Whole-genome sequence of Bacillus subtilis TP111, a potential fish probiotic that prevents motile Aeromonas septicemia in Nile tilapia (Oreochromis niloticus)

TL;DR

This paper presents the whole-genome sequence of Bacillus subtilis TP111, a fish probiotic that may help prevent a deadly disease in Nile tilapia.

Contribution

The study provides the complete genome sequence of a probiotic strain effective against motile Aeromonas septicemia in fish.

Findings

The genome of Bacillus subtilis TP111 is 4,174,638 bp with 43.48% GC content.

TP111 has 4,224 potential coding sequences and 10 predicted secondary metabolite gene clusters.

The strain was isolated from the gut of a healthy Nile tilapia in Bangladesh.

Abstract

We report the genome of fish probiotic Bacillus subtilis TP111 strain isolated from the gut of a healthy Nile tilapia (Oreochromis niloticus) in Bangladesh. TP111 has a genome size of 4,174,638 bp, 43.48% guanine-cytosine, 243.0× genome coverage with 4,224 potential coding sequences, and 10 predicted secondary metabolite biosynthetic gene clusters.

Tables

TABLE 1. Presence of secondary metabolite biosynthetic gene clusters in the genome sequence of Bacillus subtilis TP111, as predicted by antiSMASH

Region	Type	From	To	Most similar known cluster		Similarity
Scaffold 2
Region 2.1	T3PKS	94,603	135,700	1-Carbapen-2-em-3-carboxylic acid	Other	16
Scaffold 4
Region 4.1Region 4.2	NRPSSactipeptide and ranthipeptide	191,707386,206	257,095409,159	SurfactinSporulation killing factor	NRP: lipopeptideRiPP: head-to-tailcyclized peptide	82100
Scaffold 5
Region 5.1Region 5.2	SactipeptideCDPS	8,743241,961	30,354262,707	Subtilosin A Pulcherriminic acid	RiPP:thiopeptideOther	100100
Scaffold 6
Region 6.1	TransAT-PKS, PKS-like, T3PKS, and NRPS	270,063	384,234	Bacillaene	Polyketide + NRP	100
Scaffold 7
Region 7.1Region 7.2	EpipeptideOther	85,458325,496	107,156361,282	Thailanstatin ABacilysin	NRP + polyketideOther	10100
Scaffold 8
Region 8.1	NRP-metallophore,NRPs	81,682	133,459	Bacillibactin	NRP	100
Scaffold 10
Region 10.1	NRPs, betalactone	47,522	125,281	Fengycin	NRP	100

Full text

ANNOUNCEMENT

Bacillus subtilis is recognized as a probiotic bacterium frequently studied for its beneficial properties and safety profile (1, 2). Here, we report the whole-genome sequence of a promising fish probiotic strain, TP111, which exhibits in vitro antimicrobial activity against fish pathogenic Aeromonas veronii and suppresses motile Aeromonas septicemia in Nile tilapia (Oreochromis niloticus) (3).

To isolate Bacillus subtilis TP111, the abdomen of a healthy Nile tilapia was cut aseptically, and the gut was taken out. One gram of homogenates of the intestinal segments was serially diluted and spread onto de Man, Rogosa, and Sharpe (MRS) agar plates and incubated at 28°C for 48 h. TP111 was picked from the growing colonies on the MRS plate (3). To isolate high-quality genomic DNA, a single colony of TP111 was inoculated in MRS broth and incubated at 28°C for 48 h. Then, the DNA was extracted using a GeneJET genomic DNA purification kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Extracted DNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). A paired-end DNA library was prepared using a Nextera XT library prep kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions (4). Genome sequencing (600 cycles) was carried out using the Illumina MiSeq benchtop sequencer (Illumina), yielding a total of 4,339,952 paired-end reads with 1,013,366,396 bases. Quality filtering was done using PRINSEQ v.0.20.3 (5), and Trimmomatic v.0.38 (6) was used for trimming low-quality sequences. The de novo assembly was conducted using SPAdes v.3.9.0 (7) followed by gene prediction and annotation using the National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline (PGAP) (https://www.ncbi.nlm.nih.gov/refseq/annotation_prok/) (8). Secondary metabolite biosynthetic gene clusters were identified using antiSMASH v.6.0 (9). Probiotic safety-associated genes were checked using ResFinder v.4.1 (10) and PathogenFinder v.1.1 (11). Default parameters were used for all software unless otherwise noted.

The de novo assembly resulted in an estimated chromosome size of 4,174,638 bp (26 contigs), with 43.48% guanine-cytosine content from 4,339,952 paired-end reads and a total of 1,013,366,396 bases sequenced, providing 243× genome coverage. The genome contains 4,224 coding sequences and 106 RNA genes as predicted by PGAP (81 tRNA, 20 rRNA, and five non-coding RNA genes). The N50 and L50 values of the assembly were 389,041 and 5, respectively. The largest and smallest contigs were 502,094 and 669 bp, respectively. No remarkable antibiotic-resistant genes except aadk, mph(K), and tet were identified in the genome using ResFinder v.4.1 (10). PathogenFinder v.1.1 (11) predicted TP111 as a non-human pathogen (matched pathogenic families: 0, matched non-pathogenic families: 275). RAST v.2.0 (12) predicted 337 subsystems and 1,705 protein-coding genes involved in the putative functional categories of a potential probiotic bacterium. antiSMASH v.6.0 (9) predicted 10 secondary metabolite biosynthetic gene clusters (Table 1). The presented genome information will assist further specific studies of this strain to exploit its probiotic potential.