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Erwinia chrysanthemi (Ech) causes bacterial soft rot disease on several crops by secreting extracellular pectinase and cellulase. A spontaneous mutant Ech RA3B produces tremendous amounts of a black-blue pigment (indigoidine, 5,5' -diamino-4,4'-dihydroxy-3,3'-diazadiphenoquinone) on yeast-extract dextrose calciumcarbonate (YDC) medium. A 6kb EcoRI DNA fragment containing pecS, PECm, idgA, and idgB genes involved in blue pigment biosynthesis was cloned from Ech RA3B strain. Three non-polar mutants, RA3B-idgA, RA3B-IDgB, and RA3B-pecM, were constructed by insertion of nptII gene to investigate blue pigmentation. The mutants RA3B-idgA, RA3B-idgB, and RA3B-pecM cultured on YDC medium produced white, pale-blue, and dark-blue colonies, respectively. The idgA, idgB and pecS genes can be overexpressed by T7 RNA polymerase dependent system. Complementation assay, primer extension and band-shift assay yielded several observations. (1) idgA and idgB genes are likely to be organized in the same transcriptional unti (2) A transcription initiation site located at the 114th nucleotide (C) upstream of the idgA gene from the ATG site. (3) PecS protein can bind upstream region of the idgA gene specifically. Furthermore, the luciferase genes, luxA and luxB, were used as reporter genes to monitor the promoter activity The results showed that a promoter activity was detected at the upstreacm region of idgA and the activity of the promoter was inhibited by PecS protein in E. coli. Mutiple copies of pecS gene present in Ech RA3B supressed the production of the bacterial blue pigment and pectinase, indicating that PecC protein is a negative regulator of the idgA gene.
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