CHARACTERIZATION OF IS1474, AN INSERTION-SEQUENCE OF THE IS21 FAMILY ISOLATED FROM PSEUDOMONAS-ALCALIGENES NCIB-9867

A new insertion sequence (IS) designated IS1474 was isolated from Pseu domonas alcaligenes NCIB 9867 (P25X). IS1474 is a 2632 bp element whic h showed a characteristic IS structure with 12 bp inverted repeats (IR s) flanking a 2608 bp central region. IS1474 contained four open readi ng frames (ORF1-ORF4), two in each orientation. Similarities were dete cted between ORF1 and ORF2 and the putative transposases of the IS21 f amily. Sequences upstream from IS1474 were found to display up to 89% homoolgy with IS53 from Pseudomonas syringae suggesting that IS1474 ha d inserted into another related IS element designated IS1475. An open reading frame, ORF5, located at the junction of IS1474 and IS1475, sho wed similarities with the IstB protein of IS21 and could possibly be t he transposase subunit of IS1475. Transposition assays showed that IS1 474 transposed at a relatively low frequency leading to cointegration with target plasmids. Hybridization studies showed that IS1474 is pres ent in at least 13 copies in the chromosome of P25X and one copy on it s endogenous plasmid.