Large scale changes in host methylation patterns induced by IncA/C plasmid transformation in Vibrio cholerae

DNA methylation is a central epigenetic modification and has diverse biological functions in eukaryotic and prokaryotic organisms alike. The IncA/C plasmid genomes are approximately 150kb in length and harbour three methylase genes, two of which demonstrate cytosine specificity. Transformation of the Vibrio cholerae strain C6706 with the IncA/C plasmid pVC211 resulted in a significant relabelling of the methylation patterns on the host chromosomes. The new methylation patterns induced by transformation with IncA/C plasmid were accepted by the restriction enzymes of the host’s restriction modification (RM) system. These data uncover a novel mechanism by which plasmids can be compatible with a host’s RM system and suggest a possible reason that plasmids of the IncA/C family are broad-host-range. Author summary Antibiotic resistance of bacteria is a growing serious problem worldwidely and the horizontal transfer of multi-drug resistance genes mediated by plasmids within and between species of bacteria is the main reason. In the researches of multi-drug resistance of Vibrio cholerae, I have isolated several IncA/C plasmids. What impressed me most is their ability to accumulate the resistant genes. Moreover, they can transfer with high frequency and are stable in several bacterial species. There are at least three Tra regions on the IncA/C plasmid which containing components of the Type 4 Secretion System and are important for conjugative transfer of plasmids. So the horizontal transfer ability of IncA/C plasmids is reasonable. There are three methylase genes on the small genome of IncA/C plasmids, which demonstrate cytosine specificity and are seldom in bacteria. Their modification target and roles are interesting. Here, we analysed the methylation profiles of the host V. choerae induced by the plasmid pVC211 and found that they were completely changed. In addition to replicons, this may be a novel mechanism that plasmid cross the barrier of the host’s RM system and become broad-host range. Changing the activity of methylase in IncA/C plasmids may be a new way to affect the stability of IncA/C plasmids to eliminate these multidrug-resistant plasmids from bacteria.

eliminate these multidrug-resistant plasmids from bacteria.

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IncA/C family plasmids are important carriers of multi-drug resistance genes in 51 plasmid-incompatible groups (Inc) and mediate the dissemination of multi-drug 52 resistance in bacteria [1,2]. Both of the plasmid pIP1202, which has high resistance to 53 at least eight antibiotics isolated from Yersinia pestis IP275 in 1995 [3], and the 54 plasmid of NDM-1 super-resistant bacteria in India, Bangladesh, Pakistan, Britain and 55 the United States in August 2010 [4] are IncA/C family plasmids and have aroused 56 great concern in public health and bioterrorism. IncA/C plasmids have great ability to 57 accumulate antibiotic resistant genes. There are many resistance genes of rifampicin, 58 erythromycin, streptomycin, chloramphenicol, sulfonamides and disinfectants 59 routinely harbored on the plasmids [5], as well as a variety of beta-lactamase genes.

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Common methylation modifications cells are 6-methyladenine ( m6 A), 4-120 methylcytosine ( m4 C), and 5-methylcytosine ( m5 C) [14]. There are two ways to 121 measure DNA methylation on a genome-wide scale: bisulfite sequencing and single-122 molecule, real-time (SMRT) sequencing. Bisulfite sequencing is commonly used to 123 detect m5 C. In this method, treatment of DNA with bisulfite converts cytosine residues 124 to uracil, leaving 5-methylcytosine residues unaffected. The methylation status of a 125 fraction of DNA can be determined by the analysis performed on such alterations.

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SMRT sequencing identifies bases using kinetic information recorded during each 127 nucleotide addition step, which can be used to distinguish modified and native bases 128 by comparing with the kinetic reference for dynamics without modification. Different 129 epigenetic modification types have unique kinetic signatures and can be inferred, 130 including the methylation at each GATC site. But because the m5 C modification 131 produces weaker and somewhat more diffuse SMRT signals and requires 10 fold 132 sequencing coverage, only all predicted m6 A and m4 C MTases are able to be detected 133 unambiguously in this way [14,15]. In this study, the two complementary methods  Table 2). The methylation of the two chromosomes of V. cholerae 151 was considerably different. The genes on chromosome I were generally methylated, 152 dominated by m4 C-m6 A double methylation, and sites involving m4 C and m6 A 153 accounted for 84.7% (3401/4013) of the total genes. On the other hand, more than 154 half of the genes on chromosome Ⅱ exhibited non-m4 C non-m6 A methylation. In 155 particular, the methylation of the tRNA gene was the most significantly different 156 between the two chromosomes, with 92 of the 94 genes on chromosome I involved in were 0.33%, 1.74% and 0.43% in C6706 and 0.34%, 1.81% and 0.44% in CV2.

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Moreover, the graphs of the methylation levels also showed a slightly increasing trend 205 from C6706 to CV2 (Fig. 1A). For example, 50% of mCHH sites were 10% 206 methylated in C6706; this was increased in CV2 as 59% of mCHH sites were 20% 207 methylated. Notably, though the total number of mCG sites was the most similar 208 between C6706 and CV2, their methylation levels were greatly different. The 209 methylation levels at mCG sites changed from 68% mCG sites 10% methylated, 32% 210 mCG sites 20% methylated in C6706 to 19% mCG sites 10% methylated, 57% mCG 211 sites 20% methylated, and 18% mCG sites 30% methylated in CV2 (Fig. 1A).    repaired. Both sides of the DNA fragments were respectively connected with hairpin 319 adapter to get a dumbbell (set of horse ring) structure, which known as SMRTbell.