Related Experiment Videos
Inducible gene expression mediated by a repressor-operator system isolated from Lactococcus lactis bacteriophage r1t
A Nauta1, D van Sinderen, H Karsens
1Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
Molecular Microbiology
|March 1, 1996
Summary
The Lactococcus lactis bacteriophage r1t repressor (rro) gene and its operator sequences were identified. Phage repressor Rro controls the switch between lysogenic and lytic cycles by regulating gene expression.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The temperate Lactococcus lactis bacteriophage r1t has a regulatory region controlling its life cycle.
- This region contains two divergently oriented genes: rro (encoding the phage repressor) and tec.
- Key promoter and operator sequences within this region are crucial for phage regulation.
Purpose of the Study:
- To clone and characterize the regulatory region of the Lactococcus lactis bacteriophage r1t.
- To investigate the function of the rro gene and its encoded repressor protein.
- To elucidate the transcriptional regulation mechanisms governing the phage's lysogenic and lytic life cycles.
Main Methods:
- Gene cloning and characterization of the r1t regulatory region.
- Mapping of transcription start sites and identification of promoter sequences.
- Gel mobility shift assays to study Rro protein binding to operator sequences.
- Construction of a lacZ translational fusion to assess gene expression.
- Analysis of gene expression under different conditions (lysogenic vs. lytic induction).
Main Results:
- The r1t regulatory region contains three direct repeats with dyad symmetry, likely acting as operators.
- The phage repressor, Rro, specifically binds to these operator sequences.
- Rro represses promoter activity in non-induced cells, maintaining the lysogenic state.
- Mitomycin C induction triggers a 70-fold increase in gene expression, facilitating the switch to the lytic cycle.
- A frameshift mutation in rro leads to constitutive expression of downstream genes.
Conclusions:
- The rro gene product, Rro, acts as a transcriptional repressor, controlling the expression of genes in the r1t regulatory region.
- Rro plays a critical role in maintaining the lysogenic life cycle by repressing gene expression.
- The phage repressor Rro is essential for regulating the switch between the lysogenic and lytic life cycles of Lactococcus lactis bacteriophage r1t.