A medium-copy-number plasmid for insertional mutagenesis of Streptococcus mutans
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Updated: Oct 27, 2026
Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning
Published on: October 23, 2017
1Center for Oral Biology, University of Rochester, Rochester, New York, 14642, USA.
Abstract:
We have constructed a plasmid useful for insertional mutagenesis in Streptococcus mutans. The molecule, pSU20Erm, is based on a derivative of pACYC184 known as pSU20. The plasmid described here is approximately 3.7 kb in size and has the following properties: it replicates in Escherichia coli, does not replicate in S. mutans, contains an erythromycin-resistance marker which can be selected in E. coli or the streptococci, contains a multiple cloning site with few restriction sites in the remainder of the molecule, and can be screened on X-Gal-containing medium for the presence of insertions into the multiple cloning site. We have used the plasmid to construct a library of S. mutans DNA in E. coli and show that the clones can be reintegrated into the S. mutans chromosome via homologous recombination, thereby interrupting native genes. The plasmid has been used to clone part of a homologue of the E. coli drpA gene, encoding a global regulatory element for RNA synthesis. Further, we have identified an element closely linked to drpA in S. mutans with high homology to IS861.
Insights
Researchers developed pSU20Erm, a novel plasmid for insertional mutagenesis in Streptococcus mutans. This tool facilitates gene disruption and cloning, aiding in the study of bacterial genetics and gene function.
Area of Science:
- Microbiology
- Molecular Biology
- Bacteriology
Background:
- Streptococcus mutans is a key cariogenic bacterium.
- Efficient genetic tools are needed for S. mutans research.
- Insertional mutagenesis is a powerful technique for gene function analysis.
Purpose of the Study:
- To construct and characterize a novel plasmid for insertional mutagenesis in Streptococcus mutans.
- To demonstrate the utility of the plasmid for creating gene knockouts and cloning bacterial DNA.
- To identify novel genetic elements in S. mutans.
Main Methods:
- Plasmid construction (pSU20Erm) based on pACYC184 derivative.
- Characterization of plasmid replication and selection markers in E. coli and S. mutans.
- Construction of an S. mutans DNA library in E. coli.
- Analysis of gene disruption via homologous recombination.
- Cloning and identification of S. mutans genes homologous to E. coli drpA and IS861.
Main Results:
- pSU20Erm is a 3.7 kb plasmid that replicates in E. coli but not S. mutans.
- It carries an erythromycin-resistance marker and a multiple cloning site suitable for insertional mutagenesis.
- Insertions into the multiple cloning site can be screened using X-Gal.
- The plasmid was used to create an S. mutans DNA library and successfully disrupt native genes.
- Homologues of E. coli drpA and a novel element homologous to IS861 were identified and cloned.
Conclusions:
- pSU20Erm is an effective tool for insertional mutagenesis in Streptococcus mutans.
- The plasmid facilitates gene disruption and cloning, enabling functional genomics studies.
- The identification of drpA homologues and IS861-like elements provides new insights into S. mutans gene regulation and genome dynamics.
