Complete genome of Bradyrhizobium sp. strain BDV5419, representative of Australian genospecies L

International audienceWe report the complete genome sequence of Bradyrhizobium sp. strain BDV5419, representative of Bradyrhizobium genospecies L, which symbiotically associates with the Australian native legume Hardenbergia violaceae and is expected to represent a novel Bradyrhizobium species. The complete genome sequence provides a genetic reference for this Australian genospecies

Complete Genome Sequence of Bradyrhizobium sp. Strain BDV5040, Representative of Widespread Genospecies B in Australia

International audienceWe report the complete genome sequence of Bradyrhizobium sp. strain BDV5040, representative of Bradyrhizobium genospecies B, which symbiotically associates with legume hosts belonging to all three Fabaceae subfamilies across the Australian continent. The complete genome sequence provides a genetic reference for this Australian genospecies.Bradyrhizobium sp. strain BDV5040 was isolated in 1995 from a root nodule of Bossiaea ensata (Fabaceae, Faboideae, Bossiaeeae) collected in Ben Boyd National Park, New South Wales, Australia (37°12′S, 149°57′E; altitude, 140 m), in the course of a survey of rhizobia associated with native shrubby legumes in southeastern Australia (1). It is a representative of Bradyrhizobium genospecies B, which occurs under different climatic and edaphic conditions across the whole Australian continent and exhibits a broad host range encompassing all three Fabaceae subfamilies (1–4).Strain BDV5040 was grown from a lyophilized stock in 30 ml of yeast extract mannitol broth (5) at 25°C and 200 rpm for 5 days. Genomic DNA was prepared by successive phenol-chloroform extractions as described (6). DNA quantification and quality control were performed using a NanoDrop spectrophotometer, a Qubit 4 fluorometer, and agarose gel electrophoresis. The same DNA was used for Nanopore and Illumina sequencing. Illumina libraries were obtained using the Nextera XT kit following the manufacturer’s instructions, starting with 1 ng of genomic DNA, and were analyzed by paired-end 2 × 300-bp sequencing on a MiSeq instrument. Poor-quality regions (Q 1,500 bp) and quality (score of >8) using Nanofilt v2.5.0 (11), and adapters were removed using Porechop v0.2.4 (12). Long reads were further reduced to 800 Mbp as a target quantity using Filtlong v0.2.0 (13) (parameters: --min_length 2000 --keep_percent 90 --target_bases 800000000). Illumina and Nanopore reads were coassembled using Unicycler v0.4.8 (14) with default parameters, resulting in a single component with eight segments and incomplete status (length, 7,622,333 bp; N50, 7,339,313 bp). Completion was obtained by exporting the sequence path from Bandage v0.8.1 (15) and filling a last gap using Pilon v1.23 (16) and by manually comparing the sequence with Unicycler 003_long_read_assembly.fasta. The assembly and complete chromosome sequence were carefully inspected by visualizing the alignment of long and short reads using minimap2 v2.17 (17) and IGV v2.7.2 (18). Finally, the chromosome was rotated to start at dnaA.The circular chromosome is 7,622,528 bp long, with an average G+C content of 63.92%. The sequence was automatically annotated by the NCBI Prokaryote Genome Annotation Pipeline (PGAP) v4.13 (19). The genome consists of 7,092 protein-coding genes, 48 tRNAs, 1 copy each of the 5S, 16S, and 23S rRNA genes, and 88 pseudogenes.Data availability.The genome sequence of Bradyrhizobium genospecies B strain BDV5040 is available in NCBI GenBank under accession number CP061379. The raw sequence reads are available under SRA accession numbers SRX9514896 and SRX9514898 under BioProject number PRJNA662585 and BioSample number SAMN16089659

A White Campion (Silene latifolia) floral expressed sequence tag (EST) library: annotation, EST-SSR characterization, transferability, and utility for comparative mapping

<p>Abstract</p> <p>Background</p> <p>Expressed sequence tag (EST) databases represent a valuable resource for the identification of genes in organisms with uncharacterized genomes and for development of molecular markers. One class of markers derived from EST sequences are simple sequence repeat (SSR) markers, also known as EST-SSRs. These are useful in plant genetic and evolutionary studies because they are located in transcribed genes and a putative function can often be inferred from homology searches. Another important feature of EST-SSR markers is their expected high level of transferability to related species that makes them very promising for comparative mapping. In the present study we constructed a normalized EST library from floral tissue of <it>Silene latifolia </it>with the aim to identify expressed genes and to develop polymorphic molecular markers.</p> <p>Results</p> <p>We obtained a total of 3662 high quality sequences from a normalized <it>Silene </it>cDNA library. These represent 3105 unigenes, with 73% of unigenes matching genes in other species. We found 255 sequences containing one or more SSR motifs. More than 60% of these SSRs were trinucleotides. A total of 30 microsatellite loci were identified from 106 ESTs having sufficient flanking sequences for primer design. The inheritance of these loci was tested via segregation analyses and their usefulness for linkage mapping was assessed in an interspecific cross. Tests for crossamplification of the EST-SSR loci in other <it>Silene </it>species established their applicability to related species.</p> <p>Conclusion</p> <p>The newly characterized genes and gene-derived markers from our <it>Silene </it>EST library represent a valuable genetic resource for future studies on <it>Silene latifolia </it>and related species. The polymorphism and transferability of EST-SSR markers facilitate comparative linkage mapping and analyses of genetic diversity in the genus <it>Silene</it>.</p

lpxC and yafS are the Most Suitable Internal Controls to Normalize Real Time RT-qPCR Expression in the Phytopathogenic Bacteria Dickeya dadantii

