Genes and enzymes involved in the biodegradation of the quaternary carbon compound pivalate in the denitrifying Thauera humireducens strain PIV-1.

Quaternary carbon-containing compounds exist in natural and fossil oil-derived products and are used in chemical and pharmaceutical applications up to industrial scale. Due to the inaccessibility of the quaternary carbon atom for a direct oxidative or reductive attack, they are considered as persistent in the environment. Here, we investigated the unknown degradation of the quaternary carbon-containing model compound pivalate (2,2-dimethyl-propionate) in the denitrifying bacterium Thauera humireducens strain PIV-1 (formerly Thauera pivalivorans). We provide multiple evidence for a pathway comprising the activation to pivalyl-CoA and the carbon skeleton rearrangement to isovaleryl-CoA. Subsequent reactions proceed similar to the catabolic leucine degradation pathway such as the carboxylation to 3-methylglutaconyl-CoA and the cleavage of 3-methyl-3-hydroxyglutaryl-CoA to acetyl-CoA and acetoacetate. The completed genome of Thauera humireducens strain PIV-1 together with proteomic data was used to identify pivalate-upregulated gene clusters including genes putatively encoding pivalate CoA ligase and adenosylcobalamin-dependent pivalyl-CoA mutase. A pivalate-induced gene encoding a putative carboxylic acid CoA ligase was heterologously expressed, and its highly enriched product exhibited pivalate CoA ligase activity. The results provide the first experimental insights into the biodegradation pathway of a quaternary carbon-containing model compound that serves as a blueprint for the degradation of related quaternary carbon-containing compounds.

A zero inflated log-normal model for inference of sparse microbial association networks.

The advent of high-throughput metagenomic sequencing has prompted the development of efficient taxonomic profiling methods allowing to measure the presence, abundance and phylogeny of organisms in a wide range of environmental samples. Multivariate sequence-derived abundance data further has the potential to enable inference of ecological associations between microbial populations, but several technical issues need to be accounted for, like the compositional nature of the data, its extreme sparsity and overdispersion, as well as the frequent need to operate in under-determined regimes. The ecological network reconstruction problem is frequently cast into the paradigm of Gaussian Graphical Models (GGMs) for which efficient structure inference algorithms are available, like the graphical lasso and neighborhood selection. Unfortunately, GGMs or variants thereof can not properly account for the extremely sparse patterns occurring in real-world metagenomic taxonomic profiles. In particular, structural zeros (as opposed to sampling zeros) corresponding to true absences of biological signals fail to be properly handled by most statistical methods. We present here a zero-inflated log-normal graphical model (available at https://github.com/vincentprost/Zi-LN) specifically aimed at handling such "biological" zeros, and demonstrate significant performance gains over state-of-the-art statistical methods for the inference of microbial association networks, with most notable gains obtained when analyzing taxonomic profiles displaying sparsity levels on par with real-world metagenomic datasets.

Metabolic reconstruction of the genome of candidate Desulfatiglans TRIP_1 and identification of key candidate enzymes for anaerobic phenanthrene degradation.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed pollutants. As oxygen is rapidly depleted in water-saturated PAH-contaminated sites, anaerobic microorganisms are crucial for their consumption. Here, we report the metabolic pathway for anaerobic degradation of phenanthrene by a sulfate-reducing enrichment culture (TRIP) obtained from a natural asphalt lake. The dominant organism of this culture belongs to the Desulfobacteraceae family of Deltaproteobacteria and genome-resolved metagenomics led to the reconstruction of its genome along with a handful of genomes from lower abundance bacteria. Proteogenomic analyses confirmed metabolic capabilities for dissimilatory sulfate reduction and indicated the presence of the Embden-Meyerhof-Parnas pathway, a complete tricarboxylic acid cycle as well as a complete Wood-Ljungdahl pathway. Genes encoding enzymes putatively involved in the degradation of phenanthrene were identified. This includes two gene clusters encoding a multisubunit carboxylase complex likely involved in the activation of phenanthrene, as well as genes encoding reductases potentially involved in subsequent ring dearomatization and reduction steps. The predicted metabolic pathways were corroborated by transcriptome and proteome analyses, and provide the first insights into the metabolic pathway responsible for the anaerobic degradation of three-ringed PAHs.

