RESUMO
Erwinia chrysanthemi (Ech) RA3B produces a large amount of blue indigoidine. Using Tn5-induced mutagenesis, three indigoidine-deficient mutants were generated. Followed by library screening, a 5.8kb fragment complemented mutants for indigoidine synthesis was cloned. This fragment contains four complete open-reading frames (ORFs), pecS, pecM, idgA, and idgB, and two partial ORFs, argG, and idgC. These genes are nearly identical to those in strain Ech3937. Primer extension assays demonstrated a clear transcriptional start site prior to idgA, while no promoter preceding idgB and idgC was detected, suggesting that idgA, idgB, and idgC are organized as one transcription unit. In contrast, indAB is separated from indC in Ech3937. Interestingly, an ERIC sequence was present between idgB and idgC in place of the promoter region of the homolog indC, which may contribute to the loss of promoter activity in RA3B. Futhermore, idgB mutant displayed much lighter blue color, while indB mutant appeared white on media. Overexpression of pecS in RA3B resulted in significantly reduced indigoidine production and idgC transcript. Moreover, gel shift and luxAB reporter assays revealed that PecS specifically binds to the sequence preceding idgA and inhibits gene expression, which is consistent with the results observed in Ech3937.
