARN8 relied on ammonium import rather than nitrogen fixation. The expression of flagellum and chemotaxis genes hints that at least a proportion of this strain population was motile. Finally, apart from some genes related to metal resistance showing surprisingly low expression values, genes involved in stress response were well expressed as expected in AMDs.
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http://dx.doi.org/10.1016/j.resmic.2019.09.008DOI Listing
March 2020

Natural Chlordecone Degradation Revealed by Numerous Transformation Products Characterized in Key French West Indies Environmental Compartments.

Environ Sci Technol 2019 06 22;53(11):6133-6143. Epub 2019 May 22.

Génomique Métabolique, Genoscope , Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay , 91057 , Evry , France.

Production and use of the insecticide chlordecone has caused long-term environmental pollution in the James River area and the French West Indies (FWI) that has resulted in acute human-health problems and a social crisis. High levels of chlordecone in FWI soils, even after its ban decades ago, and the absence of detection of transformation products (TPs), have suggested that chlordecone is virtually nonbiodegradable in the environment. Here, we investigated laboratory biodegradation, consisting of bacterial liquid cultures and microcosms inoculated with FWI soils, using a dual nontargeted GC-MS and LC-HRMS approach. In addition to previously reported, partly characterized hydrochlordecones and polychloroindenes (families A and B), we discovered 14 new chlordecone TPs, assigned to four families (B, C, D, and E). Organic synthesis and NMR analyses allowed us to achieve the complete structural elucidation of 19 TPs. Members of TP families A, B, C, and E were detected in soil, sediment, and water samples from Martinique and include 17 TPs not initially found in commercial chlordecone formulations. 2,4,5,6,7-Pentachloroindene was the most prominent TP, with levels similar to those of chlordecone. Overall, our results clearly show that chlordecone pollution extends beyond the parent chlordecone molecule and includes a considerable number of previously undetected TPs. Structural diversity of the identified TPs illustrates the complexity of chlordecone degradation in the environment and raises the possibility of extensive worldwide pollution of soil and aquatic ecosystems by chlordecone TPs.
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http://dx.doi.org/10.1021/acs.est.8b06305DOI Listing
June 2019

Screening and Characterization of Novel Polyesterases from Environmental Metagenomes with High Hydrolytic Activity against Synthetic Polyesters.

Environ Sci Technol 2018 11 15;52(21):12388-12401. Epub 2018 Oct 15.

Department of Chemical Engineering and Applied Chemistry , University of Toronto , Toronto , ON M5S 3E5 , Canada.

The continuous growth of global plastics production, including polyesters, has resulted in increasing plastic pollution and subsequent negative environmental impacts. Therefore, enzyme-catalyzed depolymerization of synthetic polyesters as a plastics recycling approach has become a focus of research. In this study, we screened over 200 purified uncharacterized hydrolases from environmental metagenomes and sequenced microbial genomes and identified at least 10 proteins with high hydrolytic activity against synthetic polyesters. These include the metagenomic esterases MGS0156 and GEN0105, which hydrolyzed polylactic acid (PLA), polycaprolactone, as well as bis(benzoyloxyethyl)-terephthalate. With solid PLA as a substrate, both enzymes produced a mixture of lactic acid monomers, dimers, and higher oligomers as products. The crystal structure of MGS0156 was determined at 1.95 Å resolution and revealed a modified α/β hydrolase fold, with a lid domain and highly hydrophobic active site. Mutational studies of MGS0156 identified the residues critical for hydrolytic activity against both polyester and monoester substrates, with two-times higher polyesterase activity in the MGS0156 L169A mutant protein. Thus, our work identified novel, highly active polyesterases in environmental metagenomes and provided molecular insights into their activity, thereby augmenting our understanding of enzymatic polyester hydrolysis.
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http://dx.doi.org/10.1021/acs.est.8b04252DOI Listing
November 2018

Distinct Carbon Isotope Fractionation Signatures during Biotic and Abiotic Reductive Transformation of Chlordecone.

Environ Sci Technol 2018 03 6;52(6):3615-3624. Epub 2018 Mar 6.

Helmholtz Centre for Environmental Research, UFZ, Isotope Biogeochemistry, Permoserstraße 15 , 04318 Leipzig , Germany.

