Functional Characterization, Genome Assembly, and Annotation of Geobacillus sp. G4 Isolated from a Geothermal Field in Tacna, Peru

Abstract

The genome of Geobacillus sp. G4, a thermophilic bacterium isolated from a geothermal field in Peru, was sequenced and analyzed to evaluate its taxonomic and biotechnological potential. This strain exhibits optimal growth at temperatures between 50 and 70 °C and at a pH range of 6.0-7.5. Phenotypic assays demonstrated extracellular enzymatic activities, including amylases, cellulases, pectinases, and xylanases, highlighting its potential for efficient polysaccharide degradation. The assembled genome comprises approximately 3.4 Mb with a G+C content of 52.59%, containing 3,490 genes, including coding sequences, rRNAs, and tRNAs. Functional annotation revealed genes associated with key metabolic pathways such as glycogen and trehalose biosynthesis, indicating adaptation to carbohydrate-rich environments. Phylogenetic analyses based on ANI and dDDH values identified Geobacillus thermoleovorans KCTC3570 as its closest relative, suggesting a strong evolutionary relationship. Additionally, the genome harbors gene clusters for secondary metabolites such as betalactone and fengycin, suggesting potential industrial and pharmaceutical applications, including bioremediation. The identification of antibiotic resistance genes, specifically those conferring glycopeptide resistance, underscores their relevance for antimicrobial resistance studies. The presence of enzymes like amylases and pullulanase further emphasizes its biotechnological potential, particularly in starch hydrolysis and biofuel production. Overall, this research highlights the significant potential of Geobacillus species as valuable sources of thermostable enzymes and biosynthetic pathways for industrial applications.

Keywords: bacterial thermophily; biotechnological potential; industrial enzymes; phylogenomic analysis.

Figure 1

Map of the geothermal sampling area in Calientes, Candarave, using ArcGIS version 10.5. Image of the geothermal spring sampled in this study, located in the Calientes geothermal field, Candarave, Tacna, Peru.

Figure 2

Quantitative and qualitative enzymatic activity of Geobacillus sp. G4. Extracellular enzymatic activities of Geobacillus sp. G4. (A) Formation of clear halos on agar plates indicating positive extracellular enzyme production: Amy (amylase), Pec (pectinase), Xyl (xylanase), Cel (cellulase), Prot (protease), and Lip (lipase). (B) Enzyme production profiles measured in LB liquid media over time.

Figure 3

Phylogenetic tree based on the 16S rRNA gene of 13 type strains of Geobacillus and Bacillus, extracted from the complete genome using the (barrnap) tool. Accession numbers and references are available in Supplementary Table S1. Evolutionary analyses were conducted using the RAxML tool. The strain Geobacillus sp. G4, in bold, corresponds to the 16S gene from the genome assembled in this study. The green star indicates the position of the strain isolated in this study.

Figure 4

Phylogenetic tree of 13 type strains of Geobacillus and Bacillus, constructed on the online platform TYGS available at (https://tygs.dsmz.de). The green star indicates the position of the strain isolated in this study.

Figure 5

Comparison of the genomes of G. thermoleovorans KCTC3570 and G. kaustophilus NBRC102445 with the genome of the isolated strain Geobacillus sp. G4. (A) Geobacillus sp. G4 genome (gray), G. thermoleovorans KCTC3570 genome (green), and G. kaustophilus NBRC102445 genome (purple). (B) Similarity comparison between the genomes of Geobacillus sp. G4 (gray), G. thermoleovorans KCTC3570 (inner circle), and G. kaustophilus NBRC102445 (outer circle).

Figure 6

Functional gene categories derived from the functional annotation of the G. thermoleovorans G4 genome using Prokka software and the UniProt database on the Galaxy server. Categories were classified using the eggNOG-mapper online tool with the eggNOG5 database.

Figure 7

Enzyme classes were obtained through functional annotation of the assembled genome of Geobacillus thermoleovorans G4 using Prokka software on the Galaxy server (https://usegalaxy.org/) and classified using the eggNOG-mapper online tool with the eggNOG 5 database (http://eggnog-mapper.embl.de/). The “Unknown” category refers to proteins encoded in the genome that have been annotated as possible enzymes, but do not have an assigned EC number.

Figure 8

Metabolic pathway map of starch and sucrose metabolism displaying genes and enzymes of interest annotated in the genome of G. thermoleovorans G4. Annotations were performed using the KEGG Mapper tool (www.genome.jp/kegg/mapper, accessed on 20 November 2024), identifying key enzymes involved in the metabolism of sugars and complex carbohydrates.

Figure 9

Distribution of presence/absence of stress-related genes in the genome of G. thermoleovorans G4 and in reference genomes of other Geobacillus strains. Each row represents an annotated gene, while each column corresponds to a strain. Black circles indicate the presence of the gene, and white circles indicate its absence. Genes are grouped into four main functional categories: carbon starvation, DNA repair and supercoiling, heat-shock response, and oxidative stress.