Next Article in Journal
Cognitive Function, Sleep, and Neuroinflammatory Markers in Mice Exposed to Very Long-Term Intermittent Hypoxia
Next Article in Special Issue
Detection of Live Shiga Toxin-Producing Escherichia coli with Long-Read Sequencing
Previous Article in Journal
Overexpression of TCP5 or Its Dominant Repressor Form, TCP5-SRDX, Causes Male Infertility in Arabidopsis
Previous Article in Special Issue
Evidence for the Worldwide Distribution of a Bile Salt Hydrolase Gene in Enterococcus faecium Through Horizontal Gene Transfer
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 26
Issue 5
10.3390/ijms26051812
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

N4-Methylcytosine Supports the Growth of Escherichia coli Uracil Auxotrophs

by
Jaunius Urbonavičius
,
Aušrinė Čekytė
and
Daiva Tauraitė
*
Department of Chemistry and Bioengineering, Vilnius Gediminas Technical University, Saulėtekio Av. 11, 10223 Vilnius, Lithuania
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(5), 1812; https://doi.org/10.3390/ijms26051812
Submission received: 29 January 2025 / Revised: 17 February 2025 / Accepted: 18 February 2025 / Published: 20 February 2025
(This article belongs to the Special Issue Molecular Research on Bacteria)
Browse Figures
Versions Notes

Abstract

N4-methylcytosine is a modified heterocyclic base present both in RNA and DNA. The biosynthesis and function of this derivative are widely investigated. However, how the demethylation of this base occurs is not known. Here, we have investigated the growth of an Escherichia coli uracil auxotroph strain in minimal M9 medium supplemented with N4-methylcytosine. We have found that this compound, but not the related N4,N4-dimethylcytosine, well supports growth with a generation time of the bacterium being 3 h compared to 1.5 h for media supplemented with cytosine or uracil. Using high-performance liquid chromatography (HPLC), we have demonstrated that the concentration of N4-methylcytosine in the growth medium decreases by 12% after 24 h of growth. We have shown that N4-methylcytosine is not directly converted into uracil by E. coli CodA cytosine deaminase. Instead, we propose the enzymatic pathway in which N4-methylcytosine is converted into cytosine by yet unknown demethylase, whereas CodA converts the resulting cytosine to uracil, thereby supporting the growth.

1. Introduction

RNA is a molecule involved in many cellular functions. To fulfill its function, different RNA species are modified by various chemical groups. The biggest variety of modifications is observed for tRNA, where more than 170 different species are found [1]. These modifications play an important role in the maintenance of tRNA structure and stability and for their localization and transport within the cell. The links between tRNA modifications, cellular stress response, protein synthesis, and disease development have also been extensively investigated, and some modifications are being used in new types of mRNA vaccines [2,3]. Despite numerous previous works on the discovery of the genes that encode the enzymes that catalyze the formation of RNA modifications and on their role in the cell, much less is known about the degradation and return to metabolism of modified nucleosides and the corresponding heterocyclic bases. Studying such enzymes is of great importance as they can be used as biocatalysts in e.g., the biosynthesis of antiviral drugs [4]. Demodification of the heterocyclic bases is often investigated at the level of RNA or DNA as part of epigenetics studies [5]. However, degradation of the corresponding modified nucleosides was investigated only in some cases. One example is the conversion of pseudouridine into uridine and then into uracil [6]. In the case of modified heterocyclic bases, the best-studied compound is 5-methyluracil (thymine), which is known to be degraded in three distinct pathways (Rut, oxidative, and reductive ones [7,8,9].
Despite the presence of the majority of modified nucleosides in tRNA, some of the modifications are found in other RNA types or in DNA. One such modification is N4-methylcytidine (m4C), found at position 1402 of 16S rRNA in the bacterium Escherichia coli. It may also be further 2′-O-methylated to form m4Cm [10]. These authors demonstrate that loss of the N4-methyl group of m4Cm1402 in the rsmH mutant that is lacking the corresponding methyltransferase leads to a 15% increase in doubling time compared with the wild-type strain. In this mutant, the efficiency of non-AUG initiation and the fidelity of translation are also affected. For the plants, it was found that in Arabidopsis chloroplasts, m4C is present at position 1352 of 16S rRNA. Loss of CMAL protein, responsible for the introduction of such modification, results not only in impaired function of chloroplasts but also affects leaf and root development [11]. It was also demonstrated that METTL15 methyltransferase is responsible for the formation of m4C at position 839 of mitochondrial 12S rRNA in human cells, and its lack impairs the translation of human mitochondria. In addition, m42C was found in the total RNA of Zika and HCV virions during the infection of human cells [12]. Recently, m42C was observed to be present at position 918 in the 16S rRNA of the archaeon Thermococcus kodakarensis [13]. In many bacteria, m4C is found as one of three (together with m5C and m6A) DNA epigenetic modifications [14]. In Helicobacter pylori, m4C DNA methylation regulates transcription and pathogenesis [15]. Recently, it was found that such DNA modification can be used as an epigenetic mark in eukaryotes, namely bdelloid rotifers, tiny freshwater invertebrates [16].
To investigate the degradation of modified nucleotides into corresponding heterocyclic bases and further demodification and return into the metabolism, we used an E. coli uracil auxotrophy-based selection system where the inability of modified heterocyclic bases to support the bacterial growth was reversed by the enzymatic activity of proteins that convert them into uracil. Using such an approach, a gene that encodes the TudS protein catalyzing the conversion of 2-thiouracil into uracil was found [17]. Our systematic screening of several naturally occurring and synthetic modified heterocyclic bases for support of the growth of E. coli uracil auxotrophs demonstrated that N4-methylcytosine, but not N4,N4-dimethylcytosine, performs this role. Below we describe the experiments that confirm our observations and propose a mechanism for the conversion of N4-methylcytosine into uracil.

