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

Conjugation is an important mechanism for horizontal gene transfer in , the leading cause of human bacterial gastroenteritis in developed countries. However, to date, the factors that significantly influence conjugation efficiency in spp. are still largely unknown. Given that multiple recombinant loci could independently occur within one recipient cell during natural transformation, the genetic materials from a high-frequency conjugation (HFC) strain may be cotransformed with a selection marker into a low-frequency conjugation (LFC) recipient strain, creating new HFC transformants suitable for the identification of conjugation factors using a comparative genomics approach. To test this, an erythromycin resistance selection marker was created in an HFC strain; subsequently, the DNA of this strain was naturally transformed into NCTC 11168, an LFC strain, leading to the isolation of NCTC 11168-derived HFC transformants. Whole-genome sequencing analysis and subsequent site-directed mutagenesis identified Cj1051c, a putative restriction-modification enzyme ( CjeI) that could drastically reduce the conjugation efficiency of NCTC 11168 (>5,000-fold). Chromosomal complementation of three diverse HFC strains with CjeI also led to a dramatic reduction in conjugation efficiency (∼1,000-fold). The purified recombinant CjeI could effectively digest the -derived shuttle vector pRY107. The endonuclease activity of CjeI was abolished upon short heat shock treatment at 50°C, which is consistent with our previous observation that heat shock enhanced conjugation efficiency in Together, in this study, we successfully developed and utilized a unique cotransformation strategy to identify a restriction-modification enzyme that significantly influences conjugation efficiency in Conjugation is an important horizontal gene transfer mechanism contributing to the evolution of bacterial pathogenesis and antimicrobial resistance. , the leading foodborne bacterial organism, displays significant strain diversity due to horizontal gene transfer; however, the molecular components influencing conjugation efficiency in are still largely unknown. In this study, we developed a cotransformation strategy for comparative genomics analysis and successfully identified a restriction-modification enzyme that significantly influences conjugation efficiency in The new cotransformation strategy developed in this study is also expected to be broadly applied in other naturally competent bacteria for functional comparative genomics research.