Direct Xylella fastidiosa whole genome sequencing from various plant species using targeted enrichment.

A targeted enrichment method was developed to sequence Xylella fastidiosa genomic DNA directly from plant samples. The method was evaluated on various plant species infected with different strains at different levels of contamination. After enrichment, X. fastidiosa genome coverage was above 99.9% for all tested samples.

A Primer on the Analysis of High-Throughput Sequencing Data for Detection of Plant Viruses.

High-throughput sequencing (HTS) technologies have become indispensable tools assisting plant virus diagnostics and research thanks to their ability to detect any plant virus in a sample without prior knowledge. As HTS technologies are heavily relying on bioinformatics analysis of the huge amount of generated sequences, it is of utmost importance that researchers can rely on efficient and reliable bioinformatic tools and can understand the principles, advantages, and disadvantages of the tools used. Here, we present a critical overview of the steps involved in HTS as employed for plant virus detection and virome characterization. We start from sample preparation and nucleic acid extraction as appropriate to the chosen HTS strategy, which is followed by basic data analysis requirements, an extensive overview of the in-depth data processing options, and taxonomic classification of viral sequences detected. By presenting the bioinformatic tools and a detailed overview of the consecutive steps that can be used to implement a well-structured HTS data analysis in an easy and accessible way, this paper is targeted at both beginners and expert scientists engaging in HTS plant virome projects.

Diversity of bacterial communities in French chicken cuts stored under modified atmosphere packaging.

Poultry meat, the second most consumed meat in France, is commercialized mainly as portions of chicken cuts with various quality labels, stored under various modified atmosphere packaging (MAP), with shelf-life ranging from 9 to 17 days. We used 16S rDNA pyrosequencing to describe microbiota of chicken legs. Ten samples representing a wide diversity of labels and MAP available on the market were collected from local supermarkets and stored at 4 °C. Microbiota were collected, total DNA was extracted, and V1-V3 fragment of 16S rRNA genes were amplified and sequenced. For data analysis several pipelines were compared. The Qiime pipeline was chosen to cluster reads and we used a database previously developed for a meat and fish microbial ecology study. Variability between samples was observed and a listing of bacteria present on chicken meat was established. The structure of the bacterial communities were compared with traditional cultural methods and validated with quantitative real time PCR. Brochothrix thermosphacta, Pseudomonas sp., and Carnobacterium sp. were dominant and the nature of the gas used for packaging influenced the relative abundance of each suggesting a MAP gas composition dependent competition between these species. We also noticed that slaughterhouse environment may influence the nature of the contaminants.

Comparative Genomic Analysis Reveals Ecological Differentiation in the Genus Carnobacterium.

Lactic acid bacteria (LAB) differ in their ability to colonize food and animal-associated habitats: while some species are specialized and colonize a limited number of habitats, other are generalist and are able to colonize multiple animal-linked habitats. In the current study, Carnobacterium was used as a model genus to elucidate the genetic basis of these colonization differences. Analyses of 16S rRNA gene meta-barcoding data showed that C. maltaromaticum followed by C. divergens are the most prevalent species in foods derived from animals (meat, fish, dairy products), and in the gut. According to phylogenetic analyses, these two animal-adapted species belong to one of two deeply branched lineages. The second lineage contains species isolated from habitats where contact with animal is rare. Genome analyses revealed that members of the animal-adapted lineage harbor a larger secretome than members of the other lineage. The predicted cell-surface proteome is highly diversified in C. maltaromaticum and C. divergens with genes involved in adaptation to the animal milieu such as those encoding biopolymer hydrolytic enzymes, a heme uptake system, and biopolymer-binding adhesins. These species also exhibit genes for gut adaptation and respiration. In contrast, Carnobacterium species belonging to the second lineage encode a poorly diversified cell-surface proteome, lack genes for gut adaptation and are unable to respire. These results shed light on the important genomics traits required for adaptation to animal-linked habitats in generalist Carnobacterium.

Complete Genome Sequence of Lactococcus piscium CNCM I-4031, a Bioprotective Strain for Seafood Products.

