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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.

Melanin-Like Pigment Synthesis by Soil Bacillus weihenstephanensis Isolates from Northeastern Poland