Maria N. Romanenko

The development of eco-friendly multifunctional biopreparations based on microorganisms is a fundamental challenge in modern agriculture. Bacillus thuringiensis (Bt) strains are widely used for development of biopreparations, and a deeper understanding of their genomic makeup is required. Here, we used the Illumina platform and sequenced the genome of Bt strain 25, patented as an efficient biocontrol agent against coleopteran pests, including Leptinotarsa decemlineata, and fungal phytopathogens Fusarium oxysporum and Botrytis cinerea. The genome was 6,104,178 bp in length, with a low contamination (0.04 %) and a high completeness (99.01 %) level. Out of 6409 genes, 1030 loci were related to functionally important activities. The genome harboured 9 genes encoding well-known insecticidal toxins, such as InhA1/2, Bmp1, Spp1Aa, and Cry1Ea, 89 insecticidal minor virulence factors, and 560 putative proteases. It was predicted to be active against nematodes and lepidopteran pests. The genome contained a total of 24 biosynthetic gene clusters (BGCs), 11 of which are probably related to bactericidal activities, while 5 are known clusters, synthesising, e. g., lipopeptide fengycin and siderophores petrobactin and bacillibactin. The genome was enriched with mobile genetic elements and horizontally transferred regions. Our results elucidate the composition of genetic determinants controlling beneficial features of Bt strain 25.

Members of the genus Psychrobacillus are widely distributed across diverse environments, and novel representatives continue to be identified. Here, we describe the strain nc5.1 ^T , isolated from soil near Lake Syltran-Kel in the Kabardino-Balkarian Republic, Russia. Phylogenomic analysis indicated that P. glaciei PB01 ^T was its closest relative, placing the isolate within the genus Psychrobacillus . The average nucleotide identity (84.3%) and digital DNA–DNA hybridization (31%) values compared with PB01 ^T suggested that the isolate represents a novel species. The strain nc5.1 ^T was a Gram-negative, motile, rod-shaped bacterium forming circular, smooth, beige-brown colonies. No morphologically detectable endospores were observed under sporulation-inducing conditions. Optimal growth conditions were pH 6.5, 0.5% NaCl, and 30°C; however, a minor but reproducible viable fraction persisted after exposure to 80°C. Biochemical profiling revealed that the strain assimilated 46 carbon sources spanning carbohydrates, amino acids, and organic acids and exhibited growth in the presence of aztreonam, nalidixic acid, and potassium tellurite. The dominant cellular fatty acids were C _14:1 (52.8%), C _14:0 (16.0%), and C _16:0 (7.7%), which distinguished the isolate from P. glaciei . Genomic analysis identified biosynthetic gene clusters associated with the synthesis of iturin and a streptozotocin-like moiety, suggesting the capacity for biosynthesis of secondary metabolites with beneficial properties. MTT assays showed that culture supernatants did not reduce mammalian cell viability but, conversely, moderately stimulated the proliferation of PANC-1 cells. Together, the phenotypic, chemotaxonomic, and genomic data indicate that strain nc5.1 ^T represents a novel species with the proposed name Psychrobacillus syltrankelensis sp. nov. The type strain is nc5.1 ^T (=RCAM 07221). IMPORTANCE: The discovery of the P. syltrankelensis species extends our understanding of diversity within Bacillaceae and reveals traits that challenge the established views of the family. Based on genomic thresholds, nc5.1ᵀ clearly represents a novel species while also displaying atypical morphological features, such as Gram-negative staining and absence of sporulation. Despite the lack of morphologically detectable endospores, the strain retained viability after exposure to elevated temperatures, further emphasizing its unusual physiological profile. These characteristics underscore the limitations of morphology-based taxonomy and the necessity of genome-guided classification. Genome analysis revealed two adaptive groups within Psychrobacillus , distinguished by mutually exclusive biosynthetic clusters responsible for the synthesis of either iturin or bacillomycin D, with nc5.1ᵀ belonging to the iturin-producing lineage. The nc5.1ᵀ isolate also harbored a highly divergent streptozotocin-like locus, indicating unexplored secondary metabolism with potential biotechnological relevance. At the same time, culture supernatants unexpectedly stimulated the growth of human pancreatic carcinoma cells, underlining the complexity of functional predictions based solely on genomic data. Together, these findings highlight the importance of combining phenotypic, ecological, and genomic approaches for accurate species delineation and provide new insights into the hidden metabolic potential of Psychrobacillus .

