Draft genome sequence of Bacillus velezensis strain 3TSA-3, a potential probiotic for Pacific white shrimp Penaeus vannamei postlarvae isolated from commercial hatchery tanks

TL;DR

This paper presents the genome of a Bacillus strain that could act as a probiotic for shrimp larvae in aquaculture.

Contribution

The study provides a draft genome of a Bacillus velezensis strain with probiotic potential for shrimp hatcheries.

Findings

The strain 3TSA-3 lacks virulence genes and contains bacteriocin-encoding genes.

It was isolated from a hatchery with high shrimp survival despite Vibrio presence.

Abstract

We report the draft genome of Bacillus velezensis strain 3TSA-3, isolated from Pacific white shrimp Penaeus vannamei postlarvae collected from a hatchery tank with high survival despite the presence of pathogenic Vibrio. The strain possesses genes encoding bacteriocins and lacks virulence factor genes, characteristics for a potential aquaculture probiotic.

Figures

Circular genome map and Mauve alignment of the Bacillus velezensis strain 3TSA-3. (a) Multiple scaffolds (13) of the incomplete genome of 3TSA-3 showing DNA coding sequences (CDSs), tRNAs, rRNAs, regulatory, ncRNA, tmRNA, GC content, and GC skews. (b) Mauve alignment of the 3TSA-3 draft genome with the B. velezensis reference genome strain JS25R (Genbank accession number GCA_000769555.1).

Full text

ANNOUNCEMENT

Bacillus velezensis is a probiotic bacterium beneficial to adult stages of Penaeus (Litopenaeus) vannamei (1–3). B. velezensis strain 3TSA-3 was isolated from P. vannamei postlarvae collected from a commercial larviculture tank in Santa Elena province, Ecuador, on 9 March 2021. Despite being affected by vibriosis, the postlarvae exhibited high survival rates at harvest. The strain 3TSA-3 was isolated from shrimp postlarvae macerated in tryptone-casein soy broth (TSB) with 2% sodium chloride. The mixture was then cultured on tryptone-casein soy agar (TSA). After 24 h of incubation at 30°C, colonies suggestive of Bacillus were subcultured on TSA.

Genomic DNA (gDNA) of B. velezensis 3TSA-3 was extracted from an overnight culture on TSA using a DNeasy UltraClean Microbial Kit (Qiagen, The Netherlands). The quality and concentration of gDNA were assessed using a Denovix DS-11 spectrophotometer (Denovix Inc., USA).

The whole-genome library was prepared using the NEBNext ULtra DNA Prep Kit for Illumina (350 bp), and sequencing was performed by Novogene Inc. (Sacramento, USA) using the Illumina NovaSeq 6000 PE150. Whole-genome sequencing analyses were performed using the Bactopia pipeline v3.0.0 (4) with default software and parameters. The quality of the raw reads was assessed using FastQC v0.12.1. This analysis yielded a total of 8,254,750 high-quality reads with a Phred score of 36.5. De novo assembly was performed with Shovill v1.1.0. A circular genomic map (Fig. 1a) was generated using Proksee server v1.1.0 (5). 3TSA-3 was identified as B. velezensis based on the Kraken v2.0 taxonomic classification system (6) and 16S rRNA gene sequence homology using BLASTN (7). The contigs were ordered and oriented on one reference genome of B. velezensis (Strain JS25R, Genbank accession number GCA_000769555.1) using CSAR v1.1.1 (8). Average nucleotide identities with the reference genome B. velezensis strain JS25R were calculated using fastANI v1.3.3 (5), which showed a close similarity (98.05%) of strain 3TSA-3 strain to B. velezensis strain JS25R. In addition, the contigs of the circular genome were aligned to the reference genome using Mauve v20150226 (9) (Fig. 1b). Genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (10). Quality and statistics of the assembled genome sequence were performed using QUAST v5.2 (11). The assembly data revealed a total of 3,890,479 bp in 13 scaffolds of >500 bp (minimum size of 630 bp, maximum size of 2,060,921 bp, and N50 contig length of 2,060,921), with a GC content of 46.43% and an average read coverage of 312×. Homology-based gene prediction identified a total of 3,882 genes, including 3,684 protein-coding sequences (CDSs), 98 RNA genes (82 tRNA, 11 rRNA, and 5 ncRNA), and 100 pseudogenes. Several genes encoding enzymes involved in sporulation, biosynthesis of siderophores and arsenate bioremediation, biosynthesis of indole acetic acid, and biosynthesis of bacteriocins, such as the uberolysin/carnocyclin family, were detected.

Circular genome map and Mauve alignment of the Bacillus velezensis strain 3TSA-3. (a) Multiple scaffolds (13) of the incomplete genome of 3TSA-3 showing DNA coding sequences (CDSs), tRNAs, rRNAs, regulatory, ncRNA, tmRNA, GC content, and GC skews. (b) Mauve alignment of the 3TSA-3 draft genome with the B. velezensis reference genome strain JS25R (Genbank accession number GCA_000769555.1).

The discovery of effective probiotics and their genomic data are fundamental to the sustainability of the shrimp farming industry, especially considering the global threat of multi-resistance to antibiotics, which dictates the need to explore natural cost-effective alternatives such as probiotics.