position Modificati on type No. of motifs detected No. of motifs in genome % of motifs detected Mean Score Mean coverage AGGAG 4 m6A 1512 1515 99.80 112.60 83.42 CACAT 4 m6A 2437 2438 99.96 120.87 82.99 CATG 2 m6A 15320 15320 100.00 122.22 83.38 CCATC 3 m6A 2115 2115 100.00 126.46 83.66 CCGG 1 m4C 3547 3552 99.86 79.26 85.24 CCTYNA 6 m6A 269 8388 3.21 119.43 84.49 GAATTC 3 m6A 352 352 100.00 118.20 79.53 GACY 2 m6A 8225 8228 99.96 114.79 83.87 GAGG 2 m6A 5028 5055 99.47 104.19 83.55 GATC 2 m6A 10994 10994 100.00 128.41 83.91 GATGG 2 m6A 2115 2115 100.00 119.72 83.56 GCGC 2 m5C 1009 13046 7.73 51.04 86.19 GTSAC 4 m6A 250 250 100.00 117.48 85.38 TCNNGA 6 m6A 3983 3986 99.92 115.11 84.08 TGCA 4 m6A 10462 10466 99.96 118.71 84.08
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5.2. Manuscript II
The core genome
m5C methyltransferase JHP1050 (M.Hpy99III) plays an important role in
orchestrating gene expression in Helicobacter pylori
Iratxe Estibariz, Annemarie Overmann, Florent Ailloud, Juliane Krebes, Christine Josenhans, and Sebastian Suerbaum
Published in:
Nucleic Acids Research 2019; Mar 18; 47(5):2336-2348 (doi: 10.1093/nar/gky1307)
Short summary
In this article, we functionally characterized the role of a highly conserved m5C-MTase in H. pylori present and putatively active in all strains. We studied the impact of this MTase on the transcriptomes of two H. pylori wild-type and respective MTase mutant strains, and showed that the MTase had both strain-specific and conserved effects. Methylation of motifs overlapping promoter sequences had a direct impact on gene expression. Methylation affected several phenotypic traits like adhesion to host cells, natural competence, copper resistance, and bacterial morphology.
Nucleic Acids Research, 2019 1 doi: 10.1093/nar/gky1307
The core genome
m5C methyltransferase JHP1050
(M.Hpy99III) plays an important role in orchestrating
gene expression in
Helicobacter pylori
Iratxe Estibariz1,2,3, Annemarie Overmann1, Florent Ailloud1,2,3, Juliane Krebes2, Christine Josenhans1,2,3,* and Sebastian Suerbaum1,2,3,*
1Medical Microbiology and Hospital Epidemiology, Max von Pettenkofer Institute, Faculty of Medicine, LMU Munich,
M ¨unchen, Germany,2Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School,
Hannover, Germany and3German Center for Infection Research (DZIF), Munich Site, Munich, Germany
Received August 15, 2018; Revised December 18, 2018; Editorial Decision December 19, 2018; Accepted December 21, 2018
ABSTRACT
Helicobacter pylori encodes a large number of restriction–modification (R–M) systems despite its small genome. R–M systems have been described as ‘primitive immune systems’ in bacteria, but the role of methylation in bacterial gene regulation and other processes is increasingly accepted. Every H. pylori strain harbours a unique set of R–M systems resulting in a highly diverse methylome. We identi- fied a highly conserved GCGC-specific m5C MTase
(JHP1050) that was predicted to be active in all of 459H. pylorigenome sequences analyzed. Transcrip- tome analysis of two H. pylori strains and their re- spective MTase mutants showed that inactivation of the MTase led to changes in the expression of 225 genes in strain J99, and 29 genes in strain BCM- 300. Ten genes were differentially expressed in both mutated strains. Combining bioinformatic analysis and site-directed mutagenesis, we demonstrated that motifs overlapping the promoter influence the ex- pression of genes directly, while methylation of other motifs might cause secondary effects. Thus, m5C
methylation modifies the transcription of multiple genes, affecting important phenotypic traits that in- clude adherence to host cells, natural competence for DNA uptake, bacterial cell shape, and suscepti- bility to copper.