Oxydifficidin is a natural polyketide antibiotic that has long been recognized as a ribosome-targeting agent that inhibits protein synthesis. In this paper, we describe Bacillus velezensis strain EV17 and compare its complete genome sequence with that of the previously characterized B. velezensis strain K-3618 and the difficidin biosynthetic gene cluster (BGC) combined with mass spectrometry to elucidate the production of oxydifficidin by these strains. Toeprinting and small fluorescent peptide assays showed that isolated oxydifficidin induces a generalized inhibition of translation at every step of elongation in protein biosynthesis. In previous studies, it has been demonstrated that oxydifficidin targets bL12 protein. Although spontaneous mutations conferring resistance to oxydifficidin in ribosomal protein bL12 located relatively close to the thiostrepton binding site on uL11, our data show that oxydifficidin binding does not interfere with thiostrepton, thereby refining previous findings about its putative ribosomal target. We are the first to show that this compound does not affect eukaryotic translation and has two orders of magnitude lower effect on eukaryotic cells compared to bacteria. These facts are important to further investigate its potential as a bioprotectant against phytopathogens or even as a therapeutic agent.

The transition to organic farming is one of the most desirable achievements of our time. Rational use of organic farming approaches not only enables a reduction in costs and increased yields but also limits the risks associated with the use of pesticides and chemicals. Despite the widest practical application of numerous biocontrol agents based on Bacillus strains, their metabolome, including the main active substances, often remains unknown. In order to understand the basic principles of the functioning of the Bacillus velezensis K-3618 strain, widely used in organic farming, we studied its spectrum of antimicrobial metabolites in detail. It was shown that the main antimicrobial agents of B. velezensis K-3618 are representatives of the macrolactin family. The identified macrolactin A (MLN A) and its acylated analogs 7-O-malonyl macrolactin A (mal-MLN A) and 7-O-succinyl macrolactin A (suc-MLN A) are active against Gram-positive bacterial pathogens, including multidrug-resistant strains. Among them, suc-MLN A is the most potent antimicrobial, highly active (MIC = 0.1 μg/mL) against the common human pathogen methicillin-resistant Staphylococcus aureus (MRSA). It was revealed that the primary mechanism of action of MLN A-based macrolactins is protein translation inhibition. Acylated macrolactins outperform MLN A in the prokaryotic cell-free system, displaying high efficiency in low micromolar concentrations. We observed that acylated MLN A analogs undergo pathogen-mediated biotransformation into MLN F analogs, having their antimicrobial activity reduced by two orders of magnitude. Hence, both acylation of MLNs and stabilization of the MLN A core are essential for the creation of new synthetic MLNs with improved antimicrobial activity and stability. However, we speculate that these degradability modes are of prime importance for bacterial ecology, and they are highly conserved in Bacillus species from various ecological niches.

The Altai Republic remains a geographic region with an uncovered microbial diversity hiding yet undescribed potential species. Here, we describe the strain al37.1^T from the Altai soil. It showed genomic similarity with the Bacillus mycoides strain DSM 2048^T. However, the in silico DNA–DNA hybridization (DDH) was 61.6%, which satisfies the accepted threshold for delineating species. The isolate formed circular, smooth colonies, in contrast to the rhizoidal morphology typical of B. mycoides. The strain showed optimal growth under the following conditions: pH 6.5, NaCl concentration 0.5% w/v, and +30 °C. The major fraction of fatty acids was composed of C_16:0 (34.77%), C_18:1 (15.20%), C_14:0 (9.06%), and C_18:0 (7.88%), which were sufficiently lower in DSM 2048^T (C_16:0–15.6%, C_14:0–3.7%). In contrast to DSM 2048^T, al37.1^T utilized glycerol, D-mannose, and D-galactose, while being unable to assimilate D-sorbitol, D-melibiose, and D-raffinose. The strain contains biosynthetic gene clusters (BGCs) associated with the production of fengycin, bacillibactin, petrobactin, and paeninodin, as well as loci coding for insecticidal factors, such as Spp1Aa, chitinases, Bmp1, and InhA1/InhA2. The comparative analysis with the 300 closest genomes demonstrated that these BGCs and Spp1Aa could be considered core for the whole group. Most of the strains, coupled with al37.1^T, contained full nheABC and hblABC operons orchestrating the synthesis of enteric toxins. We observed a cytotoxic effect (≈19 and 22% reduction in viability) of the strain on the PANC-1 cell line. Given the unique morphological features and genome-derived data, we propose a new species, B. altaicus, represented by the type strain al37.1^T.

