Quantitative analysis of group II intron expression and splicing in Lactococcus lactis

The group II intron L1.ltrB is found within the ltrB relaxase gene of the conjugative element pRS01 in Lactococcus lactis. Precise splicing of the intron is essential for pRS01 transfer. The transcription regulation wand in vivo splicing activity of L1.ltrB have not been investigated thoroughly in L. lactis in the natural pRS01 context. We developed absolute quantitative real-time reverse transcription-PCR assays to quantify RNA levels of the 5' exon (ltrBE1) and the spliced relaxase (ItrB) and intron-encoded protein (ltrA) genes, as well as L1.ltrB splicing activity under different physiological conditions. The mRNA levels for the ATP-binding protein OppD were assayed for comparison to the ItrB transcripts. The oppD mRNA ranged from 10- to 10,000-fold higher than ItrB region genes. ltrBE1 expression was growth-phase dependent. The mRNA level of ltrA was almost constant during all growth phases and in all media tested. L1.ltrB in vivo splicing activity ranged from (6.5 +/- 2.1)% to (22.1 +/- 8.0)%. Acid challenge significantly decreased both ItrB region mRNA levels and intron splicing activity. The presence of recipient cells, different mating environments, and temperature stress had no significant effects on expression and splicing. Western blotting showed that the level of LtrB protein expressed from an intronless ItrB gene was much higher (about 20-fold) than the level of protein expressed from an intron-containing construct. Interestingly, LtrB protein showed a tendency to function in cis on its oriT target. The low level of ItrB transcript and relatively inefficient splicing of the intron may limit L1.ltrB mobility and dissemination in nature.