2. Results and Discussion

2.1. N4-Methylhcytosine but Not N4,N4-Dimethylcytosine Supports Growth of the Uracil Auxotrophs

2.1.1. Growth of Uracil Auxotrophs in M9 Solid Medium

As the initial experiments, we have investigated several naturally occurring and synthetic pyrimidine heterocyclic bases for their role in supporting the growth of uracil auxotrophs. This assay is based on the assumption that some of the tested compounds are converted by cellular enzymes into uracil from the modified uracils directly or, as in the case of modified cytosines, via cytosine by aid of the bacterial cytosine deaminases. We found that N4-methylcytosine well supported the growth of the E. coli ∆pyrF::Km auxotroph strain compared to the other compounds tested. However, the dimethylated derivative N4,N4-dimethylcytosine did not support its growth (Figure 1). Similar growth patterns were observed for the S. typhi ∆pyrF::Tn10 uracil auxotroph, suggesting that this organism uses the same strategy for conversion of N4-methylcytosine into uracil (Figure S1). However, no growth was observed in the case of the B. subtilis ∆pyrF::erm uracil auxotroph (Figure S2).

2.1.2. Growth of the E. coli ∆pyrF::Km in M9 Agar Medium After the Serial Dilutions

For better comparison of growth rates of the E. coli ∆pyrF::Km strain in M9 solid medium supplemented with the tested above-modified cytosines, serial dilutions of cells obtained from the overnight culture were plated on the agarized M9 medium with the same modified bases and controls as described above. Figure 2 demonstrates that after 1 day (approx. 24 h) of growth, a similar growth rate was observed when either uracil or cytosine was used as substrates, whereas only some growth was observed for N4-methylcytosine. At the same time, no growth was observed when N4,N4-dimethylcytosine was used as a source of uracil or any heterocyclic base was not added. However, after 2 days (approx. 48 h), clearly visible growth in the medium with N4-methylcytosine was observed, whereas both uracil and cytosine strongly supported the growth. As after 1 day, no growth was observed when N4,N4-dimethylcytosine or no heterocyclic base was added.

