Chromosomal Integration, Tandem Amplification, and Deamplification in Pseudomonas putida F1 of a 105-Kilobase Genetic Element Containing the Chlorocatechol Degradative Genes from Pseudomonas sp. Strain B13

Analysis of chlorobenzene-degrading transconjugants of Pseudomonas putida F1 which had acquired the genes for chlorocatechol degradation (clc) from Pseudomonas sp. strain B13 revealed that the clc gene cluster was present on a 105-kb amplifiable genetic element (named the clc element). In one such transconjugant, P. putida RR22, a total of seven or eight chromosomal copies of the entire genetic element were present when the strain was cultivated on chlorobenzene. Chromosomal integrations of the 105-kb clc element occurred in two different loci, and the target sites were located within the 3′ end of glycine tRNA structural genes. Tandem amplification of the clc element was preferentially detected in one locus on the F1 chromosome. After prolonged growth on nonselective medium, transconjugant strain RR22 gradually diverged into subpopulations with lower copy numbers of the clc element. Two nonadjacent copies of the clc element in different loci always remained after deamplification, but strains with only two copies could no longer use chlorobenzene as a sole substrate. This result suggests that the presence of multiple copies of the clc gene cluster was a prerequisite for the growth of P. putida RR22 on chlorobenzene and that amplification of the element was positively selected for in the presence of chlorobenzene.