INTRODUCTION
Epigenetics denotes inheritable mechanisms that regulate gene expression without altering the DNA sequence. In prokaryotes, methyltransferases (MTases) transfer methyl groups from S-adenosyl methionine to adenines or cy-
tosines within a DNA target motif and so contribute to changes of the epigenome (1–3). MTases either belong to restriction–modification (R–M) systems that include MTase and restriction endonuclease (REase) activities, or occur as orphan MTases in the absence of a cognate restric- tion enzyme (4). Three types of DNA methylation occur in bacteria, N6-methyladenine (m6A), 5-methylcytosine (m5C)
and N4-methylcytosine (m4C) (1,2). So far, the major role
allocated to bacterial R–M systems is self-DNA protection by restriction of incoming foreign un-methylated DNA (5), and they have thus been described as ‘primitive immune systems’ (6). Other functions have also been attributed to prokaryotic R–M systems (7–9). For example, methylation marks promoter sequences and alters DNA stability and structure, modifying the affinity of DNA binding proteins and influencing the expression of genes (10,11). Addition- ally, disturbance of DNA strand separation by methylation can have an effect on gene expression (12).
Methylation can be involved in multiple bacterial func- tions. In Escherichia coli, the Dam adenine MTase plays an essential role in DNA replication (13,14). Another well- studied example is the CcrM MTase from Caulobacter cres-
centus that controls the progression of the cell cycle (15). Furthermore, phase-variable MTases have been shown to control the regulation of multiple genes in several differ- ent pathogens, including Haemophilus influenzae, Neisseria
meningitidis and Helicobacter pylori (16–18). These MTase- dependent regulons were termed phasevarions (19). As de- scribed previously, adenine methylation has been shown to play a key role in transcriptional regulation but the influ- ence of cytosine methylation in gene expression has so far only been investigated in very few studies (20–22).
Helicobacter pylori infection affects half of the world’s
population and is a major cause of gastric diseases that include ulcers, gastric cancer, and MALT lymphoma (23). This gastric pathogen has coexisted with humans since, at
*To whom correspondence should be addressed. Tel: +4989218072801; Fax: +4989218072802; Email: [email protected]
Correspondence may also be addressed to Christine Josenhans. Email: [email protected]
C
The Author(s) 2019. 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 Non-Commercial 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 [email protected]
least, 88 000 years ago (24). Helicobacter pylori strains dis- play an extraordinary genetic diversity caused in part by a high mutation rate but especially by DNA recombina- tion occurring during mixed infection with other H. pylori strains within the same stomach (25–27). The very high se- quence diversity of H. pylori and the coevolution of this pathogen with its human host have caused its separation into phylogeographic populations, whose distribution re- flects human migrations (28–30).
Despite its small genome, H. pylori is one of the pathogens with the highest number of R–M systems (31). The development of Single Molecule, Real-Time (SMRT) Sequencing technology has allowed genome-wide studies of methylation patterns and strongly accelerated the func- tional elucidation of MTases and their roles in bacterial bi- ology (32,33). Methylome studies of several H. pylori strains have revealed that every strain carries a different set of R–M systems leading to highly diverse methylomes (34–37). R– M systems in H. pylori were shown to protect the bacterial chromosome against the integration of non-homologous DNA (e.g. antibiotic resistance cassettes), while they had no significant effect on recombination between highly homolo- gous sequences, permitting efficient allelic replacement (9). Despite the diversity of methylation patterns, a small num- ber of target motifs were shown to be methylated in all (one motif, GCGC) or almost all (3 motifs protected in>99% of strains) H. pylori strains in a study by Vale et al., who tested genomic DNAs purified from 221 H. pylori strains for sus- ceptibility to cleavage by 29 methylation-sensitive restric- tion enzymes, and in those studies investigating the methy- lomes of multiple H. pylori strains (34,35,37,38). R–M sys- tems have also previously been shown to contribute to gene regulation in H. pylori; the phase-variable MTase ModH5 is involved in the control of the expression of virulence- associated genes like hopG or flaA in strain P12 (39,40).
In the present study, we functionally characterized the role of a highly conserved m5C MTase (JHP1050, M.Hpy99III) in H. pylori (41). We show the MTase gene to be part of the H. pylori core genome, present and predicted to be active in all of several hundred H. pylori strains repre- sentative of all known phylogeographic populations. Tran- scriptome comparisons of two H. pylori wild-type strains and their respective knockout mutants demonstrated that JHP1050 has a strong impact on the H. pylori transcrip- tome that includes both conserved and strain-specific regu- latory effects. We show thatm5C methylation of GCGC se- quences, among others, affects metabolic pathways, compe- tence and adherence to gastric epithelial cells. Moreover, we provide evidence that methylation of GCGC motifs over- lapping with promoter sequences can play a direct role in gene expression, while the regulatory effects of methylated sites outside of promoter regions may be indirect.