GROUP-II INTRON FROM PSEUDOMONAS-ALCALIGENES NCIB-9867 (P25X) - ENTRAPMENT IN PLASMID-RP4 AND SEQUENCE-ANALYSIS

Pseudomonas alcaligenes NCIB 9867 (strain P25X), which grows on 2,5-xy lenol and harbours the plasmid RP4, was mated with a plasmid-free deri vative of Pseudomonas putida NCIB 9869, strain RA713, which cannot gro w on 2,5-xylenol. Some RA713 transconjugants, initially selected on 2, 5-xylenol, were found to carry 884 plasmids that had acquired addition al fragments (designated Xln) which ranged in size from 2 kb to approx imately 26 kb. Instability of DNA inserts in RP4::Xln hybrid plasmids was observed. The smallest insert present in a stable RP4::XIn6 hybrid plasmid, termed XIn6, yielded multiple bands when it was used as a pr obe with digested P25X chromosomal DNA. Sequence analysis of XIn6 led to the discovery of an open reading frame with homology to the maturas es of group II introns. The XIn6 insert also exhibited several feature s characteristic of a group II intron. These included the presence of the consensus sequence GUGYG at the 5' end and RAY at the 3' end of th e intron. RNA secondary structure modelling of XIn6 also revealed the presence of perfectly conserved domains V and VI. Differences were det ected in the XIn6 hybridization profiles of several P25X catabolic mut ants that have lost the ability to grow on 2,5-xylenol. In these mutan ts the loss of 2,5-xylenol degradative ability could be due to genome rearrangements mediated by sequences related to the XIn6 group II intr on. This is the first reported group II intron isolated from Pseudomon as spp, and the first time that the mobility of a bacterial group II i ntron has been demonstrated.