Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and 2′-deoxycytidine to uridine and 2′-deoxyuridine. Here, we report that prokaryotic homo-tetrameric CDAs catalyze the nucleophilic substitution at the fourth position of N ⁴ -acyl-cytidines, N ⁴ -alkyl-cytidines, and N ⁴ -alkyloxycarbonyl-cytidines, and S ⁴ -alkylthio-uridines and O ⁴ -alkyl-uridines, converting them to uridine and corresponding amide, amine, carbamate, thiol, or alcohol as leaving groups. The x-ray structure of a metagenomic CDA_F14 and the molecular modeling of the CDAs used in this study show a relationship between the bulkiness of a leaving group and the volume of the binding pocket, which is partly determined by the flexible β3α3 loop of CDAs. We propose that CDAs that are active toward a wide range of substrates participate in salvage and/or catabolism of variously modified pyrimidine nucleosides. This identified promiscuity of CDAs expands the knowledge about the cellular turnover of cytidine derivatives, including the pharmacokinetics of pyrimidine-based prodrugs.

In the present work, the impact of three new bacterial strains and their consortium on composting was evaluated using textile waste as a main substrate mixed with paper, cardboard and green waste, The effectiveness of these micro-organisms in accelerating organic matter degradation was tested. For bioaugmentation of composting, three concentrations (4%, 6% and 8%) were applied. Among the three strains tested, one strain and the consortium demonstrated high organic matter degradation potential, achieving a total organic carbon concentration between 19–21%, total Kjeldahl nitrogen between 1.29–1.56%, a C/N ratio between 13–16%, and a temperature exceeding 55 °C. In the current study, mature compost was attained in 10 weeks, instead of the 44 weeks required for conventional composting and the 12 weeks achieved with other strains previously used. Identification of the strains by 16S rRNA sequencing revealed that they belonged to Bacillus sp., Paenibacillus sp., and Enterobacter aerogenes, respectively. These strains are recognized for their remarkable potential to breakdown a broad variety of organic matter, including lignocellulosic molecules. Furthermore, incorporation of bacteria into the waste mixture (either separately or as a consortium) extended the thermophilic phase by 2 weeks in this study, especially Bacillus sp., Paenibacillus sp. and consortium, leading to a significant reduction in compost production time. It is noteworthy that the efficacy of these strains was considerably greater compared with the three previous strains (i.e., Streptomyces cellulosae, Achromobacter xylosoxidans and Serratia liquefaciens), which were isolated from compost and used for bioaugmentation in a previous study. Our results demonstrate that bioaugmentation by endogenous microbial strains and/or their consortium significantly accelerates the composting process.

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.

The synthesis of various heterocyclic base modifications of nucleic acids has been thoroughly investigated; however, much less is known about their catabolism. Also, little is known about the transport of such compounds across the microbial cell membranes. Using the Saccharomyces cerevisiae single-gene deletion library, we performed genome-wide screening for genes affecting the growth of yeast in minimal media supplemented with N⁴-acetylcytosine as a source of uracil. We found that Fcy1, Fcy21, Bud16, Gnd1, and Fur4 proteins are required for efficient growth in the tested medium. Additionally, we used several heterocyclic pyrimidine bases and Fcy homolog mutants to test their growth in respective minimal media. We found that tested permeases differently affect the growth of yeast that is dependent on the heterocyclic pyrimidine bases used as a source of uracil. The most pronounced effect was observed for the ∆fur4 mutant, which was growing much slower than the corresponding wild-type strain in the media supplemented with N⁴-acetylcytosine, 4-methylthiouracil, N⁴-methylcytosine, N⁴,N⁴-dimethylcytosine, 2-thiouracil, or 4-thiouracil. We suggest that Fur4 protein is the major yeast transporter of modified heterocyclic pyrimidine bases. Our observations might be helpful when investigating the actions of various heterocyclic base-based antifungal, anticancer, and antiviral drugs.

N⁶-methyladenine and N⁶,N⁶-dimethyladenine are the heterocyclic bases present in the RNA of eukaryotic and bacterial cells and play important regulatory roles. How the degradation of such modified nucleic acids, and the subsequent demethylation of modified heterocyclic bases, occurs in the bacterium Escherichia coli is not established. Here, we investigated the growth of adenine auxotroph strains in a minimal M9 medium supplemented with either N⁶-methyladenine or N⁶,N⁶-dimethyladenine. We found that N⁶-methyladenine supported the growth of ∆purH::Km but not that of the ∆purA::Km strain, whereas N⁶,N⁶-dimethyladenine did not support the growth of either adenine auxotroph. Similar experiments performed using structurally related 2-amino-N⁶-methylpurine and 2-amino-N⁶,N⁶-dimethylpurine bases—using ∆guaA::Km, ∆guaB::Km, and ∆purH::Km guanine auxotrophs—demonstrated that growth of only the ∆guaB::Km mutant was supported by 2-amino-N⁶-methylpurine but not by its dimethylated counterpart. We expressed and purified C-teminus 6xHis tagged E. coli adenine/adenosine deaminases AdeC and Add and tested their substrate specificity. We demonstrated that AdeC protein does not catalyse deamination of either N⁶-methyl- or N⁶,N⁶-dimethyladenine, whereas Add catalyses deamination of N⁶-methyl- but not that of N⁶,N⁶-dimethyladenosine. Based on our findings, biochemical pathways leading to the demodification and return into metabolism of N⁶-methyladenine and 2-amino-N⁶-methylpurine in E. coli are proposed.