Draft Genome Sequence of Bacillus velezensis Strain E68, Isolated from an Oil Battery.

Bacillus velezensis strain E68 is a biosurfactant-producing bacterium isolated from an oil battery near Chauvin, Alberta, Canada. Strain E68 exhibited antimicrobial activity against fungal pathogens and could potentially serve as a biological control agent. Its genome was sequenced and annotated, revealing the presence of multiple lipopeptide biosynthetic gene clusters.

ANNOUNCEMENT

Bacillus velezensis is a species of Gram-positive endospore-forming bacteria capable of promoting plant growth and producing biologically active compounds that suppress a broad spectrum of microorganisms (1). Numerous strains of B. velezensis exhibit antifungal activity against a range of human- and plant-pathogenic fungi (2). The suppression of microbial growth by B. velezensis is partly linked to the production of cyclic lipopeptides (3). Cyclic lipopeptides are biosurfactants which demonstrate antifungal activity, aid in biofilm formation, and can induce systemic resistance in host plants (4). Bacteria isolated from petroleum oil or soil contaminated with it often demonstrate elevated production of biosurfactants, which help solubilize the hydrocarbon carbon sources (5). As a result, they are attractive candidates for use as biological control agents. Bacterial strain E68, previously identified as B. subtilis, was isolated from an oil battery near Chauvin, Alberta, Canada, and was characterized as a top biosurfactant producer (6). Whole-genome sequencing of strain E68 will provide a basis for understanding its biocontrol mechanisms and facilitating its applications in the future.

A single colony of strain E68 was used to inoculate 10 ml of LB broth. It was then incubated at 37°C overnight, with shaking at 140 rpm. Genomic DNA was extracted from strain E68 using a DNeasy blood and tissue kit (Qiagen, Germany) following the protocol for Gram-positive bacteria. Library preparation and genome sequencing were performed at IDseq, Inc. (Sacramento, CA, USA). The DNA was fragmented using a Covaris E220 sonicator. Then, a paired-end sequencing library was prepared using a Nextera XT DNA library preparation kit (Illumina, USA). Genome sequencing was carried out on a HiSeq X instrument, using a 2 × 150-bp protocol, producing 24,785,553 sequencing reads. All software was used with default settings unless specified otherwise. The quality of the reads was assessed with FastQC v.0.11.8 (7). The reads were then quality trimmed and adapters were removed using Trim Galore v.0.6.5 (8). The trimmed reads were assembled de novo using SPAdes v.3.13.0 with k-mer sizes of 21, 33, 55, 77, 99, and 127 (9). This resulted in an assembly with 145 contigs. The contigs were ordered and combined into scaffolds using MeDuSa v.1.6 with the –d setting enabled to estimate gap lengths between pairs of contigs (10). Medusa was given the following set of 5 genomes of Bacillus velezensis strains (RefSeq assembly accession number) for scaffolding purposes: CBMB205 (GCF_002117165.1), FZB42 (GCF_000015785.2), JS25R (GCF_000769555.1), OB9 (GCF_001266815.1), and B26 (GCF_001266825.1). Scaffolds under 200 bp or containing contaminants as detected by NCBI’s contamination screen were removed from the assembly. This resulted in a final assembly of 69 scaffolds and a total length of 3,862,910 bp. The assembly featured a GC content of 46.65%, an N50 value of 3,717,019, and an average sequencing depth of 1,809×. Assembly statistics were provided by QUAST v.5.0.2 (11), and sequencing depth information was provided by BBMap v.37.36 (12). Strain E68 was identified as Bacillus velezensis following ribosomal multilocus sequence typing analysis (https://pubmlst.org/rmlst/) (13). The draft genome was annotated with NCBI’s Prokaryotic Genome Annotation Pipeline v.4.11 (14). Annotation detected 3,637 coding DNA sequences, 111 RNA genes, 12 complete rRNAs, 86 tRNAs, 5 noncoding RNAs (ncRNAs), and 75 pseudogenes. Biosynthetic gene clusters were identified with antiSMASH v.5.1.1 (15). The genome of strain E68 contained gene clusters for the cyclic lipopeptides surfactin, fengycin, and bacillomycin D, as well as the antimicrobials bacillaene, difficidin, macrolactin H, bacillibactin, and bacilysin.

Data availability.

This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number JAAECG000000000. The version described in this paper is the first version, JAAECG010000000. The sequencing reads have been deposited in the NCBI SRA under accession number PRJNA603554.