Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning

Area of Science:

• Molecular Biology
• Microbiology
• Plasmid Technology

Background:

• Shuttle vectors are crucial for genetic manipulation across different bacterial species.
• Understanding gene expression in Streptococcus species requires suitable cloning vectors.

Purpose of the Study:

• To describe a novel 9.2-kb plasmid vector, pVA856, capable of replication in both Escherichia coli and Streptococcus sanguis.
• To demonstrate the utility of modified shuttle plasmids for cloning and selecting sequences that confer autonomous replication in S. sanguis.

Main Methods:

• Construction and characterization of the pVA856 plasmid.
• Analysis of gene determinant expression (Cmr, Tcr, Emr) in E. coli and S. sanguis.
• Modification of the pVA838 shuttle plasmid to prevent replication in S. sanguis.
• Demonstration of cloning strategies using these plasmids.

Main Results:

• The pVA856 plasmid replicates in both E. coli and S. sanguis, expressing Emr determinant in S. sanguis.
• Cmr and Tcr determinants of pVA856 are amenable to insertional inactivation.
• A modified pVA838 plasmid was created that cannot replicate in S. sanguis.
• The modified plasmid facilitates cloning and selection of S. sanguis replication sequences.

Conclusions:

• pVA856 is a versatile tool for molecular cloning and genetic studies involving E. coli and S. sanguis.
• The modified shuttle plasmid system provides a method for identifying and isolating DNA sequences that enable autonomous replication in S. sanguis.