Complete genome sequence of Stenotrophomonas maltophilia complex strain STEN00241.

We report here the complete, closed genome sequence of Stenotrophomonas maltophilia complex strain STEN00241, which was isolated from patient sputum. The genome is a single contig with a total length of 4,751,329 nucleotides and a GC content of 66.5%.

Co-option of a conserved host glutamine transporter facilitates aphid/Buchnera metabolic integration.

Organisms across the tree of life colonize novel environments by partnering with bacterial symbionts. These symbioses are characterized by intimate integration of host/endosymbiont biology at multiple levels, including metabolically. Metabolic integration is particularly important for sap-feeding insects and their symbionts, which supplement nutritionally unbalanced host diets. Many studies reveal parallel evolution of host/endosymbiont metabolic complementarity in amino acid biosynthesis, raising questions about how amino acid metabolism is regulated, how regulatory mechanisms evolve, and the extent to which similar mechanisms evolve in different systems. In the aphid/Buchnera symbiosis, the transporter ApGLNT1 (Acyrthosiphon pisum glutamine transporter 1) supplies glutamine, an amino donor in transamination reactions, to bacteriocytes (where Buchnera reside) and is competitively inhibited by Buchnera-supplied arginine-consistent with a role regulating amino acid metabolism given host demand for Buchnera-produced amino acids. We examined how ApGLNT1 evolved a regulatory role by functionally characterizing orthologs in insects with and without endosymbionts. ApGLNT1 orthologs are functionally similar, and orthology searches coupled with homology modeling revealed that GLNT1 is ancient and structurally conserved across insects. Our results indicate that the ApGLNT1 symbiotic regulatory role is derived from its ancestral role and, in aphids, is likely facilitated by loss of arginine biosynthesis through the urea cycle. Given consistent loss of host arginine biosynthesis and retention of endosymbiont arginine supply, we hypothesize that GLNT1 is a general mechanism regulating amino acid metabolism in sap-feeding insects. This work fills a gap, highlighting the broad importance of co-option of ancestral proteins to novel contexts in the evolution of host/symbiont systems.

Application of a quantitative framework to improve the accuracy of a bacterial infection model.

Laboratory models are critical to basic and translational microbiology research. Models serve multiple purposes, from providing tractable systems to study cell biology to allowing the investigation of inaccessible clinical and environmental ecosystems. Although there is a recognized need for improved model systems, there is a gap in rational approaches to accomplish this goal. We recently developed a framework for assessing the accuracy of microbial models by quantifying how closely each gene is expressed in the natural environment and in various models. The accuracy of the model is defined as the percentage of genes that are similarly expressed in the natural environment and the model. Here, we leverage this framework to develop and validate two generalizable approaches for improving model accuracy, and as proof of concept, we apply these approaches to improve models of Pseudomonas aeruginosa infecting the cystic fibrosis (CF) lung. First, we identify two models, an in vitro synthetic CF sputum medium model (SCFM2) and an epithelial cell model, that accurately recapitulate different gene sets. By combining these models, we developed the epithelial cell-SCFM2 model which improves the accuracy of over 500 genes. Second, to improve the accuracy of specific genes, we mined publicly available transcriptome data, which identified zinc limitation as a cue present in the CF lung and absent in SCFM2. Induction of zinc limitation in SCFM2 resulted in accurate expression of 90% of P. aeruginosa genes. These approaches provide generalizable, quantitative frameworks for microbiological model improvement that can be applied to any system of interest.

Trading amino acids at the aphid-Buchnera symbiotic interface.

