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.

Pseudomonas fluorescens PCL1751 is a rod-shaped Gram-negative bacterium isolated from the rhizosphere of a greenhouse-grown tomato plant in Uzbekistan. It controls several plant root diseases caused by Fusarium fungi through the mechanism of competition for nutrients and niches (CNN). This mechanism does not rely on the production of antibiotics, so it avoids the concerns of resistance development and is environmentally safe. Additionally, this bacterium promotes plant growth by alleviating salt stress for its plant host. To investigate the genetic mechanisms that may explain these observations, we determined the complete genome sequence of this bacterium, examined its gene content, and performed comparative genomics analysis with other Pseudomonas strains. The genome of P. fluorescens PCL1751 consisted of one circular chromosome that is 6,143,950 base-pairs (bp) in size; no plasmid was found. The annotation included 19 rRNA, 70 tRNA, and 5,534 protein-coding genes. The gene content analysis identified a large number of genes involved in chemotaxis and motility, colonization of the rhizosphere, siderophore biosynthesis, and osmoprotectant production. In contrast, the pathways involved in the biosynthesis of phytohormones or antibiotics were not found. Comparison with other Pseudomonas genomes revealed extensive variations in their genome size and gene content. The presence and absence of secretion system genes were highly variable. As expected, the synteny conservation among strains decreased as a function of phylogenetic divergence. The integration of prophages appeared to be an important driver for genome rearrangements. The whole-genome gene content analysis of this plant growth-promoting rhizobacterium (PGPR) provided some genetic explanations to its phenotypic characteristics. The extensive and versatile substrate utilization pathways, together with the presence of many genes involved in competitive root colonization, provided further support for the finding that this strain achieves biological control of pathogens through effective competition for nutrients and niches.

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.

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.

Phytoplasmas and mycoplasmas are two groups of important pathogens in the bacterial class Mollicutes. Because of their economical and clinical importance, these obligate pathogens have attracted much research attention. However, difficulties involved in the empirical study of these bacteria, particularly the fact that phytoplasmas have not yet been successfully cultivated outside of their hosts despite decades of attempts, have greatly hampered research progress. With the rapid advancements in genome sequencing, comparative genome analysis provides a new approach to facilitate our understanding of these bacteria. In this study, our main focus is to investigate the evolution of gene content in phytoplasmas, mycoplasmas, and their common ancestor. By using a phylogenetic framework for comparative analysis of 12 complete genome sequences, we characterized the putative gains and losses of genes in these obligate parasites. Our results demonstrated that the degradation of metabolic capacities in these bacteria has occurred predominantly in the common ancestor of Mollicutes, prior to the evolutionary split of phytoplasmas and mycoplasmas. Furthermore, we identified a list of genes that are acquired by the common ancestor of phytoplasmas and are conserved across all strains with complete genome sequences available. These genes include several putative effectors for the interactions with hosts and may be good candidates for future functional characterization.

The protistan phylum Apicomplexa contains many important pathogens and is the subject of intense genome sequencing efforts. Based upon the genome sequences from seven apicomplexan species and a ciliate outgroup, we identified 268 single-copy genes suitable for phylogenetic inference. Both concatenation and consensus approaches inferred the same species tree topology. This topology is consistent with most prior conceptions of apicomplexan evolution based upon ultrastructural and developmental characters, that is, the piroplasm genera Theileria and Babesia form the sister group to the Plasmodium species, the coccidian genera Eimeria and Toxoplasma are monophyletic and are the sister group to the Plasmodium species and piroplasm genera, and Cryptosporidium forms the sister group to the above mentioned with the ciliate Tetrahymena as the outgroup. The level of incongruence among gene trees appears to be high at first glance; only 19% of the genes support the species tree, and a total of 48 different gene-tree topologies are observed. Detailed investigations suggest that the low signal-to-noise ratio in many genes may be the main source of incongruence. The probability of being consistent with the species tree increases as a function of the minimum bootstrap support observed at tree nodes for a given gene tree. Moreover, gene sequences that generate high bootstrap support are robust to the changes in alignment parameters or phylogenetic method used. However, caution should be taken in that some genes can infer a "wrong" tree with strong support because of paralogy, model violations, or other causes. The importance of examining multiple, unlinked genes that possess a strong phylogenetic signal cannot be overstated.