Oxydifficidin is a natural polyketide antibiotic that is a long-known ribosome-targeting antibiotic that inhibits protein synthesis. In this paper, we describe Bacillus velezensis strain EV17 and compare its complete genome sequence with that of the previously characterized strain K-3618 and the difficidin biosynthetic gene cluster (BGC) combined with mass spectrometry, to elucidate the production of oxydifficidin by these strains. Isolated oxydifficidin was determined to increase generalized inhibition of translation at each step of protein biosynthesis using toeprinting and small fluorescent peptide assays. In previous studies, it has been demonstrated that oxydifficidin targets L7/L12 protein. Although spontaneous mutations in ribosomal protein L7/L12, located relatively close to the thiostrepton binding site on uL11, confer resistance to oxydifficidin, our data show that oxydifficidin binding does not interfere with thiostrepton, thereby refining previous findings about its putative ribosomal target. We show that this compound does not affect eukaryotic translation and has no toxic effect on eukaryotic cells. These facts are important to further investigate its potential as a bioprotectant against phytopathogens or even as a therapeutic activity.

The transition to organic farming is one of the most desirable achievements of our time. Rational use of modern organic farming approaches not only enables the reduction of costs and increased yields but also limits the risks associated with the use of pesticides and chemicals. Despite the widest practical application of numerous biocontrol agents based on Bacillus strains, their metabolome, including the main active substances, often remains unknown. In order to understand the basic principles of the functioning of the Bacillus velezensis K-3618 strain, widely used in organic farming, we studied its spectrum of antimicrobial metabolites in detail. It was shown that the main antimicrobial agents of the B. velezensis K-3618 are representatives of the macrolactin family. Macrolactins are a group of 24-membered lactones, including macrolactin A (MLN A) and its acylated analogs 7-O-malonyl macrolactin A (mal-MLN A) and 7-O-succinyl macrolactin A (suc-MLN A), mostly active against Gram-positive bacterial pathogens, including multidrug-resistant strains. Suc-MLN A was the most potent antimicrobial, highly active (MIC = 0.1 μg/mL) against the common human pathogen methicillin-resistant Staphylococcus aureus (MRSA). It was shown that the primary mechanism of action of MLN A-based macrolactins is protein translation inhibition. Acylated macrolactins outperform MLN A in the prokaryotic cell-free system, displaying high efficiency in low micromolar concentrations. We observed that acylated MLN A analogs undergo pathogen-mediated biotransformation to MLN F analogs, having the antimicrobial activity reduced by two orders of magnitude. Hence, both acylation of MLNs and stabilization of the MLN A core are essential for the creation of new synthetic MLNs with improved antimicrobial activity and stability. While the counteraction against the observed biotransformations is essential to create new macrolactin-based antimicrobials for clinical practice, we speculate that these degradability modes are essential for bacterial ecology, and they are highly conserved in many Bacillus species from various ecological niches.

The dispersal of plant pathogens is a threat to the global economy and food industry which necessitates the need to discover efficient biocontrol agents such as bacteria, fungi, etc., inhibiting them. Here, we describe the Bacillus mycoides strain b12.3 isolated from the soil of Olkhon Island in Lake Baikal, Russia. By applying the co-cultivation technique, we found that the strain inhibits the growth of plant pathogens, such as the bacteria Xanthomonas campestris, Clavibacter michiganensis, and Pectobacterium atrospecticum, as well as the fungus Alternaria solani. To elucidate the genomic fundament explaining these activities, we leveraged next-generation whole-genome sequencing and obtained a high-quality assembly based on short reads. The isolate bore seven known BGCs (biosynthetic gene clusters), including those responsible for producing bacillibactin, fengycin, and petrobactin. Moreover, the genome contained insecticidal genes encoding for App4Aa1, Tpp78Ba1, and Spp1Aa1 toxins, thus implicating possible pesticidal potential. We compared the genome with the 50 closest assemblies and found that b12.3 is enriched with BGCs. The genomic analysis also revealed that genomic architecture corresponds to the experimentally observed activity spectrum implying that the combination of produced secondary metabolites delineates the range of inhibited phytopathogens Therefore, this study deepens our knowledge of the biology and ecology of B. mycoides residing in the Lake Baikal region.

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.

Soil is a classical source of beneficial microorganisms. Soil microbiomes provided the overwhelming majority of antibiotic-producing strains, biocontrol agents, probiotics, and plant-protecting bacteria. The functionality of strains isolated from various soil samples is predetermined by the biosynthetic potential encoded in their genomes. Here, we describe a novel Peribacillus simplex d27.3 strain isolated from the soil sample of a pine forest in the Republic of Dagestan, Russia. P. simplex d27.3 displayed antibiotic activity against gram-positive bacteria and fungi while being inactive against the model hypersensitive gram-negative strain E. coli ΔlptD. Metabolomic analysis revealed that antimicrobial activity was partially mediated by the fengycin lipopetides (C-16 fengycin A, C-17 fengycin A, and C-16 fengycin B). In addition, the P. simplex d27.3 strain was found to produce other hydrophilic and more hydrophobic antimicrobials yet to be described. Thus, the P. simplex d27.3 strain is a producer of useful antimicrobial compounds with a high potential for application in biotechnology and agriculture.