The class II benzoyl-coenzyme A reductase complex from the sulfate-reducing Desulfosarcina cetonica.

Benzoyl-CoA reductases (BCRs) catalyse a key reaction in the anaerobic degradation pathways of monocyclic aromatic substrates, the dearomatization of benzoyl-CoA (BzCoA) to cyclohexa-1,5-diene-1-carboxyl-CoA (1,5-dienoyl-CoA) at the negative redox potential limit of diffusible enzymatic substrate/product couples (E°' = -622 mV). A 1-MDa class II BCR complex composed of the BamBCDEGHI subunits has so far only been isolated from the Fe(III)-respiring Geobacter metallireducens. It is supposed to drive endergonic benzene ring reduction at an active site W-pterin cofactor by flavin-based electron bifurcation. Here, we identified multiple copies of putative genes encoding the structural components of a class II BCR in sulfate reducing, Fe(III)-respiring and syntrophic bacteria. A soluble 950 kDa Bam[(BC)2 DEFGHI]2 complex was isolated from extracts of Desulfosarcina cetonica cells grown with benzoate/sulfate. Metal and cofactor analyses together with the identification of conserved binding motifs gave rise to 4 W-pterins, two selenocysteines, six flavin adenine dinucleotides, four Zn, and 48 FeS clusters. The complex exhibited 1,5-dienoyl-CoA-, NADPH- and ferredoxin-dependent oxidoreductase activities. Our results indicate that high-molecular class II BCR metalloenzyme machineries are remarkably conserved in strictly anaerobic bacteria with regard to subunit architecture and cofactor content, but their subcellular localization and electron acceptor preference may differ as a result of adaptations to variable energy metabolisms.

Anaerobic degradation of phenanthrene by a sulfate-reducing enrichment culture.

Anaerobic degradation processes are very important to attenuate polycyclic aromatic hydrocarbons (PAHs) in saturated, anoxic sediments. However, PAHs are poorly degradable, leading to very slow microbial growth and thus resulting in only a few cultures that have been enriched and studied so far. Here, we report on a new phenanthrene-degrading, sulfate-reducing enrichment culture, TRIP1. Genome-resolved metagenomics and strain specific cell counting with FISH and flow cytometry indicated that the culture is dominated by a microorganism belonging to the Desulfobacteraceae family (60% of the community) and sharing 93% 16S rRNA sequence similarity to the naphthalene-degrading, sulfate-reducing strain NaphS2. The anaerobic degradation pathway was studied by metabolite analyses and revealed phenanthroic acid as the major intermediate consistent with carboxylation as the initial activation reaction. Further reduced metabolites were indicative of a stepwise reduction of the ring system. We were able to measure the presumed second enzyme reaction in the pathway, phenanthroate-CoA ligase, in crude cell extracts. The reaction was specific for 2-phenanthroic acid and did not transform other isomers. The present study provides first insights into the anaerobic degradation pathways of three-ringed PAHs. The biochemical strategy follows principles known from anaerobic naphthalene degradation, including carboxylation and reduction of the aromatic ring system.

A spectral algorithm for fast de novo layout of uncorrected long nanopore reads.

MOTIVATION: New long read sequencers promise to transform sequencing and genome assembly by producing reads tens of kilobases long. However, their high error rate significantly complicates assembly and requires expensive correction steps to layout the reads using standard assembly engines. RESULTS: We present an original and efficient spectral algorithm to layout the uncorrected nanopore reads, and its seamless integration into a straightforward overlap/layout/consensus (OLC) assembly scheme. The method is shown to assemble Oxford Nanopore reads from several bacterial genomes into good quality (∼99% identity to the reference) genome-sized contigs, while yielding more fragmented assemblies from the eukaryotic microbe Sacharomyces cerevisiae. AVAILABILITY AND IMPLEMENTATION: https://github.com/antrec/spectrassembler. CONTACT: antoine.recanati@inria.fr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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

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/ß 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.

A patchwork pathway for oxygenase-independent degradation of side chain containing steroids.