Lactococcus piscium CNCM I-4031 is a psychotrophic foodborne lactic acid bacterium showing potential interest for the biopreservation of seafood products due to its inhibition properties toward pathogenic and spoilage bacteria. The analysis of its genome will provide a better understanding of the mechanisms of interaction between these bacteria.

A method to isolate bacterial communities and characterize ecosystems from food products: Validation and utilization in as a reproducible chicken meat model.

Influenced by production and storage processes and by seasonal changes the diversity of meat products microbiota can be very variable. Because microbiotas influence meat quality and safety, characterizing and understanding their dynamics during processing and storage is important for proposing innovative and efficient storage conditions. Challenge tests are usually performed using meat from the same batch, inoculated at high levels with one or few strains. Such experiments do not reflect the true microbial situation, and the global ecosystem is not taken into account. Our purpose was to constitute live stocks of chicken meat microbiotas to create standard and reproducible ecosystems. We searched for the best method to collect contaminating bacterial communities from chicken cuts to store as frozen aliquots. We tested several methods to extract DNA of these stored communities for subsequent PCR amplification. We determined the best moment to collect bacteria in sufficient amounts during the product shelf life. Results showed that the rinsing method associated to the use of Mobio DNA extraction kit was the most reliable method to collect bacteria and obtain DNA for subsequent PCR amplification. Then, 23 different chicken meat microbiotas were collected using this procedure. Microbiota aliquots were stored at -80°C without important loss of viability. Their characterization by cultural methods confirmed the large variability (richness and abundance) of bacterial communities present on chicken cuts. Four of these bacterial communities were used to estimate their ability to regrow on meat matrices. Challenge tests performed on sterile matrices showed that these microbiotas were successfully inoculated and could overgrow the natural microbiota of chicken meat. They can therefore be used for performing reproducible challenge tests mimicking a true meat ecosystem and enabling the possibility to test the influence of various processing or storage conditions on complex meat matrices.

Draft Genome Sequence of Carnobacterium divergens V41, a Bacteriocin-Producing Strain.

In this study, we present the draft genome sequence of Carnobacterium divergens V41. This strain was previously reported as producing divercin V41, a bacteriocin of interest for food biopreservation. Its genome revealed also the presence of a gene cluster putatively involved in polyketide production, which is unique in lactic acid bacteria.

Genes associated to lactose metabolism illustrate the high diversity of Carnobacterium maltaromaticum.

The dairy population of Carnobacterium maltaromaticum is characterized by a high diversity suggesting a high diversity of the genetic traits linked to the dairy process. As lactose is the main carbon source in milk, the genetics of lactose metabolism was investigated in this LAB. Comparative genomic analysis revealed that the species C. maltaromaticum exhibits genes related to the Leloir and the tagatose-6-phosphate (Tagatose-6P) pathways. More precisely, strains can bear genes related to one or both pathways and several strains apparently do not contain homologs related to these pathways. Analysis at the population scale revealed that the Tagatose-6P and the Leloir encoding genes are disseminated in multiple phylogenetic lineages of C. maltaromaticum: genes of the Tagatose-6P pathway are present in the lineages I, II and III, and genes of the Leloir pathway are present in the lineages I, III and IV. These data suggest that these genes evolved thanks to horizontal transfer, genetic duplication and translocation. We hypothesize that the lac and gal genes evolved in C. maltaromaticum according to a complex scenario that mirrors the high population diversity.

Genomic and proteomic evidence supporting the division of the plant pathogen Ralstonia solanacearum into three species.

