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. 2004 Mar;2(3):E69.
doi: 10.1371/journal.pbio.0020069. Epub 2004 Mar 16.

Phylogenomics of the Reproductive Parasite Wolbachia Pipientis wMel: A Streamlined Genome Overrun by Mobile Genetic Elements

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Free PMC article

Phylogenomics of the Reproductive Parasite Wolbachia Pipientis wMel: A Streamlined Genome Overrun by Mobile Genetic Elements

Martin Wu et al. PLoS Biol. .
Free PMC article

Abstract

The complete sequence of the 1,267,782 bp genome of Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of wMel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of wMel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between wMel and D. melanogaster or older transfers between Wolbachia and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the alpha-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for the host, the wMel-D. melanogaster symbiosis is now an ideal system for studying the biology and evolution of Wolbachia infections.

Conflict of interest statement

The authors have declared that no conflicts of interest exist.

Figures

Figure 1
Figure 1. Circular Map of the Genome and Genome Features
Circles correspond to the following: (1) forward strand genes; (2) reverse strand genes, (3) in red, genes with likely orthologs in both R. conorii and R. prowazekii; in blue, genes with likely orthologs in R. prowazekii, but absent from R. conorii; in green, genes with likely orthologs in R. conorii but absent from R. prowazekii; in yellow, genes without orthologs in either Rickettsia (Table S3); (4) plot is of χ2 analysis of nucleotide composition; phage regions are in pink; (5) plot of GC skew (G–C)/(G+C); (6) repeats over 200 bp in length, colored by category; (7) in green, transfer RNAs; (8) in blue, ribosomal RNAs; in red, structural RNA.
Figure 2
Figure 2. Phage Alignments and Neighboring Genes
Conserved gene order between the WO phage in Wolbachia sp. wKue and prophage regions of wMel. Putative proteins in wKue (Masui et al. 2001) were searched using TBLASTN against the wMel genome. Matches with an E-value of less than 1e−15 are linked by connecting lines. CDSs are colored as follows: brown, phage structural or replication genes; light blue, conserved hypotheticals; red, hypotheticals; magenta, transposases or reverse transcriptases; blue, ankyrin repeat genes; light gray, radC; light green, paralogous genes; gold, others. The regions surrounding the phage are shown because they have some unusual features relative to the rest of the genome. For example, WO-A and WO-B are each flanked on one side by clusters of genes in two paralogous families that are distantly related to phage repressors. In each of these clusters, a homolog of the radC gene is found. A third radC homolog (WD1093) in the genome is also flanked by a member of one of these gene families (WD1095). While the connection between radC and the phage is unclear, the multiple copies of the radC gene and the members of these paralogous families may have contributed to the phage rearrangements described above.
Figure 3
Figure 3. Alignment of wMel with a 60 kbp Region of the Wolbachia from B. malayi
The figure shows BLASTN matches (green) and whole-proteome alignments (red) that were generated using the “promer” option of the MUMmer software (Delcher et al. 1999). The B. malayi region is from a BAC clone (Ware et al. 2002). Note the regions of alignment broken up by many rearrangements and the presence of repetitive sequences at the regions of the breaks.
Figure 4
Figure 4. Long Evolutionary Branches in wMel
Maximum-likelihood phylogenetic tree constructed on concatenated protein sequences of 285 orthologs shared among wMel, R. prowazekii, R. conorii, C. crescentus, and E. coli. The location of the most recent common ancestor of the α-Proteobacteria (Caulobacter, Rickettsia, Wolbachia) is defined by the outgroup E. coli. The unit of branch length is the number of changes per amino acid. Overall, the amino acid substitution rate in the wMel lineage is about 63% higher than that of C. crescentus, a free-living α-Proteobacteria. wMel has evolved at a slightly higher rate than the Rickettssia spp., close relatives that are also obligate intracellular bacteria that have undergone accelerated evolution themselves. This higher rate is likely in part to be due to an increase in the rate of slightly deleterious mutations, although we have not ruled out the possibility of G+C content effects on the branch lengths.
Figure 5
Figure 5. Mitochondrial Evolution Using Concatenated Alignments
Networks of protein LogDet distances for an alignment of 32 proteins constructed with Neighbor-Net (Bryant and Moulton 2003). The scale bar indicates 0.1 substitutions per site. Enlargements at lower right show the component of shared similarity between mitochondrial-encoded proteins and (i) their homologs from intracellular endosymbionts (red) as well as (ii) their homologs from free-living α-Proteobacteria (blue). (A) Result using 6,776 gap-free sites per genome (heavily biased in amino acid composition). (B) Result using 3,100 sites after exclusion of highly variable positions (data not biased in amino acid composition at p = 0.95). All data and alignments are available upon request. Results of phylogenetic analyses are summa-rized in Table S7. Since amino acid content bias was very severe in these datasets, protein LogDet analyses were also preformed. In neighbor-joining, parsimony, and maximum-likelihood trees generated from alignments both including and excluding highly biased positions (6,776 and 3,100 gap-free amino acid sites per genome, respectively), mitochondria usually branched basal to the Wolbachia–Rickettsia clade, but never specifically with Rickettsia (Table S7).
Figure 6
Figure 6. Genomic Organization and expression of Type IV Secretion Operons in wMel
(A) Organization of the nine vir-like CDSs (white arrows) and five adjacent CDSs that encode for either putative membrane-spanning proteins (black arrows) or non-vir CDSs (gray arrows) of wMel, R. conorii, and A. tumefaciens. Solid horizontal lines denote RT experiments that have confirmed that adjacent CDSs are expressed as part of a polycistronic transcript. Results of these RT-PCR experiments are presented in (B). Lane 1, virB3-virB4; lane 2, RT control; lane 3, virB6-WD0856; lane 4, RT control; lane 5, WD0856-WD0855; lane 6, RT control; lane 7, WD0854-WD0853; lane 8, RT control; lane 9, virB8-virB9; lane 10, RT control; lane 11, virB9-virB11; lane 12, RT control; lane 13, virB11-virD4; lane 14, RT control; lane 15, virD4-wspB; lane 16, RT control; lane 17, virB4-virB6; lane 18, RT control; lane 19, WD0855-WD0854; lane 20, RT control. Only PCRs that contain reverse transcriptase amplified the desired products. PCR primer sequences are listed in Table S9.

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