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Cloning and expression of Bacillus subtilis phage DNA methyltransferase genes in Escherichia coli and B. subtilis
Abstract:
The DNA methyltransferase (Mtase) genes of the temperate Bacillus subtilis phages SPR (wild type and various mutants), phi 3T, rho 11 and SP beta have been cloned and expressed in Escherichia coli and B. subtilis host-plasmid vector systems. Mtase activity has been quantitated in these clones by performing in vitro methylation assays of cell-free extracts. The four-phage Mtase genes differ in the amount of Mtase synthesized when transcribed from their genuine promoters. In B. subtilis as well as in E. coli the SPR Mtase is always produced in smaller amounts than the other phage Mtases. Expression levels of the SPR Mtase are dependent on the strength of the upstream vector promoter sequences. Overproduction of the SPR wild-type and mutant enzymes was achieved in E. coli (inducible expression) by fusions to the lambda pL or the tac promoter and in B. subtilis (constitutive expression) by means of the phage SP02 promoter.
Insights
Researchers cloned and expressed DNA methyltransferase (Mtase) genes from four Bacillus subtilis phages in E. coli and B. subtilis. SPR phage Mtase showed lower expression, influenced by promoter strength, but could be overproduced.
Area of Science:
- Molecular biology
- Microbial genetics
- Bacteriophage research
Background:
- Bacteriophages are viruses that infect bacteria.
- DNA methyltransferases (Mtases) are enzymes that modify DNA.
- Understanding phage gene expression is crucial for molecular biology.
Purpose of the Study:
- To clone and express DNA methyltransferase (Mtase) genes from four Bacillus subtilis phages.
- To compare the expression levels and activity of these phage Mtases.
- To investigate methods for overproducing specific phage Mtase enzymes.
Main Methods:
- Cloning and expression of phage Mtase genes in Escherichia coli and Bacillus subtilis host-plasmid vector systems.
- In vitro methylation assays using cell-free extracts to quantify Mtase activity.
- Utilizing inducible (lambda pL, tac) and constitutive (SP02) promoters for enzyme overproduction.
Main Results:
- Differential synthesis of Mtase was observed among the four cloned phage genes when using their native promoters.
- SPR phage Mtase consistently exhibited lower production levels in both B. subtilis and E. coli compared to other phage Mtases.
- SPR Mtase expression levels were found to be dependent on the strength of the upstream vector promoter sequences.
- Successful overproduction of SPR wild-type and mutant Mtases was achieved in E. coli and B. subtilis using specific promoter fusions.
Conclusions:
- The expression levels of DNA methyltransferase genes from different B. subtilis phages vary significantly.
- SPR phage Mtase expression is particularly low and sensitive to promoter strength.
- Engineered expression systems allow for the overproduction of specific phage Mtase enzymes, facilitating further biochemical and structural studies.