Polychlorinated Biphenyl Transformation, Peroxidase and Oxidase Activities of Fungi and Bacteria Isolated from a Historically Contaminated Site.

Causing major health and ecological disturbances, polychlorinated biphenyls (PCBs) are persistent organic pollutants still recovered all over the world. Microbial PCB biotransformation is a promising technique for depollution, but the involved molecular mechanisms remain misunderstood. Ligninolytic enzymes are suspected to be involved in many PCB transformations, but their assessments remain scarce. To further inventory the capabilities of microbes to transform PCBs through their ligninolytic enzymes, we investigated the role of oxidase and peroxidase among a set of microorganisms isolated from a historically PCB-contaminated site. Among 29 isolated fungi and 17 bacteria, this work reports for the first time the PCB-transforming capabilities from fungi affiliated to Didymella, Dothiora, Ilyonectria, Naganishia, Rhodoturula, Solicoccozyma, Thelebolus and Truncatella genera and bacteria affiliated to Peribacillus frigotolerans, Peribacillus muralis, Bacillus mycoides, Bacillus cereus, Bacillus toyonensis, Pseudarthrobacter sp., Pseudomonas chlororaphis, Erwinia aphidicola and Chryseobacterium defluvii. In the same way, this is the first report of fungal isolates affiliated to the Dothiora maculans specie and Cladosporium genus that displayed oxidase (putatively laccase) and peroxidase activity, respectively, enhanced in the presence of PCBs (more than 4-fold and 20-fold, respectively, compared to controls). Based on these results, the observed activities are suspected to be involved in PCB transformation.

Prokaryotic, Microeukaryotic, and Fungal Composition in a Long-Term Polychlorinated Biphenyl-Contaminated Brownfield.

Polychlorinated biphenyls (PCBs) are recognized as persistent organic pollutants and accumulate in organisms, soils, waters, and sediments, causing major health and ecological perturbations. Literature reported PCB bio-transformation by fungi and bacteria in vitro, but data about the in situ impact of those compounds on microbial communities remained scarce while being useful to guide biotransformation assays. The present work investigated for the first time microbial diversity from the three-domains-of-life in a long-term contaminated brownfield (a former factory land). Soil samples were ranked according to their PCB concentrations, and a significant increase in abundance was shown according to increased concentrations. Microbial communities structure showed a segregation from the least to the most PCB-polluted samples. Among the identified microorganisms, Bacteria belonging to Gammaproteobacteria class, as well as Fungi affiliated to Saccharomycetes class or Pleurotaceae family, including some species known to transform some PCBs were abundantly retrieved in the highly polluted soil samples.

Development and validation of a UHPLC-ESI-QTOF mass spectrometry method to analyze opines, plant biomarkers of crown gall or hairy root diseases.

Opines are low-molecular-weight metabolites specifically biosynthesized by agrobacteria-transformed plant cells when plants are struck by crown gall and hairy root diseases, which cause uncontrolled tissue overgrowth. Transferred DNA is sustainably incorporated into the genomes of the transformed plant cells, so that opines constitute a persistent biomarker of plant infection by pathogenic agrobacteria and can be targeted for crown gall/hairy root disease diagnosis. We developed a general, rapid, specific and sensitive analytical method for overall opine detection using ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-MS-QTOF), with easy preparation of samples. Based on MS, MS/MS and chromatography data, the detection selectivity of a wide range of standard opines was validated in pure solution and in different plant extracts. The method was successfully used to detect different structural types of opines, including opines for which standard compounds are unavailable, in tumors or hairy roots induced by pathogenic strains. As the method can detect a wide range of opines in a single run, it represents a powerful tool for plant gall analysis and crown gall/hairy root disease diagnosis. Using an appropriate dilution of plant extract and a matrix-based calibration curve, the quantification ability of the method was validated for three opines belonging to different families (nopaline, octopine, mannopine), which were accurately quantified in plant tissue extracts.

Anthropization level of Lascaux Cave microbiome shown by regional-scale comparisons of pristine and anthropized caves.

Limestone areas across the world develop karstic caves, which are populated by a wide range of macro- and microorganisms. Many of these caves display Paleolithic art or outstanding speleothems, and in the last century they have been subjected to anthropization due to touristic management and intense human frequentation. Despite their cultural importance and associated conservation issues, the impact of anthropization on cave biodiversity is not known. Here, we show that anthropization is associated with specific cave biota modifications. We compared diversity in four pristine caves, four anthropized show caves, and the iconic Lascaux Cave with even stronger anthropization. The predominant microbial higher taxa were the same in all caves, but the most anthropized cave (Lascaux) was unique as it differed from the eight others by a higher proportion of Bacteroidetes bacteria and the absence of Euryarchaeota and Woesearchaeota archaea. Anthropization resulted in lower diversity and altered community structure for bacteria and archaea on cave walls, especially in Lascaux, but with a more limited effect on microeukaryotes and arthropods. Our findings fill a key gap in our understanding of the response of karstic communities to anthropization, by revealing that tourism-related anthropization impacts on the prokaryotic microbiome rather than on eukaryotic residents, and that it shapes cave biota irrespective of cave natural features.

