Molecular Diversity of Legume Root-Nodule Bacteria in Kakadu National Park, Northern Territory, Australia

BACKGROUND: Symbiotic relationships between leguminous plants (family Fabaceae) and nodule-forming bacteria in Australia native ecosystems remain poorly characterized despite their importance. Most studies have focused on temperate parts of the country, where the use of molecular approaches have already revealed the presence of Bradyrhizobium, Ensifer (formerly Sinorhizobium), Mesorhizobium and Rhizobium genera of legume root-nodule bacteria. We here provide the first molecular characterization of nodulating bacteria from tropical Australia. METHODOLOGY/PRINCIPAL FINDINGS: 45 nodule-forming bacterial strains, isolated from eight native legume hosts at eight locations in Kakadu National Park, Northern Territory, Australia, were examined for their genetic diversity and phylogenetic position. Using SSU rDNA PCR-RFLPs and phylogenetic analyses, our survey identified nine genospecies, two of which, Bradyrhizobium genospp. B and P, had been previously identified in south-eastern Australia and one, Mesorhizobium genospecies AA, in southern France. Three of the five newly characterized Bradyrhizobium genospecies were more closely related to B. japonicum USDA110, whereas the other two belonged to the B. elkanii group. All five were each more closely related to strains sampled in various tropical areas outside Australia than to strains known to occur in Australia. We also characterized an entirely novel nodule-forming lineage, phylogenetically distant from any previously described rhizobial and non-rhizobial legume-nodulating lineage within the Rhizobiales. CONCLUSIONS/SIGNIFICANCE: Overall, the present results support the hypothesis of tropical areas being centres of biodiversity and diversification for legume root-nodule bacteria and confirm the widespread occurrence of Bradyrhizobium genosp. B in continental Australia

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

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

Formation of chromosome in bacteria

International audienceThe genome of bacteria is classically separated into the essential, stable and slow evolving chromosomes and the accessory, mobile and rapidly evolving plasmids. This paradigm is being questioned since the discovery of extra-chromosomal essential replicons (EER), be they called "megaplasmids", "secondary chromosomes" or "chromids". These genomic elements are thought to be indispensable based on their having a GC content identical to that of the chromosome and harboring essential genes, but plasmidic in origin because of the structure of their replication origin. However, none of these criteria are universally applicable and the true nature of these replicons is yet to be formally determined. Here, we explore the relationships of chromosomes and plasmids with reference to their genetic information inheritance systems (GIIS), under the assumption that the inheritance of EERs is integrated to the cell cycle and highly constrained in contrast to that of standard plasmids. We performed a global comparative genomic analysis including all bacterial complete genome sequences available in NCBI RefSeq. Using ACLAME and KEGG GIIS-related proteins as input, we first identified GIIS functional homologs from all bacterial genomes. These homologs were then clustered according to function and sequences homology. The relationships between all bacterial replicons were then investigated using the GIIS clusters as parameters and unsupervised analyses (projections and graphs, and clustering. Finally, identification of putative EERs and of trends in GIIS usage were performed using supervised analyses (classification, regression). This strategy clearly discriminated between chromosomes and plasmids with respect to their GIIS usage, and revealed another class of genomic elements that corresponds to EER-annotated replicons. They are characterized by a specific GIIS usage that testifies of the continuity of the genomic material in bacteria. Furthermore, whereas some are plasmidic in origin, others derive from the cleavage of an ancestral chromosome. Our study provides insights into the formation of "neo-chromosomes" and the emergence of multipartite genomes in bacteria, as well as clues about the forces shaping the genome

Classification of bacterial replicons based on the Genetic Information Transmission Systems