Plant sap-feeding insects are widespread, having evolved to occupy diverse environmental niches despite exclusive feeding on an impoverished diet lacking in essential amino acids and vitamins. Success depends exquisitely on their symbiotic relationships with microbial symbionts housed within specialized eukaryotic bacteriocyte cells. Each bacteriocyte is packed with symbionts that are individually surrounded by a host-derived symbiosomal membrane representing the absolute host-symbiont interface. The symbiosomal membrane must be a dynamic and selectively permeable structure to enable bidirectional and differential movement of essential nutrients, metabolites, and biosynthetic intermediates, vital for growth and survival of host and symbiont. However, despite this crucial role, the molecular basis of membrane transport across the symbiosomal membrane remains unresolved in all bacteriocyte-containing insects. A transport protein was immunolocalized to the symbiosomal membrane separating the pea aphid Acyrthosiphon pisum from its intracellular symbiont Buchnera aphidicola The transporter, A. pisum nonessential amino acid transporter 1, or ApNEAAT1 (gene: ACYPI008971), was characterized functionally following heterologous expression in Xenopus oocytes, and mediates both inward and outward transport of small dipolar amino acids (serine, proline, cysteine, alanine, glycine). Electroneutral ApNEAAT1 transport is driven by amino acid concentration gradients and is not coupled to transmembrane ion gradients. Previous metabolite profiling of hemolymph and bacteriocyte, alongside metabolic pathway analysis in host and symbiont, enable prediction of a physiological role for ApNEAAT1 in bidirectional host-symbiont amino acid transfer, supplying both host and symbiont with indispensable nutrients and biosynthetic precursors to facilitate metabolic complementarity.

Horizontal Transfer of Bacterial Cytolethal Distending Toxin B Genes to Insects.

Horizontal gene transfer events have played a major role in the evolution of microbial species, but their importance in animals is less clear. Here, we report horizontal gene transfer of cytolethal distending toxin B (cdtB), prokaryotic genes encoding eukaryote-targeting DNase I toxins, into the genomes of vinegar flies (Diptera: Drosophilidae) and aphids (Hemiptera: Aphididae). We found insect-encoded cdtB genes are most closely related to orthologs from bacteriophage that infect Candidatus Hamiltonella defensa, a bacterial mutualistic symbiont of aphids that confers resistance to parasitoid wasps. In drosophilids, cdtB orthologs are highly expressed during the parasitoid-prone larval stage and encode a protein with ancestral DNase activity. We show that cdtB has been domesticated by diverse insects and hypothesize that it functions in defense against their natural enemies.

Gene Family Expansions in Aphids Maintained by Endosymbiotic and Nonsymbiotic Traits.

Facilitating the evolution of new gene functions, gene duplication is a major mechanism driving evolutionary innovation. Gene family expansions relevant to host/symbiont interactions are increasingly being discovered in eukaryotes that host endosymbiotic microbes. Such discoveries entice speculation that gene duplication facilitates the evolution of novel, endosymbiotic relationships. Here, using a comparative transcriptomic approach combined with differential gene expression analysis, we investigate the importance of endosymbiosis in retention of amino acid transporter paralogs in aphid genomes. To pinpoint the timing of amino acid transporter duplications we inferred gene phylogenies for five aphid species and three outgroups. We found that while some duplications arose in the aphid common ancestor concurrent with endosymbiont acquisition, others predate aphid divergence from related insects without intracellular symbionts, and still others appeared during aphid diversification. Interestingly, several aphid-specific paralogs have conserved enriched expression in bacteriocytes, the insect cells that host primary symbionts. Conserved bacteriocyte enrichment suggests that the transporters were recruited to the aphid/endosymbiont interface in the aphid common ancestor, consistent with a role for gene duplication in facilitating the evolution of endosymbiosis in aphids. In contrast, the temporal variability of amino acid transporter duplication indicates that endosymbiosis is not the only trait driving selection for retention of amino acid transporter paralogs in sap-feeding insects. This study cautions against simplistic interpretations of the role of gene family expansion in the evolution of novel host/symbiont interactions by further highlighting that multiple complex factors maintain gene family paralogs in the genomes of eukaryotes that host endosymbiotic microbes.

Signatures of host/symbiont genome coevolution in insect nutritional endosymbioses.

The role of symbiosis in bacterial symbiont genome evolution is well understood, yet the ways that symbiosis shapes host genomes or more particularly, host/symbiont genome coevolution in the holobiont is only now being revealed. Here, we identify three coevolutionary signatures that characterize holobiont genomes. The first signature, host/symbiont collaboration, arises when completion of essential pathways requires host/endosymbiont genome complementarity. Metabolic collaboration has evolved numerous times in the pathways of amino acid and vitamin biosynthesis. Here, we highlight collaboration in branched-chain amino acid and pantothenate (vitamin B5) biosynthesis. The second coevolutionary signature is acquisition, referring to the observation that holobiont genomes acquire novel genetic material through various means, including gene duplication, lateral gene transfer from bacteria that are not their current obligate symbionts, and full or partial endosymbiont replacement. The third signature, constraint, introduces the idea that holobiont genome evolution is constrained by the processes governing symbiont genome evolution. In addition, we propose that collaboration is constrained by the expression profile of the cell lineage from which endosymbiont-containing host cells, called bacteriocytes, are derived. In particular, we propose that such differences in bacteriocyte cell lineage may explain differences in patterns of host/endosymbiont metabolic collaboration between the sap-feeding suborders Sternorrhyncha and Auchenorrhynca. Finally, we review recent studies at the frontier of symbiosis research that are applying functional genomic approaches to characterization of the developmental and cellular mechanisms of host/endosymbiont integration, work that heralds a new era in symbiosis research.