Ancestral Genome Estimation Reveals the History of Ecological Diversification in Agrobacterium.

Horizontal gene transfer (HGT) is considered as a major source of innovation in bacteria, and as such is expected to drive adaptation to new ecological niches. However, among the many genes acquired through HGT along the diversification history of genomes, only a fraction may have actively contributed to sustained ecological adaptation. We used a phylogenetic approach accounting for the transfer of genes (or groups of genes) to estimate the history of genomes in Agrobacterium biovar 1, a diverse group of soil and plant-dwelling bacterial species. We identified clade-specific blocks of cotransferred genes encoding coherent biochemical pathways that may have contributed to the evolutionary success of key Agrobacterium clades. This pattern of gene coevolution rejects a neutral model of transfer, in which neighboring genes would be transferred independently of their function and rather suggests purifying selection on collectively coded acquired pathways. The acquisition of these synapomorphic blocks of cofunctioning genes probably drove the ecological diversification of Agrobacterium and defined features of ancestral ecological niches, which consistently hint at a strong selective role of host plant rhizospheres.

Draft Genome Sequence of Chryseobacterium sp. JV274 Isolated from Maize Rhizosphere.

We report the draft genome sequence of Chryseobacterium sp. JV274. This strain was isolated from the rhizosphere of maize during a greenhouse experiment. JV274 harbors genes involved in flexirubin production (darA and darB genes), bacterial competition (type VI secretion system), and gliding (bacterial motility; type IX secretion system).

Genomic characterization of Ensifer aridi, a proposed new species of nitrogen-fixing rhizobium recovered from Asian, African and American deserts.

BACKGROUND: Nitrogen fixing bacteria isolated from hot arid areas in Asia, Africa and America but from diverse leguminous plants have been recently identified as belonging to a possible new species of Ensifer (Sinorhizobium). In this study, 6 strains belonging to this new clade were compared with Ensifer species at the genome-wide level. Their capacities to utilize various carbon sources and to establish a symbiotic interaction with several leguminous plants were examined. RESULTS: Draft genomes of selected strains isolated from Morocco (Merzouga desert), Mexico (Baja California) as well as from India (Thar desert) were produced. Genome based species delineation tools demonstrated that they belong to a new species of Ensifer. Comparison of its core genome with those of E. meliloti, E. medicae and E. fredii enabled the identification of a species conserved gene set. Predicted functions of associated proteins and pathway reconstruction revealed notably the presence of transport systems for octopine/nopaline and inositol phosphates. Phenotypic characterization of this new desert rhizobium species showed that it was capable to utilize malonate, to grow at 48 °C or under high pH while NaCl tolerance levels were comparable to other Ensifer species. Analysis of accessory genomes and plasmid profiling demonstrated the presence of large plasmids that varied in size from strain to strain. As symbiotic functions were found in the accessory genomes, the differences in symbiotic interactions between strains may be well related to the difference in plasmid content that could explain the different legumes with which they can develop the symbiosis. CONCLUSIONS: The genomic analysis performed here confirms that the selected rhizobial strains isolated from desert regions in three continents belong to a new species. As until now only recovered from such harsh environment, we propose to name it Ensifer aridi. The presented genomic data offers a good basis to explore adaptations and functionalities that enable them to adapt to alkalinity, low water potential, salt and high temperature stresses. Finally, given the original phylogeographic distribution and the different hosts with which it can develop a beneficial symbiotic interaction, Ensifer aridi may provide new biotechnological opportunities for degraded land restoration initiatives in the future.

Single acquisition of protelomerase gave rise to speciation of a large and diverse clade within the Agrobacterium/Rhizobium supercluster characterized by the presence of a linear chromid.

