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

Abstract

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.

Fig 1. Melanin-like pigment production in Luria-Bertoni broth by B. weihenstephanensis.

B. cereus ATCC 10987 was used as the negative control.

Fig 2. The phenotypic similarity and phylogeny among B. weihenstephanensis producing melanin-like pigment and reference strains.

Comparisons between strains based on biochemical API 50CH and API 20E tests were made using simple matching coefficient and clustered with the UPGMA algorithm (A). Phylogenetic trees were constructed based on 16S rDNA gene (B) and seven concatenated housekeeping loci (MLST) (C) using the Neighbor-Joining (NJ) method implemented in MEGA6 software, where branch quality was evaluated using 1,000 replicates bootstraps.

Fig 3. PFGE fingerprints of melanin-positive B. weihenstephanensis isolates and reference strains.

Genomic DNA was digested using NotI. M1, PFG Lambda Ladders; M2, PFG Yeast chromosomes. The values on the left and right are molecular weight marker in kb.

Fig 4. EPR (A) and FT-IR (B) spectra of commercial melanin and pigment obtained from B. weihenstephanensis isolates.

Fig 5. Putative pathways of melanin synthesis and the inhibition tests for pigment production by B. weihenstephanensis isolates.

TYR, tyrosinase (EC 1.14.18.1); L-DOPA, L-3,4-dihydroxyphenylalanine; DOPA-Q, Dopa-quinone; LAC, laccase (EC 1.10.3.2); TAT, tyrosine aminotransferase (EC 2.6.1.5); ArAT, aromatic amino acid aminotransferase (EC 2.6.1.57); 4-HPPA, 4-hydroxyphenylpyruvic acid; 4-HPPD, 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27); HGA, homogentisic acid; PKS-1, polyketide synthase type I; 1,3,6,8 THN, 1,3,6,8-tetrahydroxynaphthalene; THR, Hydroxynaphthalene reductase; SCD, scytalone dehydratase (EC 4.2.1.94); 1,3,8 THN, 1,3,8-trihydroxynaphthalene; 1,8 DHN, 1,8-dihydroxynaphthalene; DHN-Melanin, dihydroxynaphthalene melanin; RppA, polyketide synthase type III; P450-mel, cytochrome P-450 enzyme; HPQ, 1,4,6,7,9,12-hexahydroxyperylene-3,10-quinone; HPQ-Melanin, hexahydroxyperylenequinone melanin; C, control. The dotted line represents the non-enzymatic reactions (oxidation and/or polymerization). Abbreviations of inhibited enzymes are marked in red color.