Chlordecone is a synthetic organochlorine pesticide, extensively used in banana plantations of the French West Indies from 1972 to 1993. Due to its environmental persistence and bioaccumulation, it has dramatic public health and socio-economic impact. Here we describe a method for carbon-directed compound specific isotope analysis (CSIA) for chlordecone and apply it to monitor biotic and abiotic reductive transformation reactions, selected on the basis of their distinct product profiles (polychloroindenes versus lower chlorinated hydrochlordecones). Significant carbon isotopic enrichments were observed for all microbially mediated transformations (ε = -6.8‰ with a Citrobacter strain and ε = -4.6‰ with a bacterial consortium) and for two abiotic transformations (ε = -4.1‰ with zerovalent iron and ε = -2.6‰ with sodium sulfide and vitamin B). The reaction with titanium(III) citrate and vitamin B, which shows the product profile most similar to that observed in biotic transformation, led to low carbon isotope enrichment (ε =-0.8‰). The CSIA protocol was also applied on representative chlordecone formulations previously used in the French West Indies, giving similar chlordecone δC values from -31.1 ± 0.2‰ to -34.2 ± 0.2‰ for all studied samples. This allows the in situ application of CSIA for the assessment of chlordecone persistence.
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http://dx.doi.org/10.1021/acs.est.7b05394DOI Listing
March 2018

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families.

Sci Rep 2017 03 8;7:44103. Epub 2017 Mar 8.

Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, M5S 3E5, Canada.

Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.
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http://dx.doi.org/10.1038/srep44103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341072PMC
March 2017

Microbial Degradation of a Recalcitrant Pesticide: Chlordecone.

Front Microbiol 2016 20;7:2025. Epub 2016 Dec 20.

Commissariat à l'Energie Atomique et aux Energies Alternatives, Direction de la Recherche Fondamentale, Institut de GénomiqueEvry, France; Université d'Evry Val d'EssonneEvry, France; Centre National de la Recherche Scientifique, UMR8030, Génomique métaboliqueEvry, France.

Chlordecone (Kepone®) is a synthetic organochlorine insecticide (CClO) used worldwide mostly during the 1970 and 1980s. Its intensive application in the French West Indies to control the banana black weevil led to a massive environmental pollution. Persistence of chlordecone in soils and water for numerous decades even centuries causes global public health and socio-economic concerns. In order to investigate the biodegradability of chlordecone, microbial enrichment cultures from soils contaminated by chlordecone or other organochlorines and from sludge of a wastewater treatment plant have been conducted. Different experimental procedures including original microcosms were carried out anaerobically over long periods of time. GC-MS monitoring resulted in the detection of chlorinated derivatives in several cultures, consistent with chlordecone biotransformation. More interestingly, disappearance of chlordecone (50 μg/mL) in two bacterial consortia was concomitant with the accumulation of a major metabolite of formula CClH (named B1) as well as two minor metabolites CClHO (named A1) and CClH (named B3). Finally, we report the isolation and the complete genomic sequences of two new isolates, closely related to , and that were capable of reproducing chlordecone transformation. Further characterization of these strains should yield deeper insights into the mechanisms involved in this transformation process.
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http://dx.doi.org/10.3389/fmicb.2016.02025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167691PMC
December 2016

Reconstructing rare soil microbial genomes using in situ enrichments and metagenomics.

Front Microbiol 2015 30;6:358. Epub 2015 Apr 30.

Environmental Microbial Genomics, Laboratoire Ampere, Centre National de la Recherche Scientifique, Ecole Centrale de Lyon, Université de Lyon Ecully, France.

Despite extensive direct sequencing efforts and advanced analytical tools, reconstructing microbial genomes from soil using metagenomics have been challenging due to the tremendous diversity and relatively uniform distribution of genomes found in this system. Here we used enrichment techniques in an attempt to decrease the complexity of a soil microbiome prior to sequencing by submitting it to a range of physical and chemical stresses in 23 separate microcosms for 4 months. The metagenomic analysis of these microcosms at the end of the treatment yielded 540 Mb of assembly using standard de novo assembly techniques (a total of 559,555 genes and 29,176 functions), from which we could recover novel bacterial genomes, plasmids and phages. The recovered genomes belonged to Leifsonia (n = 2), Rhodanobacter (n = 5), Acidobacteria (n = 2), Sporolactobacillus (n = 2, novel nitrogen fixing taxon), Ktedonobacter (n = 1, second representative of the family Ktedonobacteraceae), Streptomyces (n = 3, novel polyketide synthase modules), and Burkholderia (n = 2, includes mega-plasmids conferring mercury resistance). Assembled genomes averaged to 5.9 Mb, with relative abundances ranging from rare (<0.0001%) to relatively abundant (>0.01%) in the original soil microbiome. Furthermore, we detected them in samples collected from geographically distant locations, particularly more in temperate soils compared to samples originating from high-latitude soils and deserts. To the best of our knowledge, this study is the first successful attempt to assemble multiple bacterial genomes directly from a soil sample. Our findings demonstrate that developing pertinent enrichment conditions can stimulate environmental genomic discoveries that would have been impossible to achieve with canonical approaches that focus solely upon post-sequencing data treatment.
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http://dx.doi.org/10.3389/fmicb.2015.00358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4415585PMC
May 2015

16S rRNA and As-Related Functional Diversity: Contrasting Fingerprints in Arsenic-Rich Sediments from an Acid Mine Drainage.

Microb Ecol 2015 Jul 16;70(1):154-67. Epub 2015 Jan 16.