2.1.3. Determination of the E. coli ∆pyrF::Km Generation Time

To determine the generation time, bacteria were grown overnight in M9 liquid medium with uracil. After harvesting and washing the cells in 0.9% NaCl, they were transferred into fresh medium supplemented with respective substrates. Afterwards, growth in each medium was spectrophotometrically measured to obtain the respective growth curves. Figure 3 demonstrates good growth of the tested strain in media containing either uracil or cytosine and somewhat slower growth for N4-methylcytosine. By contrast, very little, if any, growth was observed when N4,N4-dimethylcytosine was used as a substrate. For better comparison of the growth rates in different media, generation times were calculated and are presented in Table 1. The semi-logarithmic plots used for the calculation of generation times are presented in Figure S3. Our results indicate that in the presence of uracil or cytosine, the generation time of the E. coli ∆pyrF::Km strain in M9 medium is around 1.5 h, whereas in the presence of N4-methylcytosine, the generation time is around 3 h. However, when N4,N4-dimethylcytosine was used, a generation time of at least 33 h was obtained, suggesting that very little, if any, of this compound is consumed.
To demonstrate that N4-methylcytosine is consumed during the growth of the E. coli ∆pyrF::Km strain in M9, samples of the medium obtained during the growth of bacteria were collected and analyzed by HPLC. Figure 4 demonstrates that the concentration of N4-methylcytosine rapidly decreases during the first six hours of growth and continues to decrease during the rest of the observed growth period. HPLC chromatograms representing the decrease in the peak area are shown in Figure S4. After 24 h of growth, around 12% of N4-methylcytosine was consumed and presumably converted into cytosine, further into uracil, for supporting the growth.
One reason why the concentration of N4-methylcytosine decreases in the growth medium during the growth of bacteria could be the instability of this compound at the experimental conditions used. To test this, N4-methylcytosine or N4,N4-dimethylcytosine-containing medium was incubated at the same growth conditions without bacteria for 72 h, and concentrations of N4-methylcytosine, N4,N4-dimethylcytosine, cytosine, and uracil were measured by HPLC. Figure S5 demonstrates no degradation of N4-methylcytosine or N4,N4-dimethylcytosine and the appearance of additional cytosine and uracil peaks in the HPLC profile, attesting that E. coli cells are responsible for the decrease in the concentration of the heterocyclic base under investigation.
When looking for the enzymes capable of catalyzing the conversion of N4-methylcytosine into uracil in E. coli and thereby supporting the growth in M9 minimal medium, one possibility would be direct conversion without the formation of a cytosine intermediate. The candidate to perform this reaction is a well-characterized CodA deaminase that catalyzes the conversion of cytosine into uracil [18]. To test whether CodA catalyzes the conversion of N4-methylcytosine into uracil, the enzyme was purified, and its enzymatic activity was tested using the same substrates as the ones used for the growth rate determination. Figure 5 demonstrates that whereas cytosine was well converted into uracil, no such conversion was observed for N4-methylcytosine (and also for N4,N4-dimethylcytosine), suggesting that E. coli encodes the specialized enzyme to convert N4-methylcytosine into cytosine, and only then the latter is converted into uracil by CodA (Figure 6). This kind of mechanism may be conserved at least in bacteria, since similar to the E. coli ∆pyrF::Km strain, growth patterns were observed for the S. typhi ∆pyrF::Tn10 uracil auxotroph. Also, it is possible that in B. subtilis, N4-methylcytosine is also converted into cytosine, but because of the absence of cytosine deaminase in the strain tested, it cannot support the growth in minimal media requiring uracil. Indeed, we have demonstrated that in contrast to E. coli and S. typhi, cytosine does not support the growth of the B. subtilis ∆pyrF::erm uracil auxotroph (Figure S2).
How N4-methylcytosine is converted into cytosine is unclear, even at the level of nucleosides, nucleotides, or nucleic acids. It was previously demonstrated that some cytidine deaminases are capable of converting both N4-methylcytidine and N4,N4-dimethylcytidine into uridine [19]. However, it is likely that N4-methylcytidine is not a physiological substrate of cytidine deaminase isolated from E. coli B, since the observed Km/Vmax values were at least three orders of magnitude higher than those of cytidine deamination [20]. For a long time, the m4C modification was considered as being part of the bacterial restriction/modification system [21]. However, the presence of this modification in transposable elements of the freshwater invertebrate Adineta vaga was associated with their transcriptional repression [16]. In that work, N4CMT, a horizontally transferred enzyme of bacterial origin, was identified as DNA m4C methyltransferase (“writer” enzyme). However, the demethylation (“eraser”) enzyme for this modification is unknown. Recently, it was suggested [22] that its catalytic activity may be similar to that of AlkB proteins that are responsible for m6A demethylation in RNA. Indeed, both AlkB family non-heme Fe(II)/2-oxoglutarate-dependent oxygenases FTO and ALKBH5 act as oxidative m6A demethylases, but their action yields different products, hm6A and A, respectively [23]. In the case of small molecules, N-dealkylation is often performed by the cytochrome P450 superfamily of heme-containing monooxygenases [24]. Bacterial P450 was successfully used to convert drug verapamil into norverapamil [25]. Therefore, one may envision the following scenario of N4-methylcytosine demethylation to yield the cytosine and then the uracil (Figure 6). As mentioned above [19], N4,N4-dimethylcytidine may be a substrate for conversion into uridine by cytidine deaminases. Here, we propose different reaction mechanisms for the removal of N4-methyl groups. At least two N-dimethylated heterocyclic bases (m62A and m22G, see [1]) exist in RNA. It was demonstrated that ALKBH5 catalyzes demethylation of m62A using rRNA or oligoribonucleotides as substrates [26]. However, removal of the first methyl group may be the rate-limiting step (see Figure 4D in [27]). Demethylation of N4,N4-dimethylcytosine may occur in a similar manner, which is reflected in the slow, if any, conversion into N4-methylcytosine and further into cytosine and uracil to support the growth of uracil auxotrophs.
E. coli is one of the most thoroughly characterized model organisms, yet around 35% of genes in its genome [28] are poorly described. In EcoCyc, the most comprehensive curated genome database for E. coli K-12 [29], 15.5% (738) of genes are annotated as the uncharacterized ones. Therefore, several approaches may be used in search of the E. coli N4-methylcytosine demethylase gene. One way would be the use of the gene ontology (GO) resource [30] to search for the candidate genes and then test the enzymatic activity of the corresponding proteins. Another way is the use of traditional protein chromatography following the proposed enzymatic activity [31]. However, this method may be tedious since no recombinant proteins are used for the initial screening procedures. The third way is genome-wide screening for the mutants that are not able to grow in N4-methylcytosine-containing minimal media using a single gene deletion library, similar to what was previously used [32]. This approach may also be time-consuming; however, budding yeast are particularly useful since the genetic background of the traditionally used Saccharomyces cerevisiae BY4741 strain renders it the uracil auxotroph. For bacteria, a more plausible approach is to use transposon insertion sequencing, a currently well-established method [33]. Finally, the transcriptomics approach similar to what was used to discover and characterize the morphinan N-demethylase [34] may be suggested.

3. Materials and Methods

3.1. Synthesis of Methylated Cytosines

  • General information
Chemicals, reagents, solvents, and kits were purchased from Sigma-Aldrich (Merck group, Darmstadt, Germany) and Thermo Fisher Scientific Baltics (Vilnius, Lithuania) and used without further purification. N4-methylcytosine and N4,N4-dimethylcytosine were synthesized and analyzed as described below. Thin-layer chromatography (TLC) was carried out on 25 TLC aluminum sheets coated with silica gel 60 F254 (Merck, Darmstadt, Germany), and column chromatography was performed on silica gel 60 (0.063–0.200 nm) (Merck) using chloroform/methanol mixtures as a mobile phase. NMR spectra were recorded in DMSO-d6 on a Bruker Ascend 400 1H NMR—400 MHz and 13C NMR—101 MHz (Bruker BioSpin, Rheinstetten, Germany). Chemical shifts were reported in ppm relative to the solvent resonance signal as an internal standard. UV spectra were recorded on a SPECORD 210 UV/Vis spectrophotometer (analytic Jena, Jena, Germany). The HPLC analyses were performed using a high-performance liquid chromatography system (UFLC LC-20AD), equipped with a photodiode array detector (SPD-M20A, Shimadzu, Kyoto, Japan). The chromatographic separation was conducted using a Symmetry C18 column, 3.9 × 150 mm (Waters, Milford, MA, USA) at 40 °C and a mobile phase that consisted of a 0.01% formic acid (or 5 mM ammonium acetate) and water solution (solvent A) and a 0.01% formic acid (or 5 mM ammonium acetate) and methanol solution (solvent B).