Amino acid transporter expansions associated with the evolution of obligate endosymbiosis in sap-feeding insects (Hemiptera: sternorrhyncha).

BACKGROUND: Mutualistic obligate endosymbioses shape the evolution of endosymbiont genomes, but their impact on host genomes remains unclear. Insects of the sub-order Sternorrhyncha (Hemiptera) depend on bacterial endosymbionts for essential amino acids present at low abundances in their phloem-based diet. This obligate dependency has been proposed to explain why multiple amino acid transporter genes are maintained in the genomes of the insect hosts. We implemented phylogenetic comparative methods to test whether amino acid transporters have proliferated in sternorrhynchan genomes at rates grater than expected by chance. RESULTS: By applying a series of methods to reconcile gene and species trees, inferring the size of gene families in ancestral lineages, and simulating the null process of birth and death in multi-gene families, we uncovered a 10-fold increase in duplication rate in the AAAP family of amino acid transporters within Sternorrhyncha. This gene family expansion was unmatched in other closely related clades lacking endosymbionts that provide essential amino acids. CONCLUSIONS: Our findings support the influence of obligate endosymbioses on host genome evolution by both inferring significant expansions of gene families involved in symbiotic interactions, and discovering increases in the rate of duplication associated with multiple emergences of obligate symbiosis in Sternorrhyncha.

Dynamic recruitment of amino acid transporters to the insect/symbiont interface.

Symbiosis is well known to influence bacterial symbiont genome evolution and has recently been shown to shape eukaryotic host genomes. Intriguing patterns of host genome evolution, including remarkable numbers of gene duplications, have been observed in the pea aphid, a sap-feeding insect that relies on a bacterial endosymbiont for amino acid provisioning. Previously, we proposed that gene duplication has been important for the evolution of symbiosis based on aphid-specific gene duplication in amino acid transporters (AATs), with some paralogs highly expressed in the cells housing symbionts (bacteriocytes). Here, we use a comparative approach to test the role of gene duplication in enabling recruitment of AATs to bacteriocytes. Using genomic and transcriptomic data, we annotate AATs from sap-feeding and non sap-feeding insects and find that, like aphids, AAT gene families have undergone independent large-scale gene duplications in three of four additional sap-feeding insects. RNA-seq differential expression data indicate that, like aphids, the sap-feeding citrus mealybug possesses several lineage-specific bacteriocyte-enriched paralogs. Further, differential expression data combined with quantitative PCR support independent evolution of bacteriocyte enrichment in sap-feeding insect AATs. Although these data indicate that gene duplication is not necessary to initiate host/symbiont amino acid exchange, they support a role for gene duplication in enabling AATs to mediate novel host/symbiont interactions broadly in the sap-feeding suborder Sternorrhyncha. In combination with recent studies on other symbiotic systems, gene duplication is emerging as a general pattern in host genome evolution.

Horizontal gene transfer from diverse bacteria to an insect genome enables a tripartite nested mealybug symbiosis.

The smallest reported bacterial genome belongs to Tremblaya princeps, a symbiont of Planococcus citri mealybugs (PCIT). Tremblaya PCIT not only has a 139 kb genome, but possesses its own bacterial endosymbiont, Moranella endobia. Genome and transcriptome sequencing, including genome sequencing from a Tremblaya lineage lacking intracellular bacteria, reveals that the extreme genomic degeneracy of Tremblaya PCIT likely resulted from acquiring Moranella as an endosymbiont. In addition, at least 22 expressed horizontally transferred genes from multiple diverse bacteria to the mealybug genome likely complement missing symbiont genes. However, none of these horizontally transferred genes are from Tremblaya, showing that genome reduction in this symbiont has not been enabled by gene transfer to the host nucleus. Our results thus indicate that the functioning of this three-way symbiosis is dependent on genes from at least six lineages of organisms and reveal a path to intimate endosymbiosis distinct from that followed by organelles.