The denitrifying betaproteobacterium Sterolibacterium denitrificans serves as model organism for studying the oxygen-independent degradation of cholesterol. Here, we demonstrate its capability of degrading various globally abundant side chain containing zoo-, phyto- and mycosterols. We provide the complete genome that empowered an integrated genomics/proteomics/metabolomics approach, accompanied by the characterization of a characteristic enzyme of steroid side chain degradation. The results indicate that individual molybdopterin-containing steroid dehydrogenases are involved in C25-hydroxylations of steroids with different isoprenoid side chains, followed by the unusual conversion to C26-oic acids. Side chain degradation to androsta-1,4-diene-3,17-dione (ADD) via aldolytic C-C bond cleavages involves acyl-CoA synthetases/dehydrogenases specific for the respective 26-, 24- and 22-oic acids/-oyl-CoAs and promiscuous MaoC-like enoyl-CoA hydratases, aldolases and aldehyde dehydrogenases. Degradation of rings A and B depends on gene products uniquely found in anaerobic steroid degraders, which after hydrolytic cleavage of ring A, again involves CoA-ester intermediates. The degradation of the remaining CD rings via hydrolytic cleavage appears to be highly similar in aerobic and anaerobic bacteria. Anaerobic cholesterol degradation employs a composite repertoire of more than 40 genes partially known from aerobic degradation in gammaproteobacteria/actinobacteria, supplemented by unique genes that are required to circumvent oxygenase-dependent reactions.

Functional soil metagenomics: elucidation of polycyclic aromatic hydrocarbon degradation potential following 12 years of in situ bioremediation.

A culture-independent function-based screening approach was used to assess the microbial aerobic catabolome for polycyclic aromatic hydrocarbons degradation of a soil subjected to 12 years of in situ bioremediation. A total of 422 750 fosmid clones were screened for key aromatic ring-cleavage activities using 2,3-dihydroxybiphenyl as substrate. Most of the genes encoding ring-cleavage enzymes on the 768 retrieved positive fosmids could not be identified using primer-based approaches and, thus, 205 fosmid inserts were sequenced. Nearly two hundred extradiol dioxygenase encoding genes of three different superfamilies could be identified. Additional key genes of aromatic metabolic pathways were identified, including a high abundance of Rieske non-heme iron oxygenases that provided detailed information on enzymes activating aromatic compounds and enzymes involved in activation of the side chain of methylsubstituted aromatics. The gained insights indicated a complex microbial network acting at the site under study, which comprises organisms similar to recently identified Immundisolibacter cernigliae TR3.2 and Rugosibacter aromaticivorans Ca6 and underlined the great potential of an approach that combines an activity-screening, a cost-effective high-throughput sequencing of fosmid clones and a phylogenomic-routed and manually curated database to carefully identify key proteins dedicated to aerobic degradation of aromatic compounds.

Enterotypes of the human gut microbiome.

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.

A scalable assembly-free variable selection algorithm for biomarker discovery from metagenomes.

BACKGROUND: Metagenomics holds great promises for deepening our knowledge of key bacterial driven processes, but metagenome assembly remains problematic, typically resulting in representation biases and discarding significant amounts of non-redundant sequence information. In order to alleviate constraints assembly can impose on downstream analyses, and/or to increase the fraction of raw reads assembled via targeted assemblies relying on pre-assembly binning steps, we developed a set of binning modules and evaluated their combination in a new "assembly-free" binning protocol. RESULTS: We describe a scalable multi-tiered binning algorithm that combines frequency and compositional features to cluster unassembled reads, and demonstrate i) significant runtime performance gains of the developed modules against state of the art software, obtained through parallelization and the efficient use of large lock-free concurrent hash maps, ii) its relevance for clustering unassembled reads from high complexity (e.g., harboring 700 distinct genomes) samples, iii) its relevance to experimental setups involving multiple samples, through a use case consisting in the "de novo" identification of sequences from a target genome (e.g., a pathogenic strain) segregating at low levels in a cohort of 50 complex microbiomes (harboring 100 distinct genomes each), in the background of closely related strains and the absence of reference genomes, iv) its ability to correctly identify clusters of sequences from the E. coli O104:H4 genome as the most strongly correlated to the infection status in 53 microbiomes sampled from the 2011 STEC outbreak in Germany, and to accurately cluster contigs of this pathogenic strain from a cross-assembly of these 53 microbiomes. CONCLUSIONS: We present a set of sequence clustering ("binning") modules and their application to biomarker (e.g., genomes of pathogenic organisms) discovery from large synthetic and real metagenomics datasets. Initially designed for the "assembly-free" analysis of individual metagenomic samples, we demonstrate their extension to setups involving multiple samples via the usage of the "alignment-free" d2S statistic to relate clusters across samples, and illustrate how the clustering modules can otherwise be leveraged for de novo "pre-assembly" tasks by segregating sequences into biologically meaningful partitions.

