Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168

Published online 18 January 2016 Nucleic Acids Research, 2016, Vol. 44, No. 10 4581–4594 doi: 10.1093/nar/gkw019 Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168 Awais Anjum1 , Kelly J. Brathwaite2 , Jack Aidley1 , Phillippa L. Connerton2 , Nicola J. Cummings2 , Julian Parkhill3 , Ian Connerton2 and Christopher D. Bayliss1,* 1 Department of Genetics, University of Leicester, Leicester LE1 7RH, UK, 2 Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonington LE12 5RD, UK and 3 The Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UK Received September 2, 2015; Revised December 22, 2015; Accepted December 26, 2015 INTRODUCTION Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system in which the endonuclease and methyltransferase are encoded by a single gene. Using both inhibition of restriction and PacBio-derived methylome analyses of mutants and phase-variants, the cj0031c allele in C. jejuni strain NCTC11168 was demonstrated to specifically methylate adenine in 5 CCCGA and 5 CCTGA sequences. Alterations in the levels of specific transcripts were detected using RNA-Seq in phase-variants and mutants of cj0031c but these changes did not correlate with observed differences in phenotypic behaviour. Alterations in restriction of phage growth were also associated with phase variation (PV) of cj0031c and correlated with presence of sites in the genomes of these phages. We conclude that PV of a Type IIG restriction-modification system causes changes in site-specific methylation patterns and gene expression patterns that may indirectly change adaptive traits. Campylobacter jejuni is a major cause of food-borne gastroenteritis in humans with more than 400 million cases of Campylobacteriosis reported annually worldwide (1). Currently, the disease burden exerted by Campylobacters in the UK is higher than for any other foodborne pathogen (2) with chickens being the main source of infection in over 80% of disease cases (3). Campylobacters are present in a very high fraction of farmed chickens with significant numbers of bacteria in each colonized bird (4,5). Rapid microevolution may be a major mechanism that facilitates persistent colonization of and rapid transmission between individual birds. One of the characteristic mechanisms for enabling rapid adaptation is phase variation (PV) mediated by hypermutable simple sequence repeats (6). Most of phase-variable genes of C. jejuni encode surface proteins or enzymes that modify host surface structures (7). However one of these phase-variable genes encodes a restriction modification (RM) system that has the potential to act as a stochastically-regulated modifier of expression of other genes or mediate alternation between phage susceptible and resistant states (8). PV is an adaptive process characterized by high frequency, reversible ON/OFF switching of gene expression that is stochastic in nature and gives rise to heterogeneous population (9). The main mechanism of PV in C. jejuni involves insertion or deletion of repeat units during genome replication in polyG or polyC repeat tracts located in the reading frames of genes. In a recent survey, each C. jejuni genome was found to contain between 12 and 29 polyG/polyC tracts capable of mediating PV (8). Most of these phase-variable loci encode enzymes that modify the glycan components of the flagella, capsule and lipooligosaccharide. One of the tracts was present in a putative Type IIG restriction-modification system. This is a general paradigm * To whom correspondence should be addressed. Tel: +44 0 116 252 3465; Fax: +44 0 116 252 3378; Email:  C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact ABSTRACT 4582 Nucleic Acids Research, 2016, Vol. 44, No. 10 nism of methylation to the phasevarion-associated Type III RM systems, suggested that PV of cj0031 may confer fitness advantages on the bacterial population, due to alterations in expression of multiple genes mediated by changes in global methylation. To test this hypothesis, we investigated the functions, methylome, global expression patterns and phenotypic variation of mutants and phase-variants of cj0031 in C. jejuni strain NCTC11168. MATERIALS AND METHODS Bacterial strains and growth conditions Two variants of C. jejuni strain NCTC11168 were used in the present study: a motile but non-swarming variant (11168); and a chicken-adapted hypermotile variant (CH11168). The C. jejuni strains were grown on MuellerHinton agar (MHA, Oxoid, UK) supplemented with vancomycin (10 ␮g/ml Sigma Aldrich, UK) and trimethoprim (5 ␮g/ml Sigma Aldrich) in a VA500 Variable Atmosphere Incubator (Don Whitley, UK) providing 4% oxygen (v/v), 10% carbon dioxide (v/v), 86% nitrogen (v/v) and a temperature of 42◦ C. Esherichia coli strain DH5␣ was used for cloning and was cultured at 37◦ C on Luria agar plates or in Luria broth containing appropriate antibiotic as required; ampicillin, 50 ␮g/ml; kanamycin, 50 ␮g/ml; and chloramphenicol, 10 ␮g/ml. Phylogenetic analysis The cj0031 gene sequence was downloaded from the NCBI database, and then used in a BLAST search to identify homologues of this gene and of the allelic variant, A911 00150, present in C. jejuni PT14 (25). These sequences were submitted to the Phylogeny.fr platform in order to construct a phylogenetic tree. This platform uses MUSCLE for the alignment of the submitted sequences, PhyML for tree building and TreeDyn for tree rendering (26). For building phylogenetic tree of A911 00150, the default parameters set for MUSCLE, PhyML and TreeDyn were applied. Isolation of ON and OFF phase-variants Serial dilutions of a single colony of C. jejuni NCTC11168 strain were plated onto MHA plates and incubated for 3 days under micro-aerobic conditions at 42◦ C. A total of 110 colonies were randomly selected and sub-cultured. Boiled lysates were prepared in 100 ␮l of distilled water for DNA amplification. The repeat tract of cj0031 was amplified using two primers, 0031F-Fam and 0031R and then analysed by GeneScan as described elsewhere (8). A (...truncated)