Équipe Environnement et Microbiologie, EEM, UMR 5254 (IPREM, CNRS), Université de Pau et des Pays de l'Adour, IBEAS, BP 1155, 64013, Pau Cedex, France.

To gain an in-depth insight into the diversity and the distribution of genes under the particular evolutionary pressure of an arsenic-rich acid mine drainage (AMD), the genes involved in bacterial arsenic detoxification (arsB, ACR3) and arsenite oxidation (aioA) were investigated in sediment from Carnoulès (France), in parallel to the diversity and global distribution of the metabolically active bacteria. The metabolically active bacteria were affiliated mainly to AMD specialists, i.e., organisms detected in or isolated from AMDs throughout the world. They included mainly Acidobacteria and the non-affiliated "Candidatus Fodinabacter communificans," as well as Thiomonas and Acidithiobacillus spp., Actinobacteria, and unclassified Gammaproteobacteria. The distribution range of these organisms suggested that they show niche conservatism. Sixteen types of deduced protein sequences of arsenite transporters (5 ArsB and 11 Acr3p) were detected, whereas a single type of arsenite oxidase (AioA) was found. Our data suggested that at Carnoulès, the aioA gene was more recent than those encoding arsenite transporters and subjected to a different molecular evolution. In contrast to the 16S ribosomal RNA (16S rRNA) genes associated with AMD environments worldwide, the functional genes aioA, ACR3, and to a lesser extent arsB, were either novel or specific to Carnoulès, raising the question as to whether these functional genes are specific to high concentrations of arsenic, AMD-specific, or site-specific.
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http://dx.doi.org/10.1007/s00248-014-0558-3DOI Listing
July 2015

Growth of nitrite-oxidizing bacteria by aerobic hydrogen oxidation.

Science 2014 Aug;345(6200):1052-4

Department of Microbiology and Ecosystem Science, Division of Microbial Ecology, University of Vienna, 1090 Vienna, Austria.

The bacterial oxidation of nitrite to nitrate is a key process of the biogeochemical nitrogen cycle. Nitrite-oxidizing bacteria are considered a highly specialized functional group, which depends on the supply of nitrite from other microorganisms and whose distribution strictly correlates with nitrification in the environment and in wastewater treatment plants. On the basis of genomics, physiological experiments, and single-cell analyses, we show that Nitrospira moscoviensis, which represents a widely distributed lineage of nitrite-oxidizing bacteria, has the genetic inventory to utilize hydrogen (H2) as an alternative energy source for aerobic respiration and grows on H2 without nitrite. CO2 fixation occurred with H2 as the sole electron donor. Our results demonstrate a chemolithoautotrophic lifestyle of nitrite-oxidizing bacteria outside the nitrogen cycle, suggesting greater ecological flexibility than previously assumed.
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http://dx.doi.org/10.1126/science.1256985DOI Listing
August 2014

Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes.

Nat Biotechnol 2014 Aug 6;32(8):822-8. Epub 2014 Jul 6.

1] INRA, Institut National de la Recherche Agronomique, UMR 14121 MICALIS, Jouy en Josas, France. [2] INRA, Institut National de la Recherche Agronomique, US 1367 Metagenopolis, Jouy en Josas, France. [3] King's College London, Centre for Host-Microbiome Interactions, Dental Institute Central Office, Guy's Hospital, United Kingdom.

Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.
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http://dx.doi.org/10.1038/nbt.2939DOI Listing
August 2014

Enrichment and genome sequence of the group I.1a ammonia-oxidizing Archaeon "Ca. Nitrosotenuis uzonensis" representing a clade globally distributed in thermal habitats.

PLoS One 2013 20;8(11):e80835. Epub 2013 Nov 20.

Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia.

The discovery of ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota and the high abundance of archaeal ammonia monooxygenase subunit A encoding gene sequences in many environments have extended our perception of nitrifying microbial communities. Moreover, AOA are the only aerobic ammonia oxidizers known to be active in geothermal environments. Molecular data indicate that in many globally distributed terrestrial high-temperature habits a thaumarchaeotal lineage within the Nitrosopumilus cluster (also called "marine" group I.1a) thrives, but these microbes have neither been isolated from these systems nor functionally characterized in situ yet. In this study, we report on the enrichment and genomic characterization of a representative of this lineage from a thermal spring in Kamchatka. This thaumarchaeote, provisionally classified as "Candidatus Nitrosotenuis uzonensis", is a moderately thermophilic, non-halophilic, chemolithoautotrophic ammonia oxidizer. The nearly complete genome sequence (assembled into a single scaffold) of this AOA confirmed the presence of the typical thaumarchaeotal pathways for ammonia oxidation and carbon fixation, and indicated its ability to produce coenzyme F420 and to chemotactically react to its environment. Interestingly, like members of the genus Nitrosoarchaeum, "Candidatus N. uzonensis" also possesses a putative artubulin-encoding gene. Genome comparisons to related AOA with available genome sequences confirmed that the newly cultured AOA has an average nucleotide identity far below the species threshold and revealed a substantial degree of genomic plasticity with unique genomic regions in "Ca. N. uzonensis", which potentially include genetic determinants of ecological niche differentiation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080835PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3835317PMC
December 2014

Gene capture coupled to high-throughput sequencing as a strategy for targeted metagenome exploration.