3.1.1. Synthesis of N4-Methylcytosine

(i)
4-methylthiouracil
To a solution of 120 mg (0.94 mmol) 4-thiouracil and 0.94 mL 1 M NaOH in 5.0 mL 50% ethanol, 116 μL (1.88 mmol) methyl iodide was added, and the mixture was stirred at room temperature for 1 h. After the reaction was completed (TLC), the mixture was neutralized with diluted acetic acid (25% in water) to approx. pH 6. After evaporation under reduced pressure, the residue was purified by flash column chromatography (silica gel, chloroform/methanol mixture, 10/0→10/1). Yield 108 mg (81%), yellowish foam, Rf = 0.46 (CHCl3/MeOH–9/1). UV (CH3OH) λmax (ε) 297 (11.2 × 103) nm (M−1 × cm−1). 1H NMR (DMSO-d6, 400 MHz): δ = 2.43 (s, 3H, CH3), 6.32 (d, 1H, J = 6.7 Hz, CH=CH), 7.62 (d, 1H, J = 6.7, Hz, CH=CH), 11.47 (s, 1H, NH). 13C NMR (DMSO-d6, 101 MHz): δ = 12.39, 102.22, 143.60, 154.71, 177.93.
(ii)
N4-methylcytosine
To a solution of 50 mg (0.35 mmol) 4-methylthiouracil in 5 mL of water, 542 μL (7 mmol) of 40% CH3NH2 in water was added, and the mixture was stirred at 70 °C for 36 h. After the reaction was completed (TLC), the solvents were removed under reduced pressure. The residue was purified by flash column chromatography (silica gel, chloroform/methanol mixture, 10/0→10/2). Yield 39 mg (90%), white solid, Rf = 0.15 (CHCl3/MeOH–9/1). UV (CH3OH) λmax (ε) 267 (12.1 × 103) nm (M−1 × cm−1). 1H NMR (DMSO-d6, 400 MHz): δ = 2.73 (d, J = 4.5 Hz, 3H, CH3), 5.58 (d, 1H, J = 7.1 Hz, CH=CH), 7.24 (d, 1H, J = 7.1, Hz, CH=CH), 7.54 (d, 1H, J = 4.8 Hz, NH), 10.24 (s, 1H, NH). 13C NMR (DMSO-d6, 101 MHz): δ = 27.16, 93.71, 141.61, 157.26, 165.45.

3.1.2. Synthesis of N4,N4-Dimethylcytosine

To a solution of 50 mg (0.35 mmol) 4-methylthiouracil in 5 mL of water, 805 μL (7 mmol) of 40% (CH3)2NH in water was added, and the mixture was stirred at 70 °C for 36 h. After the reaction was completed (TLC), the solvents were removed under reduced pressure. The residue was purified by column chromatography (silica gel, chloroform/methanol mixture, 10/0→10/2). Yield 46 mg (95%), white solid. Rf = 0.24 (CHCl3/MeOH–9/1). UV (CH3OH) λmax (ε) 275 (13.4 × 103) nm (M−1 × cm−1). 1H NMR (DMSO-d6, 400 MHz): δ = 3.02 (s, 6H, CH3), 5.88 (d, 1H, J = 7.4 Hz, CH=CH), 7.42 (d, 1H, J = 7.4, Hz, CH=CH), 10.44 (s, 1H, NH). 13C NMR (DMSO-d6, 101 MHz): δ = 90.38, 143.19, 156.38, 164.77.

3.2. Bacterial Strains and Growth Media

The uracil auxotroph ∆pyrF::Km in Escherichia coli K-12 BW25113 genetic background was obtained from the Keio collection via Horizon Discovery (Cambridge, UK). Salmonella typhi LT2 ∆pyrF::Tn10 was a kind gift from G.R. Björk laboratory (Umeå, Sweden). The Bacillus subtilis 168 ∆pyrF::erm trpC2 strain was obtained from the Bacillus Genetic Stock Center (Columbus, OH, USA).
Bacterial strains were recovered from long-term storage at −80 °C by plating them on LB-agar plates at 37 °C. For testing the growth of bacteria in minimal media, M9 medium, containing necessary salts and 0.4% of glucose, was supplemented with 20 µg/mL (or 200 µg/mL for the determination of concentration by HPLC) of either uracil, cytosine, N4-methylcytosine, or N4,N4-dimethylcytosine. In the case of E. coli, the growth medium was also supplemented with 15 µg/mL of kanamycin. For B. subtilis, media were supplemented with 40 µg/mL tryptophan since the trpC2 mutation in the B. subtilis 168 strain renders cells the tryptophan auxotroph. For growing bacteria on solid media, 2% agar was added.

3.3. Growth of Bacteria in Minimal Media

For monitoring the bacterial growth in solid media, bacterial strains were inoculated by streaking them on M9 minimal media containing different modified cytosines and respective controls and aerobically grown for up to 7 days at 37 °C.
For better growth rate comparison in different media, the E. coli ∆pyrF::Km strain was grown in liquid M9 minimal medium supplemented with uracil and kanamycin overnight, OD600 was measured, the concentration of cells was adjusted to approximately 109/mL (assuming that OD600 = 1 corresponds to 8 × 108/mL) by washing and diluting in 0.9% NaCl, and serial 10× dilutions were made before plating 5 µL of cell suspension on M9 minimal medium agar plates supplemented with kanamycin, respective modified cytosines, and controls.

