Genomic and phenotypical potential of antimicrobial activity of a bacillus strain Bacillus megaterium В-4801

The genetic determinants of bacterial strains Bacillus sp., which determine the possibility of biosynthesis of various antimicrobial compounds, are of particular scientific interest, since thanks to them these microorganisms are widely used as the basis of probiotics. An important stage in the systemic analysis of the mechanisms of probiotic action, in particular the antimicrobial activity of microorganisms, is the reconstruction of its metabolic map, that is, the collection and visualization of all potential cell processes. In this work, for the first time, the potentially inherent genetic mechanisms for the synthesis of a number of biologically active substances in the bacterial strain Bacillus megaterium are described, in particular, the possibility of synthesizing canosamine, a bacteriocin belonging to the aminoglycoside group, which can play an important role in the implementation of probiotic properties due to its pronounced antimicrobial activity. Our goal was to study the antimicrobial activity of the strain Bacillus megaterium B-4801 against pathogenic and opportunistic bacteria, as well as to search for genes associated with antimicrobial activity based on whole genome sequencing. The B. megaterium B-4801 strain deposited in the collection of OOO BIOTROF+, possesses a pronounced probiotic activity. Its antimicrobial activity against Staphylococcus aureus , Candida tropicalis , Clostridium sp., and Escherichia coli was assessed by the method of delayed antagonism using wells. A DNA library for whole genome sequencing was generated using Nextera XT kit (Illumina, Inc., USA). Nucleotide sequences were determined using a MiSeq instrument (Illumina, Inc., USA) and MiSeq Reagent Kit v3 (300-cycle) (Illumina, Inc., USA). Invalid sequences and adapters were removed using the Trimmomatic-0.38 program. Filtered in length from 50 to 150 bp pair-terminal sequences were assembled de novo using genomic assembler SPAdes-3.11.1. Functional annotation of the genome was performed with PROKKA 1.12 and RAST 2.0 programs. The pool of genes associated with antimicrobial activity was assessed and the metabolic map was constructed using the KEGG Pathway database (http://www.genome.jp/kegg/). The antagonistic activity of B. megaterium B-4801 against pathogenic and opportunistic microorganisms was revealed by cultural methods. The growth inhibition zones of the test strains ranged from 2±0.15 to 25±1.4 mm. The genome of the B. megaterium B-4801 strain is a single circular chromosome with a size of 6,113,972 bp, containing 37.5 % GC pairs. More than 45 % of B. megaterium B-4801 genes are involved in the transport and metabolism of amino acids, transcription, translation, transport and metabolism of carbohydrates and proteins. The key genetic loci that determine the synthesis of antimicrobial metabolites have been identified. The sequenced genome of the strain contains genes ( FabD , FabF , FabG , FabZ , FabI , etc.) associated with the production of proteins involved in the synthesis of aliphatic unsaturated C3-C18 carboxylic acids, in particular, butyric, nylon, caprylic, capric, lauric, myristic, palmitic, stearic, oleic. According to the information accumulated by world science, all these substances have pronounced antimicrobial properties. The whole-genome sequencing also discovered a cluster of genes ( Asm22-24 , Asm43-45 , and Asm47 ) associated with the biosynthesis of bacteriocin kanosamin, which belongs to the aminoglycoside group, and polyketide ansamycin antibiotics from the macrolide group. The established probiotic potential indicates the role of the investigated strain as a potential probiotic candidate, in particular for use in animal husbandry. The performed genomic analysis revealed new systems of operons that control the metabolic pathways for the synthesis of antimicrobial substances, which were not previously described for B. megaterium .