Phytoplasmas are uncultivated plant-pathogenic bacteria with agricultural importance. Those belonging to the 16SrII group, represented by 'Candidatus P. aurantifolia', have a wide range of plant hosts and cause significant yield losses in valuable crops, such as pear, sweet potato, peanut, and soybean. In this study, a method that combines immunoprecipitation-based enrichment and MinION long-read DNA sequencing was developed to solve the challenge of phytoplasma genome studies. This approach produced long reads with high mapping rates and high genomic coverage that can be combined with Illumina reads to produce complete genome assemblies with high accuracy. We applied this method to strain NCHU2014 and determined its complete genome sequence, which consists of one circular chromosome with 635,584 bp and one plasmid with 4,224 bp. Although 'Ca. P. aurantifolia' NCHU2014 has a small chromosome with only 471 protein-coding genes, it contains 33 transporter genes and 27 putative effector genes, which may contribute to obtaining nutrients from hosts and manipulating host developments for their survival and multiplication. Two effectors, the homologs of SAP11 and SAP54/PHYL1 identified in 'Ca. P. aurantifolia' NCHU2014, have the biochemical activities in destabilizing host transcription factors, which can explain the disease symptoms observed in infected plants. Taken together, this study provides the first complete genome available for the 16SrII phytoplasmas and contributes to the understanding of phytoplasma pathogenicity.

The chilling requirement (CR) is the main factor controlling the peach floral bud break and subsequent reproductive growth. To date, several peach CR quantitative trait loci (QTLs) have been identified. To improve the accessibility and convenience of this genetic information for peach breeders, the aim of this study was to establish an easy-to-use genotype screening system using peach CR molecular markers as a toolkit for marker-assisted selection. Here, we integrated 22 CR-associated markers from three published QTLs and positioned them on the Prunus persica physical map. Then, we built a PCR-based genotyping platform by using high-resolution melting (HRM) analysis with specific primers and trained this platform with 27 peach cultivars. Due to ambiguous variant calls from a commercial HRM software, we developed an R-based pipeline using principal component analysis (PCA) to accurately differentiate genotypes. Based on the PCA results, this toolkit was able to determine the genotypes at the CR-related single nucleotide polymorphisms (SNPs) in all tested peach cultivars. In this study, we showed that this HRM-PCA pipeline served as a low-cost, high-throughput, and non-gel genotyping solution. This system has great potential to accelerate CR-focused peach breeding.

The economically important plant pathogen Xylella fastidiosa has been reported in multiple regions of the globe during the last two decades, threatening a growing list of plants. Particularly, X. fastidiosa subspecies fastidiosa causes Pierce's disease (PD) of grapevines, which is a problem in the USA, Spain, and Taiwan. In this work, we studied PD-causing subsp. fastidiosa populations and compared the genome sequences of 33 isolates found in Central Taiwan with 171 isolates from the USA and two from Spain. Phylogenetic relationships, haplotype networks, and genetic diversity analyses confirmed that subsp. fastidiosa was recently introduced into Taiwan from the Southeast USA (i.e. the PD-I lineage). Recent core-genome recombination events were detected among introduced subsp. fastidiosa isolates in Taiwan and contributed to the development of genetic diversity. The genetic diversity observed includes contributions through recombination from unknown donors, suggesting that higher genetic diversity exists in the region. Nevertheless, no recombination event was detected between X. fastidiosa subsp. fastidiosa and the endemic sister species Xylella taiwanensis, which is the causative agent of pear leaf scorch disease. In summary, this study improved our understanding of the genetic diversity of an important plant pathogenic bacterium after its invasion to a new region.

