Rhizobium sp. CU-A1, isolated from petroleum-contaminated soil in Thailand, is able to utilize acenaphthylene or naphthalene as sole carbon and energy sources. In order to elucidate the acenaphthylene catabolic pathways, transposon Tn5 was employed as inducer for mutagenesis in this strain via conjugation with Eschericia coli strain S17-1 harboring suicide plasmid pSUP2021. The highest transposition efficiency of 4.75 x 10-5 per recipient was obtained by mixing both donor and recipient at their early log phase with the ratio of 1 : 1, and incubating at 30 C for 24 hours, followed by selecting on CFMM agar containing 1.0 g/I protocatechuic acid and 50 ug/ml kanamycin. The random insertion of transposon Tn5 to the DNA of Rhizobium was verified bySouthern hybridization. From 15,370 transconjugants obtained, 21 mutants which are deficient in oxidation or inability to utilize acenaphthylene and naphthalene, were identified. Growth on acenaphthylene, naphthalene and other intermediates with subsequent TLC analysis of intermediates accumulated in Tn5-induced mutants revealed that strain CU-A1 may degraded acenaphthylene through acenaphthoquinone, naphthalene-1,8-dicarboxylic acid and gentisic acid respectively, and naphthalene was also oxidized to gentisic acid. Moreover, Rhizobium sp. strain CU-GFP2 containing gfp gene was successfully constructed by using the optimal conjugation conditions. The strain CU-GFP2 can degrade acenaphthylene at the same rate as strain CU-A1 and show green fluorescence.