BACKGROUND: The increased availability of genome sequences has advanced the development of genomic distance methods to describe bacterial diversity. Results of these fast-evolving methods are highly correlated with those of the historically standard DNA-DNA hybridization technique. However, these genomic-based methods can be done more rapidly and less expensively and are less prone to technical and human error. They are thus a technically accessible replacement for species delineation. Here, we use several genomic comparison methods, supported by our own proteomic analyses and metabolic characterization as well as previously published DNA-DNA hybridization analyses, to differentiate members of the Ralstonia solanacearum species complex into three species. This pathogen group consists of diverse and widespread strains that cause bacterial wilt disease on many different plants. RESULTS: We used three different methods to compare the complete genomes of 29 strains from the R. solanacearum species complex. In parallel we profiled the proteomes of 73 strains using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS). Proteomic profiles together with genomic sequence comparisons consistently and comprehensively described the diversity of the R. solanacearum species complex. In addition, genome-driven functional phenotypic assays excitingly supported an old hypothesis (Hayward et al. (J Appl Bacteriol 69:269-80, 1990)), that closely related members of the R. solanacearum could be identified through a simple assay of anaerobic nitrate metabolism. This assay allowed us to clearly and easily differentiate phylotype II and IV strains from phylotype I and III strains. Further, genomic dissection of the pathway distinguished between proposed subspecies within the current phylotype IV. The assay revealed large scale differences in energy production within the R. solanacearum species complex, indicating coarse evolutionary distance and further supporting a repartitioning of this group into separate species. CONCLUSIONS: Together, the results of these studies support the proposed division of the R. solanacearum species complex into three species, consistent with recent literature, and demonstrate the utility of proteomic and genomic approaches to delineate bacterial species.

Bacterial spoilers of food: behavior, fitness and functional properties.

Most food products are highly perishable as they constitute a rich nutrient source for microbial development. Among the microorganisms contaminating food, some present metabolic activities leading to spoilage. In addition to hygienic rules to reduce contamination, various treatments are applied during production and storage to avoid the growth of unwanted microbes. The nature and appearance of spoilage therefore depend on the physiological state of spoilers and on their ability to resist the processing/storage conditions and flourish on the food matrix. Spoilage also relies on the interactions between the microorganisms composing the ecosystems encountered in food. The recent rapid increase in publicly available bacterial genome sequences, as well as the access to high-throughput methods, should lead to a better understanding of spoiler behavior and to the possibility of decreasing food spoilage. This review lists the main bacterial species identified as food spoilers, their ability to develop during storage and/or processing, and the functions potentially involved in spoilage. We have also compiled an inventory of the available genome sequences of species encompassing spoilage strains. Combining in silico analysis of genome sequences with experimental data is proposed in order to understand and thus control the bacterial spoilage of food better.

Constraints on genome dynamics revealed from gene distribution among the Ralstonia solanacearum species.

Because it is suspected that gene content may partly explain host adaptation and ecology of pathogenic bacteria, it is important to study factors affecting genome composition and its evolution. While recent genomic advances have revealed extremely large pan-genomes for some bacterial species, it remains difficult to predict to what extent gene pool is accessible within or transferable between populations. As genomes bear imprints of the history of the organisms, gene distribution pattern analyses should provide insights into the forces and factors at play in the shaping and maintaining of bacterial genomes. In this study, we revisited the data obtained from a previous CGH microarrays analysis in order to assess the genomic plasticity of the R. solanacearum species complex. Gene distribution analyses demonstrated the remarkably dispersed genome of R. solanacearum with more than half of the genes being accessory. From the reconstruction of the ancestral genomes compositions, we were able to infer the number of gene gain and loss events along the phylogeny. Analyses of gene movement patterns reveal that factors associated with gene function, genomic localization and ecology delineate gene flow patterns. While the chromosome displayed lower rates of movement, the megaplasmid was clearly associated with hot-spots of gene gain and loss. Gene function was also confirmed to be an essential factor in gene gain and loss dynamics with significant differences in movement patterns between different COG categories. Finally, analyses of gene distribution highlighted possible highways of horizontal gene transfer. Due to sampling and design bias, we can only speculate on factors at play in this gene movement dynamic. Further studies examining precise conditions that favor gene transfer would provide invaluable insights in the fate of bacteria, species delineation and the emergence of successful pathogens.

Sequencing of K60, type strain of the major plant pathogen Ralstonia solanacearum.

Ralstonia solanacearum is a widespread and destructive plant pathogen. We present the genome of the type strain, K60 (phylotype IIA, sequevar 7). Sequevar 7 strains cause ongoing tomato bacterial wilt outbreaks in the southeastern United States. K60 generally resembles R. solanacearum CFBP2957, a Caribbean tomato isolate, but has almost 360 unique genes.

