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

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

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Convergent Evolution among Ruminant-Pathogenic Mycoplasma Involved Extensive Gene Content Changes

Convergent evolution, a process by which organisms evolved independently to have similar traits, provides opportunities to understand adaptation. The bacterial genus Mycoplasma contains multiple species that evolved independently to become ruminant pathogens, which represents an interesting study system for investigating the process. In this work, we determined the genome sequences of 11 Entomoplasma/Mesoplasma species. This new data set, together with the other available Mollicutes genomes, provided comprehensive taxon sampling for inferring the gene content evolution that led to the emergence of Mycoplasma Mycoides cluster. Our results indicated that the most recent common ancestor (MRCA) of the Mycoides-Entomoplasmataceae clade lost ∼15% of the core genes when it diverged from the Spiroplasma Apis clade. After this initial wave of genome reduction, relatively few gene gains or losses were inferred until the emergence of the Mycoides cluster. Compared with those Entomoplasmataceae lineages that maintained the association with insects, the MRCA of the Mycoides cluster experienced a second wave of gene losses, as well as acquiring \u3e100 novel genes through horizontal gene transfer. These gene acquisitions involved many with the Mycoplasma Hominis/Pneumoniae lineages as the putative donors, suggesting that gene exchanges among these vertebrate symbionts with distinct phylogenetic affiliations may be important in the emergence of the Mycoides cluster. These findings demonstrated that the gene content of bacterial genomes could be exceedingly dynamic, even for those symbionts with highly reduced genomes. Moreover, the emergence of novel pathogens may involve extensive remodeling of gene content, rather than acquisition of few virulence genes

