Bioinformatic analysis of the Bacillus velezensis KR-2 genome to reveal biotechnologically important properties

Members of the genus Bacillus are actively used to create biological preparations for agriculture due to their ability to produce a wide range of biologically active molecules with antimicrobial activity, stimulating plant growth and restoring the balance of microorganisms in the digestive system of animals. This paper presents for the first time research data on the identification of a unique pathway for intracellular synthesis of the osmoprotectant glycine betaine encoded by the BetA , BetB , BetT , and BetC genes, which has not previously been found in the genus Bacillus . The peculiarity of the B. velezensis KR-2 genome associated with the synthesis of the siderophore myxochelin A, which we have identified, is probably also unique among other strains of B. velezensis , since it has not been previously described. The aim of the study was whole genome sequencing (WGS) and bioinformatic annotation of the Bacillus velezensis KR-2 genome to identify genetic determinants capable of encoding biosynthesis of various bioactive substances for agriculture. The B. velezensis KR-2 strain isolated from the rumen of a dairy cow (the collection of OOO BIOTROF+) was examined. Its antimicrobial activity was investigated using the differed antagonism assay. DNA was isolated according to standard procedures with the Genomic DNA Purification Kit (Thermo Fisher Scientific Inc., USA). A DNA library for WGS was prepared using the Nextera XT kit (Illumina, Inc., USA). Nucleotide sequences were determined using a MiSeq instrument (Illumina, Inc., USA). Paired-end reads filtered by length not less than 50 to 150 bp were assembled de novo using the SPAdes-3.11.1 genomic assembler with appropriate keys. Comparative analysis of the B. velezensis KR-2 genome with other microorganisms was performed using the NCBI databases (https://www.ncbi.nlm.nih.gov/). For phylogenetic analysis, the 16S rRNA gene sequence was transferred to the Nucleotide BLAST web service (https://blast.ncbi.nlm.nih.gov). PROKKA 1.12 (https://bioweb.pasteur.fr/packages/pack@prokka@1.12) was used to convert contig sequences into an amino acid sequences. Functional annotation of the genome was performed using the RAST 2.0 web service (https://rast.nmpdr.org). The KEGG Pathway database (http://www.genome.jp/kegg/) was used to evaluate the pool of genes associated with biotechnologically valuable properties and build metabolic maps. WGS resulted in 16 contigs with a total length of 3936398 bp, containing 46.6 % of GC; the N50 and N75 conting size was 2109194 and N75 844068 bp, respectively. The chromosome contained 3854 coding sequences (CDS) associated with the synthesis of polypeptides. Among the 464 identified metabolic subsystems, the subsystems of amino acids and their derivatives and carbohydrates were the most numerous, 431 and 416, respectively. The B. velezensis KR-2 strain has a whole range of potential properties, including the production of antimicrobial peptides, fatty acids, vitamins, siderophores, auxins, the ability to adhere, to resist toxic compounds and stress factors, to stimulate plant growth and phosphate metabolism, the motility and chemotaxis. In particular, the B. velezensis КR-2 genome contains genes ( BacA , BacB , BacG , BacF , BacD ) involved in the production of the dipeptide bacilysine, a non-ribosomal bacteriocin. This is consistent with the phenomenon of antagonism against Staphylococcus aureus , Escherichia coli , Fusarium oxysporum , and Clostridium butyricum . In the B. velezensis КR-2 genome, we have found a unique pathway for intracellular synthesis of the osmoprotectant glycine betaine (the BetA , BetB , BetT , BetC genes) not previously detected in the genus Bacillus . B. velezensis КR-2 is a putative producer of auxins, such as indole-3 ethanol ( IAR , TO ), indole-3-acetaldehyde ( IAD , AAD , AO ) and indole-3-acetonitrile ( N3 ). Several gene clusters associated with siderophore synthesis ( DhbA , DhbB , DhbC , FeuA , FeuB , FeuC , FeuD ) were also identified in the genome of B. velezensis KP-2. Our findings indicate that B. velezensis KR-2 is a bacterial resource for agriculture. The unique biosynthesis pathway for glycine betaine ( BetA , BetB , BetT , and BetC ) that we have discovered are important for the B. velezensis KP-2 adaptation to high-osmolar stress under fluctuations in water content, for example, in dried silage and upper layers of soil.