CHROMOSOMAL INTEGRATION, TANDEM AMPLIFICATION, AND DEAMPLIFICATION INPSEUDOMONAS-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 put ida Fl which had acquired the genes for chlorocatechol degradation (cl c) from Pseudomonas sp, strain B13 revealed that the de gene cluster w as present on a 105-kb amplifiable genetic element (named the de eleme nt). In one such transconjugant, P. putida RR22, a total of seven or e ight chromosomal copies of the entire genetic element were present whe n the strain was cultivated on chlorobenzene. Chromosomal integrations of the 105-kb de element occurred in two different loci, and the targ et sites were located within the 3' end of glycine tRNA structural gen es. Tandem amplification of the de element was preferentially detected in one locus on the F1 chromosome. After prolonged growth on nonselec tive medium, transconjugant strain RR22 gradually diverged into subpop ulations,vith lower copy numbers of the de element. Two nonadjacent co pies of the de element in different loci always remained after deampli fication, but strains with only two copies could no longer use chlorob enzene as a sole substrate. This result suggests that the presence of multiple copies of the ck gene cluster was a prerequisite for the grow th of P. putida RR22 on chlorobenzene and that amplification of the el ement was positively selected for in the presence of chlorobenzene.