Campus Units

Veterinary Microbiology and Preventive Medicine

Document Type

Article

Publication Version

Published Version

Publication Date

12-2018

Journal or Book Title

Applied and Environmental Microbiology

Volume

84

Issue

23

First Page

e02004-18

DOI

10.1128/AEM.02004-18

Abstract

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 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) C. jejuni 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 C. jejuni strain; subsequently, the DNA of this strain was naturally transformed into NCTC 11168, an LFC C. jejuni 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 (aka CjeI) that could drastically reduce the conjugation efficiency of NCTC 11168 (>5,000-fold). Chromosomal complementation of three diverse HFC C. jejuni strains with CjeI also led to a dramatic reduction in conjugation efficiency (∼1,000-fold). The purified recombinant CjeI could effectively digest the Escherichia 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, in this study, we successfully developed and utilized a unique cotransformation strategy to identify a restriction-modification enzyme that significantly influences conjugation efficiency in C. jejuni.

Comments

This article is published as Zeng, Ximin, Zuowei Wu, Qijing Zhang, and Jun Lin. "A Cotransformation Method To Identify a Restriction-Modification Enzyme That Reduces Conjugation Efficiency in Campylobacter jejuni." Applied and Environmental Microbiology 84, no. 23 (2018): e02004-18. DOI: 10.1128/AEM.02004-18. Posted with permission.

Copyright Owner

American Society for Microbiology

Language

en

File Format

application/pdf

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