Isolation of Citrobacter sp. mutants defective in decolorizing malachite green

To identify genes involved in the decolorization of malachite green, random mutants generated by transposon insertion in the malachite green-decolorizing bacterium, Citrobacter sp. were isolated. The resulting mutant bank yielded 24 mutants with complete defects in their abilities to decolorize malachite green. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants, which appeared to have insertions at different sites of the chromosome. The Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. Based on a sequence database, the putative protein products encoded by the mg genes were identified as follows. mg3, an ABC transporter homolog; mg6, a LysR-type regulatory protein; m11, an oxidoreductase; mg17, a MalG protein in the maltose transport system; and mg21, a sugar kinase. The deduced sequences from two mg genes (mg7 and mg18) showed no significant similarity to any protein with a known function, suggesting that these two mg genes encode unidentified proteins that are responsible for the decolorization of malachite green.