Analysis of the probiotic activity of Bacillus velezensis RT-26 strain isolated from reindeer rumen by whole-genome sequencing

. The paper analyses the properties of Bacillus velezensis RT-26, a probiotic strain isolated from reindeer rumen, which has high activity towards fiber degradation, against bacterial and fungal pathogens. The analysis was performed using whole-genome sequencing of the strain using the Illumina platform. The study revealed that strain RT-26 possessed a complete set of metabolic pathways, including glycolysis, the tricarboxylic acid cycle, and the pentose phosphate pathway. 411 genes were involved in carbohydrate metabolism in the strain genome, 229 genes were related to vitamin and coenzyme metabolism, 149 genes were involved in fatty acid metabolism. The synthesis pathways of various amino acids, most B vitamins (thiamine, riboflavin, nicotiamide, vitamin B5) were identified in the genome. A complete pathway for synthesis of the dipeptide antibiotic bacilisin was detected in the strain. In addition, the strain is capable of synthesizing class A beta-lactamase. No genes responsible for the degradation of mycotoxins and xenobiotics were detected in the genome of the strain studied. A number of glycosyl hydrolase families were detected in the strain genome: GH 1, 3, 4, 5, 6, 11, 13, 16, 18, 20, 23, 26, 28, 30, 32, 43, 46, 51, 53, 68, 68, 73, 101, 109, 126.


Introduction
Probiotics are live microorganisms that, when introduced in adequate amounts, benefit the health of the host [1]. Products based on them have many advantages, but the issues of their safety must not be forgotten [2]. For example, inaccurate labeling can be caused by incorrect taxonomic identification of probiotic strains [3]. The product may be contaminated [4]. These problems can be caused by the limitations of classical microbiological identification and detection methods. In addition, the genome diversity of probiotic microorganisms is likely to affect the functionality of probiotics. So, quality assurance measures and stringent controls are needed to assess the stability of the genome of probiotic strains, especially mobile genetic elements [5]. It is also necessary to perform screening of common bacterial toxic metabolites harmful to human health, including hemolysins, D-lactic acid (D-lactate), biogenic amines involving key enzymes such as nitroreductase, amino acid decarboxylase enzyme and azoreductases [6][7][8].
Identification by sequencing methods at the strain level ensures that microbes stored in culture repository are correctly identified and handled [9]. It provides a genetic passport for monitoring both probiotic engraftment and probiotic-associated bacteremia, and provides a baseline sequence to assess whether the genetic composition of the strain changes over time.
Cumulatively, the whole-genome analysis is an expected method for accurate identification and safety assessment [10], which may address growing concerns about human health risks associated with probiotic products [11][12][13].
The aim of this study was to perform the whole-genome sequencing of the most promising probiotic strain RT-26 isolated from reindeer rumen and evaluate its biological activities related to the synthesis of biologically active, antimicrobial substances, vitamins, enzymes contributing to fiber degradation, mycotoxins, etc.

Materials and methods
The collection of bacterial strains was obtained by isolation from the rumen of reindeer (Rangifer tarandus L.). At the first stage, RT-26 strain with high activity against bacterial and fungal pathogens was selected from the collection. Whole-genome sequencing was performed for it to determine biological activities. Total DNA was isolated from strains using the Genomic DNA Purification Kit (Thermo Fisher scientific, USA). The Nextera DNA Flex Library Prep kit (Illumina) was used to prepare samples for whole-genome sequencing using the Illumina Miseq platform. The MiSeq Reagent Kit v2-150 (Illumina) was used to sequence the obtained libraries.
The strain genome was combined using Illumina paired-end reads. The reads were analyzed using the Fastqc program. Trimming was performed using the trimmomatic program with HEADCROP, SLIDINGWINDOW, LEADING, and TRAILING filters. After filtering, the read quality was checked again with Fastqc, and when the results achieved the proper quality, assembly was started with SPAdes from paired-end Illumina reads varying the k-measure length. The output data were then loaded by scaffolds to the QUAST assembly evaluation tool. A mapping program was used to map the reads. The output file sam was converted into a binary sorted by coordinate bam, which was more convenient for work and storage. For ease of processing, bam files were indexed using the samtools software package. The bwa mapper was used. After executing the protocol for our sample, we obtained one sorted bam file and its corresponding index file, which were further used for analysis. Using the database of 16S rRNA bacteria and the blastn (as well as makeblastdb) program, we determined the location of the 16S rRNA gene in the assembly. The maximum match of our assembly according to 16S gene was with the bacterium Bacillus velezensis. This result was obtained using the NODE 12 scaffold with a length of 4767 bp, an alignment length of 1545 bp, and a similarity of 99.93% relative to the 16S reference gene. The strain was classified as Bacillus velezensis.
After a more accurate identification of the strain species, we re-evaluated the assembly in QUAST together with the Bacillus velezensis reference genome taken from the NSBI database (https://www.ncbi.nlm.nih.gov/genome/?term=Bacillus%20velezensis[Organism]&cmd=D etailsSearch). The genome fractions score was 93.1%, which proved the high quality of our assembly. For further analysis and comparison with other databases, we used the Prokka program, a software tool for rapid annotation of genes and identification of coding sequences in prokaryotic genomes (conversion of nucleotide sequences to amino acids). For the analysis, we loaded the assembly into a functional annotation in the KEGG database using the KEGG Automatic Annotation Server program and the GHOSTX, bi-directional best hit (BBH) algorithm. It was also loaded in the SEED database (a platform to support genome comparison and annotation) and to the dbCAN2 meta-server (a web server for automated annotation of carbohydrate-active enzymes funded by the National Science Foundation). The CAZy database describes families of structurally related catalytic and carbohydrate-binding modules (or functional domains) of enzymes that degrade, modify, or create glycosidic bonds. CAZy data are available either by browsing sequence-based families or by browsing the contents of genomes in carbohydrate-active enzymes.

