Genomic insights into the taxonomic status and bioactive gene cluster profiling of Bacillus velezensis RVMD2 isolated from desert rock varnish in Ma'an, Jordan

Abstract

Extreme environments like arid and semi-arid deserts harbor unique microbial diversity, offering rich sources of specialized microbial metabolites. This study explores Bacillus velezensis RVMD2, a strain isolated from rock varnish in the Ma'an Desert, Jordan. The genome was sequenced using the Illumina NextSeq 2000 platform, resulting in a 4,212,579 bp assembly with a GC content of 45.94%. The assembled genome comprises 112 contigs and encodes 4,250 proteins, 77 tRNA genes, and 4 rRNA genes. Phylogenetic analysis of the 16S rRNA gene indicated a 99.84% similarity to previously identified B. velezensis strains. Whole-genome phylogeny using EzBiome, MiGA, and TYGS confirmed its classification as B. velezensis. Functional annotation identified genes involved in carbohydrate metabolism, including 324 carbohydrate-active enzyme (CAZyme) genes, stress response, and secondary metabolite biosynthesis. The genome also contains 50 genes associated with heavy metal resistance and plant growth promotion. Analysis using AntiSMASH identified 12 biosynthetic gene clusters involved in the production of secondary metabolites, including fengycin, surfactin, polyketides, terpenes, and bacteriocins. Notably, several clusters did not match any known sequences, suggesting the presence of potentially novel antimicrobial compounds. The genomic features of RVMD2 highlight its adaptability to extreme environments and its potential for biotechnological applications, including bioremediation and the discovery of novel bioactive metabolites.

Fig 1. Phylogenetic tree based on the 16S rRNA gene sequence of strain RVMD2, generated with Protologger, showcasing its closest relatives from the SILVA Living Tree Project.

The tree includes species with validly published names as listed by DSMZ nomenclature. The visualization was further refined using the Interactive Tree of Life (iTOL) v5 tool [23]. The scale bar represents a genetic distance of 0.01 substitutions per site.

Fig 2. Circular genome map of Bacillus velezensis strain RVMD2 generated through Proksee web-based tool.

The tracks display open reading frames (ORFs) on forward and reverse strands, backbone contigs, GC content, and GC skew (green/purple graph), illustrating the detailed genomic structure. The outermost ring shows ORFs on the positive strand, followed by backbone contigs. The third ring represents GC content with black peaks for higher GC areas, and the fourth ring shows GC skew, with green for positive (GC Skew+) and purple for negative (GC Skew-). The innermost ring shows ORFs on the negative strand.

Fig 3. Functional Characterization of the Bacillus velezensis RVMD2 Genome.

(A) Sankey diagram illustrating the distribution of genes into subsystems classified by biological functions. (B) Summary of protein annotations detailing cross-genus and genus-specific family assignments, pathway connections, GO categorizations, EC number designations, and other functional annotations, along with hypothetical proteins—all as annotated by BV-BRC [44].

Fig 4. Phylogenetic analysis of Bacillus species, including B. velezensis RVMD2, based on whole-genome comparisons using the Genome Blast Distance Phylogeny (GBDP) method, conducted via the TYGS server.

The tree was constructed with FastME 2.1.6.1 [55] using GBDP distances derived from genome sequences, with branch lengths reflecting the GBDP distance metric (d5). Numbers above branches represent GBDP pseudo-bootstrap support values exceeding 60% from 100 replications, with an average branch support of 75.6%. The tree was midpoint-rooted [56]. Leaf labels indicate species and subspecies clusters based on digital DNA-DNA hybridization (dDDH), genomic G+C content, delta values, genome length, and protein count. Lower delta values correspond to higher accuracy in tree-likeness [57].

Fig 5. ANI values (FastANI) heatmap of B. velezensis RVMD2 and its closest Bacillus strains according to Protologger, with hierarchical clustering.

The color gradient from red to blue indicates the degree of genomic similarity, with red representing higher similarity (closer species) and blue indicating lower similarity.

Fig 6. Distribution of carbohydrate-active enzymes (CAZymes) identified in the genome of Bacillus velezensis RVMD2, as determined through analysis using Protologger v0.99 [

22].

Fig 7. Genomic islands and prophage regions in B. velezensis RVMD2.

(A) Genomic islands in strain RVMD2 were predicted using IslandViewer 4. The circular plot illustrates these genomic islands, with the outer circle representing the genomic scale in Mbps. The genomic islands are depicted in different colors: red for integrated detection, blue for IslandPath-DIMOB, orange for SIGI-HMM, and green for IslandPick. (B)The linear genome viewer from PHASTEST [34] shows prophage regions in the strain RVMD2 genome, highlighting bacterial genes, attachment sites, phage-related proteins, and GC content. Key regions are annotated with hypothetical proteins, integrases, tail proteins, and other relevant phage-related proteins.

Fig 8. Secondary metabolites and gene clusters in B. velezensis RVMD2 genome.

(A) Structures of secondary metabolites and gene clusters identified in the B. velezensis RVMD2 genome. (B) Schematic representation of gene clusters with diverse functions highlighted in different colors, as shown in the legend. Predictions were made using antiSMASH software version 4.0.

Fig 9. Comprehensive pangenome analysis of B. velezensis strains, including detailed profile of strain RVMD2 and its closest 14 strains.

(A) Pangenome analysis of 615 B. velezensis strains, including strain RVMD2. The rarefaction curves compare pan-gene clusters (blue) and core gene clusters (orange), generated using IPGA. (B) Pangenome profile of B. velezensis strain RVMD2 and its closest 14 strains. COG annotation shows core and unique genes among these strains. The phylogenetic tree reflects the number of shared gene clusters.

Fig 10. Detailed visualization of similarity analysis between the assembled genome of Bacillus velezensis strain RVMD2 and its three closest Bacillus velezensis strains (Q12, CFSAN034340, and ASM-2), using the Proksee tool to determine the average nucleotide identity (FastANI).

Each red line represents a reciprocal map between the genomes, highlighting their evolutionarily conserved sequences. The red lines denote conserved genomic regions, with the color intensity indicating a high value of ANI. This whole genome alignment and comparison illustrate the genomic conservation and divergence between RVMD2 and its closest strains.