International audienceThe genome of bacteria is classically separated into the essential, stable and slow evolving chromosomes and the accessory, mobile and rapidly evolving plasmids. This distinction has been blurred in recent years by the characterization of multipartite genomes constituted of a primary "standard" chromosome and one or several additional and essential replicons adapted to the cell cycle. Depending on the authors, these genomic elements are either named "secondary chromosomes" or "megaplasmids". However, their true nature and evolution are yet to be determined. Here we investigate the relationships of these secondary essential replicons (SERs) to classical chromosomes and plasmids based on the key processes involved in the maintenance of genomes and replicons, i.e., their replication, partition and segregation, and perform a global comparative genomic analysis for all bacterial replicons available from public databases. Several classes of replicons could thus be characterized, and chromosomes, plasmids and SERs differentiated. This sets the basis to the investigation of the emergence of SER-like genomic structures

Visualization and analysis of an interconnected network of genomic elements

National audienceIn the context of studying the relationships among bacterial replicons, i.e., chromosomes and plasmids), we investigate various methodological approaches using a test dataset. Standard method fail to describe the complexity of the genetic events that occur in the evolution and adaptation of these elements. Given a set of genes of interest linked functionally and used as variables, the organization of beta-proteobacterial replicons was studied using several dimension reduction methods, graphs as well as supervised classification. Combinations of methods prove indispensable to characterize the relationships between replicons, permitting to identify both global trends among replicons as well as specific features of single replicon. Furthermore, our study underlines the inherent difficulty to explore exhaustively genomic data using a single tool

Neo-formation of chromosomes in bacteria.

ABSTRACTAlthough the bacterial secondary chromosomes/megaplasmids/chromids, first noticed about forty years ago, are commonly held to originate from stabilized plasmids, their true nature and definition are yet to be resolved. On the premise that the integration of a replicon within the cell cycle is key to deciphering its essential nature, we show that the content in genes involved in the replication, partition and segregation of the replicons and in the cell cycle discriminates the bacterial replicons into chromosomes, plasmids, and another class of essential genomic elements that function as chromosomes. These latter do not derive directly from plasmids. Rather, they arise from the fission of a multi-replicon molecule corresponding to the co-integrated and rearranged ancestral chromosome and plasmid. All essential replicons in a distributed genome are thus neochromosomes. Having a distributed genome appears to extend and accelerate the exploration of the bacterial genome evolutionary landscape, producing complex regulation and leading to novel eco-phenotypes and species diversification.</jats:p

Contribution of graphs to the analysis of bacterial genome architecture

International audienceThe genome of bacteria is classically separated into the essential, stable and slow evolving chromosomes and the accessory, mobile and rapidly evolving plasmids. Recently, a new class of genomic elements, neither chromosomes nor plasmids, has been revealed, the nature and evolution of which proves difficult to pinpoint. In the context of characterizing the different replicons forming the bacterial genomes, we investigated the analytical methodologies best able to decipher and visualize the functional and evolutionary relationships between replicons. Using databases of genes involved in genetic information transmission systems (GITS) as inputs, we performed a global comparative genomics analysis on all available bacterial genomes according to the following methodology: i) identification and assessment of functional homologs from all bacterial genomes, ii) construction of clusters of proteins linking the bacterial replicons by their GITS, with regard to the shared functions and protein sequence homologies, iii) testing of several unsupervised approaches (visualization, clustering). The comparison of these methods relied on the biological result assessment using the replicon host taxonomy and structure as external criteria as well as stability measurements as internal criteria. Bipartite graphs proved most useful for the meaningful representation of the replicons according to their GITS proteins. Community detection algorithms (INFOMAP) performed best in terms of stability with reference to the bacterial taxonomy in comparison to traditional clustering methodologies. Furthermore, our study brought about a dual functional and taxonomical structuration of the replicon space. This led to results with strong biological implications. Indeed, we were able to characterize the third class of replicons relative to chromosomes and plasmids, and to propose novel defining criteria for these genomic elements. Beyond the biological relevance, our study sets the basis for further analyses (workflow improvement/enrichment, classifications...) in order to bring to light driving forces of genome evolution