Project description:Endosymbiotic bacteria associated with eukaryotic hosts are omnipresent in nature, particularly in insects. Studying the bacterial side of host-symbiont interactions is, however, often limited by the unculturability and genetic intractability of the symbionts. Spiroplasma poulsonii is a maternally transmitted bacterial endosymbiont that is naturally associated with several Drosophila species. S. poulsonii strongly affects its host’s physiology, for example by causing male killing or by protecting it against various parasites. Despite intense work on this model since the 1950s, attempts to cultivate endosymbiotic Spiroplasma in vitro have failed so far. Here, we developed a method to sustain the in vitro culture of S. poulsonii by optimizing a commercially accessible medium. We also provide a complete genome assembly, including the first sequence of a natural plasmid of an endosymbiotic Spiroplasma species. Last, by comparing the transcriptome of the in vitro culture to the transcriptome of bacteria extracted from the host, we identified genes putatively involved in host-symbiont interactions. This work provides new opportunities to study the physiology of endosymbiotic Spiroplasma and paves the way to dissect insect-endosymbiont interactions with two genetically tractable partners.
Project description:We investigated the effect of Spiroplasma infection on Drosophila hemolymph protein content using Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS). To this end, we extracted total hemolymph from uninfected and infected 10 days old females. At this age, Spiroplasma is already present at high titers in the hemolymph but does not cause major deleterious phenotypes to the fly. Extraction was achieved by puncturing the thorax and drawing out with a microinjector. Four replicates were made
Project description:BACKGROUND: The genus Spiroplasma contains a group of helical, motile, and wall-less bacteria in the class Mollicutes. Similar to other members of this class, such as the animal-pathogenic Mycoplasma and the plant-pathogenic 'Candidatus Phytoplasma', all characterized Spiroplasma species were found to be associated with eukaryotic hosts. While most of the Spiroplasma species appeared to be harmless commensals of insects, a small number of species have evolved pathogenicity toward various arthropods and plants. In this study, we isolated a novel strain of honeybee-associated S. melliferum and investigated its genetic composition and evolutionary history by whole-genome shotgun sequencing and comparative analysis with other Mollicutes genomes. RESULTS: The whole-genome shotgun sequencing of S. melliferum IPMB4A produced a draft assembly that was ~1.1 Mb in size and covered ~80% of the chromosome. Similar to other Spiroplasma genomes that have been studied to date, we found that this genome contains abundant repetitive sequences that originated from plectrovirus insertions. These phage fragments represented a major obstacle in obtaining a complete genome sequence of Spiroplasma with the current sequencing technology. Comparative analysis of S. melliferum IPMB4A with other Spiroplasma genomes revealed that these phages may have facilitated extensive genome rearrangements in these bacteria and contributed to horizontal gene transfers that led to species-specific adaptation to different eukaryotic hosts. In addition, comparison of gene content with other Mollicutes suggested that the common ancestor of the SEM (Spiroplasma, Entomoplasma, and Mycoplasma) clade may have had a relatively large genome and flexible metabolic capacity; the extremely reduced genomes of present day Mycoplasma and 'Candidatus Phytoplasma' species are likely to be the result of independent gene losses in these lineages. CONCLUSIONS: The findings in this study highlighted the significance of phage insertions and horizontal gene transfer in the evolution of bacterial genomes and acquisition of pathogenicity. Furthermore, the inclusion of Spiroplasma in comparative analysis has improved our understanding of genome evolution in Mollicutes. Future improvements in the taxon sampling of available genome sequences in this group are required to provide further insights into the evolution of these important pathogens of humans, animals, and plants.
Project description:Spiralin is the major membrane protein of the helical mollicute Spiroplasma citri. A similar protein occurs in the membrane of Spiroplasma melliferum, an organism related to S. citri. The gene encoding spiralin has been sequenced. A restriction fragment of the spiralin gene has been used as a probe to detect the gene encoding S. melliferum spiralin. A 4.6-kilobase-pair ClaI DNA fragment from S. melliferum strongly hybridized with the probe. This fragment was inserted in pBR322 and cloned in Escherichia coli. It was further subcloned in the replicative forms of M13mp18 and M13mp19, and its nucleotide sequence was determined (GenBank accession number M33991). An open reading frame showing 88.6% base sequence homology with the S. citri spiralin gene could be identified and was assumed to be the gene encoding S. melliferum spiralin. The deduced amino acid sequence of the protein had 75% homology with the spiralin sequence. In particular, the two proteins possess a stretch of 20 amino acids which can form an alpha-helix, in which all polar amino acids occupy approximately one-third of the axial projection down the helix. On the basis of these data and published data, we propose a topological model for the structural organization of the spiralin in the cell membrane of spiroplasmas.