3.4. Determination of Growth Rate of E. coli ∆pyrF::Km Strain

The strain was grown in M9 nutrient minimal medium supplemented with uracil and kanamycin at 37 °C overnight. Cells were washed with a 0.9% NaCl solution and transferred to the same medium supplemented with kanamycin and 200 µg/mL of either uracil, cytosine, N4-methylcytosine, or N4,N4-dimethylcytosine, and cells were aerobically grown at 37 °C. Samples for OD600 measurements were taken each hour. Experiments were performed 3 times in triplicates. Generation times in different media were calculated by plotting the values obtained at the exponential growth conditions in a semi-logarithmic scale (Figure S3). From the obtained curves, the generation time g was calculated according to the formula
g = t × ln2/(lnXt − lnX0),
where t is the time in hours, Xt is OD600 at time t, and X0 is OD600 at time 0.

3.5. Determination of N4-Methylcytosine and N4,N4-Dimethylcytosine Concentration and Chemical Stability Using HPLC

Samples for HPLC analysis were taken under the same frequency as for the measurement of OD600. A total of 500 µL of sample was centrifuged, and 0.6 mL of methanol was added to 0.3 mL of the obtained supernatant in order to kill the remaining cells and to precipitate the salts. Samples were centrifuged again, and 10 μL of the solution was loaded on a C18, 3.9 × 150 mm column. HPLC analysis was performed as described above in Section 3.1.
For chemical stability analysis, 200 µg/mL N4-methylcytosine or N4,N4-dimethylcytosine was incubated in the M9 liquid medium supplemented with kanamycin at 37 °C for 72 h without the E. coli ∆pyrF::Km strain. Samples were taken each day (approx. every 24 h) and analyzed by HPLC.

3.6. Purification of E. coli CodA Protein and Determination of Its Enzymatic Activity

Recombinant CodA protein was purified using the pQE-70-codA plasmid [35]. The plasmid was transformed into the E. coli DH10B strain (Thermo Fisher Scientific Baltics). The resulting bacteria were grown in LB medium with 100 µg/mL of ampicillin at 37 °C until OD600 reached around 0.8; the IPTG inducer was added to the final concentration of 0.5 mM, and the culture was grown for another 3 h. Cells were collected by centrifugation and resuspended in 20 mM Tris-HCl, pH 7.9, with 0.3 M NaCl. Then cells were disrupted by 5 min. sonication at 22 kHz in 10 s periods with 10 s cooling intervals, using 40% amplitude. Cell debris was removed by centrifugation at 12,000× g for 30 min. The resulting supernatant was loaded onto an Ni-NTA column (Cytiva Sweden AB, Uppsala, Sweden), previously equilibrated with 20 mM Tris-HCl, pH 7.9, with 0.3 M NaCl. Proteins were applied to the column and washed with 2 column volumes of the equilibration buffer. The absorbed proteins were eluted with 20 mM Tris-HCl, pH 7.9, with 0.3 M NaCl using a linear gradient of 0–250 mM of imidazole. Protein-containing fractions were pooled and desalted against 20 mM Tris-HCl, pH 7.9. Purity of recombinant CodA was confirmed by SDS-PAGE electrophoresis (Figure S6). The concentration of protein was measured using the Bradford method.
The enzymatic reactions were carried out at 25 °C in 20 mM Tris-HCl, pH 7.9, for 30 min. A 30 µL final reaction mixture contained 1 µg (leading to the final concentration of around 0.6 µM) of purified protein and 20 mM substrate. After incubation, 1 µL of the reaction mixture was used for thin-layer chromatography analysis that was performed on aluminum sheets coated with silica gel 60 F254 using a chloroform/methanol mixture (5:1) as a mobile phase. The spots were visualized under 254 nm UV light.

4. Conclusions

We demonstrated in this study that N4-methylcytosine, but not N4,N4-dimethylcytosine, supports the growth of the E. coli ∆pyrF::Km auxotroph in minimal M9 media. Further experiments demonstrated that the generation time of this strain in M9 medium supplemented with N4-methylcytosine is approximately 3 h compared to approximately 1.5 h for cytosine or uracil. In contrast, little or no growth was observed for N4,N4-dimethylcytosine, with a calculated generation time of at least 40 h. We found that around 12% of N4-methylcytosine is consumed and presumably converted into cytosine and further into uracil after 24 h of growth in M9 medium. Purification of the CodA deaminase and determination of its enzymatic activity demonstrated that, in contrast to cytosine, neither N4-methylcytosine nor N4,N4-dimethylcytosine is converted into uracil. Therefore, we propose that N4-methylcytosine is not directly converted into uracil by CodA to support the growth of E. coli, but instead it is converted first into cytosine by yet unknown enzyme(s). The enzymatic pathway for such conversion is proposed. Further experiments to determine the gene encoding the N-demethylase enzyme are described.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26051812/s1.