DNA Res 2013 Apr 30;20(2):185-96. Epub 2013 Jan 30.

Centre de Recherche en Nutrition Humaine Auvergne, Clermont Université, Université d'Auvergne, EA 4678, Conception, Ingénierie et Développement de l'Aliment et du Médicament, BP 10448, Clermont-Ferrand 63000, France.

Next-generation sequencing (NGS) allows faster acquisition of metagenomic data, but complete exploration of complex ecosystems is hindered by the extraordinary diversity of microorganisms. To reduce the environmental complexity, we created an innovative solution hybrid selection (SHS) method that is combined with NGS to characterize large DNA fragments harbouring biomarkers of interest. The quality of enrichment was evaluated after fragments containing the methyl coenzyme M reductase subunit A gene (mcrA), the biomarker of methanogenesis, were captured from a Methanosarcina strain and a metagenomic sample from a meromictic lake. The methanogen diversity was compared with direct metagenome and mcrA-based amplicon pyrosequencing strategies. The SHS approach resulted in the capture of DNA fragments up to 2.5 kb with an enrichment efficiency between 41 and 100%, depending on the sample complexity. Compared with direct metagenome and amplicons sequencing, SHS detected broader mcrA diversity, and it allowed efficient sampling of the rare biosphere and unknown sequences. In contrast to amplicon-based strategies, SHS is less biased and GC independent, and it recovered complete biomarker sequences in addition to conserved regions. Because this method can also isolate the regions flanking the target sequences, it could facilitate operon reconstructions.
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http://dx.doi.org/10.1093/dnares/dst001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3628448PMC
April 2013

The structure and spatio-temporal distribution of the Archaea in a horizontal subsurface flow constructed wetland.

Sci Total Environ 2012 Oct 9;435-436:465-71. Epub 2012 Aug 9.

Laboratoire d'Analyses, Traitement et Valorisation des Polluants de l'Environnement et des Produits, Faculté de Pharmacie, rue Avicenne 5000 Monastir, Université de Monastir, Tunisia.

In this study, archaeal community structure and temporal dynamics were monitored, using 16S rRNA clone libraries construction from a horizontal subsurface flow constructed wetland. Phylogenetic assignation of 1026 16S rRNA gene sequences shows that 96.2% of the total operational taxonomic units (OTUs) were affiliated with Thaumarchaeota, a newly proposed archaeal phylum and 3.7% with unclassified Archaea. Among the total sequences, 42% and 40.2% were affiliated with Candidatus Nitrososphaera and unclassified Nitrosopumilus respectively with more than 99% similarity. Results suggest that several dominant and active nitrifiers may benefit from the micro-aerobic conditions around the reed roots to perform ammonia oxidation. The archaeal diversity detected in the rhizosphere zone is clearly different from that detected in the bottom basin. This engineered habitat revealed the reed root and the water composition effects on the archaeal diversity.
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http://dx.doi.org/10.1016/j.scitotenv.2012.07.047DOI Listing
October 2012

Characterization of rhizosphere prokaryotic diversity in a horizontal subsurface flow constructed wetland using a PCR cloning-sequencing based approach.

Appl Microbiol Biotechnol 2013 May 11;97(9):4221-31. Epub 2012 Jul 11.

Institut de Génomique/CEA//CNRS-UMR 8030, Genoscope, 2, rue Gaston Crémieux, 91057 Evry, France.

Performance of biological wastewater treatment systems may be related to the composition and activity of microbial populations they contain. However, little information is known regarding microbial community inhabiting these ecosystems. The purpose of this study was to investigate archaeal and bacterial diversity, using cultivation-independent molecular techniques, in a constructed wetland receiving domestic wastewater. Two 16S rRNA gene libraries were constructed using total genomic DNA and amplified by PCR using primers specific for archaeal and bacterial domains. A high microbial diversity was detected. The Proteobacteria phylum is the most abundant and diversified phylogenetic group representing 31.3 % of the OTUs, followed by the Bacteroidetes (14.8 %), Planctomycetales (13.8 %), Actinobacteria (12 %), and Chloroflexi (8.2 %). Sequences affiliated with minor phylogenetic divisions such as the TM7, Nitrospira, OP10, and BRC1 are represented by <6 % of total OTUs. The Archaea domain was represented by the Thaumarchaeota phylum dominated by the Candidatus Nitrososphaera genus.
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http://dx.doi.org/10.1007/s00253-012-4239-1DOI Listing
May 2013

Nitrification expanded: discovery, physiology and genomics of a nitrite-oxidizing bacterium from the phylum Chloroflexi.

