Identification of a cytosine methyltransferase that improves transformation efficiency in Methylomonas sp. DH-1

Background Industrial biofuels and other value-added products can be produced from metabolically engineered microorganisms. Methylomonas sp. DH-1 is a candidate platform for bioconversion that uses methane as a carbon source. Although several genetic engineering techniques have been developed to work with Methylomonas sp. DH-1, the genetic manipulation of plasmids remains difficult because of the restriction-modification (RM) system present in the bacteria. Therefore, the RM system in Methylomonas sp. DH-1 must be identified to improve the genetic engineering prospects of this microorganism. Results We identified a DNA methylation site, TGGCCA, and its corresponding cytosine methyltransferase for the first time in Methylomonas sp. DH-1 through whole-genome bisulfite sequencing. The methyltransferase was confirmed to methylate the fourth nucleotide of TGGCCA. In general, methylated plasmids exhibited better transformation efficiency under the protection of the RM system than non-methylated plasmids did. As expected, when we transformed Methylomonas sp. DH-1 with plasmid DNA harboring the psy gene, the metabolic flux towards carotenoid increased. The methyltransferase-treated plasmid exhibited an increase in transformation efficiency of 2.5 × 103 CFU/μg (124%). The introduced gene increased the production of carotenoid by 26%. In addition, the methyltransferase-treated plasmid harboring anti-psy sRNA gene exhibited an increase in transformation efficiency by 70% as well. The production of carotenoid was decreased by 40% when the psy gene was translationally repressed by anti-psy sRNA. Conclusions Plasmid DNA methylated by the discovered cytosine methyltransferase from Methylomonas sp. DH-1 had a higher transformation efficiency than non-treated plasmid DNA. The RM system identified in this study may facilitate the plasmid-based genetic manipulation of methanotrophs.

For efficient genetic engineering, genetic manipulation tools have been developed to work with methanotrophs [14][15][16][17]. Recently, the type l Methylomonas sp. DH-1 was isolated from brewery waste sludge, and several engineering tools have been developed [18]. This bacterium has been favored in diverse examples of metabolic engineering: the conversion of methane to methanol [18] and the production of value-added chemicals such as acetone [19,20], succinate [21], and d-lactate [22].
The first hurdle in genetic engineering is to develop an efficient transformation method. In prokaryotes, DNA methylation and degradation by restrictionmodification (RM) systems, which are rudimentary bacterial immune systems, are yet to be identified [23]. Usually, foreign DNA is not methylated and is thus destroyed by host restriction enzymes. The methylation of particular sequences in the host genome protects those sequences from cleavage by host restriction enzymes [24,25]. A previous study showed that 88% of bacterial genomes contain RM systems and that 44% of bacterial genomes carry four or more RM systems [26]. Recently, the process of DNA methylation is utilized for epigenetic regulation [27] and nanopore sequencing [28].
Although several genetic manipulation techniques have been developed to metabolically engineer Methylomonas sp. DH-1 [29], the low transformation efficiency due to the inherent RM system has been an obstacle. In this study, we aimed to identify the RM system in Methylomonas sp. DH-1 and use it for enhanced genetic manipulation with plasmid DNA. Discovering the RM system of Methylomonas sp. DH-1 would enable the establishment of transformation techniques for efficient genetic manipulation.

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 TGG CCA motif was identified (Fig. 1a). In the REBASE database [30,31], Methylomonas sp. DH-1 contains 12 RM systems in its genome and two in its native plasmid (Fig. 1b). According to REBASE, it was predicted that the cytosine methyltransferase AYM39_01025 (Additional file 1) would recognize the GGCC sequence for methylation, which is similar to the identified methylation site TGG CCA, in which the fourth nucleotide (C) was methylated in our results. Therefore, this cytosine methyltransferase was selected as a potential methylase for TGG CCA .

