The mosaic genome structure of the Wolbachia wRi strain infecting Drosophila simulans

Abstract

The obligate intracellular bacterium Wolbachia pipientis infects around 20% of all insect species. It is maternally inherited and induces reproductive alterations of insect populations by male killing, feminization, parthenogenesis, or cytoplasmic incompatibility. Here, we present the 1,445,873-bp genome of W. pipientis strain wRi that induces very strong cytoplasmic incompatibility in its natural host Drosophila simulans. A comparison with the previously sequenced genome of W. pipientis strain wMel from Drosophila melanogaster identified 35 breakpoints associated with mobile elements and repeated sequences that are stable in Drosophila lines transinfected with wRi. Additionally, 450 genes with orthologs in wRi and wMel were sequenced from the W. pipientis strain wUni, responsible for the induction of parthenogenesis in the parasitoid wasp Muscidifurax uniraptor. The comparison of these A-group Wolbachia strains uncovered the most highly recombining intracellular bacterial genomes known to date. This was manifested in a 500-fold variation in sequence divergences at synonymous sites, with different genes and gene segments supporting different strain relationships. The substitution-frequency profile resembled that of Neisseria meningitidis, which is characterized by rampant intraspecies recombination, rather than that of Rickettsia, where genes mostly diverge by nucleotide substitutions. The data further revealed diversification of ankyrin repeat genes by short tandem duplications and provided examples of horizontal gene transfer across A- and B-group strains that infect D. simulans. These results suggest that the transmission dynamics of Wolbachia and the opportunity for coinfections have created a freely recombining intracellular bacterial community with mosaic genomes.

Fig. 1.

Circular map of the Wolbachia pipientis wRi genome. Each circle confined by the gray lines except for the 2 innermost circles illustrates different features on the plus (outer region) and minus (inner region) strands. Lines and boxes in the 3 outermost circles are colored according to the Clusters of Orthologous Groups (COG) categories. First (Outer) circle: protein-coding genes (CDSs). Second circle: pseudogenes. Third circle: unique CDSs compared to wMel. Fourth circle: ANK genes in blue and gene synteny breakpoints compared to wMel in red. Fifth circle: prophage regions in green (not affiliated to a strand) and IS elements color-coded as described in Table S5. Sixth circle: a diagram showing the synonymous substitution frequency (Ks) between wRi and wMel potential orthologs; the maximum cut-off was set to Ks = 1. Seventh circle: GC-skew of the wRi genome.

Fig. 2.

Ternary plot showing substitution frequency variation across (A) 410 genes in A-group Wolbachia (40 genes with no synonymous substitutions in all 3 strains were excluded); (B) 818 genes in Spotted-Fever Group Rickettsia; (C) 1,529 genes in Neisseria meningitidis; and (D) 2,207 genes in Staphylococcus aureus. Each dot in the diagram represents 1 gene. Absolute Ks-values have been transformed to relative values between 0 and 1. The mean relative Ks-value for each pair is shown on each axis. Numbers in bold represent the median distance to the average point. The color of each dot represents the maximum absolute Ks-value among the 3 pairs, ranging from light yellow (low values) to red (high values). Median Ks-values for all pairs are reported in Table S8. Rr, Rickettsia rickettsii; Rc, Rickettsia conorii; Rm, Rickettsia massiliae.

Fig. 3.

Recombination within leuRS and virB10. (A) The diagram shows the Ks-values calculated for the leuRS gene pair segments consisting of a 99-base window sliding over the gene alignment with 15-base steps. The boxes under the x-axis indicate regions where wRi (red box) and wUni (black box) are highly diverged from the other two strains. (B and C) Inference of sequence relationships based on segments in the virB10 gene between (B) positions 72 and 892 and (C) positions 967 and 1526, respectively, of the gene alignment. The gray circle shows the position of wRi in the tree. The strains wKueYo, wMel, wUni, wAtab3, and wRi belong to supergroup A (names in red), whereas strains wPip and wTai belong to supergroup B (names in blue).

Fig. 4.

Clustering of ankyrin repeat domains from wRi, wMel, and wUni using Bayesian phylogenetic inference. The colors on the branches indicate different intervals of posterior probability: (red) 0.95–1.0, (yellow) 0.90–0.94, and (blue) 0.89–0.80 (nonsignificant), and (black) <0.80.