Wen-Sui Lo

Comparative genome analysis of Spiroplasma melliferum IPMB4A, a honeybee-associated bacterium

BackgroundThe 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.ResultsThe 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.ConclusionsThe 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.

Complete Genomes of Two Dipteran-Associated Spiroplasmas Provided Insights into the Origin, Dynamics, and Impacts of Viral Invasion in Spiroplasma

Spiroplasma is a genus of wall-less, low-GC, Gram-positive bacteria with helical morphology. As commensals or pathogens of plants, insects, ticks, or crustaceans, they are closely related with mycoplasmas and form a monophyletic group (Spiroplasma–Entomoplasmataceae–Mycoides) with Mycoplasma mycoides and its relatives. In this study, we report the complete genome sequences of Spiroplasma chrysopicola and S. syrphidicola from the Chrysopicola clade. These species form the sister group to the Citri clade, which includes several well-known pathogenic spiroplasmas. Surprisingly, these two newly available genomes from the Chrysopicola clade contain no plectroviral genes, which were found to be highly repetitive in the previously sequenced genomes from the Citri clade. Based on the genome alignment and patterns of GC-skew, these two Chrysopicola genomes appear to be relatively stable, rather than being highly rearranged as those from the Citri clade. Phylogenetic analyses suggest that the susceptibility to plectroviral invasion probably originated in the common ancestor of the Citri clade or one of its subclades. This susceptibility may be attributed to the absence of antiviral systems found in the Chrysopicola clade. Using the virus-free genomes of the Chrysopicola clade as references, we inferred the putative viral integration sites in the Citri genomes. Comparisons of syntenic regions suggest that the extensive viral invasion in the Citri clade promoted genome rearrangements and expansions. More importantly, the viral invasion may have facilitated horizontal gene transfers that contributed to adaptation in the Citri clade.

Genome-Wide Patterns of Genetic Variation in Two Domestic Chickens

Domestic chickens are excellent models for investigating the genetic basis of phenotypic diversity, as numerous phenotypic changes in physiology, morphology, and behavior in chickens have been artificially selected. Genomic study is required to study genome-wide patterns of DNA variation for dissecting the genetic basis of phenotypic traits. We sequenced the genomes of the Silkie and the Taiwanese native chicken L2 at ∼23- and 25-fold average coverage depth, respectively, using Illumina sequencing. The reads were mapped onto the chicken reference genome (including 5.1% Ns) to 92.32% genome coverage for the two breeds. Using a stringent filter, we identified ∼7.6 million single-nucleotide polymorphisms (SNPs) and 8,839 copy number variations (CNVs) in the mapped regions; 42% of the SNPs have not found in other chickens before. Among the 68,906 SNPs annotated in the chicken sequence assembly, 27,852 were nonsynonymous SNPs located in 13,537 genes. We also identified hundreds of shared and divergent structural and copy number variants in intronic and intergenic regions and in coding regions in the two breeds. Functional enrichments of identified genetic variants were discussed. Radical nsSNP-containing immunity genes were enriched in the QTL regions associated with some economic traits for both breeds. Moreover, genetic changes involved in selective sweeps were detected. From the selective sweeps identified in our two breeds, several genes associated with growth, appetite, and metabolic regulation were identified. Our study provides a framework for genetic and genomic research of domestic chickens and facilitates the domestic chicken as an avian model for genomic, biomedical, and evolutionary studies.

Comparison of Metabolic Capacities and Inference of Gene Content Evolution in Mosquito-associated Spiroplasma diminutum and S. taiwanense

Mosquitoes are hosts of several Spiroplasma species that belong to different serogroups. To investigate the genetic mechanisms that may be involved in the utilization of similar hosts in these phylogenetically distinct bacteria, we determined the complete genome sequences of Spiroplasma diminutum and S. taiwanense for comparative analysis. The genome alignment indicates that their chromosomal organization is highly conserved, which is in sharp contrast to the elevated genome instabilities observed in other Spiroplasma lineages. Examination of the substrate utilization strategies revealed that S. diminutum can use a wide range of carbohydrates, suggesting that it is well suited to living in the gut (and possibly the circulatory system) of its mosquito hosts. In comparison, S. taiwanense has lost several carbohydrate utilization genes and acquired additional sets of oligopeptide transporter genes through tandem duplications, suggesting that proteins from digested blood meal or lysed host cells may be an important nutrient source. Moreover, one glycerol-3-phosphate oxidase gene (glpO) was found in S. taiwanense but not S. diminutum. This gene is linked to the production of reactive oxygen species and has been shown to be a major virulence factor in Mycoplasma mycoides. This finding may explain the pathogenicity of S. taiwanense observed in previous artificial infection experiments, while no apparent effect was found for S. diminutum. To infer the gene content evolution at deeper divergence levels, we incorporated other Mollicutes genomes for comparative analyses. The results suggest that the losses of biosynthetic pathways are a recurrent theme in these host-associated bacteria.