Digestion protection assay
To investigate whether the selected cytosine methyltransferase (AYM39_01025) was able to recognize the identified sequence (TGG CCA ) instead of the predicted sequence (GGCC), we conducted a DNA protection assay against digestion, using several restriction enzymes. When the cytosine methyltransferase protein was overexpressed 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 Fig. 1 Identification of a methylation site and its corresponding methyltransferase in Methylomonas sp. DH-1. a The TGG CCA methylation site was the only site discovered by WGBS. b The potential methyltransferases in Methylomonas sp. DH-1. The REBASE-predicted methyltransferases in its genome and native plasmid are shown methyltransferase gene and TGG CCA sites into the E. coli 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 E. coli JM110 strain.
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 TGG CCA . To confirm that the cytosine methyltransferase recognized TGG CCA instead of GGCC, several restriction enzymes that contain GGCC in their restriction sites were used: MscI (TGGCC A), ApaI (GGGCC C), and NotI (GCGGCC GC). We also used EcoRl (GAA TTC ) and Xbal (TCT AGA ) 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 TGG CCA 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, TGG CCA in the non-methylated plasmid was converted to TGG TTA, indicating that the cytosines were non-methylated, as expected. In the methylated plasmid, only the fifth cytosine in TGG CCA 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 Additional file 2) and cotransformed into E. coli JM110 with the plasmid harboring the cytosine methyltransferase gene. 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 (Fig. 3b). The non-methylated plasmid was also extracted from the cell without 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. Since there are no artificial plasmids that exist separate from the genome of Methylomonas sp. DH-1, we measured the genome integration efficiencies of the psy gene involved in the carotenoid biosynthetic pathway (Fig. 4a) to deduce the transformation efficiency. The transformation efficiency of the methylated DNA of the psy plasmid was The conversion of non-methylated and methylated TGG CCA sequences during bisulfite sequencing. Only non-methylated cytosines are converted to thymines during bisulfite sequencing and PCR. The left-hand figure shows that the two cytosines were converted to uracils, which means there were no methylated cytosines. Conversely, the right-hand figure shows that the fourth cytosine was not changed to uracil, indicating that it was methylated by the cytosine methyltransferase 2.5 × 10 3 CFU/μg of DNA. The efficiency was increased by 124% compared with that of the non-methylated plasmid DNA (Fig. 4b). Despite of methylation, the efficiency increase was not dramatic. We think that the introduced plasmids were easily integrated into the genome of Methylomonas sp. DH-1 by recombinases and thus the protection of plasmids by methylation was not essential in genome integration experiments. However, to date there are no artificial plasmids available for Methylomonas sp. DH-1, which is independent from its genome. For the development of artificial plasmids, the protection of plasmids by methylation is essential and the identification of a cytosine methylation system is the first step for the development. In this regard, the newly identified methylation system would facilitate the development of artificial plasmids as well as other biotechnological techniques based on plasmids.
For further evaluation of methylation effect on transformation, we removed the three methylation sites in the psy plasmid by mutating nucleotides: one in an intergenic region and two in the coding region of psy. The former site was converted from TGG CCA to TGT CCA , and the latter two were converted from GTG GCC AAT to GTA GCG AAT and from CTG GCC AAA to CTA GCG AAA based on codon degeneracy not to mutate amino acids (Additional file 3: Fig S1a). As shown in Additional file 3: Fig. S1b, the deletion of the methylation sites increased transformation efficiency similar to that of the methylated psy plasmid. This indicates that methylation of plasmid DNA by the identified cytosine methyltransferase protects plasmid DNAs from the RM system of Methylomonas sp. DH-1 and increases the transformation efficiency, which may facilitate the genetic manipulation of Methylomonas sp. DH-1.
To investigate the effects of plasmid size and methylation on transformation efficiency, we constructed three different plasmids with a different length (5-7 kb). As shown in Additional file 3: Fig. S2, methylation increased transformation efficiency while plasmid length did not show any significant effect on transformation efficiency. This indicates that transformation efficiency is dependent on methylation, not plasmid size. Furthermore, we measured the growths of non-transformed wild-type cells and transformed cells with methylated plasmids (the psy plasmid or anti-psy sRNA plasmid). As shown in Additional   Fig. 3 Plasmid maps of the constructed plasmids. a The cytosine methyltransferase-containing plasmid and psy-containing plasmid are shown. The psy-containing plasmid was constructed in a proof-of-concept metabolic engineering process to increase the metabolic flux towards carotenoid, and the cytosine methyltransferase-containing plasmid was used to methylate the psy-containing plasmid. The psy gene was under the control of the mxaF promoter, encoding subunit of methanol dehydrogenase [32], which was predicted using Promoter Hunter [37]. b The overall strategy of plasmid methylation in E. coli (JM110) and transformation into Methylomonas sp. DH-1. The first step was to methylate the target plasmid using cytosine methyltransferase, and the second step was to transform it into Methylomonas sp. DH-1 after separation from the methylase-containing plasmid file 3: Fig. S3, their growth rates are very similar, showing that there could be no significant changes in cellular physiology.
Methylomonas sp. DH-1 carries a complete MEP pathway for carotenoid production [19]. 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 [32] (Fig. 4a). When the plasmid containing the psy gene was introduced into Methylomonas sp. DH-1, the psy gene was integrated into the genome by homologous recombination. The additional copy of the psy gene increased carotenoid biosynthesis by 26% (Fig. 4c).
Synthetic sRNAs have been utilized to increase the production of desired substances by regulating gene expression [33]. Synthetic sRNAs were designed to bind to the nucleotides in the translation initiation regions of mRNAs, and thereby they repressed the translation of mRNAs by preventing the binding of ribosomes in assistance of Hfq protein [34]. In this study, we constructed a plasmid containing an anti-psy synthetic sRNA gene to investigate the methylation effect on transformation efficiency and also to investigate the knock-down effect of the psy gene on carotenoid production ( Fig. 5a and Additional file 4).
Synthetic sRNAs are composed of two elements: a target binding region and a scaffold. In previous studies, various scaffolds originated from E. coli were used including MicC and SgrS [33,35]. Of many inherent sRNA genes, RyhB was known to operate in the Fig. 4 The transformation efficiency of methylated plasmid DNAs. a The overall biosynthetic pathway towards carotenoid. The psy gene is indicated. b Transformation efficiencies of non-methylated plasmids (light gray bar) and methylated plasmids (dark gray bar) in Methylomonas sp. DH-1. The maps of the two plasmids are shown in Fig. 3a. Standard deviations were calculated from triplicates. The asterisk (*) denotes p values < 0.05. c Carotenoid intensity in Methylomonas sp. DH-1 cells after transformation with the methylated psy plasmid. The intensity was measured using multi-detection microplate reader, and the carotenoid intensity was obtained 8 h after cultivation. Standard deviations were calculated from triplicates absence of Hfq protein [36] and thus was expected to work in various bacterial species. We designed an antipsy synthetic sRNA using the scaffold of RyhB sRNA. The anti-psy synthetic sRNA gene was under the control of tac promoter.
When the plasmid harboring the anti-psy synthetic sRNA gene was transformed after methylation, its transformation efficiency was enhanced by 70% compared with the non-methylated plasmid (Fig. 5b). In addition, when the synthetic sRNA gene was integrated into the genome of Methylomonas sp. DH-1, the synthetic sRNA decreased carotenoid production by 40% compared with that of wild-type Methylomonas sp. DH-1 (Fig. 5c). These results indicate that methylation of plasmids can improve transformation efficiency as well as that synthetic sRNAs based on a RyhB scaffold can be used to regulate the expression of genes in Methylomonas sp. DH-1.

