From garden soil, we isolated and sequenced Massilia sp. strain ONC3, a new member of the Oxalobacteraceae within the Massilia genus. Sequence analysis showed an assembled genome size of 5,622,601 bp, with a predicted total of 5,104 protein-coding sequences, 3,194 functionally assigned genes, 2 rRNA operons, and 56 tRNAs.
ABSTRACT
From garden soil, we isolated and sequenced Massilia sp. strain ONC3, a new member of the Oxalobacteraceae within the Massilia genus. Sequence analysis showed an assembled genome size of 5,622,601 bp, with a predicted total of 5,104 protein-coding sequences, 3,194 functionally assigned genes, 2 rRNA operons, and 56 tRNAs.
ANNOUNCEMENT
Members of the genus Massilia have been isolated from soil, air, and water samples, as well as from the plant rhizosphere and endosphere, and they are typically Gram-negative, rod-shaped aerobes (1, 2). Certain species of Massilia can promote plant growth through their ability to solubilize recalcitrant phosphate sources in soils (3) or through their positive impact on the colonization of plants by beneficial root symbionts such as arbuscular mycorrhizal fungi. Massilia sp. strain RK4, for example, increases root colonization by arbuscular mycorrhizal fungi and their nutritional benefits to maize plants under salt stress (4). Other Massilia species are highly resistant to heavy metals and have been isolated from mines (5).
We isolated a putative new species of Massilia from unplanted garden soil (pH 5.2) near Maxton, NC (34.6494, −79.4327). The isolate was an interesting target for whole-genome sequencing, since Massilia strains often show plant growth-promoting capabilities. The soil samples were dried for 3 days prior to bacterial isolation, mixed with 1× phosphate-buffered saline, streaked out several times until single colonies were isolated on R2A medium, and cultured at 30°C. Cultures were grown in R2A broth at 30°C for 2 days before genomic DNA (gDNA) was extracted using the Agencourt GenFind v2 kit (Beckman Coulter Life Sciences, Indianapolis, IN) and the protocol for bacterial gDNA extraction. The genomic library was prepared using the Illumina Nextera platform (San Diego, CA), size selected to an average fragment length of 475 bp, and sequenced using Illumina NextSeq paired-end v2 chemistry on v2.5 flow cells at 150 bp per read. The target coverage for the reads was 20×. We used the default settings of BayesHammer and SPAdes 3.13.0 (6) for quality trimming and de novo assembly of the 10,528,120 total reads. This resulted in 155 contigs with an N50 value of 56,531 bp (contig size range, 942 to 207,795 bp) and a total assembled size of 5,622,601 bp, with a GC content of 63.82%. Assembly quality with 40 reference proteins through BUSCO (7, 8) revealed a measured completeness (40 single-copy BUSCOs) of 100%. Genome assembly and annotation were carried out using the PATRIC 3.5.28 pipeline (9) and confirmed by Galaxy (10) and RAST 2.0 (11), which identified 5,104 protein-coding sequences, 3,194 proteins with functional assignments, 2 rRNA operons, and 56 tRNA genes.
Based on NCBI BLAST (12) searches of the 16S rRNA gene sequences, Massilia sp. strain ONC3 was found to have the highest similarities (98% sequence identity) with Massilia solisilvae J18 and Massilia terrae J11. The Genome-to-Genome Distance Calculator (GGDC) (13) calculates intergenomic distances using three formulas (the sum of all identities found in aligned high-scoring segment pairs divided by the total genome length and expressed as a percentage) and was used to compare the ONC3 genome to the 5 most related genomes (Massilia albidiflava DSM 17472, Massilia armeniaca ZMN-3, Massilia putida 6NM-7T, Massilia sp. strain NR 4-1, and Massilia sp. strain WG5 [14]). The results for the ONC3 genome relative to the other selected genomes showed a 0% match (or less than 70% of the scientific community threshold) (15, 16). This suggests that Massilia sp. ONC3 is a novel species within the Massilia genus.
Data availability.
This genome has been deposited at DDBJ/EMBL/GenBank under the BioProject number PRJNA529408, BioSample number SAMN11265771, accession number NZ_SPUM00000000, and SRA accession number SRR9320523.
ACKNOWLEDGMENTS
This project was funded by Novozymes North America.
We thank Novozymes North America for providing the novel isolate and whole-genome sequencing and Timothy Lilburn (Novozymes North America), Deborah Springer (Novozymes North America), and Alex Soupir (South Dakota State University) for technical guidance and support throughout this project. We also acknowledge the support of the Agricultural Experiment Station at South Dakota State University to H.B.
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Associated Data
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Data Availability Statement
This genome has been deposited at DDBJ/EMBL/GenBank under the BioProject number PRJNA529408, BioSample number SAMN11265771, accession number NZ_SPUM00000000, and SRA accession number SRR9320523.