Author Contributions

Conceptualization, J.U. and D.T.; methodology, A.Č., D.T. and J.U.; investigation, D.T. and J.U.; data curation, A.Č., D.T. and J.U.; writing—original draft preparation, J.U. and D.T.; writing—review and editing, J.U. and D.T.; supervision, J.U. and D.T.; project administration, D.T.; funding acquisition, D.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Research Council of Lithuania (LMTLT), grant No. [S-MIP-22-71]. The APC was funded by Vilnius Gediminas Technical University.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of this study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

References

  1. Cappannini, A.; Ray, A.; Purta, E.; Mukherjee, S.; Boccaletto, P.; Moafinejad, S.N.; Lechner, A.; Barchet, C.; Klaholz, B.P.; Stefaniak, F.; et al. MODOMICS: A database of RNA modifications and related information. 2023 update. Nucleic Acids Res. 2024, 52, D239–D244. [Google Scholar] [CrossRef] [PubMed]
  2. Suzuki, T. The expanding world of tRNA modifications and their disease relevance. Nat. Rev. Mol. Cell Biol. 2021, 22, 375–392. [Google Scholar] [CrossRef] [PubMed]
  3. Arevalo, C.P.; Bolton, M.J.; Le Sage, V.; Ye, N.; Furey, C.; Muramatsu, H.; Alameh, M.G.; Pardi, N.; Drapeau, E.M.; Parkhouse, K.; et al. A multivalent nucleoside-modified mRNA vaccine against all known influenza virus subtypes. Science 2022, 378, 899–904. [Google Scholar] [CrossRef] [PubMed]
  4. Van Giesen, K.J.D.; Thompson, M.J.; Meng, Q.; Lovelock, S.L. Biocatalytic synthesis of antiviral nucleosides, cyclic dinucleotides, and oligonucleotide therapies. JACS Au 2023, 3, 13–24. [Google Scholar] [CrossRef]
  5. Traube, F.R.; Carell, T. The chemistries and consequences of DNA and RNA methylation and demethylation. RNA Biol. 2017, 14, 1099–1107. [Google Scholar] [CrossRef]
  6. Preumont, A.; Snoussi, K.; Stroobant, V.; Collet, J.F.; Van Schaftingen, E. Molecular identification of pseudouridine-metabolizing enzymes. J. Biol. Chem. 2008, 283, 25238–25246. [Google Scholar] [CrossRef]
  7. Loh, K.D.; Gyaneshwar, P.; Papadimitriou, E.M.; Fong, R.; Kim, K.S.; Parales, R.; Zhou, Z.; Inwood, W.; Kustu, S. A previously underscribed pathway for pyrimidine catabolism. Proc. Natl. Acad. Sci. USA 2006, 103, 5114–5119. [Google Scholar] [CrossRef]
  8. Vogels, G.D.; Van Der Drift, C. Degradation of purines and pyrimidines by microorganisms. Bacteriol. Rev. 1976, 40, 403–468. [Google Scholar] [CrossRef]
  9. Soong, C.L.; Ogawa, J.; Shimizu, S. Novel amidohydrolytic reactions in oxidative pyrimidine metabolism: Analysis of the barbiturase reaction and discovery of a novel enzyme, ureidomalonase. Biochem. Biophys. Res. Commun. 2001, 286, 222–226. [Google Scholar] [CrossRef]
  10. Kimura, S.; Suzuki, T. Fine-tuning of the ribosomal decoding center by conserved methyl-modifications in the Escherichia coli 16S RRNA. Nucleic Acids Res. 2010, 38, 1341–1352. [Google Scholar] [CrossRef]
  11. Zou, M.; Mu, Y.; Chai, X.; Ouyang, M.; Yu, L.J.; Zhang, L.; Meurer, J.; Chi, W. The critical function of the plastid rRNA methyltransferase, CMAL, in ribosome biogenesis and plant development. Nucleic Acids Res. 2020, 48, 3195–3210. [Google Scholar] [CrossRef] [PubMed]
  12. McIntyre, W.; Netzband, R.; Bonenfant, G.; Biegel, J.M.; Miller, C.; Fuchs, G.; Henderson, E.; Arra, M.; Canki, M.; Fabris, D.; et al. Positive-sense RNA viruses reveal the complexity and dynamics of the cellular and viral epitranscriptomes during infection. Nucleic Acids Res. 2018, 46, 5776–5791. [Google Scholar] [CrossRef] [PubMed]
  13. Fluke, K.A.; Dai, N.; Wolf, E.J.; Fuchs, R.T.; Ho, P.S.; Talbott, V.; Elkins, L.; Tsai, Y.-L.; Schiltz, J.; Febvre, H.P.; et al. A novel N4,N4-dimethylcytidine in the archaeal ribosome enhances hyperthermophily. Proc. Natl. Acad. Sci. USA 2024, 121, e2405999121. [Google Scholar] [CrossRef] [PubMed]
  14. Sánchez-Romero, M.A.; Cota, I.; Casadesús, J. DNA methylation in bacteria: From the methyl group to the methylome. Curr. Opin. Microbiol. 2015, 25, 9–16. [Google Scholar] [CrossRef]
  15. Kumar, S.; Karmakar, B.C.; Nagarajan, D.; Mukhopadhyay, A.K.; Morgan, R.D.; Rao, D.N. N4-cytosine DNA methylation regulates transcription and pathogenesis in Helicobacter pylori. Nucleic Acids Res. 2018, 46, 3429–3445. [Google Scholar] [CrossRef]
  16. Rodriguez, F.; Yushenova, I.A.; DiCorpo, D.; Arkhipova, I.R. Bacterial N4-methylcytosine as an epigenetic mark in eukaryotic DNA. Nat. Commun. 2022, 13, 1072. [Google Scholar] [CrossRef]