Novel male-biased expression in paralogs of the aphid slimfast nutrient amino acid transporter expansion.

BACKGROUND: A major goal of molecular evolutionary biology is to understand the fate and consequences of duplicated genes. In this context, aphids are intriguing because the newly sequenced pea aphid genome harbors an extraordinary number of lineage-specific gene duplications relative to other insect genomes. Though many of their duplicated genes may be involved in their complex life cycle, duplications in nutrient amino acid transporters appear to be associated rather with their essential amino acid poor diet and the intracellular symbiosis aphids rely on to compensate for dietary deficits. Past work has shown that some duplicated amino acid transporters are highly expressed in the specialized cells housing the symbionts, including a paralog of an aphid-specific expansion homologous to the Drosophila gene slimfast. Previous data provide evidence that these bacteriocyte-expressed transporters mediate amino acid exchange between aphids and their symbionts. RESULTS: We report that some nutrient amino acid transporters show male-biased expression. Male-biased expression characterizes three paralogs in the aphid-specific slimfast expansion, and the male-biased expression is conserved across two aphid species for at least two paralogs. One of the male-biased paralogs has additionally experienced an accelerated rate of non-synonymous substitutions. CONCLUSIONS: This is the first study to document male-biased slimfast expression. Our data suggest that the male-biased aphid slimfast paralogs diverged from their ancestral function to fill a functional role in males. Furthermore, our results provide evidence that members of the slimfast expansion are maintained in the aphid genome not only for the previously hypothesized role in mediating amino acid exchange between the symbiotic partners, but also for sex-specific roles.

Genome expansion and differential expression of amino acid transporters at the aphid/Buchnera symbiotic interface.

In insects, some of the most ecologically important symbioses are nutritional symbioses that provide hosts with novel traits and thereby facilitate exploitation of otherwise inaccessible niches. One such symbiosis is the ancient obligate intracellular symbiosis of aphids with the γ-proteobacteria, Buchnera aphidicola. Although the nutritional basis of the aphid/Buchnera symbiosis is well understood, the processes and structures that mediate the intimate interactions of symbiotic partners remain uncharacterized. Here, using a de novo approach, we characterize the complement of 40 amino acid polyamine organocation (APC) superfamily member amino acid transporters (AATs) encoded in the genome of the pea aphid, Acyrthosiphon pisum. We find that the A. pisum APC superfamily is characterized by extensive gene duplications such that A. pisum has more APC superfamily transporters than other fully sequenced insects, including a ten paralog aphid-specific expansion of the APC transporter slimfast. Detailed expression analysis of 17 transporters selected on the basis of their phylogenetic relationship to five AATs identified in an earlier bacteriocyte expressed sequence tag study distinguished a subset of eight transporters that have been recruited for amino acid transport in bacteriocyte cells at the symbiotic interface. These eight transporters include transporters that are highly expressed and/or highly enriched in bacteriocytes and intriguingly, the four AATs that show bacteriocyte-enriched expression are all members of gene family expansions, whereas three of the four that are highly expressed but not enriched in bacteriocytes retain one-to-one orthology with transporters in other genomes. Finally, analysis of evolutionary rates within the large A. pisum slimfast expansion demonstrated increased rates of molecular evolution coinciding with two major shifts in expression: 1) a loss of gut expression and possibly a gain of bacteriocyte expression and 2) loss of expression in all surveyed tissues in asexual females. Taken together, our characterization of nutrient AATs at the aphid/Buchnera symbiotic interface provides the first examination of the processes and structures operating at the interface of an obligate intracellular insect nutritional symbiosis, offering unique insight into the types of genomic change that likely facilitated evolutionary maintenance of the symbiosis.

Diversity of Loxosceles spiders in Northwestern Africa and molecular support for cryptic species in the Loxosceles rufescens lineage.

