Whole-Genome Sequencing of Peribacillus frigoritolerans Strain d21.2 Isolated in the Republic of Dagestan, Russia

Abstract

Pesticide-free agriculture is a fundamental pillar of environmentally friendly agriculture. To this end, there is an active search for new bacterial strains capable of synthesizing secondary metabolites and toxins that protect crops from pathogens and pests. In this study, we isolated a novel strain d21.2 of Peribacillus frigoritolerans from a soil sample collected in the Republic of Dagestan, Russia. Leveraging several bioinformatic approaches on Illumina-based whole-genome assembly, we revealed that the strain harbors certain insecticidal loci (coding for putative homologs of Bmp and Vpa) and also contains multiple BGCs (biosynthetic gene clusters), including paeninodin, koranimine, schizokinen, and fengycin. In total, 21 BGCs were predicted as synthesizing metabolites with bactericidal and/or fungicidal effects. Importantly, by applying a re-scaffolding pipeline, we managed to robustly predict MGEs (mobile genetic elements) associated with BGCs, implying high genetic plasticity. In addition, the d21.2's genome was free from genes encoding for enteric toxins, implying its safety in use. A comparison with available genomes of the Peribacillus frigoritolerans strain revealed that the strain described here contains more functionally important loci than other members of the species. Therefore, strain d21.2 holds potential for use in agriculture due to the probable manifestation of bactericidal, fungicidal, growth-stimulating, and other useful properties. The assembled genome is available in the NCBI GeneBank under ASM4106054v1.

Keywords: Illumina sequencing; Peribacillus frigoritolerans; bactericidal activity; biosynthetic gene clusters; draft genome; fungicidal properties; mobile genetic elements.

Figure 1

Soil sampling location.

Figure 2

Protocol for sampling using the “envelope” method. Green circles indicate sampling areas.

Figure 3

The morphology of the d21.2 strain colonies after 24 h of cultivation on LB medium (a) and (b) transition from vegetative to sporulating culture after 48 h of cultivation on CCY medium stained with Coomassie brilliant blue (1000× magnification, scale bar corresponding 10 µm). The black solid arrow shows the vegetative cell, the black dotted arrow—the spore inside the cell, and the red solid arrow—the spore.

Figure 4

Overall and feature-wise genomic characterization of P. frigoritolerans d21.2’s assembly corrected according to the closest reference (GCF_030122925.1). (a). Comparison between raw and refined assemblies in terms of basic properties (number of contigs and CDS, quality metrics, etc.), and the abundance of certain loci, i.e., genes coding for insecticidal toxins, and BGCs as well as MGEs, namely, prophages, ISs, and GIs. The colored parts of the bar plots represent the ratio between the numeric values of the plotted feature. The exact figures are provided in Table S2. (b). The whole genome map of the corrected assembly. The inner circles demonstrate the GC distribution, while the outer tracks show the position of distinct loci colorized according to their types. (c). Relationships between the locations of BGCs and MGEs on the refined genome. The regions are considered overlapped in case the respective coordinates of compared loci intersect. The inspected coordinates are present in Table S3.

Figure 5

Comparative genomic analysis of the strain d21.2 with the closest 50 genomes of P. frigoritolerans isolates exhibiting the highest ANI values in terms of insecticidal loci and clusters responsible for the synthesis of secondary metabolites. (a) The composition of toxins predicted to be produced by the analyzed strains. The columns are organized according to the phylogeny reconstructed on the sequences of concatenated core genes. The adjacent tree is placed above. The size of the dots is proportional to the number of paralogs of a certain moiety. The shape indicates the method that detected a toxin sequence, namely, HMM (Hidden Markov Models) and BLAST with the color corresponding to the E-value and the similarity to the known homolog, respectively. (b) The total fraction of toxins identified in P. frigoritolerans genomes. The blocks are colorized according to the toxin group. (c) Known BGCs found in the genomic dataset. The order of columns corresponds to strains as in the toxins-based heatmap above. The circles are colorized according to the mean similarity with core genes of the known cluster. (d) The composition of BGCs detected with antiSMASH and DeepBGC in P. frigoritolerans strains. The color represents the classified BGC, i.e., predicted biological activity. If no activity was predicted, the BGC is marked as a known cluster (in case it exists) or other (lacking known hits and putative activities). (e) The frequency of known clusters found in the dataset. The color reflects the name of the product. (f). The overall distribution of BGCs present in P. frigoritolerans genomes. The blocks are colored according to the activity-wise classification mentioned above.