DNA plugs were prepared by mixing bacterial suspension with 2% LMP agarose at 1:1 ratio and placed into slots of a plug mold. After solidification, plugs were treated according to Gaviria Rivera and Priest, DNA was digested with 30 U of NotI restrictase and electrophoresed in the CHEF-MAPPER System following the protocol of Swiecicka et al.. PFG Lambda Ladders and PFG Yeast chromosomes from New England BioLabs were used as markers. Gels were stained with ethidium bromide solution and visualized in ChemiDOC XRS System. Extraction, A-83-01 biological activity purification, and chemical characteristics of melanin-like pigment The pigment was isolated from the four most productive strains: JAS 39/1, JAS 81/4, JAS 83/3, and JAS 86/1. Isolates were inoculated into 200 ml of nutrient broth and incubated at 30C on rotary shaker at 180 rpm for 96 h until the liquid medium became dark-brown. When the cell mass was removed, the supernatant was acidified by lowering a pH to 2.0 using 1N HCl and incubated at room temperature for one week, followed by boiling of the suspension for 1 h and centrifuging at 14,000 x g for 15 min. The pigment pellet was washed with ethanol as described by Sajjan et al.. The modified method of Fava et al. was used to perform chemical analyses of the extracted melanin-like pigment. The pigment solubility was checked in deionized water, 1 N NaOH, 1 N HCl, acetone, benzene, chloroform, ethanol, and phenol. In addition, the reactions of melanin with 30% hydrogen peroxide, 1% iron chloride, and 5% sodium hydrosulfite, were tested. Synthetic melanin was used as a reference. Electron paramagnetic resonance measurement The electron paramagnetic resonance measurement for the tested melanin-like pigments and synthetic melanin were performed with the X-band 
EPR spectrometer and the Rapid Scan Unit. Each sample was placed in a thin-walled glass tube free of paramagnetic impurities. Microwave frequency was obtained by the MCM101 Recorder at magnetic modulation of 100 kHz. The total microwave power of the klystron was 70 mW. The numerical acquisitions of the first-derivative EPR spectra were done at low microwave power of 11 mW. The spectroscopic programs SWAMP and LabVIEW 8.5 were used. Fourier transform infrared spectroscopy To quantify the extracted melanin-like pigment, IR spectra were recorded by Nicolet 6700 infrared spectrometer. A small amount of pigment 4 / 15 Melanin Produced by Bacillus weihenstephanensis was placed on a diamond window of the spectrometer, and a measurement was done in reflection mode, at a room temperature, by summary of 32 scans with a resolution of 4 cm-1. The available spectra range was 4004000 cm-1. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19783938 Synthetic melanin was used as a standard. Mechanisms of pigment production LB was inoculated with melanin-positive B. cereus s.l. isolates and incubated at 30C on a rotary shaker at 180 rpm for 24 h. After the preincubation, arginine or kojic acid as tyrosinase inhibitors were added to a final concentration of 0.010.5 mM and 10100g/ml, respectively. As a laccase inhibitor, sodium azide was added to a final concentration of 0.010.2 mM. In addition, the sulcotrione and tricyclazole were used for 4-hydroxyphenylpyruvate dioxygenase and hydroxynaphthalene reductase inhibition, respectively, to confirm/exclude the HGA PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786154 or DHN pathways of melanin synthesis. Then, cultures were incubated at 30C on the rotary shaker at 180 rpm for 96 h till the dark color occurred in the control culture. Next generation whole genome sequencing Genomic DNA of B.