Utilizing natural competence to genetically manipulate Lactobacillus iners

Area of Science:

• Microbiology
• Genetics
• Human Microbiome

Background:

• The human vaginal microbiota is dominated by Lactobacillus species, with L. iners being the most globally prevalent.
• L. iners is fastidious, has a small genome, produces less lactic acid, and may act as a pathobiont due to its unique cytolysin, inerolysin.
• Understanding L. iners biology is crucial for over a billion females, but is hindered by a lack of genetic tools.

Purpose of the Study:

• To establish the first genetic manipulation method for L. iners.
• To enable targeted genetic studies of L. iners to understand its physiology and role in the vaginal microbiome.
• To provide a foundation for potentially engineering L. iners for probiotic applications.

Main Methods:

• Demonstrated that L. iners is naturally competent and can be transformed with exogenous DNA.
• Utilized natural competence to disrupt the iny gene (encoding inerolysin) and the comGA gene (encoding a competence pilus component).
• Employed PCR-assembled DNA fragments with drug resistance markers (tetM or ermB) and homology arms for targeted gene deletion.

Main Results:

• Successfully disrupted the iny and comGA genes in L. iners using the developed transformation method.
• Confirmed that the comGA gene is essential for L. iners transformation.
• Established a straightforward method for targeted gene deletions in L. iners using in vitro generated DNA templates.

Conclusions:

• This study presents the first method for genetically manipulating L. iners.
• The ability to perform targeted deletions provides a vital tool for investigating L. iners genetics and physiology.
• This work facilitates future research into the vaginal microbiome and the development of L. iners-based probiotics.