Ralstonia syzygii, the Blood Disease Bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles.

The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical evolution than to the acquisition of DNA by horizontal transfer.

Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence.

BACKGROUND: The Ralstonia solanacearum species complex includes thousands of strains pathogenic to an unusually wide range of plant species. These globally dispersed and heterogeneous strains cause bacterial wilt diseases, which have major socio-economic impacts. Pathogenicity is an ancestral trait in R. solanacearum and strains with high genetic variation can be subdivided into four phylotypes, correlating to isolates from Asia (phylotype I), the Americas (phylotype IIA and IIB), Africa (phylotype III) and Indonesia (phylotype IV). Comparison of genome sequences strains representative of this phylogenetic diversity can help determine which traits allow this bacterium to be such a pathogen of so many different plant species and how the bacteria survive in many different habitats. RESULTS: The genomes of three tomato bacterial wilt pathogens, CFBP2957 (phy. IIA), CMR15 (phy. III) and PSI07 (phy. IV) were sequenced and manually annotated. These genomes were compared with those of three previously sequenced R. solanacearum strains: GMI1000 (tomato, phy. I), IPO1609 (potato, phy. IIB), and Molk2 (banana, phy. IIB). The major genomic features (size, G+C content, number of genes) were conserved across all of the six sequenced strains. Despite relatively high genetic distances (calculated from average nucleotide identity) and many genomic rearrangements, more than 60% of the genes of the megaplasmid and 70% of those on the chromosome are syntenic. The three new genomic sequences revealed the presence of several previously unknown traits, probably acquired by horizontal transfers, within the genomes of R. solanacearum, including a type IV secretion system, a rhi-type anti-mitotic toxin and two small plasmids. Genes involved in virulence appear to be evolving at a faster rate than the genome as a whole. CONCLUSIONS: Comparative analysis of genome sequences and gene content confirmed the differentiation of R. solanacearum species complex strains into four phylotypes. Genetic distances between strains, in conjunction with CGH analysis of a larger set of strains, revealed differences great enough to consider reclassification of the R. solanacearum species complex into three species. The data are still too fragmentary to link genomic classification and phenotypes, but these new genome sequences identify a pan-genome more representative of the diversity in the R. solanancearum species complex.

Potential of a 16S rRNA-based taxonomic microarray for analyzing the rhizosphere effects of maize on Agrobacterium spp. and bacterial communities.

Bacterial diversity is central to ecosystem sustainability and soil biological function, for which the role of roots is important. The high-throughput analysis potential of taxonomic microarray should match the breadth of bacterial diversity. Here, the power of this technology was evidenced through methodological verifications and analysis of maize rhizosphere effect based on a 16S rRNA-based microarray developed from the prototype of H. Sanguin et al. (Environ. Microbiol. 8:289-307, 2006). The current probe set was composed of 170 probes (41 new probes in this work) that targeted essentially the Proteobacteria. Cloning and sequencing of 16S rRNA amplicons were carried out on maize rhizosphere and bulk soil DNA. All tested clones that had a perfect match with corresponding probes were positive in the hybridization experiment. The hierarchically nested probes were reliable, but the level of taxonomic identification was variable, depending on the probe set specificity. The comparison of experimental and theoretical hybridizations revealed 0.91% false positives and 0.81% false negatives. The microarray detection threshold was estimated at 0.03% of a given DNA type based on DNA spiking experiments. A comparison of the maize rhizosphere and bulk soil hybridization results showed a significant rhizosphere effect, with a higher predominance of Agrobacterium spp. in the rhizosphere, as well as a lower prevalence of Acidobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes, a new taxon of interest in soil. In addition, well-known taxonomic groups such as Sphingomonas spp., Rhizobiaceae, and Actinobacteria were identified in both microbial habitats with strong hybridization signals. The taxonomic microarray developed in the present study was able to discriminate and characterize bacterial community composition in related biological samples, offering extensive possibilities for systematic exploration of bacterial diversity in ecosystems.