ISME J 2012 Dec 5;6(12):2245-56. Epub 2012 Jul 5.

Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia.

Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, a major process of the biogeochemical nitrogen cycle, but the recognized diversity of this guild is surprisingly low and only two bacterial phyla contain known NOB. Here, we report on the discovery of a chemolithoautotrophic nitrite oxidizer that belongs to the widespread phylum Chloroflexi not previously known to contain any nitrifying organism. This organism, named Nitrolancetus hollandicus, was isolated from a nitrifying reactor. Its tolerance to a broad temperature range (25-63 °C) and low affinity for nitrite (K(s)=1 mM), a complex layered cell envelope that stains Gram positive, and uncommon membrane lipids composed of 1,2-diols distinguish N. hollandicus from all other known nitrite oxidizers. N. hollandicus grows on nitrite and CO(2), and is able to use formate as a source of energy and carbon. Genome sequencing and analysis of N. hollandicus revealed the presence of all genes required for CO(2) fixation by the Calvin cycle and a nitrite oxidoreductase (NXR) similar to the NXR forms of the proteobacterial nitrite oxidizers, Nitrobacter and Nitrococcus. Comparative genomic analysis of the nxr loci unexpectedly indicated functionally important lateral gene transfer events between Nitrolancetus and other NOB carrying a cytoplasmic NXR, suggesting that horizontal transfer of the NXR module was a major driver for the spread of the capability to gain energy from nitrite oxidation during bacterial evolution. The surprising discovery of N. hollandicus significantly extends the known diversity of nitrifying organisms and likely will have implications for future research on nitrification in natural and engineered ecosystems.
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http://dx.doi.org/10.1038/ismej.2012.70DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3504966PMC
December 2012

Structure, fluctuation and magnitude of a natural grassland soil metagenome.

ISME J 2012 Sep 2;6(9):1677-87. Epub 2012 Feb 2.

Environmental Microbial Genomics, Ecole Centrale de Lyon, Université de Lyon, Ecully, France.

The soil ecosystem is critical for human health, affecting aspects of the environment from key agricultural and edaphic parameters to critical influence on climate change. Soil has more unknown biodiversity than any other ecosystem. We have applied diverse DNA extraction methods coupled with high throughput pyrosequencing to explore 4.88 × 10(9) bp of metagenomic sequence data from the longest continually studied soil environment (Park Grass experiment at Rothamsted Research in the UK). Results emphasize important DNA extraction biases and unexpectedly low seasonal and vertical soil metagenomic functional class variations. Clustering-based subsystems and carbohydrate metabolism had the largest quantity of annotated reads assigned although <50% of reads were assigned at an E value cutoff of 10(-5). In addition, with the more detailed subsystems, cAMP signaling in bacteria (3.24±0.27% of the annotated reads) and the Ton and Tol transport systems (1.69±0.11%) were relatively highly represented. The most highly represented genome from the database was that for a Bradyrhizobium species. The metagenomic variance created by integrating natural and methodological fluctuations represents a global picture of the Rothamsted soil metagenome that can be used for specific questions and future inter-environmental metagenomic comparisons. However, only 1% of annotated sequences correspond to already sequenced genomes at 96% similarity and E values of <10(-5), thus, considerable genomic reconstructions efforts still have to be performed.
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http://dx.doi.org/10.1038/ismej.2011.197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3498926PMC
September 2012

Betaproteobacteria dominance and diversity shifts in the bacterial community of a PAH-contaminated soil exposed to phenanthrene.

Environ Pollut 2012 Mar 21;162:345-53. Epub 2011 Dec 21.

CEA, DSV, iRTSV, Laboratoire de Chimie et Biologie des Métaux, Grenoble, France.

In this study, the PAH-degrading bacteria of a constructed wetland collecting road runoff has been studied through DNA stable isotope probing. Microcosms were spiked with (13)C-phenanthrene at 34 or 337 ppm, and bacterial diversity was monitored over a 14-day period. At 337 ppm, PAH degraders became dominated after 5 days by Betaproteobacteria, including novel Acidovorax, Rhodoferax and Hydrogenophaga members, and unknown bacteria related to Rhodocyclaceae. The prevalence of Betaproteobacteria was further demonstrated by phylum-specific quantitative PCR, and was correlated with a burst of phenanthrene mineralization. Striking shifts in the population of degraders were observed after most of the phenanthrene had been removed. Soil exposed to 34 ppm phenanthrene showed a similar population of degraders, albeit only after 14 days. Results demonstrate that specific Betaproteobacteria are involved in the main response to soil PAH contamination, and illustrate the potential of SIP approaches to investigate PAH biodegradation in soil.
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http://dx.doi.org/10.1016/j.envpol.2011.11.032DOI Listing
March 2012

Metabolic diversity among main microorganisms inside an arsenic-rich ecosystem revealed by meta- and proteo-genomics.