The human metagenome: our other genome?

For about a decade, the human microbiota has been investigated using molecular procedures that are now systematized via metagenomics. Several large scale studies are underway with the goal of establishing a set of reference data, such as catalogues of genes, microbial species and complete genome sequences of strains colonizing the various body sites. A first series of conclusions can be drawn from this 'natural history' approach that will also lay the ground for further studies aiming at understanding--in an ecological perspective--the mechanisms ensuring stable operation of the microbiota in healthy individuals, and how changes in its composition (dysbiosis) may result in diseases.

A placenta-specific receptor for the fusogenic, endogenous retrovirus-derived, human syncytin-2.

Syncytin-2 is an envelope gene from the human endogenous retrovirus FRD (HERV-FRD) co-opted by an ancestral primate host, conserved in evolution over >40 Myr, specifically expressed in the placenta, and with a cell-cell fusogenic activity likely contributing to placenta morphogenesis. Here, using the GeneBridge4 human/Chinese hamster radiation hybrid panel, we mapped and identified the human receptor for syncytin-2. This receptor-namely Major Facilitator Superfamily Domain Containing 2 (MFSD2)-belongs to a large family of presumptive carbohydrate transporters with 10-12 membrane-spanning domains, is located at chromosomal position 1p34.2, and is conserved in evolution. An expression vector for MFSD2 confers fusogenicity to otherwise insusceptible cells upon transfection of syncytin-2. It also confers infectivity to syncytin-2 pseudotypes, consistent with this protein being the receptor for the ancestrally acquired HERV-FRD family of endogenous retroviruses. At variance with the human gene, neither mouse nor rat MFSD2 can mediate membrane fusion, which is consistent with the fact that the envelope-derived syncytin genes co-opted by rodents during evolution are not orthologous to the human syncytin genes. Remarkably, a real-time quantitative RT-PCR analysis of MFSD2 in various human tissues demonstrates specific expression in the placenta, as well as in the human BeWo choriocarcinoma cell line, which discloses enhancement of receptor expression upon induction by forskolin of cell-cell fusion and syncytium formation. In situ hybridization of human placental tissue using an MFSD2-specific probe further unambiguously demonstrates receptor expression at the level of the syncytiotrophoblast, again consistent with a role in placenta morphogenesis.

Binning unassembled short reads based on k-mer abundance covariance using sparse coding.

BACKGROUND: Sequence-binning techniques enable the recovery of an increasing number of genomes from complex microbial metagenomes and typically require prior metagenome assembly, incurring the computational cost and drawbacks of the latter, e.g., biases against low-abundance genomes and inability to conveniently assemble multi-terabyte datasets. RESULTS: We present here a scalable pre-assembly binning scheme (i.e., operating on unassembled short reads) enabling latent genome recovery by leveraging sparse dictionary learning and elastic-net regularization, and its use to recover hundreds of metagenome-assembled genomes, including very low-abundance genomes, from a joint analysis of microbiomes from the LifeLines DEEP population cohort (n = 1,135, >1010 reads). CONCLUSION: We showed that sparse coding techniques can be leveraged to carry out read-level binning at large scale and that, despite lower genome reconstruction yields compared to assembly-based approaches, bin-first strategies can complement the more widely used assembly-first protocols by targeting distinct genome segregation profiles. Read enrichment levels across 6 orders of magnitude in relative abundance were observed, indicating that the method has the power to recover genomes consistently segregating at low levels.

