ISOLATION AND CHARACTERIZATION OF A NOVEL INSERTION-SEQUENCE ELEMENT,IS1248, IN PARACOCCUS-DENITRIFICANS

A new suicide vector, pRVS3, was constructed to facilitate gene replac ements in the genome of Paracoccus denitrificans. In control experimen ts, incorporation of this suicide vector into the genome did not depen d on the presence of homologous DNA. Using appropriate restriction enz ymes, the suicide vector and flanking DNA were recovered from the geno mic DNA. Sequence analysis demonstrated that both up- and downstream o f the ex-integrant vector there was an element that showed high homolo gy with bacterial insertion sequences (IS). Southern blot analysis of wild-type and integrant strains revealed that at least four copies of this IS element reside in the P. denitrificans genome, one of which, d esignated IS1248, had been involved in the transpositional event descr ibed here. IS1248 is 830 bp long, has 13-bp imperfect inverted repeats at the borders, and contains five open reading frames. With respect t o the organization and primary sequences of the open reading frames, I S1248 closely resembles IS869 and IS427 of Agrobacterium tumefaciens, IS402 of Pseudomonas cepacia, and ISmyco found in Mycobacterium tuberc ulosis. (C) 1995 Academic Press, Inc.