ISME J 2011 Nov 12;5(11):1735-47. Epub 2011 May 12.

Génétique Moléculaire, Génomique et Microbiologie, UMR7156 CNRS and UdS, Strasbourg, France.

By their metabolic activities, microorganisms have a crucial role in the biogeochemical cycles of elements. The complete understanding of these processes requires, however, the deciphering of both the structure and the function, including synecologic interactions, of microbial communities. Using a metagenomic approach, we demonstrated here that an acid mine drainage highly contaminated with arsenic is dominated by seven bacterial strains whose genomes were reconstructed. Five of them represent yet uncultivated bacteria and include two strains belonging to a novel bacterial phylum present in some similar ecosystems, and which was named 'Candidatus Fodinabacter communificans.' Metaproteomic data unravelled several microbial capabilities expressed in situ, such as iron, sulfur and arsenic oxidation that are key mechanisms in biomineralization, or organic nutrient, amino acid and vitamin metabolism involved in synthrophic associations. A statistical analysis of genomic and proteomic data and reverse transcriptase-PCR experiments allowed us to build an integrated model of the metabolic interactions that may be of prime importance in the natural attenuation of such anthropized ecosystems.
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http://dx.doi.org/10.1038/ismej.2011.51DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197163PMC
November 2011

Enterotypes of the human gut microbiome.

Nature 2011 May 20;473(7346):174-80. Epub 2011 Apr 20.

European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

Our knowledge of species and functional composition of the human gut microbiome is rapidly increasing, but it is still based on very few cohorts and little is known about variation across the world. By combining 22 newly sequenced faecal metagenomes of individuals from four countries with previously published data sets, here we identify three robust clusters (referred to as enterotypes hereafter) that are not nation or continent specific. We also confirmed the enterotypes in two published, larger cohorts, indicating that intestinal microbiota variation is generally stratified, not continuous. This indicates further the existence of a limited number of well-balanced host-microbial symbiotic states that might respond differently to diet and drug intake. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species, highlighting the importance of a functional analysis to understand microbial communities. Although individual host properties such as body mass index, age, or gender cannot explain the observed enterotypes, data-driven marker genes or functional modules can be identified for each of these host properties. For example, twelve genes significantly correlate with age and three functional modules with the body mass index, hinting at a diagnostic potential of microbial markers.
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http://dx.doi.org/10.1038/nature09944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728647PMC
May 2011

Characterization of the active bacterial community involved in natural attenuation processes in arsenic-rich creek sediments.

Microb Ecol 2011 May 12;61(4):793-810. Epub 2011 Feb 12.

Laboratoire HydroSciences Montpellier, UMR5569 (CNRS-IRD-Universités Montpellier I et II), Université Montpellier II, CC MSE, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France.

Acid mine drainage of the Carnoulès mine (France) is characterized by acid waters containing high concentrations of arsenic and iron. In the first 30 m along the Reigous, a small creek draining the site, more than 38% of the dissolved arsenic was removed by co-precipitation with Fe(III), in agreement with previous studies, which suggest a role of microbial activities in the co-precipitation of As(III) and As(V) with Fe(III) and sulfate. To investigate how this particular ecosystem functions, the bacterial community was characterized in water and sediments by 16S rRNA encoding gene library analysis. Based on the results obtained using a metaproteomic approach on sediments combined with high-sensitivity HPLC-chip spectrometry, several GroEL orthologs expressed by the community were characterized, and the active members of the prokaryotic community inhabiting the creek sediments were identified. Many of these bacteria are β-proteobacteria such as Gallionella and Thiomonas, but γ-proteobacteria such as Acidithiobacillus ferrooxidans and α-proteobacteria such as Acidiphilium, Actinobacteria, and Firmicutes were also detected.
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http://dx.doi.org/10.1007/s00248-011-9808-9DOI Listing
May 2011

Clostridium sticklandii, a specialist in amino acid degradation:revisiting its metabolism through its genome sequence.

BMC Genomics 2010 Oct 11;11:555. Epub 2010 Oct 11.

Institut de Génomique, Genoscope, 2 Rue Gaston Crémieux, F-91057 Evry, France.

Background: Clostridium sticklandii belongs to a cluster of non-pathogenic proteolytic clostridia which utilize amino acids as carbon and energy sources. Isolated by T.C. Stadtman in 1954, it has been generally regarded as a "gold mine" for novel biochemical reactions and is used as a model organism for studying metabolic aspects such as the Stickland reaction, coenzyme-B12- and selenium-dependent reactions of amino acids. With the goal of revisiting its carbon, nitrogen, and energy metabolism, and comparing studies with other clostridia, its genome has been sequenced and analyzed.

