Although melanin is known for protecting living organisms from harmful physical and chemical factors, its synthesis is rarely observed among endospore-forming Bacillus cereus sensu lato. Here, for the first time, we reported that psychrotolerant Bacillus weihenstephanensis from Northeastern Poland can produce melanin-like pigment. We assessed physicochemical properties of the pigment and the mechanism of its synthesis in relation to B. weihenstephanensis genotypic and phenotypic characteristics. Electron paramagnetic resonance (EPR) spectroscopy displayed a stable free radical signal of the pigment from environmental isolates which are consistent with the commercial melanin. Fourier transform infrared spectroscopy (FT-IR) and physicochemical tests indicated the phenolic character of the pigment. Several biochemical tests showed that melanin-like pigment synthesis by B. weihenstephanensis was associated with laccase activity. The presence of the gene encoding laccase was confirmed by the next generation whole genome sequencing of one B. weihenstephanensis strain. Biochemical (API 20E and 50CHB tests) and genetic (Multi-locus Sequence Typing, 16S rRNA sequencing, and Pulsed-Field Gel Electrophoresis) characterization of the isolates revealed their close relation to the psychrotrophic B. weihenstephanensis DSMZ 11821 reference strain. The ability to synthesize melanin-like pigment by soil B. weihenstephanensis isolates and their psychrotrophic character seemed to be a local adaptation to a specific niche. Detailed genetic and biochemical analyses of melanin-positive environmental B. weihenstephanensis strains shed some light on the evolution and ecological adaptation of these bacteria. Moreover, our study raised new biotechnological possibilities for the use of water-soluble melanin-like pigment naturally produced by B. weihenstephanensis as an alternative to commercial non-soluble pigment.

The Bacillus cereus group, which includes entomopathogens and etiologic agents of foodborne illness or anthrax, persists in various environments. The basis of their ecological diversification remains largely undescribed. Here we present the genetic structure and phylogeny of 273 soil B. cereus s.l. isolates from diverse habitats in northeastern Poland, with samplings acquired from the last European natural forest (Białowieża National Park), the largest marshes in Europe (Biebrza National Park), and a farm. In multi-locus sequence typing (MLST), despite negative selection in seven housekeeping loci, the isolates exhibited high genetic diversity (325 alleles), mostly resulting from mutation events, and represented 148 sequencing types (131 STs new and 17 STs already described) grouped into 19 complexes corresponding with bacterial clones, and 80 singletons. Phylogenetic analyses showed that 74% of the isolates clustered with B. cereus s.l. environmental references (clade III), while only 11 and 15%, respectively, grouped with isolates of clinical origin (clade I), and B. cereus ATCC 14579 and reference B. thuringiensis (clade II). Predominantly within clade III, we found lineages adapted to low temperature (thermal ecotypes), while putative toxigenic isolates (cytK-positive) were scattered in all clades of the marsh and farm samplings. The occurrence of 92% of STs in bacilli originating from one habitat, and the description of new STs for 78% of the isolates, strongly indicate the existence of specific genotypes within the natural B. cereus s.l. populations. In contrast to the human-associated B. cereus s.l. that exhibit a significant level of similarity, the environmental isolates appear more complex. Thus we propose dividing B. cereus s.l. into two groups, the first including environmental isolates, and the second covering those that are of clinical relevance.

In this study we reconstructed the architecture of Bacillus cereus sensu lato population based on ribosomal proteins, and identified a link between the ribosomal proteins’ variants and thermal groups (thermotypes) of the bacilli. The in silico phyloproteomic analysis of 55 ribosomal proteins (34 large and 21 small subunit r-proteins) of 421 strains, representing 14 well-established or plausible B. cereus sensu lato species, revealed several ribosomal clusters (r-clusters), which in general were well correlated with the strains’ affiliation to phylogenetic/thermal groups I–VII. However, a conformity and possibly a thermal characteristic of certain phylogenetic groups, e.g. the group IV, were not supported by a distribution of the corresponding r-clusters, and consequently neither by the analysis of cold-shock proteins (CSPs) nor by a content of heat shock proteins (HSPs). Furthermore, a preference for isoleucine and serine over valine and alanine in r-proteins along with a lack of HSP16.4 were recognized in non-mesophilic thermotypes. In conclusion, we suggest that the observed divergence in ribosomal proteins may be connected with an adaptation of B. cereus sensu lato members to various thermal niches.

Bacillus cereus sensu lato (B. cereus s.l.) comprises gram-positive spore-forming bacteria producing toxins associated with foodborne diseases. Three pore-forming enterotoxins, non-haemolytic enterotoxin (Nhe), haemolysin BL (Hbl), and cytotoxin K (CytK), are considered as the primary factors in B. cereus s.l. diarrhoea. The aim of this study was to determine the potential risk of enterotoxicity among soil B. cereus s.l. isolates representing various phylogroups and originated from different geographic locations varying in climate (Burkina Faso, Kenya, Argentina, Kazakhstan, and Poland). While, the nheA- and hblA-positive isolates are present among all B. cereus s.l. populations and distributed across all phylogenetic groups, the cytK-2-positive strains predominate in geographic regions with arid hot climate (Africa) and clustered together on a phylogenetic tree mainly within mesophilic groups III and IV. The highest in vitro cytotoxicity to Caco-2 and HeLa cells was demonstrated by the strains clustered within phylogroups II and IV. Overall, our results suggest that B. cereus s.l. pathogenicity is a comprehensive process conditioned by many intracellular factors and diverse environmental conditions. IMPORTANCE This research offers a new route for a wider understanding of the dependency between pathogenicity and phylogeny of natural bacterial population, specifically within B. cereus s.l., that are widely distributed around the world and are easily transferred into food products. Our study indicates differences in the phylogenetic and geographical distribution of potential enterotoxigenic B. cereus s.l. strains. Hence these bacilli possess a risk for human health, rapid testing methods for their identification are greatly needed. Especially detection of the CytK enterotoxin should be a supporting strategy for the identification of pathogenic B. cereus s.l.

Ecological diversification of Bacillus thuringiensis soil isolates was examined to determine whether bacteria adapted to grow at low temperature and/o

Bacillus cereus s.l., Gram-positive endospore-forming bacilli, persist ubiquitously in different natural habitats and play various ecological roles. Nevertheless, although chitin is one of the most abundant polymer on Earth, the study of the ability of B. cereus s.l. to hydrolyze this polymer were limited to individual B. cereus and B. thuringiensis strains only. Thus, to fill this gap in this research we focused on (i) the linkage between the capability to chitin degradation and the phylogenetic relatedness of B. cereus s.l. strains, and (ii) the genetic background of chitinolytic properties of these bacilli. Our results showed that chitin degradation is common among the B. cereus group members, yet strains clustered into particular phylogenetic groups differ in their chitinolytic capacity. Separate clustering of chitinolytic and non-chitinolytic strains in the phylogenetic tree indicates the ecotypic structure of these isolates. Two proteins belonging to subfamily A (ChiA) and subfamily B (ChiB) of the glycoside hydrolase GH18 family exhibited simultaneous chitobiosidase and endochitinase activities, and are responsible for chitin utilization by environmental B. cereus s.l. isolates.