Complete genome sequence of Bacillus thuringiensis strain RC340, isolated from a temperate forest soil sample in New England

ABSTRACT The complete genome sequence of Bacillus thuringiensis strain RC340, isolated from an environmental microbiology experiment soil sample is presented here. B. thuringiensis strain RC340 sequenced by GridION consists of a single genome consisting of 5.86 million bases, 8,152 predicted genes, and 0.23% contamination.

S oil microenvironments are highly competitive, and bacteria challenge one another for the limited resources available.B. thuringiensis has several mechanisms to gain a competitive advantage including bacteriocin production and antimicrobial resistance (AMR) proteins (1).Soil warming may induce the prevalence of AMR genes (2), so the focus of our genomics analysis was gene signatures of bacitracin resistance mechanisms.
Soils from the Harvard Forest soil warming experiment were experimentally heated 5°C above ambient temperature with unheated controls (3).Soil containing RC340 was isolated in 2022 from a heated soil plot, and then a streak of the soil sample was grown on actinobacteria isolation agar (AIA) (4) with 100 mg of L-1 cycloheximide.The isolate colony was then selected and grown on 10% tryptic soy broth at 30°C with shaking at 150 rpm until cultures reached an OD of 0.5, then spun at 4,000 rpm for 15 min.Genomic DNA was extracted from cell pellets using the CTAB method (5).16S rRNA sequence was sequenced as previously described (6) to determine the genome size of the species with the highest percent identity and estimate the target 40X coverage for the genome assembly.DNA was not sheared or size-selected for ONT library preparation.
To assess the presence of bacitracin resistance genes in the RC340 genome and the nine closest related neighbors, we used Compare Genomes from Pangenomes v0.0.7 (22).The pangenome output contained five bacitracin resistance genes only present in the RC340 genome.These were bacitracin export, resistance, and transport proteins.The Bt strain RC340 bacitracin resistance genes may provide a competitive advantage in the battle for nutrients in nutrient-depleted soils due to climate change stress (23,24).

ACKNOWLEDGMENTS
We are grateful to Drs.Serita Frey and Mel Knorr for maintaining this infrastructure for our community.
This project was conducted with the support of a grant from the National Science Foundation (No. DEB-1749206).

FIG 1
FIG 1 Insert Genome Into SpeciesTree v2.2.0 program created tree with Prokka file as input and a neighbor public genome count of 9, closest phylogenetic neighbors shown above.B. mycoides, B. cereus, and B. thuringiensis are closest neighbors.
The soil warming experiments at Harvard Forest are maintained with support from the National Science Foundation Long Term Ecological Research Program (DEB-1832110) and a Long Term Research in Environmental Biology grant (DEB-1456610).

TABLE 1
Genome assembly quality assessment values shown, data obtained through QUAST and CheckM a a Quality assessment metrics are largest contig, completion, and contamination.