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Quantitative analysis of group II intron expression and splicing in Lactococcus lactis
Yuqing Chen1, Joanna R Klein, Larry L McKay
1Department of Microbiology, University of Minnesota, Minneapolis, MN 55455, USA.
Applied and Environmental Microbiology
|May 5, 2005
Summary
This study quantifies group II intron Ll.ltrB splicing and transcription in Lactococcus lactis. Acid conditions reduced splicing and gene expression, potentially limiting Ll.ltrB mobility.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Group II introns, like Ll.ltrB, are mobile genetic elements crucial for conjugative element transfer.
- The Ll.ltrB intron resides within the ltrB relaxase gene of the pRS01 element in Lactococcus lactis, essential for pRS01 transfer.
- Thorough investigation of Ll.ltrB transcription and splicing in L. lactis within the native pRS01 context is lacking.
Purpose of the Study:
- To develop and apply quantitative assays for measuring Ll.ltrB RNA levels and splicing activity in L. lactis.
- To investigate the impact of physiological conditions on Ll.ltrB transcription and splicing.
- To analyze the expression of the intron-encoded LtrA protein and the relaxase LtrB protein.
Main Methods:
- Absolute quantitative real-time reverse transcription-PCR (qRT-PCR) was used to quantify ltrBE1, ltrB, and ltrA mRNA levels.
- Ll.ltrB splicing activity was assessed in vivo under various physiological conditions.
- Western blotting was employed to compare LtrB protein levels from intron-containing and intronless constructs.
Main Results:
- OppD mRNA levels were significantly higher (10- to 10,000-fold) than ltrB region genes.
- ltrBE1 expression varied with growth phase, while ltrA mRNA levels remained relatively constant.
- In vivo splicing activity ranged from 6.5% to 22.1%; acid challenge significantly reduced mRNA levels and splicing.
- LtrB protein expression was approximately 20-fold higher from an intronless construct compared to an intron-containing one.
- LtrB protein demonstrated a cis-preference for its oriT target.
Conclusions:
- Acid stress negatively impacts Ll.ltrB transcription and splicing in Lactococcus lactis.
- Low ltrB transcript levels and inefficient splicing may restrict the mobility and spread of Ll.ltrB in natural environments.
- The study provides quantitative insights into the regulation and activity of a mobile group II intron in its natural host context.