Group II intron from Pseudomonas alcaligenes NCIB 9867 (P25X): entrapment in plasmid RP4 and sequence analysis

Abstract

Summary: Pseudomonas alcaligenes NCIB 9867 (strain P25X), which grows on 2,5-xylenol and harbours the plasmid RP4, was mated with a plasmid-free derivative of Pseudomonas putida NCIB 9869, strain RA713, which cannot grow on 2,5-xylenol. Some RA713 transconjugants, initially selected on 2,5-xylenol, were found to carry RP4 plasmids that had acquired additional fragments (designated XIn) which ranged in size from 2 kb to approximately 26 kb. Instability of DNA inserts in RP4::XIn 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 probe with digested P25X chromosomal DNA. Sequence analysis of XIn6 led to the discovery of an open reading frame with homology to the maturases of group II introns. The XIn6 insert also exhibited several features 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 the intron. RNA secondary structure modelling of XIn6 also revealed the presence of perfectly conserved domains V and VI. Differences were detected in the XIn6 hybridization profiles of several P25X catabolic mutants that have lost the ability to grow on 2,5-xylenol. In these mutants the loss of 2,5-xylenol degradative ability could be due to genome rearrangements mediated by sequences related to the XIn6 group II intron. This is the first reported group II intron isolated from Pseudomonas spp. and the first time that the mobility of a bacterial group II intron has been demonstrated.