Genomic Characterization of Bacillus sp. THPS1: A Hot Spring-Derived Species with Functional Features and Biotechnological Potential

Abstract

Bacillus sp. THPS1 is a novel strain isolated from a high-temperature hot spring in Thailand, exhibiting distinctive genomic features that enable adaptation to an extreme environment. This study aimed to characterize the genomic and functional attributes of Bacillus sp. THPS1 to understand its adaptation strategies and evaluate its potential for biotechnological applications. The draft genome is 5.38 Mbp with a GC content of 35.67%, encoding 5606 genes, including those linked to stress response and sporulation, which are essential for survival in high-temperature conditions. Phylogenetic analysis and average nucleotide identity (ANI) values confirmed its classification as a distinct species within the Bacillus genus. Pangenome analysis involving 19 others closely related thermophilic Bacillus species identified 1888 singleton genes associated with heat resistance, sporulation, and specialized metabolism, suggesting adaptation to nutrient-deficient, high-temperature environments. Genomic analysis revealed 12 biosynthetic gene clusters (BGCs), including those for polyketides and non-ribosomal peptides, highlighting its potential for synthesizing secondary metabolites that may facilitate its adaptation. Additionally, the presence of three Siphoviridae phage regions and 96 mobile genetic elements (MGEs) suggests significant genomic plasticity, whereas the existence of five CRISPR arrays implies an advanced defense mechanism against phage infections, contributing to genomic stability. The distinctive genomic features and functional capacities of Bacillus sp. THPS1 make it a promising candidate for biotechnological applications, particularly in the production of heat-stable enzymes and the development of resilient bioformulations.

Keywords: Bacillus sp.; aquaculture; genomic adaptation; pangenome analysis; sporulation genes.

Figure 1

Hemolytic activity on blood agar of THPS1.

Figure 2

Storage stability of Bacillus sp. THPS1 spores at different temperatures over time. The survival rates (%) of Bacillus sp. THPS1 spores were measured at 4 °C, 25 °C, 37 °C, and 45 °C over storage durations of 7, 14, 30, and 60 days.

Figure 3

(A) Phylogenetic analysis based on single-copy genes of Bacillus sp. THPS1 and related species were performed by OrthoFinder and visualized using the ITOOL web platform (https://itol.embl.de/, accessed on 20 June 2024). OrthoFinder utilizes MAFFT v7.526 [64] for the light trimming of multiple sequence alignments to optimize runtime. FastTree v2.1 [65] was then used to construct a phylogenetic tree based on single-copy genes, providing insights into the evolutionary relationships of Bacillus sp. THPS1 with other Bacillus species and closely related thermophilic strains. (B) A whole-genome phylogenomic tree was constructed using RAxML v8.0.0 [66] to infer evolutionary relationships among various Bacillus species and related genera. The tree was visualized as a circular cladogram using ITOOL (https://itol.embl.de/, accessed on 20 June 2024). Distinct color-coded branches indicated different taxonomic groupings or species clusters. Each label corresponds to a species or strain used in the analysis, highlighting their phylogenetic positioning and comparative relationships based on whole-genome data.

Figure 4

Genomic map of Bacillus sp. THPS1. From the outermost to the innermost circle, the map displays coding sequences (CDSs), rRNA and tRNA on the positive and negative strands, contig lengths (alternatively depicted in dark and light gray shades), GC content, and GC skew. The sixth and seventh circles illustrate the mobile genetic elements distribution on the positive and negative strands.

Figure 5

Gene arrangement of three phage regions identified in the genome, classified under the Siphoviridae family. (A) illustrates the gene structure and functional annotations, highlighting genes involved in lysis, integration, replication, coat, tail, portal, and other functions. (B) focuses on a specific gene cluster, showcasing replication-associated genes alongside other functional genes. (C) represents a phage region with a similar arrangement, emphasizing the presence of terminase, coat, tail, portal, and additional genes.

Figure 6

AntiSMASH analysis of the novel Bacillus genome identified 12 biosynthetic gene clusters (BGCs) of various types, each showing varying degrees of similarity to known clusters. This figure displays the BGCs identified in the analyzed genome, including terpenes, NRPS, chemical hybrids, β-lactones, lanthipeptides (class II), RiPP-like compounds, opine-like metallophores, siderophores, and trans-AT PKS clusters. Each BGC is color-coded to represent its classification, with core biosynthetic genes, regulatory elements, and transport genes highlighted in distinct colors. The horizontal axis denotes genomic coordinates, illustrating gene arrangements within each cluster.

Figure 7

Average nucleotide identity (ANI) heatmap of Bacillus sp. THPS1 compared with closely related thermophilic Bacillus species. The ANI values range from 0.00 (low similarity, shown in blue) to 1.00 (high similarity, shown in red). The dendrogram on both axes represents the clustering of species based on their ANI values.

Figure 8

Summary of KO assignments with color coding of KEGG functional categories.

Figure 9

Pangenome study conducted using Anvi’o for genomes of 19 distinct Bacillus species, including thermophilic Bacillus species. The concentric rings represent functional gene clusters, including singletons and core genes. Heatmaps indicate genome metrics such as cluster counts, GC content, and genome length, highlighting both conserved and unique features across species, with Bacillus sp. THPS1 displaying distinctive singleton gene clusters indicative of specific adaptations.

Figure 10

The rarefaction curve illustrates the quantity of newly identified genes resulting from random additions to a single genome. The power law fit equation is represented by γ = 0.519 ± 0.004.