Biological Characterization and Whole-Genome Analysis of Bacillus subtilis MG-1 Isolated from Mink Fecal Samples

Abstract

Bacillus subtilis is an important part of the gut microbiota and a commonly used probiotic. In the present study, to assess the biological characteristics and probiotic properties of B. subtilis derived from mink, we isolated B. subtilis MG-1 isolate from mink fecal samples, characterized its biological characteristics, optimized the hydrolysis of casein by its crude extract, and comprehensively analyzed its potential as a probiotic in combination with whole-genome sequencing. Biological characteristics indicate that, under low-pH conditions (pH 2), B. subtilis MG-1 can still maintain a survival rate of 64.75%; under the conditions of intestinal fluid, gastric acid, and a temperature of 70 °C, the survival rate was increased by 3, 1.15 and 1.17 times compared with the control group, respectively. This shows that it can tolerate severe environments. The results of hydrolyzed casein in vitro showed that the crude bacterial extract of isolate MG-1 exhibited casein hydrolyzing activity (21.56 U/mL); the enzyme activity increased to 32.04 U/mL under optimized reaction conditions. The complete genome sequencing of B. subtilis MG-1 was performed using the PacBio third-generation sequencing platform. Gene annotation analysis results revealed that B. subtilis MG-1 was enriched in several Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways, and most genes were related to Brite hierarchy pathways (1485-35.31%) and metabolism pathways (1395-33.17%). The egg-NOG annotation revealed that most genes were related to energy production and conversion (185-4.10%), amino acid transport and metabolism (288-6.38%), carbohydrate transport and metabolism (269-5.96%), transcription (294-6.52%), and cell wall/membrane/envelope biogenesis (231-5.12%). Gene Ontology (GO) annotation elucidated that most genes were related to biological processes (8230-45.62%), cellular processes (3582-19.86%), and molecular processes (6228-34.52%). Moreover, the genome of B. subtilis MG-1 was predicted to possess 77 transporter-related genes. This study demonstrates that B. subtilis MG-1 has potential for use as a probiotic, and further studies should be performed to develop it as a probiotic additive in animal feed to promote animal health.

Keywords: Bacillus subtilis; biological characteristics; mink; probiotics; whole genome.

Figure 1

Colony morphology of the Bacillus subtilis isolate.

Figure 2

(a) Growth curve of B. subtilis MG-1. The abscissa is the incubation time, and the ordinate is the OD₆₀₀ value. (b) Acid tolerance analysis of B. subtilis MG-1. The abscissa is three different gradients of pH 2, 3, and 4, and the ordinate is the OD₆₀₀ value. (c) Intestinal fluid tolerance analysis of B. subtilis MG-1. The abscissa is the control group (black) and the experimental group (gray), and the ordinate is the OD₆₀₀ value. In all figures, black represents the control group, and gray represents the experimental group.

Figure 3

(a) Gastric acid tolerance analysis of B. subtilis MG-1. The abscissa is the incubation time, and the ordinate is the OD₆₀₀ value. (b) Heat tolerance analysis of B. subtilis MG-1. The abscissa is three different temperature gradients of 50, 60, and 70 °C, and the ordinate is the OD₆₀₀ value. In all figures, black represents the control group and gray represents the experimental group.

Figure 4

(a) Standard curve of L-tyrosine. The abscissa is the tyrosine concentration, and the ordinate is the OD₆₀₀ value. (b) Effect of pH on enzyme activity of B. subtilis MG-1 extract. The abscissa is different gradients of pH, and the ordinate is the enzyme activity. (c) Effect of reaction temperature on enzyme activity of B. subtilis MG-1 extract. The abscissa is different gradients of reaction temperature, and the ordinate is the enzyme activity. (d) Effect of temperature on enzyme activity of B. subtilis MG-1. The abscissa is different gradients of temperature, and the ordinate is the enzyme activity.

Figure 4

(a) Standard curve of L-tyrosine. The abscissa is the tyrosine concentration, and the ordinate is the OD₆₀₀ value. (b) Effect of pH on enzyme activity of B. subtilis MG-1 extract. The abscissa is different gradients of pH, and the ordinate is the enzyme activity. (c) Effect of reaction temperature on enzyme activity of B. subtilis MG-1 extract. The abscissa is different gradients of reaction temperature, and the ordinate is the enzyme activity. (d) Effect of temperature on enzyme activity of B. subtilis MG-1. The abscissa is different gradients of temperature, and the ordinate is the enzyme activity.

Figure 5

Chromosome of B. subtilis MG-1. From the inside out, the first circle represents the scale. The second circle represents GC Skew. The third circle represents GC content. The fourth and seventh circles represent the COG to which each CDS belongs. The fifth and sixth circles represent the positions of CDS, tRNA, and rRNA on the genome.

Figure 6

The genomic island predictions for B. subtilis MG-1. The red color represents the genomic island prediction results after the integration of the three methods (predicted with at least one method). Blue represents the prediction result of Island Path-DIMOB. Yellow represents the prediction results of SIGI-HMM. Green represents the prediction result of Island Pick. Light blue represents the Islander predictions.

Figure 7

Prediction of carbohydrate-active enzymes (CAZys) in B. subtilis MG-1 genome. The abscissa is the function class, and the ordinate is the number of matched genes.

Figure 8

egg-NOG annotation of genome of B. subtilis MG-1. Different letters on the abscissa represent different function classes, and the ordinate is the number of matched genes.

Figure 9

Kyoto Encyclopedia for Genes and Genomes (KEGG) pathway enrichment analysis of the genome of B. subtilis MG-1. The abscissa is the number of matched genes, and the ordinate is the function class.

Figure 10

Gene Ontology (GO) annotation of the genome of B. subtilis MG-1. The abscissa is the number of matched genes, and the ordinate is the function class.

Figure 11

Transporter Classification Database (TCDB) annotation of the B. subtilis genome of MG-1. The abscissa represents TCDB first-order classification type, and the ordinate represents the number of genes on the annotation.