Channeling C1 Metabolism toward S-Adenosylmethionine-Dependent Conversion of Estrogens to Androgens in Estrogen-Degrading Bacteria.

Bacterial degradation of endocrine disrupting and carcinogenic estrogens is essential for their elimination from the environment. Recent studies of the denitrifying, estrogen-degrading Denitratisoma strain DHT3 revealed the conversion of estrogens to androgens by a putative cobalamin-dependent methyltransferase encoded by the emtABCD genes. The methyl donor and its continuous regeneration to initiate estradiol catabolism have remained unknown. Here, large-scale cultivation of the denitrifying bacterium Denitratisoma oestradiolicum with estrogen provided the biomass required for quantitative biochemical analyses. Soluble fractions of extracts from estradiol-grown cells catalyzed the S-adenosyl-l-methionine (SAM)- and Ti(III)-citrate-dependent conversion of 17ß-estradiol/estrone to the respective androgens at 0.15 nmol min-1 mg-1 Kinetic studies of 17ß-estradiol methylation and reverse 1-dehydrotestosterone demethylation reactions indicated that the exergonic methyl transfer from SAM to the putative cobalamin drives the endergonic methyl transfer from the methylcobalamin intermediate to the phenolic ring A. Based on a high-quality circular genome from D. oestradiolicum, proteogenomic analyses identified a 17ß-estradiol-induced gene cluster comprising emtABCD genes together with genes involved in SAM regeneration via l-serine and l-methionine. Consistent with this finding, l-methionine/ATP or l-serine/ATP/tetrahydrofolate/l-homocysteine substituted for SAM as methyl donors, further confirmed by the incorporation of the 13C-methyl-group from 13C-l-methonine into methyl(III)cobalamine and the estrone methylation product androsta-1,4-diene-3-one. This work demonstrates that during bacterial estrogen catabolism, the C1 pool is channeled toward the initiating methyl transfer to ring A. The effective cellular SAM regeneration system may serve as a model for whole-cell SAM-dependent methylation reactions of biotechnological interest.IMPORTANCE Estrogens comprise a group of related hormones occurring in predominantly female vertebrates, with endocrine disrupting and carcinogenic potential. Microbial biodegradation of estrogens is essential for their elimination from surface waters and wastewater. Aerobic bacteria employ oxygenases for the initial cleavage of the aromatic ring A. In contrast, anaerobic degradation of estrogens is initiated by methyl transfer-dependent conversion into androgens involving a putative cobalamin-dependent methyltransferase system. The methyl donor for this unprecedented reaction and its stoichiometric regeneration have remained unknown. With the biomass obtained from large-scale fermentation of an estrogen-degrading denitrifying bacterium, we identified S-adenosyl-methionine (SAM) as the methyl donor for the cobalamin-mediated methyl transfer to estrogens. To continuously supply C1 units to initiate estrogen degradation, genes for SAM regeneration from estradiol-derived catabolites are highly upregulated. Data presented here shed light into biochemical processes involved in the globally important microbial degradation of estrogens.

Shared Nearest Neighbor Clustering in a Locality Sensitive Hashing Framework.

We present a new algorithm to cluster high-dimensional sequence data and its application to the field of metagenomics, which aims at reconstructing individual genomes from a mixture of genomes sampled from an environmental site, without any prior knowledge of reference data (genomes) or the shape of clusters. Such problems typically cannot be solved directly with classical approaches seeking to estimate the density of clusters, for example, using the shared nearest neighbors (SNN) rule, due to the prohibitive size of contemporary sequence datasets. We explore here a new approach based on combining the SNN rule with the concept of locality sensitive hashing (LSH). The proposed method, called LSH-SNN, works by randomly splitting the input data into smaller-sized subsets (buckets) and employing the SNN rule on each of these buckets. Links can be created among neighbors sharing a sufficient number of elements, hence allowing clusters to be grown from linked elements. LSH-SNN can scale up to larger datasets consisting of millions of sequences, while achieving high accuracy across a variety of sample sizes and complexities.

Fermentative Spirochaetes mediate necromass recycling in anoxic hydrocarbon-contaminated habitats.

