Unmarked gene modification in Streptococcus mutans by a cotransformation strategy with a thermosensitive plasmid

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Constructing unmarked gene modifications is crucial for genetic analyses but challenging without selectable markers.
• Streptococcus mutans is a key bacterium in oral health research, necessitating efficient genetic manipulation tools.

Purpose of the Study:

• To introduce a new, efficient genetic method for creating unmarked mutants in Streptococcus mutans.
• To demonstrate the broad applicability of this method in transformable low G+C Gram-positive bacteria.

Main Methods:

• Developed a cotransformation method using the thermosensitive plasmid pGhost4.
• Introduced desired mutant alleles into Streptococcus mutans strains.
• Utilized pGhost4's broad-host-range thermosensitive replicon for efficient gene modification.

Main Results:

• Successfully constructed unmarked mutants in Streptococcus mutans with high frequency.
• Modified two distinct genetic loci via insertion and deletion.
• Demonstrated the method's efficacy in S. mutans, a significant oral bacterium.

Conclusions:

• The described genetic method enables the efficient construction of unmarked mutants in Streptococcus mutans.
• The pGhost4-based system is applicable to various transformable low G+C Gram-positive bacteria, including oral streptococci.
• This technique provides a valuable tool for genetic studies in relevant bacterial species.