Genome sequence of the Drosophila melanogaster male-killing Spiroplasma strain MSRO endosymbiont

Abstract

Spiroplasmas are helical and motile members of a cell wall-less eubacterial group called Mollicutes. Although all spiroplasmas are associated with arthropods, they exhibit great diversity with respect to both their modes of transmission and their effects on their hosts; ranging from horizontally transmitted pathogens and commensals to endosymbionts that are transmitted transovarially (i.e., from mother to offspring). Here we provide the first genome sequence, along with proteomic validation, of an endosymbiotic inherited Spiroplasma bacterium, the Spiroplasma poulsonii MSRO strain harbored by Drosophila melanogaster. Comparison of the genome content of S. poulsonii with that of horizontally transmitted spiroplasmas indicates that S. poulsonii has lost many metabolic pathways and transporters, demonstrating a high level of interdependence with its insect host. Consistent with genome analysis, experimental studies showed that S. poulsonii metabolizes glucose but not trehalose. Notably, trehalose is more abundant than glucose in Drosophila hemolymph, and the inability to metabolize trehalose may prevent S. poulsonii from overproliferating. Our study identifies putative virulence genes, notably, those for a chitinase, the H2O2-producing glycerol-3-phosphate oxidase, and enzymes involved in the synthesis of the eukaryote-toxic lipid cardiolipin. S. poulsonii also expresses on the cell membrane one functional adhesion-related protein and two divergent spiralin proteins that have been implicated in insect cell invasion in other spiroplasmas. These lipoproteins may be involved in the colonization of the Drosophila germ line, ensuring S. poulsonii vertical transmission. The S. poulsonii genome is a valuable resource to explore the mechanisms of male killing and symbiont-mediated protection, two cardinal features of many facultative endosymbionts.

Importance: Most insect species, including important disease vectors and crop pests, harbor vertically transmitted endosymbiotic bacteria. These endosymbionts play key roles in their hosts' fitness, including protecting them against natural enemies and manipulating their reproduction in ways that increase the frequency of symbiont infection. Little is known about the molecular mechanisms that underlie these processes. Here, we provide the first genome draft of a vertically transmitted male-killing Spiroplasma bacterium, the S. poulsonii MSRO strain harbored by D. melanogaster. Analysis of the S. poulsonii genome was complemented by proteomics and ex vivo metabolic experiments. Our results indicate that S. poulsonii has reduced metabolic capabilities and expresses divergent membrane lipoproteins and potential virulence factors that likely participate in Spiroplasma-host interactions. This work fills a gap in our knowledge of insect endosymbionts and provides tools with which to decipher the interaction between Spiroplasma bacteria and their well-characterized host D. melanogaster, which is emerging as a model of endosymbiosis.

FIG 1

Draft assembly of the S. poulsonii MSRO genome. (A) Locations of the 12 contigs on the S. poulsonii chromosome as determined by optical mapping with MapSolver software (OpGen). Contigs A and B could not be placed on the chromosome. (B) Schematic representation of the S. poulsonii chromosome. Contigs (in yellow) were placed in order on the basis of the in silico alignment by optical mapping technology. The estimated length of the chromosome is ~1.89 Mb. GC content in depicted in blue. (C) Genome assembly statistics. The table shown was modified from reference . The superscript letter a indicates that for S. chrysopicola, S. melliferum, and S. poulsonii, putative pseudogenes were annotated with the “/pseudo” tag in gene feature as suggested by the NCBI GenBank guidelines and were not included in the total number of protein-coding genes. For S. citri, putative pseudogenes were annotated by adding the term “truncated” in the CDS product description field and were included in the total number of protein-coding genes. The superscript letter b indicates that most of the plectrovirus-related regions were excluded from the S. melliferum IPMB4A assembly because of unresolvable polymorphism, resulting in a lower number of plectroviral genes. The genome of S. chrysopicola does not contain any identifiable plectroviral fragments; this lineage was likely to have diverged prior to the plectroviral invasion of the common ancestor of S. citri, S. melliferum, and S. poulsonii. a.a., amino acids.

FIG 2

Phylogenetic tree of Spiroplasma species. This maximum-likelihood phylogenetic tree was inferred by using the 16S rRNA gene. GenBank accession numbers are listed in Materials and Methods. The values on internal branches are percentages of bootstrap support based on 1,000 replicates. Color codes represent the different clades, and the black arrow denotes the probable point of viral invasion of the lineage (15). Representative images of Spiroplasma hosts are shown to the right of each clade. Species previously sequenced are underlined. Solid underlining denotes an infectious pathogen, and dashed underlining denotes a mutualistic relationship with the insect host. Of note, all of the genomes sequenced are from horizontally transmitted spiroplasmas. The following pictures were obtained from Wikipedia: mosquito, http://zh.wikipedia.org/wiki/%E8%9A%8A; tulip, http://en.wikipedia.org/wiki/Liriodendron_tulipifera; honey bee, http://en.wikipedia.org/wiki/Western_honey_bee; tick, http://en.wikipedia.org/wiki/Haemaphysalis_longicornis; Syrphidae, http://en.wikipedia.org/wiki/File:Syrphidae_poster.jpg; deer fly, http://commons.wikimedia.org/wiki/File:Chrysops_callidus.jpg; giant tiger prawn, http://commons.wikimedia.org/wiki/File:CSIRO_ScienceImage_2992_The_Giant_Tiger_Prawn.jpg; scarlet pimpernel, http://commons.wikimedia.org/wiki/File:Flower_poster_2.jpg. The remaining images are from our personal lab collection.

FIG 3

Only 25% of protein-coding genes have a functional classification. The functional categorization of each protein-coding gene was done according to the COG assignment; genes that did not have any inferred COG annotation were assigned to a custom category (X). Among the total of 2,263 genes, only the 401 protein-coding genes that have specific functional category assignments are represented in the pie chart.

FIG 4

Numbers of shared and genome-specific homologous gene clusters. The Venn diagram shows the numbers of shared and genome-specific homologous gene clusters among the S. poulsonii, S. citri, S. melliferum, and S. chrysopicola genomes.

FIG 5

S. poulsonii sugar metabolism and virulence genes. (A) Metabolic scheme representing enzymes and transporters encoded by the S. poulsonii genome that are involved in sugar metabolism. Enzymes or transporters that are absent from or nonfunctional in S. poulsonii are red. (B) Potential entomopathogenic factors and surface proteins encoded by the S. poulsonii genome. Three potential virulence factors (chitinase A, GlpO, and cardiolipin) may play a role in S. poulsonii-mediated protection against parasitoid wasps. Two spiralin-like proteins and one ARP might play important roles in endosymbiosis.

FIG 6

Deformation test confirming the limited metabolic capacity of S. poulsonii. Drosophila hemolymph containing S. poulsonii was extracted and incubated in phosphate buffer alone or complemented with glucose, fructose, trehalose, or arginine. Bacterial shape was then monitored by fluorescence microscopy. Representative images (A) and percentages of helical cells compared to the total cell counts after 7 days (B) are shown. S. poulsonii maintains its helical shape for 1 week in the presence of glucose but not fructose, trehalose, or arginine. Shown is the mean ± the standard error of the mean of data pooled from three independent experiments (n = >100; ***, P < 0.0001).