Identification of Methylomonas sp. DH-1 methylation site
To identify the RM system, the genome of Methylomonas sp. DH-1 was analyzed by whole-genome bisulfite sequencing (WGBS). Interestingly, only the TGGCCA motif (Fig. 1) was identified. In the REBASE database [28, 29], Methylomonas sp. DH-1 contains twelve RM systems in its genome and two in its native plasmid (Fig. 1). According to REBASE, it was predicted that the cytosine methyltransferase AYM39_01025 would recognize the GGCC sequence for methylation, which is similar to the identified methylation site TGGC*CA, in which the fourth nucleotide (C) was methylated in our results. Therefore, this cytosine methyltransferase was selected as a potential methylase for TGGCCA.
Digestion protection assay
To investigate whether the selected cytosine methyltransferase (AYM39_01025) was able to recognize the identified sequence (TGGCCA) instead of the predicted sequence (GGCC), we conducted a DNA protection assay against digestion, using several restriction enzymes. When the cytosine methyltransferase protein was over-expressed in E. coli BL21 (DE3), the protein formed an inclusion body even though it was co-expressed with chaperones (pGro7 and pTf16). Thus, we could not perform the in vitro assay requiring purified methyltransferase. Instead, we introduced a plasmid harboring the methyltransferase gene and TGGCCA sites into the JM110 strain (dam and dcm methylase genes were deleted). Since the cytosine methyltransferase was under the control of the T5 promoter with a lac operator, we could obtain a non-methylated or methylated plasmid by IPTG. For further analysis, the plasmid was extracted from JM110.
According to the REBASE annotations, the cytosine methyltransferase of Methylomonas sp. DH-1 was predicted to methylate the GGCC sequence, while the only methylation site identified in Methylomonas sp. DH-1 by WGBS was TGGCCA. To confirm that the cytosine methyltransferase recognized TGGCCA instead of GGCC, several restriction enzymes that contain GGCC in their restriction sites were used: MscI (TGGCCA), ApaI (GGGCCC), and NotI (GCGGCCGC). We also used EcoRl (GAATTC) and Xbal (TCTAGA) restriction enzymes as negative controls. The plasmid harboring the cytosine methyltransferase gene contained all of the above-mentioned restriction sites, as well. If the methylation site was GGCC, the restriction enzymes (MscI, ApaI, and NotI) would not be able to cleave the plasmid DNA. As shown in (Fig. 2a), most restriction enzymes were able to cleave both the non-methylated and methylated plasmids, but MscI failed to cleave the methylated plasmid. This result indicated that the cytosine methyltransferase recognized TGGCCA and not GGCC.
To identify the cytosine nucleotide methylated by the cytosine methyltransferase, the methylated plasmid was analyzed by bisulfite sequencing. In bisulfite sequencing, only non-methylated cytosines are converted to uracil, and during PCR, the uracil is converted to T. Methylated cytosines are not changed by bisulfite sequencing. As shown in (Fig. 2b), TGGCCA in the non-methylated plasmid was converted to TGGTTA, indicating that the cytosines were non-methylated, as expected. In the methylated plasmid, only the fifth cytosine in TGGCCA was converted to T, indicating that the fourth cytosine was methylated by the cytosine methyltransferase.
Methylation of plasmid DNA increased transformation efficiency
The plasmid harboring the psy (phytoene synthase) gene was constructed (Fig. 3a) and co-transformed into E. coli JM110 with the plasmid harboring the cytosine methyltransferase gene psy. The psy gene is involved in the biosynthetic pathway that produces carotenoids. For the methylation of the plasmid containing psy, the media were supplemented with 0.1 mM IPTG to induce the expression of the cytosine methyltransferase. Since E. coli contains two plasmids (psy plasmid + cytosine methyltransferase plasmid), the plasmids were separated by gel electrophoresis, and the psy plasmid was extracted from the gel. The non-methylated plasmid was also extracted from the cell without added IPTG to create a control sample in which the expression of the cytosine methyltransferase was not induced.
The extracted plasmids were transformed into Methylomonas sp. DH-1 by electroporation. The transformation efficiency of the methylated DNA of the psy plasmid was 3.9 × 103 CFU/µg. The efficiency was increased by 110% compared with that of the non-treated plasmid DNA (Fig. 4b). This result indicated that the methylation of plasmid DNA by the identified cytosine methyltransferase protected the plasmid from the RM system of Methylomonas sp. DH-1. Consequently, the methylation increased the transformation efficiency, which may facilitate the genetic manipulation of Methylomonas sp. DH-1.
Methylomonas sp. DH-1 carries a complete MEP pathway for carotenoid production . The selected gene, psy, is involved in the carotenoid biosynthetic pathway. The gene was designed to be expressed by the promoter of the mxaF gene  (Fig. 4a). When the gene was introduced into the genome of Methylomonas sp. DH-1, psy increased carotenoid synthesis by 7.2% (Fig. 4c). This result indicated that the methylation of plasmid DNA by cytosine methyltransferase would be useful in the metabolic engineering of Methylomonas sp. DH-1.
(a) The overall biosynthetic pathway towards carotenoid. The psy gene is indicated. (b) Transformation efficiency of non-methylated plasmids (light gray bar) and methylated plasmid (dark gray bar) in Methylomonas sp. DH-1. The map of the two plasmids is shown in Fig. 3a. Standard deviations were calculated from triplicates. The asterisk (*) denotes p-values < 0.01. (c) Carotenoid intensity in Methylomonas sp. DH-1 cells after transformation with the methylated plasmid. The intensity was measured by using multi-detection micro-plate reader, and the carotenoid intensity was obtained 8 h after cultivation. Standard deviations were calculated from triplicates.