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.

Biomedical Center Faculty of Medicine Charles University Pilsen Czechia

Department of Biomedical and Clinical Sciences Pediatric Clinical Research Center Romeo and Enrica Invernizzi University of Milan Milan Italy

Istituto Nazionale di Genetica Molecolare Romeo and Enrica Invernizzi University of Milan Milan Italy

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