Repeated sequences including RS1100 from Pseudomonas cepacia AC1100 function as IS elements

Abstract

Several lines of evidence were obtained that the previously identified, repeated sequence RS1100 of Pseudomonas cepacia strain AC1100 undergoes transposition events. DNA sequences flanking the chlorohydroxy hydroquinone (CHQ) degradative genes of this organism were examined from sources, including several independently isolated cosmid clones from an AC1100 genomic library and genomic DNAs of two independently maintained wild-type AC1100 isolates. Hybridization and restriction endonuclease mapping studies revealed these sequences to be similar except for their numbers and distributions of RS1100 copies. A recombinant plasmid containing the immediate chq gene region and excluding any copies of RS1100 was conjugated into AC1100 mutant RHA5 which was shown to have undergone a deletion of its corresponding DNA. Hybridization and restriction mapping analyses of several reisolated plasmids revealed the presence of RS1100 sequences at different positions within either the vector or insert portions. One such plasmid contained tandem copies of RS1100 with an intervening DNA sequence also of AC1100 origin. Similar experiments involving introduction of the promoter probe plasmid pKT240 into wild-type AC1100 cells resulted in the acquisition of high-concentration streptomycin resistance by a number of recipients. The reisolated plasmids in most cases also conferred streptomycin resistance to Escherichia coli transformants and in each case were found to contain insertions close to the upstream portion of the aphC structural gene. These insertions alternatively contained RS1100 sequences for a newly identified 3400 bp repeated sequence from AC1100. Based on these results, RS1100 has been redesignated as insertion sequence IS931 and the 3400 bp repeated sequence has been designated as IS932.