The release of the first Wolbachia
Mel strain) revealed that it contains two DNA methyltransferase genes met1
, encoded by two prophages, WO-A and WO-B respectively 
. This finding is intriguing in the light of the fact that Wolbachia
-induced CI involves modification of the insect host chromosome 
. The presence of phage-like particles in Wolbachia
-infected hosts 
suggests an active role of the phage in Wolbachia
biology. Thus, it is tempting to speculate that, beyond controlling lysogeny of the phage, the methyltransferases might be involved in triggering reproductive alterations imposed by Wolbachia
on its host. We therefore undertook a survey of Wolbachia
strains of known CI status for the presence of the methyltransferases, determined their sequences and reconstructed their phylogeny.
gene is only present in a few of the tested strains; there is no correlation with CI. Wolbachia
No and w
Pip induce CI in permissive hosts 
, and they all contain at least one functional copy of the met2
gene. In contrast, strain w
does not induce or rescue CI and its only met2
ORF is disrupted. Wolbachia
Mau and w
Ma do not contain the met2
gene and also do not induce CI in their hosts 
. On the other hand, w
Tei and w
San have recently been shown to fully rescue the w
Ri modification, while they are unable to induce CI in their native hosts 
Tei is, however, able to induce 100% CI after transfer into the permissive host D. simulans 
. In this context, it is interesting to note that our data suggest a double or multiple infection of the original host of w
Tei, of which not all Wolbachia
strain(s) were transferred upon transinfection of D. simulans
(). Cloning and sequencing of PCR products, using different methyltransferase primer sets, supports the presence of a hidden double infection of D. teissieri
. This could explain the phenotypic shift in CI properties that is observed between the natural host and the engineered strains.
When the rescue properties of all A group strains are examined, resc− strains lack a functional A-group-like met2 gene, which is always present in resc+ strains (); a possible correlation between met2 and CI rescue should therefore be considered. B group strains (wNo, wMa, wMau) are all resc+, nevertheless they do not possess an A-group-like met2 gene. The genomes of these strains have not been sequenced and the presence or absence of prophage copies has not yet been documented. While wMa and wMau do not contain any met gene, wNo has a B-group-like met2 ORF; this could reflect a different mechanism regulating CI in B group Wolbachia strains.
Distribution of phage methyltransferases in resc+ and resc− Wolbachia strains.
Transgenic expression of w
(WD0594) in D. melanogaster
was recently reported using the UAS/GAL4 system 
. This study revealed no modification of phenotype in flies expressing met2
ubiquitously and, similarly, when expressed specifically in the ovaries, no rescue phenotype was apparent in CI crosses. Although these data suggest that constitutive expression of the met2
gene does not alone drive the CI phenotype, it is still unclear what type of regulation met2
or any of the phage-related genes are subject to and how this affects the mechanism of CI.
Southern blot analysis indicates the presence of a met-like gene also in the Wolbachia strain wUni, which is known to induce parthenogenesis in the parasitic wasp Muscidifurax uniraptor (data not shown). The distribution of the met gene in parthenogenesis-inducing Wolbachia strains remains to be investigated. Interestingly, the mutualistic Wolbachia strain, which is present in the filarial nematode Brugia malayi, neither induces reproductive alterations nor carries a copy of the DNA methyltransferase genes.
Additionally, and important for any interpretation of the role of met2
, we demonstrated expression of the gene in all Wolbachia
strains with RT-PCR (). The Wolbachia
phage DNA methyltransferase may be involved in the methylation of phage, bacterial, insect host genes or a combination of them. Although Drosophila
had for a long time been considered to be free of DNA methylation, both the presence of methyltransferase genes in its genome 
, and of 5-methylcytosine residues in the early stages of embryonic development 
have been demonstrated. Interestingly, a Dam-like methyltransferase has been implicated in male sterility in plants 
Base modification in bacterial genomes is performed by two classes of DNA methyltransferases: (i) those associated with restriction-modification systems, and (ii) solitary methyltransferases that do not have a restriction enzyme counterpart. Examples of the latter are the N6-adenine methyltransferases Dam and CcrM 
. In α-Proteobacteria, CcrM methylation regulates the cell cycle in Caulobacter crescentus
, Rhizobium meliloti
and Agrobacterium tumefaciens
and plays a role in Brucella abortus
infection (reviewed in 
). Overexpression of CcrM in these bacteria results in the accumulation of multiple chromosomes, indicative of overinitiation of DNA replication 
prophage methyltransferase could regulate several aspects of the symbiont's cell cycle by imposing a specific epigenetic signal.
analysis of Wolbachia
prophage methyltransferase has predicted an N-terminal ParB-like nuclease domain (data not shown) similar to the ParB of the parCBA
operon in E. coli
, which is important for plasmid stability and resolving dimeric or multimeric plasmids. ParB nucleases have also been reported in several other plasmid genomes. ParB nucleases are Ca++ dependent endonucleases with 5′ -3′ exonuclease activity 
The methyltransferase genes, met1 and met2, are closely related (). The phylogenetic clustering of the methyltransferase genes, in particular of the met2 gene, is similar to the currently accepted clustering of the arthropod Wolbachia strains, both on the level of the major division of the Wolbachia strains into two supergroups, A and B, as well as on the lower level of clades and strains. Specifically, the met2-based tree is similar to the respective wsp-based tree (data not shown). This suggests a long association of the methyltransferases, and consequently of the phages carrying them, with the harbouring Wolbachia chromosomes (). However, translocation of met2 gene from the phage genome to Wolbachia chromosome cannot be excluded for any of the strains studied; such an event could explain why met2 phylogeny correlates with Wolbachia phylogeny.
Amino acid alignment of Met1 and Met2 proteins of Wolbachia strain wMel.
Wolbachia exhibits a fascinating array of host manipulations. The elucidation of the molecular basis of the host-symbiont interaction will allow insight in the regulation of fundamental cell biological processes. Future studies will address any potential direct or indirect effect of the methyltransferase(s) in the establishment of symbiosis and/or the induction of reproductive manipulations.