Sequencing & Characterisation of the Rhizobial Isolate WSM1721, the Proposed Type Strain of Ensifer yinggardensis sp. nov.

Adam Walker

Currently, very little is known about the rhizobia that nodulate with native Australian legumes, with significant diversity stored away from widespread access. In 1996, isolate WSM1721 was obtained from a root nodule of a native Indigofera sp. plant growing approximately 60 km south-south-east of Carnarvon, Western Australia, in the traditional lands of the Yinggarda people. This isolate was shown to belong to the genus Sinorhizobium (syn. Ensifer) on the basis of 16S rRNA gene sequence identity. Later, in 2010, the genome of this strain was sequenced as part of the Joint Genome Institute’s (JGI) Genomic Encyclopedia of Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) sequencing initiative, to expand understanding of the genetic diversity of symbiosis. Short-read Next Generation Sequencing (NGS) technology was used to produce a permanent draft genome of 6,292,184 bp across 68 scaffolds. This incomplete genome was deposited into the JGI’s Integrated Microbial Genomes (IMG) database and automatically classified as a putative novel species on the basis of genome-wide Average Nucleotide Identity (gANI), with an extremely close relation to the effectively published species Ensifer aridi. Hence, a thorough polyphasic approach was undertaken in this study to determine if Ensifer sp. WSM1721 could be assigned to a new species based on established classification standards. The best practice in modern taxonomy is that, if possible, a novel prokaryotic species is initially described with multiple member strains. For this reason, previously identified putative relatives of Ensifer sp. WSM1721 were assessed using their 16S rRNA gene sequences. However, all tested isolates were shown to belong to different species clusters from WSM1721. Consequently, phenotypic characterisation was performed on the Ensifer sp. WSM1721 singleton. Of note was the finding that Ensifer sp. WSM1721 could utilise L-phenylalanine for growth and was unable to grow at 44 °C, characteristics that distinguish it from Ensifer aridi LMR001T. In addition to extensive phenotypic tests, the genome of Ensifer sp. WSM1721 was re-sequenced using long-read Oxford Nanopore Technology (ONT) and was completely assembled using the hybrid assembly program Unicycler. The new assembly revealed the genome size to be 6,326,810 bp, over four circular replicons, the number and sizes of the three plasmids matching its Eckhardt plasmid profile. Genetic analysis via BUSCO identified 638/639 tested genes as present and complete. The 16S rRNA gene sequences from the genome confirmed the close relationship of WSM1721 to E. aridi, while multiple Overall Genome Related Indices (OGRIs) confirmed that it was a separate species. Digital DNA-DNA Hybridisation (dDDH) between the genome sequence established in this study and the genome of E. aridi LMR001T provided 58.7% to 62.4% identity depending on the formulae used (well below the 70% cut-off). The highest dDDH identity (63.1%) was found to be to the genome of E. aridi LEM457. Average Nucleotide Identity comparisons using ANIb, orthoANIu and ANIm provided values of 93.94%, 94.33%, and 94.88% sequence identity to E. aridi LMR001T, respectively. The highest ANI (ANIm) value was obtained to E. aridi LEM457 at 94.9% identity. However, this value is still below the 95-96% cut-off for Intraspecific delimitation. Therefore, Ensifer sp. WSM1721 represents a new species based on phenotypic markers and multiple OGRIs. It is proposed in this thesis, with the permission of the Yinggarda Aboriginal Corporation, that the species be given the name Ensifer yinggardensis, after the Yinggarda country of the traditional aboriginal landowners. The designated type strain of this species is Ensifer yinggardensis WSM1721T.

Sequencing & Characterisation of the Rhizobial Isolate WSM1721, the Proposed Type Strain of Ensifer yinggardensis sp. nov.

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