Spirochaetes are frequently detected in anoxic hydrocarbon- and organohalide-polluted groundwater, but their role in such ecosystems has remained unclear. To address this, we studied a sulfate-reducing, naphthalene-degrading enrichment culture, mainly comprising the sulfate reducer Desulfobacterium N47 and the rod-shaped Spirochete Rectinema cohabitans HM. Genome sequencing and proteome analysis suggested that the Spirochete is an obligate fermenter that catabolizes proteins and carbohydrates, resulting in acetate, ethanol, and molecular hydrogen (H2) production. Physiological experiments inferred that hydrogen is an important link between the two bacteria in the enrichment culture, with H2 derived from fermentation by R. cohabitans used as reductant for sulfate reduction by Desulfobacterium N47. Differential proteomics and physiological experiments showed that R. cohabitans utilizes biomass (proteins and carbohydrates) released from dead cells of Desulfobacterium N47. Further comparative and community genome analyses indicated that other Rectinema phylotypes are widespread in contaminated environments and may perform a hydrogenogenic fermentative lifestyle similar to R. cohabitans. Together, these findings indicate that environmental Spirochaetes scavenge detrital biomass and in turn drive necromass recycling at anoxic hydrocarbon-contaminated sites and potentially other habitats.

An unusual strategy for the anoxic biodegradation of phthalate.

In the past two decades, the study of oxygen-independent degradation of widely abundant aromatic compounds in anaerobic bacteria has revealed numerous unprecedented enzymatic principles. Surprisingly, the organisms, metabolites and enzymes involved in the degradation of o-phthalate (1,2-dicarboxybenzene), mainly derived from phthalate esters that are annually produced at the million ton scale, are sparsely known. Here, we demonstrate a previously unknown capacity of complete phthalate degradation in established aromatic compound-degrading, denitrifying model organisms of the genera Thauera, Azoarcus and 'Aromatoleum'. Differential proteome analyses revealed phthalate-induced gene clusters involved in uptake and conversion of phthalate to the central intermediate benzoyl-CoA. Enzyme assays provided in vitro evidence for the formation of phthaloyl-CoA by a succinyl-CoA- and phthalate-specific CoA transferase, which is essential for the subsequent oxygen-sensitive decarboxylation to benzoyl-CoA. The extreme instability of the phthaloyl-CoA intermediate requires highly balanced CoA transferase and decarboxylase activities to avoid its cellular accumulation. Phylogenetic analysis revealed phthaloyl-CoA decarboxylase as a novel member of the UbiD-like, (de)carboxylase enzyme family. Homologs of the encoding gene form a phylogenetic cluster and are found in soil, freshwater and marine bacteria; an ongoing global distribution of a possibly only recently evolved degradation pathway is suggested.

Microbial communities in pyrene amended soil-compost mixture and fertilized soil.

Polycyclic aromatic hydrocarbons are distributed ubiquitously in the environment and form metabolites toxic to most organisms. Organic amendment of PAH contaminated soil with compost and farmyard manure has proven to be efficient for PAH bioremediation mediated by native microorganisms, even though information on the identity of PAH degraders in organic-amended soil is still scarce. Here we provide molecular insight into the bacterial communities in soil amended with compost or farmyard manure for which the degradation mass balances of 13C-labeled pyrene have been recently published and assess the relevant bacterial genera capable of degrading pyrene as a model PAH. We performed statistical analyses of bacterial genera abundance data based on total DNA and RNA (for comparison) extracted from the soil samples. The results revealed complex pyrene degrading communities with low abundance of individual degraders instead of a limited number of abundant key players. The bacterial degrader communities of the soil-compost mixture and soil fertilized with farmyard manure differed considerably in composition albeit showing similar degradation kinetics. Additional analyses were carried out on enrichment cultures and enabled the reconstruction of several nearly complete genomes, thus allowing to link microcosm and enrichment experiments. However, pyrene mineralizing bacteria enriched from the compost or unfertilized soil-compost samples did not dominate pyrene degradation in the soils. Based on the present findings, evaluations of PAH degrading microorganisms in complex soil mixtures with high organic matter content should not target abundant key degrading species, since the specific degraders may be highly diverse, of low abundance, and masked by high bacterial background.