A markerless protocol for genetic analysis of Aggregatibacter actinomycetemcomitans

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• Aggregatibacter actinomycetemcomitans strains possess unique genes and genomic islands with unknown functions.
• Genetic analysis of these elements is hindered by a lack of suitable genetic tools for A. actinomycetemcomitans.

Purpose of the Study:

• To test a novel genetic approach for gene deletion and restoration in A. actinomycetemcomitans.
• To overcome limitations in genetic manipulation for studying strain-specific genomic elements.

Main Methods:

• Utilized a markerless loxP/Cre system for precise gene deletion and restoration.
• Employed loxP sites with distinct spacers for sequential deletion of multiple genes, preventing unwanted recombination.
• Successfully constructed single-gene deletion mutants, multiple-gene deletion mutants, and gene-restored mutants.

Main Results:

• Generated eight single-gene deletion mutants, four multiple-gene deletion mutants, and two restored mutants.
• Demonstrated the protocol's specificity, with no unintended non-specific deletions observed.
• Confirmed that the genetic manipulation did not adversely impact A. actinomycetemcomitans growth or biofilm formation.

Conclusions:

• The developed protocol is an efficient and specific method for genetic manipulation of A. actinomycetemcomitans.
• This approach facilitates the functional analysis of multiple genes within A. actinomycetemcomitans strains.
• The method is suitable for exploring the roles of strain-specific genes and genomic islands.