Table_1_Isolation, culture and characterization of Arsenophonus symbionts from two insect species reveal loss of infectious transmission and extended host range.doc

Vertically transmitted “Heritable” microbial symbionts represent an important component of the biology and ecology of invertebrates. These symbioses evolved originally from ones where infection/acquisition processes occurred within the environment (horizontal transmission). However, the pattern of evolution that follows transition from horizontal to vertical transmission is commonly obscured by the distant relationship between microbes with differing transmission modes. In contrast, the genus Arsenophonus provides an opportunity to investigate these processes with clarity, as it includes members that are obligate vertically transmitted symbionts, facultative vertically transmitted symbionts, strains with mixed modes of transmission and ones that are purely horizontally transmitted. Significantly, some of the strains are culturable and amenable to genetic analysis. We first report the isolation of Arsenophonus nasoniae strain aPv into culture from the ectoparasitic wasp Pachycrepoideus vindemmiae and characterize the symbiosis. We demonstrate maternal vertical transmission and find no evidence for paternal inheritance, horizontal transmission or reproductive parasitism phenotypes. This leads us to conclude this strain, in contrast to related strains, is a facultative heritable symbiont which is likely to be beneficial. We then report the serendipitous discovery and onward culture of a strain of Arsenophonus (strain aPb) from the blue butterfly, Polyommatus bellargus. This association extends the range of host species carrying Arsenophonus nasoniae/Arsenophonus apicola symbionts beyond the Hymenoptera for the first time. We perform basic metabolic analysis of the isolated strains using Biolog plates. This analysis indicates all strains utilize a restricted range of carbon sources, but these restrictions are particularly pronounced in the A. nasoniae aPv strain that is solely vertically transmitted. Finally, we demonstrate the Arsenophonus sp. strain aPb from the blue butterfly can infect Galleria waxworms, providing a model system for investigating the functional genetics of Arsenophonus-insect interactions. These results are consistent with a model of reduced metabolic competence in strains evolving under vertical transmission only. The data also broadens the range of host species infected with nasoniae/apicola clade strains beyond the Hymenoptera, and indicate the potential utility of the Galleria model for investigation of symbiosis mechanism.</p

Repetitive DNA sequences and ankyrin repeat domain number polymorphism.

<p>The example of WD0766. A) Only the ankyrin repeat domain containing regions are shown. Blue rectangles represent individual ankyrin repeat domains. Dark gray rectangles with dotted outline represent ankyrin repeat domain remnants. The light gray shading between the ANK clusters indicates homologies between the different strains. Orange rectangles represent putative chimeric ankyrin repeat domains, and double arrows represent identical duplications. Small black arrows indicate direct repeats capable of engaging into illegitimate recombination. The reconstructed structure of disrupted <i>w</i>Yak and <i>w</i>San ANK homologs is also presented. The asterisk and the double yellow arrows correspond to a frame shift mutation and the position of the IS5 element, respectively. B<b>)</b> example of chimeric origin of <i>w</i>Mel (and <i>w</i>MelPop) ankyrin repeat domains 2. Identities with the parental ankyrin repeat domains 2 and 4 from <i>w</i>Au are shaded. Box shows the position of the repeated site between the three sequences. The vertical arrow indicates the loop between the two α-helices of the ankyrin repeat domain.</p

The Diversity and Evolution of <em>Wolbachia</em> Ankyrin Repeat Domain Genes

<div><p>Ankyrin repeat domain-encoding genes are common in the eukaryotic and viral domains of life, but they are rare in bacteria, the exception being a few obligate or facultative intracellular Proteobacteria species. Despite having a reduced genome, the arthropod strains of the alphaproteobacterium <em>Wolbachia</em> contain an unusually high number of ankyrin repeat domain-encoding genes ranging from 23 in <em>w</em>Mel to 60 in <em>w</em>Pip strain. This group of genes has attracted considerable attention for their astonishing large number as well as for the fact that ankyrin proteins are known to participate in protein-protein interactions, suggesting that they play a critical role in the molecular mechanism that determines host-<em>Wolbachia</em> symbiotic interactions. We present a comparative evolutionary analysis of the <em>w</em>Mel-related ankyrin repeat domain-encoding genes present in different <em>Drosophila</em>-<em>Wolbachia</em> associations. Our results show that the ankyrin repeat domain-encoding genes change in size by expansion and contraction mediated by short directly repeated sequences. We provide examples of intra-genic recombination events and show that these genes are likely to be horizontally transferred between strains with the aid of bacteriophages. These results confirm previous findings that the <em>Wolbachia</em> genomes are evolutionary mosaics and illustrate the potential that these bacteria have to generate diversity in proteins potentially involved in the symbiotic interactions.</p> </div

<i>Drosophila</i> lines and <i>Wolbachia</i> strains.

<p><i>Drosophila</i> lines and <i>Wolbachia</i> strains.</p

Density of direct repeats larger than 8nt per ANK gene.

<p>Box plot graph showing that ANK genes display ankyrin repeat number variations have greater DR density compared with genes that do not display variations (**P<0.005, Mann-Whitney test).</p

Shimodaira-Hasegawa test for the statistical significance of the topological incongruence between alternative chromosomal- and prophage-associated ankyrin genes.

<p>Values are the likelihood score (-ln L) of a given data set across its own ML tree (boldface), as well as across the alternative tree topologies. Significance levels are based on full optimization.</p>***<p><i>P</i><0.0001.</p

Phylogeny of four chromosomal- (WD0441, WD0498, WD0754 and WD1213) and three prophage-associated (WD0596, WD0636, WD0637) ANK genes.

<p>The trees are midpoint-rooted and inferred using maximum likelihood. ML bootstrap support values inferred from 100 replicates are also presented. Bootstrap values lower than 50 are omitted. The discordant positions of strains <i>w</i>Yak, <i>w</i>San and <i>w</i>Ha between the chromosomal- and prophage-associated ANK gene phylogenies are highlighted with asterisks. Evolutionary model parameters were estimated with Modeltest under the Akaike Information Criterion: HKY (WD0441,WD1213); TrN+I (WD0498, WD0596); TVM+G (WD0754); TrN+G (WD0636); GTR+I (WD0637).</p

Major evolutionary events shaping <i>Wolbachia</i> ANK genes.

a<p>Prophage associated ANK genes are presented in bold face.</p>b<p>Strains potentially involved in recombination are shown in parenthesis.</p

Comparative analysis of genetic diversity.

<p>The graph represents the patterns of synonymous substitutions within A-supergroup strains. (*: <i>w</i>Ri is an outlier, **: <i>w</i>Ha is an outlier).</p

New criteria for selecting the origin of DNA replication in and closely related bacteria-1

<p><b>Copyright information:</b></p><p>Taken from "New criteria for selecting the origin of DNA replication in and closely related bacteria"</p><p>http://www.biomedcentral.com/1471-2164/8/182</p><p>BMC Genomics 2007;8():182-182.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1914354.</p><p></p>e sequences of the region from the A and B Wolbachia. Subgroups are denoted A1 and A2, and B1 and B2