Plant endophytic bacteria live inside host plants, can be isolated from surface-sterilized plant tissues, and are non-pathogenic. These bacteria can assist host plants in obtaining more nutrients and can improve plant growth via multiple mechanisms. Certain Gram-negative Burkholderia species, including rhizobacteria, bioremediators, and biocontrol strains, have been recognized for their plant-growth-promoting abilities, while other isolates have been identified as opportunistic plant or human pathogens. In this study, we observed the auxin production, siderophore synthesis, and phosphate solubilization abilities of B. seminalis strain 869T2. Our results demonstrated that strain 869T2 promoted growth in Arabidopsis, ching chiang pak choi, pak choi, loose-leaf lettuce, romaine lettuce, red leaf lettuce, and Chinese amaranth. Leafy vegetables inoculated with strain 869T2 were larger, heavier, and had more and larger leaves and longer and heavier roots than mock-inoculated plants. Furthermore, inoculations of strain 869T2 into hot pepper caused increased flower and fruit production, and a higher percentage of fruits turned red. Inoculation of strain 869T2 into okra plants resulted in earlier flowering and increased fruit weight. In conclusion, the plant endophytic bacterium Burkholderia seminalis 869T2 exerted positive effects on growth and production in several plant species.

'Candidatus Phytoplasma meliae' is a pathogen associated with chinaberry yellowing disease, which has become a major phytosanitary problem for chinaberry forestry production in Argentina. Despite its economic impact, no genome information of this phytoplasma has been published, which has hindered its characterization at the genomic level. In this study, we used a metagenomics approach to analyze the draft genome of the 'Ca. P. meliae' strain ChTYXIII. The draft assembly consisted of twenty-one contigs with a total length of 751.949 bp, and annotation revealed 669 CDSs, 34 tRNAs, and 1 set of rRNA operons. The metabolic pathways analysis showed that ChTYXIII contains the complete core genes for glycolysis and a functional Sec system for protein translocation. Our phylogenomic analysis based on 133 single-copy genes and genome-to-genome metrics supports the classification as unique 'Ca. P. species' within the MPV clade. We also identified 31 putative effectors, including a homolog to SAP11 and others that have only been described in this pathogen. Our ortholog analysis revealed 37 PMU core genes in the genome of 'Ca. P. meliae' ChTYXIII, leading to the identification of 2 intact PMUs. Our work provides important genomic information for 'Ca. P. meliae' and others phytoplasmas for the 16SrXIII (MPV) group.

Multiple factors over the lifetime of an individual, including diet, geography, and physiologic state, will influence the microbial communities within the primate gut. To determine the source of variation in the composition of the microbiota within and among species, we investigated the distal gut microbial communities harbored by great apes, as present in fecal samples recovered within their native ranges. We found that the branching order of host-species phylogenies based on the composition of these microbial communities is completely congruent with the known relationships of the hosts. Although the gut is initially and continuously seeded by bacteria that are acquired from external sources, we establish that over evolutionary timescales, the composition of the gut microbiota among great ape species is phylogenetically conserved and has diverged in a manner consistent with vertical inheritance.

Pseudogenes are usually considered to be completely neutral sequences whose evolution is shaped by random mutations and chance events. It is possible, however, for disrupted genes to generate products that are deleterious due either to the energetic costs of their transcription and translation or to the formation of toxic proteins. We found that after their initial formation, the youngest pseudogenes in Salmonella genomes have a very high likelihood of being removed by deletional processes and are eliminated too rapidly to be governed by a strictly neutral model of stochastic loss. Those few highly degraded pseudogenes that have persisted in Salmonella genomes correspond to genes with low expression levels and low connectivity in gene networks, such that their inactivation and any initial deleterious effects associated with their inactivation are buffered. Although pseudogenes have long been considered the paradigm of neutral evolution, the distribution of pseudogenes among Salmonella strains indicates that removal of many of these apparently functionless regions is attributable to positive selection.

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.