Linear chromosomes are atypical in bacteria and likely a secondary trait derived from ancestral circular molecules. Within the Rhizobiaceae family, whose genome contains at least two chromosomes, a particularity of Agrobacterium fabrum (formerly A. tumefaciens) secondary chromosome (chromid) is to be linear and hairpin-ended thanks to the TelA protelomerase. Linear topology and telA distributions within this bacterial family was screened by pulse field gel electrophoresis and PCR. In A. rubi, A. larrymoorei, Rhizobium skierniewicense, A. viscosum, Agrobacterium sp. NCPPB 1650, and every genomospecies of the biovar 1/A. tumefaciens species complex (including R. pusense, A. radiobacter, A. fabrum, R. nepotum plus seven other unnamed genomospecies), linear chromid topologies were retrieved concomitantly with telA presence, whereas the remote species A. vitis, Allorhizobium undicola, Rhizobium rhizogenes and Ensifer meliloti harbored a circular chromid as well as no telA gene. Moreover, the telA phylogeny is congruent with that of recA used as a marker gene of the Agrobacterium phylogeny. Collectively, these findings strongly suggest that single acquisition of telA by an ancestor was the founding event of a large and diverse clade characterized by the presence of a linear chromid. This clade, characterized by unusual genome architecture, appears to be a relevant candidate to serve as a basis for a possible redefinition of the controversial Agrobacterium genus. In this respect, investigating telA in sequenced genomes allows to both ascertain the place of concerned strains into Agrobacterium spp. and their actual assignation to species/genomospecies in this genus.

Genomic species are ecological species as revealed by comparative genomics in Agrobacterium tumefaciens.

The definition of bacterial species is based on genomic similarities, giving rise to the operational concept of genomic species, but the reasons of the occurrence of differentiated genomic species remain largely unknown. We used the Agrobacterium tumefaciens species complex and particularly the genomic species presently called genomovar G8, which includes the sequenced strain C58, to test the hypothesis of genomic species having specific ecological adaptations possibly involved in the speciation process. We analyzed the gene repertoire specific to G8 to identify potential adaptive genes. By hybridizing 25 strains of A. tumefaciens on DNA microarrays spanning the C58 genome, we highlighted the presence and absence of genes homologous to C58 in the taxon. We found 196 genes specific to genomovar G8 that were mostly clustered into seven genomic islands on the C58 genome-one on the circular chromosome and six on the linear chromosome-suggesting higher plasticity and a major adaptive role of the latter. Clusters encoded putative functional units, four of which had been verified experimentally. The combination of G8-specific functions defines a hypothetical species primary niche for G8 related to commensal interaction with a host plant. This supports that the G8 ancestor was able to exploit a new ecological niche, maybe initiating ecological isolation and thus speciation. Searching genomic data for synapomorphic traits is a powerful way to describe bacterial species. This procedure allowed us to find such phenotypic traits specific to genomovar G8 and thus propose a Latin binomial, Agrobacterium fabrum, for this bona fide genomic species.

Rapid and efficient identification of Agrobacterium species by recA allele analysis: Agrobacterium recA diversity.

The analysis of housekeeping recA gene sequences from 138 strains from 13 species or genomic species of Agrobacterium, nine being biovar 1 genomospecies, and the others Agrobacterium larrymoorei, Agrobacterium rubi, Agrobacterium sp. NCPPB 1650, and Agrobacterium vitis and one "former" Agrobacterium species, Rhizobium rhizogenes, led to the identification of 50 different recA alleles and to a clear delineation of the 14 species or genomospecies entirely consistent with that obtained by amplified fragment length polymorphism (AFLP) analysis. The relevance of a recA sequencing approach for epidemiological analyses was next assessed on agrobacterial Tunisian isolates. All Tunisian isolates were found to belong to the Agrobacterium tumefaciens/biovar 1 species complex by both biochemical tests and rrs sequencing. recA sequence analysis further permitted their unambiguous assignment to A. tumefaciens genomospecies G4, G6, G7, and G8 in total agreement with the results of an AFLP-based analysis. At subspecific level, several Tunisian recA alleles were novel, indicating the power and accuracy of recA-based typing for studies of Agrobacterium spp.

Identification of genomic species in Agrobacterium biovar 1 by AFLP genomic markers.

Biovar 1 of the genus Agrobacterium consists of at least nine genomic species that have not yet received accepted species names. However, rapid identification of these organisms in various biotopes is needed to elucidate crown gall epidemiology, as well as Agrobacterium ecology. For this purpose, the AFLP methodology provides rapid and unambiguous determination of the genomic species status of agrobacteria, as confirmed by additional DNA-DNA hybridizations. The AFLP method has been proven to be reliable and to eliminate the need for DNA-DNA hybridization. In addition, AFLP fragments common to all members of the three major genomic species of agrobacteria, genomic species G1 (reference strain, strain TT111), G4 (reference strain, strain B6, the type strain of Agrobacterium tumefaciens), and G8 (reference strain, strain C58), have been identified, and these fragments facilitate analysis and show the applicability of the method. The maximal infraspecies current genome mispairing (CGM) value found for the biovar 1 taxon is 10.8%, while the smallest CGM value found for pairs of genomic species is 15.2%. This emphasizes the gap in the distribution of genome divergence values upon which the genomic species definition is based. The three main genomic species of agrobacteria in biovar 1 displayed high infraspecies current genome mispairing values (9 to 9.7%). The common fragments of a genomic species are thus likely "species-specific" markers tagging the core genomes of the species.