GENETIC-CHARACTERIZATION OF INSERTION-SEQUENCE ISJP4 ON PLASMID PJP4 FROM RALSTONIA-EUTROPHA JMP134

Directly adjacent to the (tfdT-) tfdCDEF gene cluster for chlorocatech ol breakdown on plasmid pJP4 of Ralstonia eutropha (formerly Alcaligen es eutrophus) JMP134, we identified a 0.9-kb DNA element, designated I SJP4, with the typical features of a bacterial insertion sequence. ISJ P4 occurs as a single complete copy on plasmid pJP4. About 9 kb away f rom this copy, in the tfdA-tfdS intergenic region, we found a 71-bp du plication of the ISJP4 right-hand extremity. In addition, we discovere d a complete copy of ISJP4 on the chromosome of the R. eutropha JMP134 strain that we use routinely in our laboratory. We suppose that this copy resulted from a recent transposition of the plasmid-borne ISJP4, since it was shown to be lacking from the chromosomes of R. eutropha J MP222 and JMP289, two previously pJP4-cured derivatives of JMP134. By comparing both complete copies and their flanking regions, we could es tablish that element ISJP4 has a size of 915 bp and is bordered by 18- bp inverted repeats with one mismatch. Based on sequence similarity of its coding regions, ISJP4 could be classified into the IS5 group of t he IS4 family of bacterial insertion sequences, where it is mostly rel ated to IS402 of Burkholderia cepacia. A TAA direct repeat, presumably resulting from a duplication of the target site, flanked the chromoso mal copy of ISJP4. We could demonstrate that a piece of DNA that is fl anked by two complete copies of ISJP4 can be transposed. Even more so, one complete ISJP4 plus its tfdA-tfdS intergenic remnant were suffici ent to mediate transposition of intervening DNA. A possible role of IS JP4 in the formation of the tfd pathway genes will be discussed. (C) 1 997 Elsevier Science B.V.