Conclusions
In this study, we identified a novel cytosine methyltransferase and its methylation site for the first time in Methylomonas sp. DH-1. The methylase was utilized to increase transformation efficiency by protecting plasmid DNAs from the RM system of Methylomonas sp. DH-1. Transformation is the first barrier in the genetic manipulation of bacteria, and with the aid of the methylase, the transformation barrier was effectively overcome. The use of the methylase for methylating insertional genes may facilitate the metabolic engineering of value-added products in Methylomonas sp. DH-1.

Strains, antibiotics, primers, and culture conditions
The E. coli DH5α strain was used for gene cloning and plasmid preparation, and the E. coli JM110 strain, traD36 lacI q Δ (lacZ) M15 proA + B + IrpsL (Str r ) thr leu thi lacY galK galT ara fhuA dam dcm glnV44 Δ(lac-proAB) was used for plasmid methylation. E. coli cells were cultured in Luria-Bertani (LB) broth (1% tryptone, 0.5% yeast extract, and 1% NaCl) or on LB plates (1.5% w/v agar) at 37 °C in the presence of appropriate antibiotics (25 μg/ mL of chloramphenicol and/or 10 μg/mL of Ampicillin). A cytosine methyltransferase (Additional file 1) expression vector was then constructed, and its expression was regulated by IPTG. For methylation, 0.1 mM IPTG was used. Next, the psy plasmid was constructed.
Methylomonas sp. DH-1 was cultured in a nitrate mineral salt (NMS) medium containing 10 μM CuCl 2 ·2H 2 O as described previously [18]. Methylomonas sp. DH-1 cells were cultured in a 500 mL baffled flask sealed with a screw cap containing 100 mL of NMS medium at 30 °C and 250 rpm. Methane was supplied to a final concentration of 30% (v/v) by gas substitution using a gas-tight syringe, and the headspace was refreshed daily. During carotenoid intensity measurement, methanol (0.1%) was used as a carbon source instead of methane because the Methylomonas sp. DH-1 cells were cultured in a 96-well plate.

