SMRT sequencing of the Campylobacter coli BfR-CA-9557 genome sequence reveals unique methylation motifs

Zautner et al. BMC Genomics (2015) 16:1088 DOI 10.1186/s12864-015-2317-3 RESEARCH ARTICLE Open Access SMRT sequencing of the Campylobacter coli BfR-CA-9557 genome sequence reveals unique methylation motifs Andreas E. Zautner1* , Anne-Marie Goldschmidt1, Andrea Thürmer2, Jörg Schuldes2, Oliver Bader1, Raimond Lugert1, Uwe Groß1, Kerstin Stingl3, Gabriela Salinas4 and Thomas Lingner4 Abstract Background: Campylobacter species are the most prevalent bacterial pathogen causing acute enteritis worldwide. In contrast to Campylobacter jejuni, about 5 % of Campylobacter coli strains exhibit susceptibility to restriction endonuclease digestion by DpnI cutting specifically 5’-GmATC-3’ motifs. This indicates significant differences in DNA methylation between both microbial species. The goal of the study was to analyze the methylome of a C. coli strain susceptible to DpnI digestion, to identify its methylation motifs and restriction modification systems (RM-systems), and compare them to related organisms like C. jejuni and Helicobacter pylori. Results: Using one SMRT cell and the PacBio RS sequencing technology followed by PacBio Modification and Motif Analysis the complete genome of the DpnI susceptible strain C. coli BfR-CA-9557 was sequenced to 500-fold coverage and assembled into a single contig of 1.7 Mbp. The genome contains a CJIE1-like element prophage and is phylogenetically closer to C. coli clade 1 isolates than clade 3. 45,881 6-methylated adenines (ca. 2.7 % of genome positions) that are predominantly arranged in eight different methylation motifs and 1,788 4-methylated cytosines (ca. 0.1 %) have been detected. Only two of these motifs correspond to known restriction modification motifs. Characteristic for this methylome was the very high fraction of methylation of motifs with mostly above 99 %. Conclusions: Only five dominant methylation motifs have been identified in C. jejuni, which have been associated with known RM-systems. C. coli BFR-CA-9557 shares one (RAATTY) of these, but four ORFs could be assigned to putative Type I RM-systems, seven ORFs to Type II RM-systems and three ORFs to Type IV RM-systems. In accordance with DpnI prescreening RM-system IIP, methylation of GATC motifs was detected in C. coli BfR-CA-9557. A homologous IIP RM-system has been described for H. pylori. The remaining methylation motifs are specific for C. coli BfR-CA-9557 and have been neither detected in C. jejuni nor in H. pylori. The results of this study give us new insights into epigenetics of Campylobacteraceae and provide the groundwork to resolve the function of RM-systems in C. coli. Keywords: Campylobacter coli, Genome, Methylation, Motifs, Methylome, Restriction modification systems, Isoschizomer digestion assay, SMRT sequencing, PacBio * Correspondence: 1 Institute for Medical Microbiology, University Medical Center Göttingen, Kreuzbergring 57, D-37075 Göttingen, Germany Full list of author information is available at the end of the article © 2015 Zautner et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zautner et al. BMC Genomics (2015) 16:1088 Background Campylobacteriosis is the most prevalent form of bacterial acute enteritis worldwide. In symptomatic cases it is characterized by a prodromal phase with fever, vomiting, and headaches followed by watery or bloody diarrhea and abdominal cramps [1, 2]. In consequence of acute enteritis, extraintestinal post-infectious sequelae, namely, the Guillain-Barré syndrome, inflammatory bowel disease, and reactive arthritis may occur [3, 4]. The average incidence reported in the European Union was 64.8 per 100,000 population in 2013 [5], in the USA 14.3 cases per 100,000 population in 2012, and in China 161 cases per 100,000 population in urban areas compared to 37 cases per 100,000 population in rural areas [6]. In Europe, 80.6 % were reported to have been caused by Campylobacter jejuni and 7.1 % by Campylobacter coli [5]. C. coli is phylogenetically subdivided into three clades [7, 8]: clade 1 isolates commonly colonize swine but can also be isolated from poultry and humans, although less frequently. Clades 2 and 3 are typically isolated from environmental waters [8, 9]. At the moment, seven completed C. coli chromosomal genome sequences [10–13], several scaffold genomes, and various contigs have been deposited in the NCBI Genome database [14–17]. The completed genome sequences, range from 1.685 to 1.872 Mb, have a G + C content of about 31 to 32 %, and contain 1715 – 1970 predicted genes including 1642 – 1861 protein coding ORFs [10–13]. One of the major epigenetic mechanisms in prokaryotes is DNA methylation [18]. DNA methylation patterns influence gene expression [19], through silencing of transcription [20, 21] as well as DNA replication initiation [22, 23] and mismatch repair [24]. DNA methylation also serves as a protection of the host genome against extraneous DNA [18] through restrictionmodification systems (RM-systems). RM-systems consist of two components: (i) a restriction endonuclease that recognizes a specific DNA motif and (ii) a cognate DNA methyltransferase that methylates the same DNA, preventing its cleavage by the restriction endonuclease [25]. The majority of RM-systems can be categorized into four types [25–29]: Type I RM-systems typically consist of three types of subunits: two restriction endonuclease subunits (R), which facilitate DNA cleavage, one specificity subunit (S) for recognition of specific DNA sequence motifs, and two DNA methylase subunits (M) that catalyse N6 adenine methylation [30, 31]. This composition enables Type I RM-systems to digest unmethylated DNA, whereas hemimethylated DNA is further methylated and fully methylated DNA is insusceptible to restriction [32]. Type II RM-systems are mostly composed of two homodimeric R subunits and a separated M subunit. The R Page 2 of 12 and M subunits recognize the same DNA motif, which is typically a 4–8 bp palindrome [33]. Type III RM-systems are comprised of two modification (Mod) subunits and two restriction (R) subunits. Type III RM-systems must bind to two inversely oriented copies of its 5–6 bp asymmetric recognition motif. Cleavage of unmethylated DNA typically occurs 25– 27 bp away from the binding sites [34]. Type IV RM-systems consist of two separate R subunits cleaving DNA that contains methylated, hydroxymethylated or glucosyl-hydroxymethylated cytosines. Cleavage typically occurs 30 bp away from (...truncated)