Until recently, Loxosceles rufescens was the only species known from a geographic range including Northern Africa, Mediterranean Europe and the Middle East. Rich Loxosceles diversity in the New World suggests either that L. rufescens is a young lineage or that its diversity is underappreciated. We use a molecular phylogenetic and morphological approach to examine diversity in L. rufescens and other Loxosceles lineages in Northwestern Africa. Molecular analyses of one nuclear and two mitochondrial genes strongly support a monophyletic clade including L. rufescens, the Northern Brazilian L. amazonica and three other divergent Northwestern African lineages, though relationships among them remain unresolved. A genetically divergent Moroccan individual morphologically consistent with L. rufescens was strongly supported as sister to all other putative L. rufescens, consistent with the presence of at least 2 species in this lineage. COI p-distances and population structuring among remaining putative L. rufescens clades further suggest the absence of gene flow between clades and the possibility that they represent multiple species. Morphological characters of preserved Loxosceles collected in a range of African countries provide additional indication that Loxosceles are more diverse and have a deeper history in Africa than has been previously understood.

Genetic modifiers of dFMR1 encode RNA granule components in Drosophila.

Mechanisms of neuronal mRNA localization and translation are of considerable biological interest. Spatially regulated mRNA translation contributes to cell-fate decisions and axon guidance during development, as well as to long-term synaptic plasticity in adulthood. The Fragile-X Mental Retardation protein (FMRP/dFMR1) is one of the best-studied neuronal translational control molecules and here we describe the identification and early characterization of proteins likely to function in the dFMR1 pathway. Induction of the dFMR1 in sevenless-expressing cells of the Drosophila eye causes a disorganized (rough) eye through a mechanism that requires residues necessary for dFMR1/FMRP's translational repressor function. Several mutations in dco, orb2, pAbp, rm62, and smD3 genes dominantly suppress the sev-dfmr1 rough-eye phenotype, suggesting that they are required for dFMR1-mediated processes. The encoded proteins localize to dFMR1-containing neuronal mRNPs in neurites of cultured neurons, and/or have an effect on dendritic branching predicted for bona fide neuronal translational repressors. Genetic mosaic analyses indicate that dco, orb2, rm62, smD3, and dfmr1 are dispensable for translational repression of hid, a microRNA target gene, known to be repressed in wing discs by the bantam miRNA. Thus, the encoded proteins may function as miRNA- and/or mRNA-specific translational regulators in vivo.

Phylogenetic relationships of Loxosceles and Sicarius spiders are consistent with Western Gondwanan vicariance.

The modern geographic distribution of the spider family Sicariidae is consistent with an evolutionary origin on Western Gondwana. Both sicariid genera, Loxosceles and Sicarius are diverse in Africa and South/Central America. Loxosceles are also diverse in North America and the West Indies, and have species described from Mediterranean Europe and China. We tested vicariance hypotheses using molecular phylogenetics and molecular dating analyses of 28S, COI, 16S, and NADHI sequences. We recover reciprocal monophyly of African and South American Sicarius, paraphyletic Southern African Loxosceles and monophyletic New World Loxosceles within which an Old World species group that includes L. rufescens is derived. These patterns are consistent with a sicariid common ancestor on Western Gondwana. North American Loxosceles are monophyletic, sister to Caribbean taxa, and resolved in a larger clade with South American Loxosceles. With fossil data this pattern is consistent with colonization of North America via a land bridge predating the modern Isthmus of Panama.

Convergent setal morphology in sand-covering spiders suggests a design principle for particle capture.

Sicarius and Homalonychus are unrelated, desert-dwelling spiders that independently evolved the ability to cover themselves in fine sand particles, making them cryptic against their background. Observations that particles associate with these spiders' setae inspired us to investigate the role of setal microstructure in particle capture and retention. Here we report that Sicarius and Homalonychus convergently evolved numerous high aspect ratio, flexible fibres that we call 'hairlettes' protruding from the setal shaft. We demonstrate that particles attach more densely to regions of Homalonychus with hairlettes than to other regions of the same animal where hairlettes are absent, and document close contact of hairlettes to sand particles that persists after applying force. Mathematical models further suggest that adhesion of hairlettes to sand particles is a sufficient mechanism of particle capture and retention. Together, these data provide the first evidence that hairlettes facilitate sand retention through intermolecular adhesion to particles. Their independent evolutionary origins in Sicarius and Homalonychus suggest that the unique setal structure is adaptive and represents a general biomechanical mechanism for sand capture to cuticle. This discovery has implications for the design of inventions inspired by this system, from camouflage to the management of granular systems.