Whole-genome bisulfite sequencing and plasmid construction
A whole-genome bisulfite sequencing of Methylomonas sp. DH-1 was carried out according to the manufacturer's instruction [39]. Briefly, genomic DNA of Methylomonas sp. DH-1 was extracted and was treated with bisulfite to convert unmethylated cytosines to uracils while retaining those which were methylated. Bisulfite-treated ssDNA  [38]. b Transformation efficiencies of the non-methylated plasmid (light gray bar) and methylated plasmid (dark gray bar) in Methylomonas sp. DH-1. Standard deviations were calculated from triplicates. The asterisk (*) denotes p values < 0.05. c Carotenoid intensity (arbitrary unit) in Methylomonas sp. DH-1 cells. The intensity was measured using multi-detection microplate reader, and the carotenoid intensity was obtained 8 h after cultivation. Standard deviations were calculated from triplicates. The asterisk (*) denotes p values < 0.05 fragments were randomly primed using a polymerase that can read uracil nucleotides to synthesize DNA strands containing a specific sequence tag. The 3′ ends of the newly synthesized DNA strands were then selectively tagged with a second sequence tag. This process generated di-tagged DNA strands with known tags at their 5′ and 3′ ends. The di-tagged DNA strands were enriched by PCR, resulting in dsDNA strands.
After sequencing, the raw sequence reads were filtered based on quality. Trimming process was done to eliminate adapter sequences and bases with low quality from each read using Trimmomatic program [40]. The bases with low quality or N bases less than quality 3 from the ends of reads were trimmed. Also, using a sliding window method, bases of reads that did not qualify for a 4-base wide sliding window, and the average quality per window below 15 were trimmed. Afterwards, reads with length shorter than 36 bp were dropped to produce cleaned data. The trimmed reads were mapped to a reference genome (Methylomonas sp. DH-1, ASM164468v1) with BSMAP based on SOAP (Short Oligo Alignment Program), which is a short reads mapping program for bisulfite sequencing in DNA methylation study [41]. The nucleotides around methylated cytosines (− 10 ~ + 10 nt) were extracted and consensus sequence motifs were identified using MEME [42]. As a result, one single motif was identified (TGG CCA ). The raw data files obtained from bisulfite sequencing are available to download at http://ssbio .cau.ac.kr/publi c/DH-1_1.fastq .gz and http:// ssbio .cau.ac.kr/publi c/DH-1_2.fastq .gz.

Methylomonas sp. DH-1 electroporation
Methylomonas sp. DH-1 was grown in a nitrate mineral salt (NMS) plate containing 10 μM CuCl 2 ·2H 2 O as described previously [23]. Cells were collected from plates using a spreader and resuspended in distilled water (DW) to make OD 600 = 0.8. 10 mL of the resuspended cells were harvested by centrifugation at 5000 rpm at 4 °C for 10 min. The pellet was washed with 10 mL of DW, transferred to a 15 mL conical tube, and centrifuged again at 5000 rpm at 4 °C for 10 min. The resulting pellet was resuspended in 100 μL of DW and placed on ice. Fifty microliters of the resuspended cells were gently mixed with plasmid 500 ng of DNA (3-5 μL), and the mixture was transferred to an ice-cold 1 mm micropulser electroporation cuvettes (Bio-Rad, Hercules, California 94547, United States). Electroporation was performed using a micropulser electroporator (Gene Pulser II System, Bio-Rad, Herules, California 94547, United States) at 25 μF and 200 Ω. After electrical discharge, 1 mL of NMS medium was immediately added to the cells. The cells were transferred into a 250 mL serum bottle with 10 mL of NMS medium and then incubated with 0.02% methane gas. After incubation at 30 °C for 4 h, the cells were collected by centrifugation at 5000 rpm for 10 min at 25 °C. The cells were resuspended with 1 mL of NMS medium and spread onto selective NMS plates.

Carotenoid measurement
Methylomonas sp. DH-1 transformed with the psy plasmid (Additional file 2) was cultured in NMS medium containing methanol (0.1%) at 30 °C until the stationary phase was reached. The Methylomonas sp. DH-1 was transferred to a 96-well plate containing 200 μL of NMS medium and grown in a shaking format at 30 °C. The OD 450 absorbance at 8 h was measured to infer the relative carotenoid concentration using a multi-detection microplate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA, USA).