Winding paths to simplicity: genome evolution in facultative insect symbionts

Symbiosis between organisms is an important driving force in evolution. Among the diverse relationships described, extensive progress has been made in insect–bacteria symbiosis, which improved our understanding of the genome evolution in host-associated bacteria. Particularly, investigations on several obligate mutualists have pushed the limits of what we know about the minimal genomes for sustaining cellular life. To bridge the gap between those obligate symbionts with extremely reduced genomes and their non-host-restricted ancestors, this review focuses on the recent progress in genome characterization of facultative insect symbionts. Notable cases representing various types and stages of host associations, including those from multiple genera in the family Enterobacteriaceae (class Gammaproteobacteria), Wolbachia (Alphaproteobacteria) and Spiroplasma (Mollicutes), are discussed. Although several general patterns of genome reduction associated with the adoption of symbiotic relationships could be identified, extensive variation was found among these facultative symbionts. These findings are incorporated into the established conceptual frameworks to develop a more detailed evolutionary model for the discussion of possible trajectories. In summary, transitions from facultative to obligate symbiosis do not appear to be a universal one-way street; switches between hosts and lifestyles (e.g. commensalism, parasitism or mutualism) occur frequently and could be facilitated by horizontal gene transfer.

Molecular Evolution of the Substrate Utilization Strategies and Putative Virulence Factors in Mosquito-Associated Spiroplasma Species

Comparative genomics provides a powerful tool to characterize the genetic differences among species that may be linked to their phenotypic variations. In the case of mosquito-associated Spiroplasma species, such approach is useful for the investigation of their differentiations in substrate utilization strategies and putative virulence factors. Among the four species that have been assessed for pathogenicity by artificial infection experiments, Spiroplasma culicicola and S. taiwanense were found to be pathogenic, whereas S. diminutum and S. sabaudiense were not. Intriguingly, based on the species phylogeny, the association with mosquito hosts and the gain or loss of pathogenicity in these species appears to have evolved independently. Through comparison of their complete genome sequences, we identified the genes and pathways that are shared by all or specific to one of these four species. Notably, we found that a glycerol-3-phosphate oxidase gene (glpO) is present in S. culicicola and S. taiwanense but not in S. diminutum or S. sabaudiense. Because this gene is involved in the production of reactive oxygen species and has been demonstrated as a major virulence factor in Mycoplasma, this distribution pattern suggests that it may be linked to the observed differences in pathogenicity among these species as well. Moreover, through comparative analysis with other Spiroplasma, Mycoplasma, and Mesoplasma species, we found that the absence of glpO in S. diminutum and S. sabaudiense is best explained by independent losses. Finally, our phylogenetic analyses revealed possible recombination of glpO between distantly related lineages and local rearrangements of adjacent genes.

Found and lost: the fates of horizontally acquired genes in arthropod-symbiotic Spiroplasma

Horizontal gene transfer (HGT) is an important mechanism that contributed to biological diversity, particularly in bacteria. Through acquisition of novel genes, the recipient cell may change its ecological preference and the process could promote speciation. In this study, we determined the complete genome sequence of two Spiroplasma species for comparative analyses and inferred the putative gene gains and losses. Although most Spiroplasma species are symbionts of terrestrial insects, Spiroplasma eriocheiris has evolved to be a lethal pathogen of freshwater crustaceans. We found that approximately 7% of the genes in this genome may have originated from HGT and these genes expanded the metabolic capacity of this organism. Through comparison with the closely related Spiroplasma atrichopogonis, as well as other more divergent lineages, our results indicated that these HGT events could be traced back to the most recent common ancestor of these two species. However, most of these horizontally acquired genes have been pseudogenized in S. atrichopogonis, suggesting that they did not contribute to the fitness of this lineage that maintained the association with terrestrial insects. Thus, accumulation of small deletions that disrupted these foreign genes was not countered by natural selection. On the other hand, the long-term survival of these horizontally acquired genes in the S. eriocheiris genome hinted that they might play a role in the ecological shift of this species. Finally, the implications of these findings and the conflicts among gene content, 16S rRNA gene sequencing, and serological typing, are discussed in light of defining bacterial species.

Analysis of the gut microbiota of walking sticks (Phasmatodea)

BackgroundLittle is known about the Phasmatodea gut microbial community, including whether phasmids have symbiotic bacteria aiding in their digestion. While symbionts are near ubiquitous in herbivorous insects, the Phasmatodea’s distinctively thin body shape precludes the gut enlargements needed for microbial fermentation. High-throughput sequencing was used to characterize the entire microbiota of the fat bodies, salivary glands, and anterior and posterior midguts of two species of walking stick.ResultsMost bacterial sequences belonged to a strain of Spiroplasma (Tenericutes) found primarily in the posterior midgut of the parthenogenetic species Ramulus artemis (Phasmatidae). Beyond this, no significant differences were found between the R. artemis midgut sections or between that species and Peruphasma schultei (Pseudophasmatidae). Histological analysis further indicated a lack of bacteriocytes.ConclusionsPhasmids are unlikely to depend on bacteria for digestion, suggesting they produce enzymes endogenously that most other herbivorous insects obtain from symbionts. This conclusion matches predictions based on phasmid anatomy. The role of Spiroplasma in insects warrants further study.

Complete Genome Sequence of Spiroplasma apis B31T (ATCC 33834), a Bacterium Associated with May Disease of Honeybees (Apis mellifera)

ABSTRACT Spiroplasma apis B31T (ATCC 33834) is a wall-less bacterium in the class Mollicutes that has been linked to May disease of honeybees (Apis mellifera). Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its virulence factors.

Complete Genome Sequence of Serratia marcescens WW4

ABSTRACT Serratia marcescens WW4 is a biofilm-forming bacterium isolated from paper machine aggregates. Under conditions of phosphate limitation, this bacterium exhibits intergeneric inhibition of Pseudomonas aeruginosa. Here, the complete genome sequence of S. marcescens WW4, which consists of one circular chromosome (5,241,455 bp) and one plasmid (pSmWW4; 3,248 bp), was determined.