Background: Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria Dickeya dadantii and Pectobacterium atrosepticum, which are stable in a wide range of growth conditions. [br/] Results: We extracted, from a D. dadantii micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process in planta. The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions. [br/] Conclusions: We have identified at least two genes, lpxC (ABF-0017965) and yafS (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria D. dadantii and P. atrosepticum and, probably, of other Enterobacteriaceae. Moreover, we defined general criteria to select good reference genes in bacteria

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

Metatranscriptomics Reveals the Diversity of Genes Expressed by Eukaryotes in Forest Soils

Eukaryotic organisms play essential roles in the biology and fertility of soils. For example the micro and mesofauna contribute to the fragmentation and homogenization of plant organic matter, while its hydrolysis is primarily performed by the fungi. To get a global picture of the activities carried out by soil eukaryotes we sequenced 2×10,000 cDNAs synthesized from polyadenylated mRNA directly extracted from soils sampled in beech (Fagus sylvatica) and spruce (Picea abies) forests. Taxonomic affiliation of both cDNAs and 18S rRNA sequences showed a dominance of sequences from fungi (up to 60%) and metazoans while protists represented less than 12% of the 18S rRNA sequences. Sixty percent of cDNA sequences from beech forest soil and 52% from spruce forest soil had no homologs in the GenBank/EMBL/DDJB protein database. A Gene Ontology term was attributed to 39% and 31.5% of the spruce and beech soil sequences respectively. Altogether 2076 sequences were putative homologs to different enzyme classes participating to 129 KEGG pathways among which several were implicated in the utilisation of soil nutrients such as nitrogen (ammonium, amino acids, oligopeptides), sugars, phosphates and sulfate. Specific annotation of plant cell wall degrading enzymes identified enzymes active on major polymers (cellulose, hemicelluloses, pectin, lignin) and glycoside hydrolases represented 0.5% (beech soil)–0.8% (spruce soil) of the cDNAs. Other sequences coding enzymes active on organic matter (extracellular proteases, lipases, a phytase, P450 monooxygenases) were identified, thus underlining the biotechnological potential of eukaryotic metatranscriptomes. The phylogenetic affiliation of 12 full-length carbohydrate active enzymes showed that most of them were distantly related to sequences from known fungi. For example, a putative GH45 endocellulase was closely associated to molluscan sequences, while a GH7 cellobiohydrolase was closest to crustacean sequences, thus suggesting a potentially significant contribution of non-fungal eukaryotes in the actual hydrolysis of soil organic matter

Complete Genome Sequence of the Hyperthermophilic Piezophilic Archaeon Pyrococcus kukulkanii NCB100 Isolated from the Rebecca’s Roost Hydrothermal Vent in the Guaymas Basin

Members of the order Thermococcales are common inhabitants of high-temperature hydrothermal vent systems (black smokers) that are represented in clone libraries mostly by isolates from the Thermococcus genus. We report the complete sequence of a novel species from the Pyrococcus genus, P. kukulkanii strain NCB100, which has been isolated from a flange fragment of the Rebecca’s Roost hydrothermal vent system in the Guaymas Basin

PecS Is a Global Regulator of the Symptomatic Phase in the Phytopathogenic Bacterium Erwinia chrysanthemi 3937▿ †

Pathogenicity of the enterobacterium Erwinia chrysanthemi (Dickeya dadantii), the causative agent of soft-rot disease in many plants, is a complex process involving several factors whose production is subject to temporal regulation during infection. PecS is a transcriptional regulator that controls production of various virulence factors. Here, we used microarray analysis to define the PecS regulon and demonstrated that PecS notably regulates a wide range of genes that could be linked to pathogenicity and to a group of genes concerned with evading host defenses. Among the targets are the genes encoding plant cell wall-degrading enzymes and secretion systems and the genes involved in flagellar biosynthesis, biosurfactant production, and the oxidative stress response, as well as genes encoding toxin-like factors such as NipE and hemolysin-coregulated proteins. In vitro experiments demonstrated that PecS interacts with the regulatory regions of five new targets: an oxidative stress response gene (ahpC), a biosurfactant synthesis gene (rhlA), and genes encoding exported proteins related to other plant-associated bacterial proteins (nipE, virK, and avrL). The pecS mutant provokes symptoms more rapidly and with more efficiency than the wild-type strain, indicating that PecS plays a critical role in the switch from the asymptomatic phase to the symptomatic phase. Based on this, we propose that the temporal regulation of the different groups of genes required for the asymptomatic phase and the symptomatic phase is, in part, the result of a gradual modulation of PecS activity triggered during infection in response to changes in environmental conditions emerging from the interaction between both partners

Identification de fonctions « spécifiques d'espèce » impliquées dans l'individualisation en écovar de l’espèce G8 du complexe Agrobacterium tumefaciens

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