Identification and characterization of a restriction-modification enzyme reducing conjugation efficiency in Campylobacter jejuni using a co-transformation method.

Conjugation is an important mechanism for horizontal gene transfer in Campylobacter jejuni , the leading cause of human bacterial gastroenteritis in developed countries. However, to date, the factors that significantly influence conjugation efficiency in Campylobacter are still largely unknown. Given that multiple recombinant loci could independently occur within one recipient cell during natural transformation, the genetic materials from high frequency conjugation ( HFC ) C. jejuni strain may be co-transformed with a selection marker into a low frequency conjugation ( LFC ) recipient strain, creating new HFC transformants suitable for identification of conjugation factor using comparative genomics approach. To test this, an erythromycin resistance selection marker was created in a HFC C. jejuni strain; subsequently, the DNA of this strain was naturally transformed into NCTC11168, a LFC C. jejuni strain, leading to isolation of NCTC11168-derived HFC transformants. Whole genome sequencing analysis and subsequent site-directed mutagenesis identified Cj1051c, a putative restriction-modification enzyme ( aka CjeI) that could drastically reduce conjugation efficiency of NCTC11168 (> 5,000 fold). Chromosomal complementation of three diverse HFC C. jejuni strains with CjeI also led to dramatic reduction in conjugation efficiency (∼1,000 fold). The purified recombinant CjeI could effectively digest the E. coli -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 C. jejuni Together, this study successfully developed and utilized a unique co-transformation strategy to identify a restriction-modification enzyme that significantly influences conjugation efficiency in C. jejuni Importance Conjugation is an important horizontal gene transfer mechanism contributing to the evolution of bacterial pathogenesis and antimicrobial resistance. Campylobacter jejuni , the leading foodborne bacterial organism, displays significant strain diversity due to horizontal gene transfer; however, the molecular components influencing conjugation efficiency in C. jejuni are still largely unknown. In this study, we developed a co-transformation strategy for comparative genomics analysis and successfully identified a restriction-modification enzyme that significantly influences conjugation efficiency in C. jejuni The new co-transformation strategy developed in this study is also expected to be broadly applied in other naturally competent bacteria for functional comparative genomics research.