Analysis of biosynthetic gene clusters of Bacillus velezensis ONU 553 in silico

Key words: Bacillus velezensis, biosynthetic gene clusters (BGC), bioinformatic analysis

Bacillus velezensis belongs to the operational group of B. amyloliquefaciens (OGBa), it is found in different environments, but first of all in soils and marine bottom sediments [16].

The practical interest to the members of Bacillus velezensis is induced due to their ability to the major production of secondary metabolites, rapid growth of strains, as well as their significant resistance to adverse environmental conditions [2; 21; 25]. The mentioned advantages characterize B. velezensis as a promising producer of biologically active compounds and an object of pharmaceutical biotechnology [27].

However, the morphology and physiological properties of the strains show significant heterogeneity depending on the primary genesis of the isolate. Only certain strains have a complete set of signs and properties that define them as leaders and promising producers of industrial biotechnology. In this regard, the study of complete genomes provides a comprehensive characterization of the genes of the target clusters and reveals the genetically determined potential of the obtained strains. Within the framework of the actual research, we performed a bioinformatics study of the genome of B. velezensis ONU 553 in order to substantiate its synthetic potential.

Materials and methods

biosynthetic gene bacillus velezensis

The strain Bacillus velezensis ONU 553 was isolated from the bottom sediments of the Black Sea and deposited in the Collection of marine and practically useful microorganisms of Odesa National University named after I. I. Mechnikov, and the genomic sequence was deposited in the GenBank (www.ncbi.nlm.nih.gov) under inventory number CP043416.

The annotation of the genome was conducted using he PATRIC server (Pa- thosystems Resource Integration Center) of the BV-BRC network resource (Bacterial and Viral Bioinformatics Resource Center - www.bv-brc.org) [23].

The TYGS service (Type (Strain) Genome Server - https://tygs.dsmz.de/) was used to reconstruct the phylogenetic tree based on complete genomic sequences [15]. The correctness of the topology of the tree was based on the average values of branch support and b statistics [11]. Average nucleotide identity (OrthoANI) was calculated using the tool ANI (Average Nucleotide Identity Tool) calculator on the EzBioCloud platform (www.ezbiocloud.net) [13; 26].

Mobile genetic elements, including CpG islands and prophages of B. velezensis ONU 553, were identified using Islandviewer 4 (https://www.pathogenomics. sfu.ca/islandviewer/browse/) and PHASTER (PHAge Search Tool Enhanced Release - https://phaster.ca/) [1].

Bioinformatics tools antiSMASH ("antibiotics and secondary metabolite analysis shell" (https://antismash.secondarymetabolites.org/) version 7.0.0 [4] and BAGEL4 (http://bagel.molgenrug.nl/) [22]) were used to search for gene clusters (BGC) involved in the synthesis of polyketides and bacteriocins.

The MEGAX program (Molecular Evolutionary Genetics Analysis version X) [12], CLUSTAL W multiple alignment and the Neighbor-Joining method were used to reconstruct the phylogenetic tree of bacteriocins. The ITOL server (https:// itol.embl.de/) was used to visualize the obtained tree.

To compare existing gene clusters in the genomes of the most closely related representatives of Bacillus, we used the "pheatmap" package implemented in the R 4.2.2 program.

Results and discussion

General genome annotation of Bacillus velezensis ONU 553

Genomic analysis of Bacillus velezensis ONU 553 using the PATRIC server (table 1) determined that the genome consisted of a circular chromosome that contains 3,934,563 base pairs (bp) and has an average content GC content of 46.69%.

In general, the presence presence of 3953 protein-coding DNA sequences (CDS) that were distributed along both strands was determined. Also, 86 tRNAs and 27 rRNAs, as well as 4 possible phages [20] and 11 CpG islands were detected (table 2, fig. 1).

Taxonomic status of Bacillus velezensis ONU 553

Clarification of the systematic position of B. velezensis ONU 553 carried out using a complex of methods, including a search using BLAST+ (version 2.9) and relevant databases (ref_prok_rep_genomes), calculations of the identity of nucleotide sequences (OrthoANI) and dDDH of the complete genome. In general, the obtained results were agreed among themselves. According to the results of phy- logenetic clustering which was performed using the TYGS service, B. velezensis ONU 553 is a close relative of B. amyloliquefaciens FZB42 (CP000560.2) (fig. 2).

Table 2

Characterization of CpG islands in the genome of Bacillus velezensis ONU 553

According to the recommended threshold values of OrthoANI [13], the obtained results confirm the relatedness of the genomes of Bacillus velezensis ONU 553 and Bacillus amyloliquefaciens FZB42 (98.87%)

Gene clusters of secondary metabolites in the genome of Bacillus velezensis ONU 553

Gene clusters associated with the biosynthesis of secondary metabolites in the genome of B. velezensis ONU 553 were determined by using the antiSMASH tool [14]. Among 12 clusters, four non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), hybrid clusters of NRPS/PKS and terpenes were detected (Table 3). The generalized results of the analysis of the organization of the determined clusters of genes associated with biosynthesis are shown in figure 3.

Cluster 5 encodes an atypical polyketide-nonribosomal peptide synthase, which could be potentially related to the synthesis of a new antibiotic of the bacil- laene series. The last is a polyene antibiotic first discovered in B. subtilis and largely uncharacterized due to its notorious instability [5]. It is known that the effect of bacillaene is to disrupt protein synthesis, the mechanism of which is still unclear. It is noteworthy that recently an almost identical cluster of genes was described for B. amyloliquefaciens FZB42 [24] (Table 3, Fig. 3).

Fig. 1. The result of searching for CpG- islands in the genome of Bacillus velezensis ONU 553 using the Islandviewer 4 server

Fig. 2. Phylogenetic position of Bacillus velezensis ONU 553

Table З

Gene clusters associated with the synthesis of secondary metabolites (BGC) in Bacillus velezensis ONU 553 (according to the results of analysis in antiSMASH)