Evidence that host-mediated epigenetic modifications regulate gene expression in Wolbachia pipientis

Wolbachia pipientis is an obligate intracellular bacterium, associated with several arthropods and filarial nematodes. Wolbachia establishes strict symbiotic relationships with its hosts, with the consequent loss of many genes and regulatory regions. Despite this, experimental studies show that Wolbachia gene expression is coordinated to host needs, but the mechanism is still unknown. The first published RNA-Seq study on Wolbachia evidenced a strong differential expression of a DNA methyltransferase (MTase). In bacteria, this enzyme methylates either adenines or cytosines on specific motifs, contributing to the regulation of gene expression. In this work, we tested the hypothesis that the activity of MTase modulates the expression of Wolbachia genes. We first determined the methylation motif of the Wolbachia MTase by expressing it in Escherichia coli. Surprisingly, the experiment revealed that the Wolbachia MTase methylates both adenine and cytosine, without recognising highly specific motifs. Then, re-analysing data from six RNA-Seq studies, we found that the nucleotide content of Wolbachia genes correlates with their expressions, with a pattern compatible to be a consequence of the DNA methylation. Lastly, we identified MTase as the Wolbachia gene with the most conserved binding site for the Ccka/CtrA signalling transduction system, a mechanism likely involved the host-bacterium communication. Overall, these findings suggest a cascade mechanism in which the host activates the Wolbachia Ccka/CtrA signalling system, thus inducing the expression of the MTase gene. Then, the subsequent DNA methylation will affect the expression of several Wolbachia genes on the basis of their cytosine and adenine content.