Results: C. sticklandii is one of the best biochemically studied proteolytic clostridial species. Useful additional information has been obtained from the sequencing and annotation of its genome, which is presented in this paper. Besides, experimental procedures reveal that C. sticklandii degrades amino acids in a preferential and sequential way. The organism prefers threonine, arginine, serine, cysteine, proline, and glycine, whereas glutamate, aspartate and alanine are excreted. Energy conservation is primarily obtained by substrate-level phosphorylation in fermentative pathways. The reactions catalyzed by different ferredoxin oxidoreductases and the exergonic NADH-dependent reduction of crotonyl-CoA point to a possible chemiosmotic energy conservation via the Rnf complex. C. sticklandii possesses both the F-type and V-type ATPases. The discovery of an as yet unrecognized selenoprotein in the D-proline reductase operon suggests a more detailed mechanism for NADH-dependent D-proline reduction. A rather unusual metabolic feature is the presence of genes for all the enzymes involved in two different CO2-fixation pathways: C. sticklandii harbours both the glycine synthase/glycine reductase and the Wood-Ljungdahl pathways. This unusual pathway combination has retrospectively been observed in only four other sequenced microorganisms.

Conclusions: Analysis of the C. sticklandii genome and additional experimental procedures have improved our understanding of anaerobic amino acid degradation. Several specific metabolic features have been detected, some of which are very unusual for anaerobic fermenting bacteria. Comparative genomics has provided the opportunity to study the lifestyle of pathogenic and non-pathogenic clostridial species as well as to elucidate the difference in metabolic features between clostridia and other anaerobes.
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http://dx.doi.org/10.1186/1471-2164-11-555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091704PMC
October 2010

A Nitrospira metagenome illuminates the physiology and evolution of globally important nitrite-oxidizing bacteria.

Proc Natl Acad Sci U S A 2010 Jul 12;107(30):13479-84. Epub 2010 Jul 12.

Department of Microbial Ecology, Ecology Centre, University of Vienna, 1090 Vienna, Austria.

Nitrospira are barely studied and mostly uncultured nitrite-oxidizing bacteria, which are, according to molecular data, among the most diverse and widespread nitrifiers in natural ecosystems and biological wastewater treatment. Here, environmental genomics was used to reconstruct the complete genome of "Candidatus Nitrospira defluvii" from an activated sludge enrichment culture. On the basis of this first-deciphered Nitrospira genome and of experimental data, we show that Ca. N. defluvii differs dramatically from other known nitrite oxidizers in the key enzyme nitrite oxidoreductase (NXR), in the composition of the respiratory chain, and in the pathway used for autotrophic carbon fixation, suggesting multiple independent evolution of chemolithoautotrophic nitrite oxidation. Adaptations of Ca. N. defluvii to substrate-limited conditions include an unusual periplasmic NXR, which is constitutively expressed, and pathways for the transport, oxidation, and assimilation of simple organic compounds that allow a mixotrophic lifestyle. The reverse tricarboxylic acid cycle as the pathway for CO2 fixation and the lack of most classical defense mechanisms against oxidative stress suggest that Nitrospira evolved from microaerophilic or even anaerobic ancestors. Unexpectedly, comparative genomic analyses indicate functionally significant lateral gene-transfer events between the genus Nitrospira and anaerobic ammonium-oxidizing planctomycetes, which share highly similar forms of NXR and other proteins reflecting that two key processes of the nitrogen cycle are evolutionarily connected.
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http://dx.doi.org/10.1073/pnas.1003860107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2922143PMC
July 2010

Nitrite-driven anaerobic methane oxidation by oxygenic bacteria.

Nature 2010 Mar;464(7288):543-8

Radboud University Nijmegen, IWWR, Department of Microbiology, Heyendaalseweg 135, 6525 AJ, Nijmegen, The Netherlands.

Only three biological pathways are known to produce oxygen: photosynthesis, chlorate respiration and the detoxification of reactive oxygen species. Here we present evidence for a fourth pathway, possibly of considerable geochemical and evolutionary importance. The pathway was discovered after metagenomic sequencing of an enrichment culture that couples anaerobic oxidation of methane with the reduction of nitrite to dinitrogen. The complete genome of the dominant bacterium, named 'Candidatus Methylomirabilis oxyfera', was assembled. This apparently anaerobic, denitrifying bacterium encoded, transcribed and expressed the well-established aerobic pathway for methane oxidation, whereas it lacked known genes for dinitrogen production. Subsequent isotopic labelling indicated that 'M. oxyfera' bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen and oxygen, which was used to oxidize methane. These results extend our understanding of hydrocarbon degradation under anoxic conditions and explain the biochemical mechanism of a poorly understood freshwater methane sink. Because nitrogen oxides were already present on early Earth, our finding opens up the possibility that oxygen was available to microbial metabolism before the evolution of oxygenic photosynthesis.
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http://dx.doi.org/10.1038/nature08883DOI Listing
March 2010

A human gut microbial gene catalogue established by metagenomic sequencing.

Nature 2010 Mar;464(7285):59-65

BGI-Shenzhen, Shenzhen 518083, China.