  17. Aučynaitė, A.; Rutkienė, R.; Gasparavičiūtė, R.; Meškys, R.; Urbonavičius, J. A gene encoding a DUF523 domain protein is involved in the conversion of 2-thiouracil into uracil. Environ. Microbiol. Rep. 2018, 10, 49–56. [Google Scholar] [CrossRef]
  18. Hall, R.S.; Fedorov, A.A.; Xu, C.; Fedorov, E.V.; Almo, S.C.; Raushel, F.M. Three-dimensional structure and catalytic mechanism of cytosine deaminase. Biochemistry 2011, 50, 5077–5085. [Google Scholar] [CrossRef]
  19. Urbeliene, N.; Tiškus, M.; Tamulaitiene, G.; Gasparavičiute, R.; Lapinskaite, R.; Jauniškis, V.; Sudžius, J.; Meškiene, R.; Tauraite, D.; Skrodenyte, E.; et al. Cytidine deaminases catalyze the conversion of N(S,O)4-substituted pyrimidine nucleosides. Sci. Adv. 2023, 9, eade4361. [Google Scholar] [CrossRef]
  20. Cohen, R.M.; Wolfenden, R. Cytidine deaminase from Escherichia coli. Purification, properties and inhibition by the potential transition state analog 3,4,5,6-tetrahydrouridine. J. Biol. Chem. 1971, 246, 7561–7565. [Google Scholar] [CrossRef]
  21. Casadesús, J.; Sánchez-Romero, M.A. DNA methylation in prokaryotes. In DNA Methyltransferases—Role and Function; Advances in Experimental Medicine and Biology; Jeltsch, A., Jurkowska, R.Z., Eds.; Springer: Cham, Switzerland, 2022; Volume 1389, pp. 21–43. [Google Scholar]
  22. Meng, W.Y.; Wang, Z.X.; Zhang, Y.; Hou, Y.; Xue, J.H. Epigenetic marks or not? The discovery of novel DNA modifications in eukaryotes. J. Biol. Chem. 2024, 300, 106791. [Google Scholar] [CrossRef] [PubMed]
  23. Toh, J.D.W.; Crossley, S.W.M.; Bruemmer, K.J.; Ge, E.J.; He, D.; Iovan, D.A.; Chang, C.J. Distinct RNA N-demethylation pathways catalyzed by conheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates. Proc. Natl. Acad. Sci. USA 2020, 117, 25284–25292. [Google Scholar] [CrossRef] [PubMed]
  24. Najmi, A.A.; Bischoff, R.; Permentier, H.P. N-Dealkylation of amines. Molecules 2022, 27, 3293. [Google Scholar] [CrossRef] [PubMed]
  25. Shen, C.; Liu, H.; Dai, W.; Liu, X.; Liu, J.; Yu, B. Specific N-demethylation of verapamil by Cytochrome P450 from Streptomyces griseus ATCC 13273. Eng. Life Sci. 2019, 19, 292–301. [Google Scholar] [CrossRef]
  26. Ensfelder, T.T.; Kurz, M.Q.; Iwan, K.; Geiger, S.; Matheisl, S.; Müller, M.; Beckmann, R.; Carell, T. ALKBH5-induced demethylation of mono- and dimethylated adenosine. Chem. Comm. 2018, 54, 8591–8593. [Google Scholar] [CrossRef]
  27. Shen, D.; Wang, B.; Gao, Y.; Zhao, L.; Bi, Y.; Zhang, J.; Wang, N.; Kang, H.; Pang, J.; Liu, Y.; et al. Detailed resume of RNA M6A demethylases. Acta Pharm. Sin. B 2022, 12, 2193–2205. [Google Scholar] [CrossRef]
  28. Moore, L.R.; Caspi, R.; Boyd, D.; Berkmen, M.; Mackie, A.; Paley, S.; Karp, P.D. Revisiting the Y-ome of Escherichia coli. Nucleic Acids Res. 2024, 52, 12201–12207. [Google Scholar] [CrossRef]
  29. Karp, P.D.; Paley, S.; Caspi, R.; Kothari, A.; Krummenacker, M.; Midford, P.E.; Moore, L.R.; Subhraveti, P.; Gama-Castro, S.; Tierrafria, V.H.; et al. The EcoCyc database (2023). EcoSal Plus 2023, 11, esp-0002. [Google Scholar] [CrossRef]
  30. Carbon, S.; Douglass, E.; Good, B.M.; Unni, D.R.; Harris, N.L.; Mungall, C.J.; Basu, S.; Chisholm, R.L.; Dodson, R.J.; Hartline, E.; et al. The gene ontology resource: Enriching a GOld mine. Nucleic Acids Res. 2021, 49, D325–D334. [Google Scholar] [CrossRef]
  31. Stanislauskienė, R.; Laurynėnas, A.; Rutkienė, R.; Aučynaitė, A.; Tauraitė, D.; Meškienė, R.; Urbelienė, N.; Kaupinis, A.; Valius, M.; Kaliniene, L.; et al. YqfB protein from Escherichia coli: An atypical amidohydrolase active towards N4-acylcytosine derivatives. Sci. Rep. 2020, 10, 788. [Google Scholar] [CrossRef]
  32. Serviene, E.; Lukša, J.; Orentaite, I.; Lafontaine, D.L.J.; Urbonavičius, J. Screening the budding yeast genome reveals unique factors affecting K2 toxin susceptibility. PLoS ONE 2012, 7, e50779. [Google Scholar] [CrossRef] [PubMed]
  33. Van Opijnen, T.; Levin, H.L. Transposon insertion sequencing, a global measure of gene function. Annu. Rev. Genet. 2020, 54, 337–365. [Google Scholar] [CrossRef] [PubMed]
  34. Augustin, M.M.; Augustin, J.M.; Brock, J.R.; Kutchan, T.M. Enzyme morphinan N-demethylase for more sustainable opiate processing. Nat. Sustain. 2019, 2, 465–474. [Google Scholar] [CrossRef]
  35. Aučynaite, A.; Rutkiene, R.; Tauraite, D.; Meškys, R.; Urbonavičius, J. Discovery of bacterial deaminases that convert 5-fluoroisocytosine into 5-fluorouracil. Front. Microbiol. 2018, 9, 2375. [Google Scholar] [CrossRef]
