Wolbachia Prophage DNA Adenine Methyltransferase Genes in Different Drosophila-Wolbachia Associations

Wolbachia is an obligatory intracellular bacterium which often manipulates the reproduction of its insect and isopod hosts. In contrast, Wolbachia is an essential symbiont in filarial nematodes. Lately, Wolbachia has been implicated in genomic imprinting of host DNA through cytosine methylation. The importance of DNA methylation in cell fate and biology calls for in depth studing of putative methylation-related genes. We present a molecular and phylogenetic analysis of a putative DNA adenine methyltransferase encoded by a prophage in the Wolbachia genome. Two slightly different copies of the gene, met1 and met2, exhibit a different distribution over various Wolbachia strains. The met2 gene is present in the majority of strains, in wAu, however, it contains a frameshift caused by a 2 bp deletion. Phylogenetic analysis of the met2 DNA sequences suggests a long association of the gene with the Wolbachia host strains. In addition, our analysis provides evidence for previously unnoticed multiple infections, the detection of which is critical for the molecular elucidation of modification and/or rescue mechanism of cytoplasmic incompatibility

The bacteriophage WORiC is the active phage element in <it>w</it>Ri of <it>Drosophila simulans </it>and represents a conserved class of WO phages

Abstract Background The alphaproteobacterium Wolbachia pipientis, the most common endosymbiont in eukaryotes, is found predominantly in insects including many Drosophila species. Although Wolbachia is primarily vertically transmitted, analysis of its genome provides evidence for frequent horizontal transfer, extensive recombination and numerous mobile genetic elements. The genome sequence of Wolbachia in Drosophila simulans Riverside (wRi) is available along with the integrated bacteriophages, enabling a detailed examination of phage genes and the role of these genes in the biology of Wolbachia and its host organisms. Wolbachia is widely known for its ability to modify the reproductive patterns of insects. One particular modification, cytoplasmic incompatibility, has previously been shown to be dependent on Wolbachia density and inversely related to the titer of lytic phage. The wRi genome has four phage regions, two WORiBs, one WORiA and one WORiC. Results In this study specific primers were designed to distinguish between these four prophage types in wRi, and quantitative PCR was used to measure the titer of bacteriophages in testes, ovaries, embryos and adult flies. In all tissues tested, WORiA and WORiB were not found to be present in excess of their integrated prophages; WORiC, however, was found to be present extrachromosomally. WORiC is undergoing extrachromosomal replication in wRi. The density of phage particles was found to be consistent in individual larvae in a laboratory population. The WORiC genome is organized in conserved blocks of genes and aligns most closely with other known lytic WO phages, WOVitA and WOCauB. Conclusions The results presented here suggest that WORiC is the lytic form of WO in D. simulans, is undergoing extrachromosomal replication in wRi, and belongs to a conserved family of phages in Wolbachia.</p

Southern blot analysis of <i>met</i> genes using genomic DNA from different <i>Wolbachia</i> infected species.

<p>The blot was hybridized with a probe corresponding to the full length <i>met1</i> (<b>A</b>) and <i>met2</i> (<b>B</b>) genes from <i>w</i>Mel.</p

PCR primers used in the present study.

<p>PCR primers used in the present study.</p

PCR analysis of <i>met2</i> copies in <i>Wolbachia</i> strain <i>w</i>Tei.

<p>PCR was performed using primers met_102F and met_269R (<b>A</b>), met_1024R (<b>B</b>) or TeiB_1024R (<b>C</b>). Primers met_102F and met_269R detect both A-group- and B-group-like <i>met2</i>, met_1024R detects only A-group-like <i>met2</i>, while TeiB_1024R detects only B-group-like <i>met2</i>. M: 100 base pair molecular weight DNA marker (New England Biolabs).</p

Insect lines and <i>Wolbachia</i> strains used in the present study.

<p>Insect lines and <i>Wolbachia</i> strains used in the present study.</p

Phylogenetic tree of <i>Wolbachia</i> based on <i>met</i> gene sequences.

<p>The tree was constructed by Maximum Likelihood analysis. Numbers on the nodes indicate bootstrap values.</p

Distribution of phage methyltransferases in <i>resc</i>+ and <i>resc</i>− <i>Wolbachia</i> strains.

<p>Distribution of phage methyltransferases in <i>resc</i>+ and <i>resc</i>− <i>Wolbachia</i> strains.</p