Analysis of the Campylobacter jejuni Genome by SMRT DNA Sequencing Identifies Restriction-Modification Motifs

February Analysis of the Campylobacter jejuni Genome by SMRT DNA Sequencing Identifies Restriction-Modification Motifs Jason L. O'Loughlin 0 1 2 3 Tyson P. Eucker 0 1 2 3 Juan D. Chavez 0 1 2 3 Derrick R. Samuelson 0 1 2 3 Jason Neal-McKinney 0 1 2 3 Christopher R. Gourley 0 1 2 3 James E. Bruce 0 1 2 3 Michael E. Konkel 0 1 2 3 0 Funding: This work was supported by funds awarded by the National Institutes of Health (NIH) to Michael E. Konkel (Award Number R56 AI088518- 01A1). Jason L. O'Loughlin was supported, in part, by funds awarded by the National Institutes of Health T32 Training program in Infectious Diseases and Microbial Immunology (Award Number 5 T32 AI 1 Data Availability Statement: All the data for this paper is available from the following sources: the genome is available from NCBI (BioProject ID: PRJNA222831) and proteomic analysis is available from UniProt (entry CAMJE) 2 Academic Editor: Qijing Zhang, Iowa State University , UNITED STATES 3 1 School of Molecular Biosciences, College of Veterinary Medicine, Washington State University , Pullman, Washington , United States of America, 2 Department of Genome Sciences, University of Washington , Seattle, Washington , United States of America Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. The goal of this study was to analyze the C. jejuni F38011 strain, recovered from an individual with severe enteritis, at a genomic and proteomic level to gain insight into microbial processes. The C. jejuni F38011 genome is comprised of 1,691,939 bp, with a mol.% (G+C) content of 30.5%. PacBio sequencing coupled with REBASE analysis was used to predict C. jejuni F38011 genomic sites and enzymes that may be involved in DNA restriction-modification. A total of five putative methylation motifs were identified as well as the C. jejuni enzymes that could be responsible for the modifications. Peptides corresponding to the deduced amino acid sequence of the C. jejuni enzymes were identified using proteomics. This work sets the stage for studies to dissect the precise functions of the C. jejuni putative restrictionmodification enzymes. Taken together, the data generated in this study contributes to our knowledge of the genomic content, methylation profile, and encoding capacity of C. jejuni. - Campylobacter jejuni is an important pathogen causing significant morbidity and mortality. C. jejuni is a Gram-negative, comma-shaped, microaerophilic bacterium, and is motile by means of unipolar or bipolar flagella. The genus Campylobacter was proposed in 1963, separating these bacteria from Vibrio-like organisms based on morphology, DNA composition, microaerobic growth requirement, and non-fermentive metabolism [1]. C. jejuni was first isolated from human feces in 1972 [2]. Human infection, also called campylobacteriosis, often occurs after handling or ingesting food contaminated by raw poultry products. Clinical infection with C. jejuni presents as diarrhea with blood and leukocytes, fever, nausea, and severe abdominal cramps that occur 25 days following ingestion [3, 4]. In recent years, infection rates with Campylobacter spp. has been comparable to or has exceeded that of other enteric pathogens, including Salmonella spp. and Shigella spp. The highest prevalence of Campylobacter-mediated disease is among children less than 7025-33). Tyson P. Eucker was supported, in part, by funds awarded by the National Institute of General Medical Sciences (NIGMS, Award Number T32GM008336 and T32GM083864). Derrick R. Samuelson was supported, in part, by funds awarded by NIGMS (T32GM083864). James E. Bruce and Juan D. Chavez were supported by funds awarded by the NIH (Award Number 5R01GM086688). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 5 years of age [5]. Trials with human volunteers have demonstrated that the infectious dose of C. jejuni is as few as 500 organisms, although there is no clear correlation between dose and disease severity. Although campylobacteriosis is usually self-limiting, erythromycin and azithromycin can be used to treat individuals with severe enteritis. Infection with some C. jejuni isolates correlates with a higher incidence of Guillain-Barr syndrome (GBS), an autoimmune disease and the most common cause of flaccid paralysis in the post-polio era [6]. The association between C. jejuni infections and GBS is due to molecular mimicry resulting in a cross-reactive immune response between Campylobacter lipo-oligosaccharides (LOS) and neural gangliosides [7, 8]. In the United States, treatment of C. jejuni infections and GBS is estimated to cost $1.2 billion per year. Advances in sequencing technology have resulted in an increase in the number of C. jejuni genomes available to researchers [7, 9, 10]. Deciphering the C. jejuni pan genome, which is comprised of both the core and variable genes, coupled with the epidemiology and ecology data has led to a deeper understanding of the genomic content and how this can influence the environmental niche and transmission of individual isolates. For example, comparative genomic analysis has demonstrated variability in the genomic content of C. jejuni strains, possibly influenced by the host from which the isolate was recovered (e.g., human, chicken, ruminant, etc.) and geographical location. More specifically, the genomes of C. jejuni human isolates are more similar to non-livestock isolates (environmental) than to livestock isolates, as assessed by DNA microarray analysis of whole-genome comparisons of 111 isolates [11]. This type of analysis will also allow for improved risk-based approaches for implementing efficient C. jejuni-mediated disease control measures in the environment, at the farm, and in retail consumer meat [12, 13]. Methylation of the genome plays important biological roles in bacteria. Several decades ago it was determined that methylation is an important component of restriction-modification (RM) systems that prevent foreign DNA, including phage DNA, from integrating into the bacterial genome. The DNA in the host is marked through sequence-specific methylation of adenine and cytosine nucleotides. Most RM systems fall into one of four categories, Types IIV, although non-classical RM systems have been discovered that are not as easily categorized [14]. Classification is based on the number of genes/subunits, methylation and restriction activities performed, co-factors required for activity, recognition sequence, cleavage site, and mechanism of cleavage. Type I RM systems are composed of three different enzymes, designated R, S or M, which are responsible for endonuclease activity, determining the sequence specificity of methylation and cleavage, and methylation of the DNA substrate, respectively. The recognition sequence is both bipartite and asymmetrical with cleavage occur (...truncated)