Ijms 26 01812 g001
Figure 1. Growth of the E. coli ∆pyrF::Km strain on the M9 agar minimal medium supplemented with either uracil, cytosine, N4-methylcytosine, N4,N4-dimethylcytosine, or without any heterocyclic base (control). The agar plates were incubated at 37 °C for 72 h.
Figure 1. Growth of the E. coli ∆pyrF::Km strain on the M9 agar minimal medium supplemented with either uracil, cytosine, N4-methylcytosine, N4,N4-dimethylcytosine, or without any heterocyclic base (control). The agar plates were incubated at 37 °C for 72 h.
Ijms 26 01812 g001
Ijms 26 01812 g002
Figure 2. Growth of the E. coli ∆pyrF::Km strain in the M9 agar medium supplemented with either uracil, cytosine, N4-methylcytosine, or N4,N4-dimethylcytosine after serial dilutions.
Figure 2. Growth of the E. coli ∆pyrF::Km strain in the M9 agar medium supplemented with either uracil, cytosine, N4-methylcytosine, or N4,N4-dimethylcytosine after serial dilutions.
Ijms 26 01812 g002
Ijms 26 01812 g003
Figure 3. Growth of the E. coli ∆pyrF::Km strain in the M9 liquid medium supplemented with uracil Ijms 26 01812 i001; cytosine Ijms 26 01812 i002; N4-methylcytosine Ijms 26 01812 i003; or N4,N4-dimethylcytosine Ijms 26 01812 i004. Error bars show the standard deviation of the three experiments.
Figure 3. Growth of the E. coli ∆pyrF::Km strain in the M9 liquid medium supplemented with uracil Ijms 26 01812 i001; cytosine Ijms 26 01812 i002; N4-methylcytosine Ijms 26 01812 i003; or N4,N4-dimethylcytosine Ijms 26 01812 i004. Error bars show the standard deviation of the three experiments.
Ijms 26 01812 g003
Ijms 26 01812 g004
Figure 4. Decrease in N4-methylcytosine concentration during the growth of the E. coli ∆pyrF::Km strain in M9 liquid medium. The results are represented as peak area obtained during HPLC analysis. Error bars show the standard deviation of the three experiments.
Figure 4. Decrease in N4-methylcytosine concentration during the growth of the E. coli ∆pyrF::Km strain in M9 liquid medium. The results are represented as peak area obtained during HPLC analysis. Error bars show the standard deviation of the three experiments.
Ijms 26 01812 g004
Ijms 26 01812 g005
Figure 5. Cytosine deaminase CodA does not convert N4-methylcytosine or N4,N4-dimethylcytosine into uracil. Thin-layer chromatography (TLC) plate of cytosine, N4-methylcytosine, or N4,N4-dimethylcytosine enzymatic reactions with CodA.
Figure 5. Cytosine deaminase CodA does not convert N4-methylcytosine or N4,N4-dimethylcytosine into uracil. Thin-layer chromatography (TLC) plate of cytosine, N4-methylcytosine, or N4,N4-dimethylcytosine enzymatic reactions with CodA.
Ijms 26 01812 g005
Ijms 26 01812 g006
Figure 6. Proposed mechanism of N4-methylcytosine conversion into uracil.
Figure 6. Proposed mechanism of N4-methylcytosine conversion into uracil.
Ijms 26 01812 g006
Table 1. Generation time of the E. coli ∆pyrF::Km strain in liquid M9 media with different heterocyclic bases. Values represent the average of 3 experiments and the standard deviation.
Table 1. Generation time of the E. coli ∆pyrF::Km strain in liquid M9 media with different heterocyclic bases. Values represent the average of 3 experiments and the standard deviation.
UracilCytosineN4-MethylCN4,N4-DimethylC
Generation time, h1.53 ± 0.041.49 ± 0.102.99 ± 0.5933.03 ± 11.41
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Urbonavičius, J.; Čekytė, A.; Tauraitė, D. N4-Methylcytosine Supports the Growth of Escherichia coli Uracil Auxotrophs. Int. J. Mol. Sci. 2025, 26, 1812. https://doi.org/10.3390/ijms26051812

AMA Style

Urbonavičius J, Čekytė A, Tauraitė D. N4-Methylcytosine Supports the Growth of Escherichia coli Uracil Auxotrophs. International Journal of Molecular Sciences. 2025; 26(5):1812. https://doi.org/10.3390/ijms26051812

Chicago/Turabian Style

Urbonavičius, Jaunius, Aušrinė Čekytė, and Daiva Tauraitė. 2025. "N4-Methylcytosine Supports the Growth of Escherichia coli Uracil Auxotrophs" International Journal of Molecular Sciences 26, no. 5: 1812. https://doi.org/10.3390/ijms26051812

APA Style

Urbonavičius, J., Čekytė, A., & Tauraitė, D. (2025). N4-Methylcytosine Supports the Growth of Escherichia coli Uracil Auxotrophs. International Journal of Molecular Sciences, 26(5), 1812. https://doi.org/10.3390/ijms26051812

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop