Horizontally Acquired Cellulases Assist the Expansion of Dietary Range in Pristionchus Nematodes.

Horizontal gene transfer (HGT) enables the acquisition of novel traits via non-Mendelian inheritance of genetic material. HGT plays a prominent role in the evolution of prokaryotes, whereas in animals, HGT is rare and its functional significance is often uncertain. Here, we investigate horizontally acquired cellulase genes in the free-living nematode model organism Pristionchus pacificus. We show that these cellulase genes 1) are likely of eukaryotic origin, 2) are expressed, 3) have protein products that are secreted and functional, and 4) result in endo-cellulase activity. Using CRISPR/Cas9, we generated an octuple cellulase mutant, which lacks all eight cellulase genes and cellulase activity altogether. Nonetheless, this cellulase-null mutant is viable and therefore allows a detailed analysis of a gene family that was horizontally acquired. We show that the octuple cellulase mutant has associated fitness costs with reduced fecundity and slower developmental speed. Furthermore, by using various Escherichia coli K-12 strains as a model for cellulosic biofilms, we demonstrate that cellulases facilitate the procurement of nutrients from bacterial biofilms. Together, our analysis of cellulases in Pristionchus provides comprehensive evidence from biochemistry, genetics, and phylogeny, which supports the integration of horizontally acquired genes into the complex life history strategy of this soil nematode.

Evolution and Diversity of TGF-ß Pathways are Linked with Novel Developmental and Behavioral Traits.

Transforming growth factor-ß (TGF-ß) signaling is essential for numerous biologic functions. It is a highly conserved pathway found in all metazoans including the nematode Caenorhabditis elegans, which has also been pivotal in identifying many components. Utilizing a comparative evolutionary approach, we explored TGF-ß signaling in nine nematode species and revealed striking variability in TGF-ß gene frequency across the lineage. Of the species analyzed, gene duplications in the DAF-7 pathway appear common with the greatest disparity observed in Pristionchus pacificus. Specifically, multiple paralogues of daf-3, daf-4 and daf-7 were detected. To investigate this additional diversity, we induced mutations in 22 TGF-ß components and generated corresponding double, triple, and quadruple mutants revealing both conservation and diversification in function. Although the DBL-1 pathway regulating body morphology appears highly conserved, the DAF-7 pathway exhibits functional divergence, notably in some aspects of dauer formation. Furthermore, the formation of the phenotypically plastic mouth in P. pacificus is partially influenced through TGF-ß with the strongest effect in Ppa-tag-68. This appears important for numerous processes in P. pacificus but has no known function in C. elegans. Finally, we observe behavioral differences in TGF-ß mutants including in chemosensation and the establishment of the P. pacificus kin-recognition signal. Thus, TGF-ß signaling in nematodes represents a stochastic genetic network capable of generating novel functions through the duplication and deletion of associated genes.

Nematode biphasic 'boom and bust' dynamics are dependent on host bacterial load while linking dauer and mouth-form polyphenisms.

Cross-kingdom interactions involve dynamic processes that shape terrestrial ecosystems and represent striking examples of co-evolution. The multifaceted relationships of entomopathogenic nematodes with their insect hosts and symbiotic bacteria are well-studied cases of co-evolution and pathogenicity. In contrast, microbial interactions in soil after the natural death of insects and other invertebrates are minimally understood. In particular, the turnover and succession of nematodes and bacteria during insect decay have not been well documented - although it represents a rich ecological niche with multiple species interactions. Here, we utilize developmentally plastic nematode Pristionchus pacificus and its associated scarab beetles as models. On La Réunion Island, we collected rhinoceros beetle Oryctes borbonicus, induced death, and placed carcasses in cages both on the island and in a mock-natural environment in the laboratory controlling for high spatial and temporal resolution. Investigating nematode population density and dispersal dynamics, we were able to connect two imperative plasticities, dauer and mouth form. We observed a biphasic 'boom and bust' dispersal dynamic of dauer larvae that corresponds to bacterial load on carcasses but not bacterial type. Strikingly, all post-dauer adults have the predatory mouth form, demonstrating novel intricate interactions on decaying insect hosts. Thus, ecologically relevant survival strategies incorporate critical plastic traits.

Avian phenotypic convergence is subject to low genetic constraints based on genomic evidence.

BACKGROUND: Phenotypic convergence between distinct species provides an opportunity to examine the predictability of genetic evolution. Unrelated species sharing genetic underpinnings for phenotypic convergence suggests strong genetic constraints, and thus high predictability of evolution. However, there is no clear big picture of the genomic constraints on convergent evolution. Genome-based phylogenies have confirmed many cases of phenotypic convergence in birds, making them a good system for examining genetic constraints in phenotypic convergence. In this study, we used hierarchical genomic approaches to estimate genetic constraints in three convergent avian traits: nocturnality, raptorial behavior and foot-propelled diving. RESULTS: Phylogeny-based hypothesis tests and positive selection tests were applied to compare 16 avian genomes, representing 14 orders, and identify genes with strong convergence signals. We found 43 adaptively convergent genes (ACGs) associated with the three phenotypic convergence cases and assessed genetic constraints in all three cases, from (amino acid) site mutations to genetic pathways. We found that the avian orders shared few site mutations in the ACGs that contributed to the convergent phenotypes, and that these ACGs were not enriched in any genetic pathways. In addition, different pairs of orders with convergent foot-propelled diving or raptorial behaviors shared few ACGs. We also found that closely related orders that shared foot-propelled diving behavior did not share more ACGs than did distinct orders, suggesting that convergence among these orders could not be explained by their initial genomic backgrounds. CONCLUSIONS: Our analyses of three avian convergence events suggest low constraints for phenotypic convergence across multiple genetic levels, implying that genetic evolution is unpredictable at the phylogenetic level of avian order. Ours is one of first studies to apply hierarchical genomic examination to multiple avian convergent cases to assess the genetic constraints in life history trait evolution.

The chicken frizzle feather is due to an α-keratin (KRT75) mutation that causes a defective rachis.

Feathers have complex forms and are an excellent model to study the development and evolution of morphologies. Existing chicken feather mutants are especially useful for identifying genetic determinants of feather formation. This study focused on the gene F, underlying the frizzle feather trait that has a characteristic curled feather rachis and barbs in domestic chickens. Our developmental biology studies identified defects in feather medulla formation, and physical studies revealed that the frizzle feather curls in a stepwise manner. The frizzle gene is transmitted in an autosomal incomplete dominant mode. A whole-genome linkage scan of five pedigrees with 2678 SNPs revealed association of the frizzle locus with a keratin gene-enriched region within the linkage group E22C19W28_E50C23. Sequence analyses of the keratin gene cluster identified a 69 bp in-frame deletion in a conserved region of KRT75, an α-keratin gene. Retroviral-mediated expression of the mutated F cDNA in the wild-type rectrix qualitatively changed the bending of the rachis with some features of frizzle feathers including irregular kinks, severe bending near their distal ends, and substantially higher variations among samples in comparison to normal feathers. These results confirmed KRT75 as the F gene. This study demonstrates the potential of our approach for identifying genetic determinants of feather forms.

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.

Microbiota succession influences nematode physiology in a beetle microcosm ecosystem.

Unravelling the multifaceted and bidirectional interactions between microbiota and host physiology represents a major scientific challenge. Here, we utilise the nematode model, Pristionchus pacificus, coupled to a laboratory-simulated decay process of its insect host, to mimic natural microbiota succession and investigate associated tripartite interactions. Metagenomics reveal that during initial decay stages, the population of vitamin B-producing bacteria diminishes, potentially due to a preferential selection by nematodes. As decay progresses to nutrient-depleted stages, bacteria with smaller genomes producing less nutrients become more prevalent. Lipid utilisation and dauer formation, representing key nematode survival strategies, are influenced by microbiota changes. Additionally, horizontally acquired cellulases extend the nematodes' reproductive phase due to more efficient foraging. Lastly, the expressions of Pristionchus species-specific genes are more responsive to natural microbiota compared to conserved genes, suggesting their importance in the organisms' adaptation to its ecological niche. In summary, we show the importance of microbial successions and their reciprocal interaction with nematodes for insect decay in semi-artificial ecosystems.

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.

Vitamin B12 and predatory behavior in nematodes.

The round worms or nematodes are the largest phylum of animals with an estimated species number of more than one million. Nematodes have invaded all ecosystems and are known from all continents including Antarctica. Parasitic species infest plants, animals and humans often with high host-specificity. Free-living species are known from marine, fresh water and soil systems, the latter of which contain many culturable species. This includes Caenorhabditis elegans, a species that was developed as one of the most prominent model systems in modern biology since the 1960ies. Pristionchus pacificus is a second nematode model organism that can easily be cultured in the laboratory. This species shows a number of complex traits including omnivorous feeding and the capability of predation on other nematodes. Predation depends on the formation of teeth-like denticles in the mouth of P. pacificus, structures unknown from C. elegans and most other nematodes. Here, we review the current knowledge about the role of vitamin B12 for the predatory behavior in P. pacificus and correlate its role with that on the physiology and development in C. elegans.

Synergistic interaction of gut microbiota enhances the growth of nematode through neuroendocrine signaling.

Animals are associated with a diverse bacterial community that impacts host physiology. It is well known that nutrients and enzymes synthesized by bacteria largely expand host metabolic capacity. Bacteria also impact a wide range of animal physiology that solely depends on host genetics through direct interaction. However, studying the synergistic effects of the bacterial community remains challenging due to its complexity. The omnivorous nematode Pristionchus pacificus has limited digestive efficiency on bacteria. Therefore, we established a bacterial collection that represents the natural gut microbiota that are resistant to digestion. Using this collection, we show that the bacterium Lysinibacillus xylanilyticus by itself provides limited nutritional value, but in combination with Escherichia coli, it significantly promotes life-history traits of P. pacificus by regulating the neuroendocrine peptide in sensory neurons. This gut-to-brain communication depends on undigested L. xylanilyticus providing Pristionchus nematodes a specific fitness advantage to compete with nematodes that rupture bacteria efficiently. Using RNA-seq and CRISPR-induced mutants, we show that 1-h exposure to L. xylanilyticus is sufficient to stimulate the expression of daf-7-type TGF-ß signaling ligands, which induce a global transcriptome change. In addition, several effects of L. xylanilyticus depend on TGF-ß signaling, including olfaction, body size regulation, and a switch of energy allocation from lipid storage to reproduction. Our results reveal the beneficial effects of a gut bacterium to modify life-history traits and maximize nematode survival in natural habitats.

Host-associated microbe PCR (hamPCR) enables convenient measurement of both microbial load and community composition.

The ratio of microbial population size relative to the amount of host tissue, or 'microbial load', is a fundamental metric of colonization and infection, but it cannot be directly deduced from microbial amplicon data such as 16S rRNA gene counts. Because existing methods to determine load, such as serial dilution plating, quantitative PCR, and whole metagenome sequencing add substantial cost and/or experimental burden, they are only rarely paired with amplicon sequencing. We introduce host-associated microbe PCR (hamPCR), a robust strategy to both quantify microbial load and describe interkingdom microbial community composition in a single amplicon library. We demonstrate its accuracy across multiple study systems, including nematodes and major crops, and further present a cost-saving technique to reduce host overrepresentation in the library prior to sequencing. Because hamPCR provides an accessible experimental solution to the well-known limitations and statistical challenges of compositional data, it has far-reaching potential in culture-independent microbiology.

Improving Transgenesis Efficiency and CRISPR-Associated Tools Through Codon Optimization and Native Intron Addition in Pristionchus Nematodes.

A lack of appropriate molecular tools is one obstacle that prevents in-depth mechanistic studies in many organisms. Transgenesis, clustered regularly interspaced short palindromic repeats (CRISPR)-associated engineering, and related tools are fundamental in the modern life sciences, but their applications are still limited to a few model organisms. In the phylum Nematoda, transgenesis can only be performed in a handful of species other than Caenorhabditis elegans, and additionally, other species suffer from significantly lower transgenesis efficiencies. We hypothesized that this may in part be due to incompatibilities of transgenes in the recipient organisms. Therefore, we investigated the genomic features of 10 nematode species from three of the major clades representing all different lifestyles. We found that these species show drastically different codon usage bias and intron composition. With these findings, we used the species Pristionchus pacificus as a proof of concept for codon optimization and native intron addition. Indeed, we were able to significantly improve transgenesis efficiency, a principle that may be usable in other nematode species. In addition, with the improved transgenes, we developed a fluorescent co-injection marker in P. pacificus for the detection of CRISPR-edited individuals, which helps considerably to reduce associated time and costs.

Correction: Bacterial vitamin B12 production enhances nematode predatory behavior.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bacterial vitamin B12 production enhances nematode predatory behavior.

Although the microbiota is known to affect host development, metabolism, and immunity, its impact on host behavior is only beginning to be understood. In order to better characterize behavior modulation by host-associated microorganisms, we investigated how bacteria modulate complex behaviors in the nematode model organism Pristionchus pacificus. This nematode is a predator that feeds on the larvae of other nematodes, including Caenorhabditis elegans. By growing P. pacificus on different bacteria and testing their ability to kill C. elegans, we reveal large differences in killing efficiencies, with a Novosphingobium species showing the strongest enhancement. This enhanced killing was not accompanied by an increase in feeding, which is a phenomenon known as surplus killing, whereby predators kill more prey than necessary for sustenance. Our RNA-seq data demonstrate widespread metabolic rewiring upon exposure to Novosphingobium, which facilitated screening of bacterial mutants with altered transcriptional responses. We identified bacterial production of vitamin B12 as an important cause of such enhanced predatory behavior. Although vitamin B12 is an essential cofactor for detoxification and metabolite biosynthesis, shown previously to accelerate development in C. elegans, supplementation with this enzyme cofactor amplified surplus killing in P. pacificus, whereas mutants in vitamin B12-dependent pathways reduced surplus killing. By demonstrating that production of vitamin B12 by host-associated microbiota can affect complex host behaviors, we reveal new connections between animal diet, microbiota, and nervous system.

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 >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.

Complete Genome Sequence of Spiroplasma floricola 23-6T (ATCC 29989), a Bacterium Isolated from a Tulip Tree (Liriodendron tulipifera L.).

Spiroplasma floricola 23-6T (ATCC 29989) was isolated from the flower surface of a tulip tree (Liriodendron tulipifera L.). Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its biology and the comparative genomics among Spiroplasma species.

Complete Genome Sequence of Spiroplasma monobiae MQ-1T (ATCC 33825), a Bacterium Isolated from the Vespid Wasp (Monobia quadridens).

Spiroplasma monobiae MQ-1T (ATCC 33825) was isolated from the hemolymph of an adult vespid wasp (Monobia quadridens) collected in Maryland. Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its biology and the comparative genomics among Spiroplasma species.

Horizontal Acquisition and Transcriptional Integration of Novel Genes in Mosquito-Associated Spiroplasma.

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.

Complete Genome Sequence of Spiroplasma corruscae EC-1T (DSM 19793), a Bacterium Isolated from a Lampyrid Beetle (Ellychnia corrusca).

Spiroplasma corruscae EC-1T (DSM 19793) was isolated from the gut of a lampryid beetle (Ellychnia corrusca) collected in Beltsville, MD, USA, in 1983. Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its biology and the comparative genomics among Spiroplasma species.

Complete Genome Sequence of Spiroplasma sp. TU-14.

Spiroplasma sp. TU-14 was isolated from a contaminated sample of Entomoplasma lucivorax PIPN-2T obtained from the International Organization for Mycoplasmology collection. Here, we report the complete genome sequence of this bacterium to facilitate the investigation of its biology and the comparative genomics among Spiroplasma spp.