UNLABELLED: Wolbachia pipientis is an obligate intracellular bacterium, associated with several arthropods and filarial nematodes. Wolbachia establishes a variety of symbiotic relationships with its hosts, with consequent genomic rearrangements, variation in gene content, and loss of regulatory regions. Despite this, experimental studies show that Wolbachia gene expression is coordinated with host developmental stages, but the mechanism is still unknown. In this work, we analyzed published RNA-seq data of four Wolbachia strains, finding a correlation between gene nucleotide composition and gene expression. The strength and direction of this phenomenon changed with the expression of the S-adenosyl-methionine-dependent methyltransferase midA. Specifically, when midA is overexpressed, there is a negative relationship between gene adenine content and gene expression, while downregulation of midA reverses this trend. MidA is known to methylate protein arginine, with potential effect on protein affinity for substrates, including nucleic acids. To expand our understanding of this poorly characterized enzyme, we investigated its ability to methylate DNA expressing it in Escherichia coli. The experiment revealed that the Wolbachia MidA can methylate both adenine and cytosine. Lastly, we found upstream the midA gene, a conserved binding site for the Ccka/CtrA signaling transduction system, and we hypothesize that this mechanism could be involved in the communication between the host and the bacterium. Overall, these findings suggest a cascade mechanism in which the host activates the bacterium Ccka/CtrA signaling system, thus inducing the expression of the midA gene, with subsequent effect on the expression of several Wolbachia genes on the basis of their nucleotide composition. IMPORTANCE: Wolbachia pipientis is one of the most common intracellular bacteria in insects, and it is currently utilized as a tool for the control of vector-borne diseases. As for many other endosymbiont bacteria, Wolbachia experienced important genome rearrangements, gene content changes, and the loss of several regulatory sequences, affecting the integrity of operons and promoters. Nevertheless, experimental studies have shown that Wolbachia gene expression is coordinated with the host physiology (e.g., developmental stages), although the underlying mechanism remains unclear. In this work, based on in silico analyses and an experimental study on wOo methyltransferase, we propose that bacterial DNA methylation could be a key mechanism regulating Wolbachia gene expression. Additionally, we found evidence suggesting that the DNA methylation process in Wolbachia can be activated by the host.

• Keywords
• MidA, Wolbachia pipientis, endosymbionts, gene expression, regulation of gene expression,
• MeSH
• Bacterial Proteins * genetics   metabolism   MeSH
• Methyltransferases * genetics   metabolism   MeSH
• DNA Methylation MeSH
• Nucleotides * genetics   MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Wolbachia * genetics   enzymology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins * MeSH
• Methyltransferases * MeSH
• Nucleotides * MeSH

DNA modifications on pyrimidine nucleobases play diverse roles in biology such as protection of bacteriophage DNA from enzymatic cleavage, however, their role in the regulation of transcription is underexplored. We have designed and synthesized a series of uracil 2'-deoxyribonucleosides and 5'-O-triphosphates (dNTPs) bearing diverse modifications at position 5 of nucleobase, including natural nucleotides occurring in bacteriophages, α-putrescinylthymine, α-glutaminylthymine, 5-dihydroxypentyluracil, and methylated or non-methylated 5-aminomethyluracil, and non-natural 5-sulfanylmethyl- and 5-cyanomethyluracil. The dNTPs bearing basic substituents were moderate to poor substrates for DNA polymerases, but still useful in primer extension synthesis of modified DNA. Together with previously reported epigenetic pyrimidine nucleotides, they were used for the synthesis of diverse DNA templates containing a T7 promoter modified in the sense, antisense or in both strands. A systematic study of the in vitro transcription with T7 RNA polymerase showed a moderate positive effect of most of the uracil modifications in the non-template strand and some either positive or negative influence of modifications in the template strand. The most interesting modification was the non-natural 5-cyanomethyluracil which showed significant positive effect in transcription.

• Publication type
• Journal Article MeSH