The Complete Genome Sequence of the Microsymbiont Rhizobium leguminosarum WSM937 Reveals this Strain has Acquired Biovar Specific Symbiotic Genes Following Horizontal Gene Transfer of an Integrative Conjugative Element

Paige Warburton

The acid-tolerant Rhizobium leguminosarum strain WSM937 was collected from the Greek Island of Tinos and identified as a biovar viciae strain based on its ability to effectively nodulate with peas (Pisum), vetch (Vicia), and lentil (Lens). Although this strain displayed excellent saprophytic competence in acidic soils, it appeared to contain an unstable genotype based on the loss of nodulation capacity over time. This genome of this strain was therefore completely sequenced in this study to investigate the reason for this nodulation instability. A combination of Illumina HiSeq and Oxford Nanopore Technology (ONT) MinION data was used to create a hybrid assembly of strain WSM937. A bioinformatics pipeline was created to analyse the quality of sequence reads (using FastQC and Nanoplot) and perform assembly (using Flye v.2.7 and Geneious Prime 2022.0.1). The quality of the assembled genome was checked using BUSCO and QUAST and then annotated using RAST and the JGI IMG annotation pipeline (IMGAP v5.1.5, Gene calling program: CRT 1.8.2). The annotated genome of the completely finished genome of WSM937 was deposited into the JGI IMG database under the IMG Genome ID 2953896481. The total genome size obtained was 7,672,600 bp and consisted of six replicons: a chromosome of 4,855,555 bp and five plasmids of size 786,145, 700,105, 499,700, 421,535 and 409,560 bp. The annotated genome was found to encode 7,505 protein coding genes and 72 RNA encoding genes. The nodulation and fixation genes were located on the 499,700 bp pSym plasmid which was designated as pRL937c. Since WSM937 lost the ability to nodulate hosts in the field over time, mobile genetic elements (the mobilome) which could be involved in DNA rearrangements, and hence genome plasticity, were identified. This mobilome analysis identified the presence of a 19.8 Kbp chromosomal phage, 112 small mobile genetic elements and a 123 Kbp integrative conjugative element (ICE) flanked by duplicated IS21 insertion sequences on pRL937c. The ICE region contained the Rhizobium leguminosarum bv. viciae nodulation and nitrogen fixation genes along with multiple small mobile elements of varying transposase families. This thesis reveals for the first time that this ICE element has been horizontally transferred from Rhizobium leguminosarum bv. viciae into the chromosomal genotype of Rhizobium leguminosarum bv. trifolii. This transfer event has enabled WSM937 to form a symbiotic relationship with legumes that are normally non-permissive for the Rhizobium leguminosarum bv. trifolii genotype. The nature of this ICE element reveals that the WSM937 genetic basis of nodulation instability may result from genomic recombination mediated rearrangements.

The Complete Genome Sequence of the Microsymbiont Rhizobium leguminosarum WSM937 Reveals this Strain has Acquired Biovar Specific Symbiotic Genes Following Horizontal Gene Transfer of an Integrative Conjugative Element

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