To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, approximately 150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively.
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http://dx.doi.org/10.1038/nature08821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779803PMC
March 2010

Molecular analyses of the microbial community composition of an anoxic basin of a municipal wastewater treatment plant reveal a novel lineage of proteobacteria.

Microb Ecol 2010 Aug 20;60(2):272-81. Epub 2010 Feb 20.

Faculté des Sciences de Bizerte, Université 7 Novembre à Carthage, Bizerte, Tunisia.

A culture-independent molecular phylogenetic approach was used to study prokaryotic diversity in an anoxic activated sludge from a municipal wastewater treatment plant. Two 16S rRNA gene libraries were constructed using total genomic DNA and amplified by polymerase chain reaction using primers specific for archaeal or bacterial domains. Phylogenetic analysis of 132 and 249 almost full-length 16S rRNA genes for Archaea and Bacteria, respectively, was done using the ARB software package. Phylogenetic groups affiliated with the Archaea belong to Euryarchaeota (93.8% of the operational taxonomic units [OTUs]) and Crenarchaeota (6.2% of the OTUs). Within the bacterial library, 84.8% of the OTUs represent novel putative phylotypes never described before and affiliated with ten divisions. The Proteobacteria phylum is the most abundant and diversified phylogenetic group representing 60.4% of the OTUs, followed by Bacteroidetes (22.1%) and gram-positives (6.1%). Interestingly, we detected a novel Proteobacteria monophyletic group distinct from the five known subclasses, which we named New Lineage of Proteobacteria (NLP) lineage, and it is composed of eight clones representing 4.6% of the Proteobacteria. A new 16S rRNA-targeted hybridization probe was designed and fluorescent in situ hybridization analyses shows representatives of NLP as cocci-shaped microorganisms. The Chloroflexi, Acidobacterium, and Nitrospira phyla and TM7 candidate division are each represented by ≤3% of clone sequences. A comprehensive set of eight 16S and 23S rRNA-targeted oligonucleotide probes was used to quantify these major groups by dot blot hybridization within 12 samples. The Proteobacteria accounted for 82.5 ± 4.9%, representing the most abundant phyla. The Bacteroidetes and Planctomycetales groups accounted for 4.9 ± 1.3% and 4 ± 1.7%, respectively. Firmicutes and Actinobacteria together accounted for only 1.9 ± 0.5%. The set of probes covers 93.4 ± 14% of the total bacterial population rRNA within the anoxic basin.
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http://dx.doi.org/10.1007/s00248-009-9632-7DOI Listing
August 2010

A conserved gene cluster rules anaerobic oxidative degradation of L-ornithine.

J Bacteriol 2009 May 27;191(9):3162-7. Epub 2009 Feb 27.

CEA, DSV, IG, Genoscope, 2 rue Gaston Crémieux, Evry F-91057, France.

For the ornithine fermentation pathway, described more than 70 years ago, genetic and biochemical information are still incomplete. We present here the experimental identification of the last four missing genes of this metabolic pathway. They encode L-ornithine racemase, (2R,4S)-2,4-diaminopentanoate dehydrogenase, and the two subunits of 2-amino-4-ketopentanoate thiolase. While described only for the Clostridiaceae to date, this pathway is shown to be more widespread.
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http://dx.doi.org/10.1128/JB.01777-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2681807PMC
May 2009

Reverse dissimilatory sulfite reductase as phylogenetic marker for a subgroup of sulfur-oxidizing prokaryotes.

Environ Microbiol 2009 Feb 26;11(2):289-99. Epub 2008 Sep 26.

Department of Microbial Ecology, Universität Wein, Wein, Austria.

Sulfur-oxidizing prokaryotes (SOP) catalyse a central step in the global S-cycle and are of major functional importance for a variety of natural and engineered systems, but our knowledge on their actual diversity and environmental distribution patterns is still rather limited. In this study we developed a specific PCR assay for the detection of dsrAB that encode the reversely operating sirohaem dissimilatory sulfite reductase (rDSR) and are present in many but not all published genomes of SOP. The PCR assay was used to screen 42 strains of SOP (most without published genome sequence) representing the recognized diversity of this guild. For 13 of these strains dsrAB was detected and the respective PCR product was sequenced. Interestingly, most dsrAB-encoding SOP are capable of forming sulfur storage compounds. Phylogenetic analysis demonstrated largely congruent rDSR and 16S rRNA consensus tree topologies, indicating that lateral transfer events did not play an important role in the evolutionary history of known rDSR. Thus, this enzyme represents a suitable phylogenetic marker for diversity analyses of sulfur storage compound-exploiting SOP in the environment. The potential of this new functional gene approach was demonstrated by comparative sequence analyses of all dsrAB present in published metagenomes and by applying it for a SOP census in selected marine worms and an alkaline lake sediment.
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http://dx.doi.org/10.1111/j.1462-2920.2008.01760.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2702494PMC
February 2009