Genome evolution in Spiroplasma

The extensive studies on the insect obligate symbionts have demonstrated that the intracellular symbiosis resulted in the massive genome reduction, yet the evolution of facultative symbionts is less clear. The genus Spiroplasma contains a group of facultative symbionts that are associated with a variety of insects in natural environments, rendering them a good system to study the host adaptation and the genome evolution. Using comparative genomics approaches, I have identified several enzyme-coding substrate metabolism genes associated with pathogenicity and host adaptation in Spiroplasma, and showed that Spiroplasma continuously acquired foreign genetic fragments. The genome of the honeybee pathogen Spiroplasma melliferum IPMB4A contains a gene encodes for chitinase, which may contribute to its pathogenicity. It also contains abundant plectroviral sequences, which are associated with the extensive genome rearrangements. In freshwater crustaceans pathogen S. eriocheiris, its genome harbors 7% of the protein-coding genes that were acquired through horizontal gene transfer (HGT). Several of these genes are involved in substrate utilization and may be associated with the adaptation to aquatic environments. In addition, the pathogenicity of Spiroplasma toward mosquitoes is associated with the presence of a gene encoding for glycerol-3-phosphate oxidase (GlpO), which produces reactive oxygen species during the glycerol metabolism. In S. taiwanense, the glpO gene was acquired through HGT from species closely related to Mycoplasma mycoides, in which the knockout of glpO alleviated the pathogenicity. Moreover, the transcriptome analyses showed that the horizontally acquired genes, including the glpO gene and several of the substrate utilization genes, were expressed in the comparative level of those for glycolysis, suggesting their important roles in metabolism. In conclusion, this study shows that the Spiroplasma species frequently acquired functional genes and mobile genetic elements through HGT, which may counteract the genome degradation during the symbiosis.摘要 i ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLE v LIST OF FIGURES vi 1 INTRODUCTION 1 1.1 THE GENOME EVOLUTION IN BACTERIAL SYMBIONTS 1 1.1.1 The genomic changes following host restriction in bacteria 1 1.1.2 Mechanisms of genome reduction 2 1.1.3 Difference between obligate and facultative insect symbionts 3 1.2 THE ECOLOGY AND EVOLUTION OF SPIROPLASMA 3 1.2.1 General features of Spiroplasma 3 1.2.2 Phylogenetic classification and host range 4 1.2.3 Spiroplasma genome evolution 6 1.3 RESEARCH PROPOSAL AND RATIONALE 7 2 Comparative genome analysis of Spiroplasma melliferum IPMB4A, a honeybee-associated bacterium 10 2.1 INTRODUCTION 10 2.2 METHODS 11 2.2.1 Strain isolation and DNA preparation 11 2.2.2 Pulsed-field gel electrophoresis 12 2.2.3 Molecular phylogenetic inference 12 2.2.4 Serology test 13 2.2.5 Whole-genome shotgun sequencing 14 2.2.6 Genome assembly and annotation 14 2.2.7 Comparative analysis with other genomes 15 2.3 RESULTS AND DISCUSSION 16 2.3.1 Species identification and phylogenetic inference 16 2.3.2 Genome assembly and annotation 17 2.3.3 Comparative analysis with S. Melliferum KC3 and S. Citri 18 2.3.4 Comparative analysis with mycoplasma and phytoplasma 20 2.4 CONCLUSIONS 23 3 The fates of horizontally acquired genes in Spiroplasma atrichopogonis and Spiroplasma eriocheiris 33 3.1 INTRODUCTION 33 3.2 MATERIALS AND METHODS 34 3.2.1 Bacterial strains 34 3.2.2 Genome sequencing, assembly, and annotation 35 3.2.3 Phylogenetic and comparative analyses 36 3.3 RESULTS 37 3.3.1 Phylogenetic placement of S. Atrichopogonis and S. Eriocheiris 37 3.3.2 Genome features of S. Atrichopogonis and S. Eriocheiris 38 3.3.3 Comparison of gene content and substrate utilization strategies 39 3.3.4 Horizontal gene transfer in S. Eriocheiris 41 3.3.5 Gene acquisition in S. Atrichopogonis mediated by mobile genetic elements 42 3.4 DISCUSSION 44 3.4.1 A model for the molecular evolution events in the mirum clade 44 3.4.2 Other examples of genome degradation in arthropod symbionts 44 3.4.3 Horizontally transferred genes: source, function, and potential link to adaptation 45 3.4.4 Implications on bacterial species description and identification 45 4 Comparison of metabolic capacities and inference of gene content evolution in mosquito-associated Spiroplasma diminutum and S. taiwanense 56 4.1 INTRODUCTION 56 4.2 MATERIALS AND METHODS 58 4.2.1 Molecular phylogenetic inference 58 4.2.2 Strain source and DNA preparation 58 4.2.3 Genome sequencing and assembly 59 4.2.4 Annotation and comparative analysis 59 4.3 RESULTS AND DISCUSSION 61 4.3.1 Molecular phylogeny of mosquito-associated Spiroplasma Species 61 4.3.2 Genome sequences of S. Diminutum and S. Taiwanense 62 4.3.3 Comparison of substrate utilization strategies 64 4.3.4 Gene content comparison with the honeybee-associated S. Melliferum 65 4.3.5 Comparison with the Mycoides-Entomoplasmataceae clade and inference of gene content evolution 67 4.4 CONCLUSIONS 68 5 Transcriptome profiling provides insights into the genome evolution and carbohydrate utilization preference of mosquito-associated Spiroplasma 78 5.1 INTRODUCTION 78 5.2 MATERIALS AND METHODS 80 5.2.1 Bacterial strains and growth conditions 80 5.2.2 RNA extraction and transcriptome sequencing 81 5.2.3 Sequence analysis 81 5.2.4 Validation of the RNA-Seq results by qrt-PCR 82 5.2.5 Comparative genomics and molecular phylogenetics 82 5.3 RESULTS AND DISCUSSION 83 5.3.1 Overview of the RNA-Seq data sets 83 5.3.2 The non-coding rnas in S. Diminutum and S. Taiwanense 84 5.3.3 Expression profiles of S. Diminutum 84 5.3.4 Expression profile of S. Taiwanense 86 5.3.5 Gene content evolution in the Apis clade 88 5.3.6 Expression of horizontally acquired genes and the implication on facultative symbiont genome evolution 89 5.4 CONCLUSIONS 90 6 Conclusion 100 7 References 10

Molecular evolution of the actin-like MreB protein gene family in wall-less bacteria

The mreB gene family encodes actin-like proteins that determine cell shape by directing cell wall synthesis and often exists in one to three copies in the genomes of non-spherical bacteria. Intriguingly, while most wall-less bacteria do not have this gene, five to seven mreB homologs are found in Spiroplasma and Haloplasma, which are both characterized by cell contractility. To investigate the molecular evolution of this gene family in wall-less bacteria, we sampled the available genome sequences from these two genera and other related lineages for comparative analysis. The gene phylogenies indicated that the mreB homologs in Haloplasma are more closely related to those in Firmicutes, whereas those in Spiroplasma form a separate clade. This finding suggests that the gene family expansions in these two lineages are the results of independent ancient duplications. Moreover, the Spiroplasma mreB homologs can be classified into five clades, of which the genomic positions are largely conserved. The inference of gene gains and losses suggests that there has been an overall trend to retain only one homolog from each of the five mreB clades in the evolutionary history of Spiroplasma

Complete Genome Sequence of Spiroplasma turonicum Tab4cT, a Bacterium Isolated from Horse Flies (Haematopota sp.)

Spiroplasma turonicum Tab4cT was isolated from a horse fly (Haematopota sp.; probably Haematopota pluvialis) collected at Champchevrier, Indre-et-Loire, Touraine, France, in 1991. Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its biology and the comparative genomics among Spiroplasma spp

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