The reverse reaction of ubiquitylation is catalyzed by different classes of deubiquitylation enzymes (DUBs), including ovarian tumor domain (OTU)-containing DUBs; experiments using Homo sapiens proteins have demonstrated that OTU DUBs modulate various cellular processes. With the exception of OTLD1, plant OTU DUBs have not been characterized. We identified 12 Arabidopsis thaliana OTU loci and analyzed 11 of the encoded proteins in vitro to determine their preferences for the ubiquitin (UB) chains of M1, K48, and K63 linkages as well as the UB-/RUB-/SUMO-GST fusions. The A. thaliana OTU DUBs were shown to be cysteine proteases and classified into four groups with distinct linkage preferences: OTU1 (M1 = K48 > K63), OTU3/4/7/10 (K63 > K48 > M1), OTU2/9 (K48 = K63), and OTU5/11/12/OTLD1 (inactive). Five active OTU DUBs (OTU3/4/7/9/10) also cleaved RUB fusion. OTU1/3/4 cleaved M1 UB chains, suggesting a possible role for M1 chains in plant cellular signaling. The different substrate specificities of the various A. thaliana OTU DUBs indicate the involvement of distinct structural elements; for example, the OTU1 oxyanion residue D89 is essential for cleaving isopeptide bond-linked chains but dispensable for M1 chains. UB-binding activities were detected only for OTU2 and OTLD1, with distinct linkage preferences. These differences in biochemical properties support the involvement of A. thaliana OTU DUBs in different functions. Moreover, based on the established phylogenetic tree, plant- and H. sapiens-specific clades exist, which suggests that the proteins within these clades have taxa-specific functions. We also detected five OTU clades that are conserved across species, which suggests that the orthologs in different species within each clade are involved in conserved cellular processes, such as ERAD and DNA damage responses. However, different linkage preferences have been detected among potential cross-species OTU orthologs, indicating functional and mechanistic differentiation.

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.

Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1(T), a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change.

Genetic differentiation among symbiotic bacteria is important in shaping biodiversity. The genus Spiroplasma contains species occupying diverse niches and is a model system for symbiont evolution. Previous studies have established that two mosquito-associated species have diverged extensively in their carbohydrate metabolism genes despite having a close phylogenetic relationship. Notably, although the commensal Spiroplasma diminutum lacks identifiable pathogenicity factors, the pathogenic Spiroplasma taiwanense was found to have acquired a virulence factor glpO and its associated genes through horizontal transfer. However, it is unclear if these acquired genes have been integrated into the regulatory network. In this study, we inferred the gene content evolution in these bacteria, as well as examined their transcriptomes in response to glucose availability. The results indicated that both species have many more gene acquisitions from the Mycoides-Entomoplasmataceae clade, which contains several important pathogens of ruminants, than previously thought. Moreover, several acquired genes have higher expression levels than the vertically inherited homologs, indicating possible functional replacement. Finally, the virulence factor and its functionally linked genes in S. taiwanense were up-regulated in response to glucose starvation, suggesting that these acquired genes are under expression regulation and the pathogenicity may be a stress response. In summary, although differential gene losses are a major process for symbiont divergence, gene gains are critical in counteracting genome degradation and driving diversification among facultative symbionts.

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 >100 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.

Colubridae represents the most phenotypically diverse and speciose family of snakes, yet no well-assembled and annotated genome exists for this lineage. Here, we report and analyze the genome of the garter snake, Thamnophis sirtalis, a colubrid snake that is an important model species for research in evolutionary biology, physiology, genomics, behavior, and the evolution of toxin resistance. Using the garter snake genome, we show how snakes have evolved numerous adaptations for sensing and securing prey, and identify features of snake genome structure that provide insight into the evolution of amniote genomes. Analyses of the garter snake and other squamate reptile genomes highlight shifts in repeat element abundance and expansion within snakes, uncover evidence of genes under positive selection, and provide revised neutral substitution rate estimates for squamates. Our identification of Z and W sex chromosome-specific scaffolds provides evidence for multiple origins of sex chromosome systems in snakes and demonstrates the value of this genome for studying sex chromosome evolution. Analysis of gene duplication and loss in visual and olfactory gene families supports a dim-light ancestral condition in snakes and indicates that olfactory receptor repertoires underwent an expansion early in snake evolution. Additionally, we provide some of the first links between secreted venom proteins, the genes that encode them, and their evolutionary origins in a rear-fanged colubrid snake, together with new genomic insight into the coevolutionary arms race between garter snakes and highly toxic newt prey that led to toxin resistance in garter snakes.