A Cotransformation Method To Identify a Restriction-Modification Enzyme That Reduces Conjugation Efficiency in...

Area of Science:

• Microbiology
• Genetics

Background:

• Conjugation is a key mechanism for horizontal gene transfer in Campylobacter jejuni, a primary cause of bacterial gastroenteritis.
• Factors influencing conjugation efficiency in Campylobacter spp. remain largely unknown, hindering research into bacterial evolution and pathogenesis.

Purpose of the Study:

• To identify factors that significantly influence conjugation efficiency in Campylobacter jejuni.
• To develop and apply a novel cotransformation strategy for comparative genomics analysis.

Main Methods:

• Developed a cotransformation strategy by integrating an erythromycin resistance marker into a high-frequency conjugation (HFC) C. jejuni strain.
• Naturally transformed the DNA into a low-frequency conjugation (LFC) C. jejuni strain (NCTC 11168) to isolate new HFC transformants.
• Utilized whole-genome sequencing and site-directed mutagenesis to identify the Cj1051c gene (CjeI) responsible for reduced conjugation efficiency.

Main Results:

• Identified Cj1051c (CjeI), a restriction-modification enzyme, which drastically reduced conjugation efficiency in NCTC 11168 (>5,000-fold).
• Complementation experiments confirmed CjeI's significant reduction of conjugation efficiency in diverse HFC C. jejuni strains (∼1,000-fold).
• Purified CjeI digested an E. coli shuttle vector, and its endonuclease activity was heat-labile, consistent with heat shock enhancing C. jejuni conjugation.

Conclusions:

• Successfully developed and employed a unique cotransformation strategy to identify a key regulator of conjugation efficiency in C. jejuni.
• The identified restriction-modification enzyme, CjeI, plays a critical role in modulating horizontal gene transfer efficiency in C. jejuni.
• The developed cotransformation strategy is applicable to other naturally competent bacteria for functional comparative genomics research.