Results and discussion
The genome size of Bacillus velezensis strain RT-26 was 3,980,359 bp, with an average GC content of 46.3%. In the strain genome we identified all major groups of genes for proteins that jointly implement a particular biological process, more than 48 % of B. velezensis genes were involved in the functions of amino acid transport and metabolism; as well as transcription, translation, transport and metabolism of carbohydrate and proteins (Fig. 1). The strain had products for the functioning of a full set of metabolic pathways, including glycolysis, the tricarboxylic acid cycle, and the pentose phosphate pathway. 411 genes were involved in carbohydrate metabolism in the strain genome, 229 genes were related to vitamin and coenzyme metabolism, 149 genes were involved in fatty acid metabolism. Bacillus velezensis is currently quite actively studied as a probiotic [14][15][16][17]. Bacillus velezensis is a plant growth-promoting bacterium that can also inhibit plant pathogens. However, there are several experiments with B. velezensis used as a substitute for feed antibiotics [15,16]. It has been suggested that these bacteria can inhibit the adhesion, replication, and virulence of intestinal pathogens [18]. In addition, it can play an important role in modulating the immune system [19]. Thus, B. velezensis can be used as a probiotic agent for animals. Now the probiotic characteristics of B. velezensis are the subjects of ongoing discussions on its potential probiotic utilization for fish and animal feed industry. Various strains of B. velezensis isolated from different sources have the ability to produce antimicrobial compounds and have a beneficial effect on the gut microbiota, which could become a probiotic in the feed industry [17,20]. Pathways for the synthesis of various amino acids and most B vitamins were identified in the genome. Identified were genes of bacterial secretion systems (11 genes) annotated in the ko03070 pathway, mostly belonging to the Sec-SRP type. Bacterial secretion systems are protein complexes located in the cell membrane of bacteria that serve to secrete various proteins. Various types of ABC transporters have been identified, annotated in the ko02010 pathway (90 genes). Quorum sensing systems (54 genes) annotated in the ko02024 pathway, as well as various biofilm formation pathways have been identified.
The full synthesis pathway of the dipeptide antibiotic bacilisin (Fig. 2) and betalactamase of class A [EC:3.5.2.6] were detected in the strain. No genes responsible for the degradation of mycotoxins and xenobiotics were detected in the genome of the studied strain.

Conclusion
The study revealed that strain RT-26 possessed a complete set of metabolic pathways, including glycolysis, the tricarboxylic acid cycle, and the pentose phosphate pathway. 411 genes were involved in carbohydrate metabolism in the strain genome, 229 genes were related to vitamin and coenzyme metabolism, 149 genes were involved in fatty acid metabolism.
In the annotations of KEGG B. velezensis RT-26 metabolism, carbohydrate and amino acid metabolism, which are considered its main functions, contain a large number of genes. It was also shown that the strain has a broad capacity for fatty acid biosynthesis. We identified all the major enzymes responsible for fatty acid formation (C3-C18): FabD, FabF, FabG, FabZ, FabI (FabK, FabL), etc. The synthesis pathways of various amino acids, and most B vitamins (thiamine, riboflavin, nicotiamide, vitamin B5) were identified in the genome.
The strain was found to have a complete synthesis pathway of the dipeptide antibiotic bacilisin. Despite its simple structure, it is active against a wide range of bacteria and fungi, including Candida albicans. In addition, the strain is capable of synthesizing class A betalactamase. No genes responsible for the degradation of mycotoxins and xenobiotics were detected in the genome of the strain studied.