Isolation and Characterization of Tn-5 Induced Insertion Mutants in the 3-Chlorobenzoate Degradation Pathway of Pseudomonas Sp. B13

Abstract

Chemical mutagenesis with such chemical substances as EMS (ethyl-methyl-sulfonic acid) and NTG (nitrosoguanidine) has been until now the only way to isolate mutants from p.sp.B13 defective in the 3-chlorobenzoate degradative pathway. This mutagenic technique produces point mutations. Very often multiple mutations are encountered in a single cell. To overcome this problem and get mutants with a more stable pheno- and genotype, it was necessary to develop another mutagenic method for P.sp.B13. Transposonmutagenesis is one of the most powerful techniques available for genetic analysis in bacteria. It also allows mapping of the genes using restriction en-donucleases. Transposons such as Tn-7 and Tn-5 previously were used successfully to mutagenize P. putida mt-2 (TOL plasmid) and P. aeruginosa. Tn-5 (encoding kanamycin resistance) is known to insert with a relatively high frequency in the genomes of prokaryotes and eukaryotes independent of the recA function; usually only one Tn-5 element is inserted per genome. It seemed reasonable to try trans-poson mutagenesis on P.sp.B13 with Tn-5 after Tn-7 and Tn-3 had been used without success.