Sandy J. MacDonald

Genomic insight into the amino acid relations of the pea aphid, Acyrthosiphon pisum, with its symbiotic bacterium Buchnera aphidicola.

The pea aphid genome includes 66 genes contributing to amino acid biosynthesis and 93 genes to amino acid degradation. In several respects, the pea aphid gene inventory complements that of its symbiotic bacterium, Buchnera aphidicola (Buchnera APS). Unlike other insects with completely sequenced genomes, the pea aphid lacks the capacity to synthesize arginine, which is produced by Buchnera APS. However, consistent with other insects, it has genes coding for individual reactions in essential amino acid biosynthesis, including threonine dehydratase and branched‐chain amino acid aminotransferase, which are not coded in the Buchnera APS genome. Overall the genome data suggest that the biosynthesis of certain essential amino acids is shared between the pea aphid and Buchnera APS, providing the opportunity for precise aphid control over Buchnera metabolism.

A fragile metabolic network adapted for cooperation in the symbiotic bacterium Buchnera aphidicola

BackgroundIn silico analyses provide valuable insight into the biology of obligately intracellular pathogens and symbionts with small genomes. There is a particular opportunity to apply systems-level tools developed for the model bacterium Escherichia coli to study the evolution and function of symbiotic bacteria which are metabolically specialised to overproduce specific nutrients for their host and, remarkably, have a gene complement that is a subset of the E. coli genome.ResultsWe have reconstructed and analysed the metabolic network of the γ-proteobacterium Buchnera aphidicola (symbiont of the pea aphid) as a model for using systems-level approaches to discover key traits of symbionts with small genomes. The metabolic network is extremely fragile with > 90% of the reactions essential for viability in silico; and it is structured so that the bacterium cannot grow without producing the essential amino acid, histidine, which is released to the insect host. Further, the amount of essential amino acid produced by the bacterium in silico can be controlled by host supply of carbon and nitrogen substrates.ConclusionThis systems-level analysis predicts that the fragility of the bacterial metabolic network renders the symbiotic bacterium intolerant of drastic environmental fluctuations, whilst the coupling of histidine production to growth prevents the bacterium from exploiting host nutrients without reciprocating. These metabolic traits underpin the sustained nutritional contribution of B. aphidicola to the host and, together with the impact of host-derived substrates on the profile of nutrients released from the bacteria, point to a dominant role of the host in controlling the symbiosis.

The central role of the host cell in symbiotic nitrogen metabolism

Symbiotic nitrogen recycling enables animals to thrive on nitrogen-poor diets and environments. It traditionally refers to the utilization of animal waste nitrogen by symbiotic micro-organisms to synthesize essential amino acids (EAAs), which are translocated back to the animal host. We applied metabolic modelling and complementary metabolite profiling to investigate nitrogen recycling in the symbiosis between the pea aphid and the intracellular bacterium Buchnera, which synthesizes EAAs. The results differ from traditional notions of nitrogen recycling in two important respects. First, aphid waste ammonia is recycled predominantly by the host cell (bacteriocyte) and not Buchnera. Host cell recycling is mediated by shared biosynthetic pathways for four EAAs, in which aphid transaminases incorporate ammonia-derived nitrogen into carbon skeletons synthesized by Buchnera to generate EAAs. Second, the ammonia substrate for nitrogen recycling is derived from bacteriocyte metabolism, such that the symbiosis is not a sink for nitrogenous waste from other aphid organs. Host cell-mediated nitrogen recycling may be general among insect symbioses with shared EAA biosynthetic pathways generated by the loss of symbiont genes mediating terminal reactions in EAA synthesis.

Genetic and metabolic determinants of nutritional phenotype in an insect–bacterial symbiosis

The pervasive influence of resident microorganisms on the phenotype of their hosts is exemplified by the intracellular bacterium Buchnera aphidicola, which provides its aphid partner with essential amino acids (EAAs). We investigated variation in the dietary requirement for EAAs among four pea aphid (Acyrthosiphon pisum) clones. Buchnera‐derived nitrogen contributed to the synthesis of all EAAs for which aphid clones required a dietary supply, and to none of the EAAs for which all four clones had no dietary requirement, suggesting that low total dietary nitrogen may select for reduced synthesis of certain EAAs in some aphid clones. The sequenced Buchnera genomes showed that the EAA nutritional phenotype (i.e. the profile of dietary EAAs required by the aphid) cannot be attributed to sequence variation of Buchnera genes coding EAA biosynthetic enzymes. Metabolic modelling by flux balance analysis demonstrated that EAA output from Buchnera can be determined precisely by the flux of host metabolic precursors to Buchnera. Specifically, the four EAA nutritional phenotypes could be reproduced by metabolic models with unique profiles of host inputs, dominated by variation in supply of aspartate, homocysteine and glutamate. This suggests that the nutritional phenotype of the symbiosis is determined principally by host metabolism and transporter genes that regulate nutrient supply to Buchnera. Intraspecific variation in the nutritional phenotype of symbioses is expected to mediate partitioning of plant resources among aphid genotypes, potentially promoting the genetic subdivision of aphid populations. In this way, microbial symbioses may play an important role in the evolutionary diversification of phytophagous insects.

Bone marrow-derived and resident liver macrophages display unique transcriptomic signatures but similar biological functions

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Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

Genomic evidence for complementary purine metabolism in the pea aphid, Acyrthosiphon pisum, and its symbiotic bacterium Buchnera aphidicola

The purine salvage pathway recycles purines to nucleotides, promoting efficient utilization of purine nucleotides. Exceptionally among animals with completely sequenced genomes, the pea aphid lacks key purine recycling genes that code for purine nucleoside phosphorylase and adenosine deaminase, indicating that the aphid can neither metabolize nucleosides to the corresponding purines, nor adenosine to inosine. Purine metabolism genes in the symbiotic bacterium Buchnera complement aphid genes, and Buchnera can meet its nucleotide requirement from aphid‐derived guanosine. Buchnera demand for nucleosides may have relaxed the selection for purine recycling in the aphid, leading to the loss of key aphid purine salvage genes. Further, the coupled purine metabolism of aphid and Buchnera could contribute to the dependence of the pea aphid on this symbiosis.

The Schistosome Esophagus Is a ‘Hotspot’ for Microexon and Lysosomal Hydrolase Gene Expression: Implications for Blood Processing

Background The schistosome esophagus is divided into anterior and posterior compartments, each surrounded by a dense cluster of gland cell bodies, the source of distinct secretory vesicles discharged into the lumen to initiate the processing of ingested blood. Erythrocytes are lysed in the lumen, leucocytes are tethered and killed and platelets are eliminated. We know little about the proteins secreted from the two glands that mediate these biological processes. Methodology/Principal Findings We have used subtractive RNA-Seq to characterise the complement of genes that are differentially expressed in a head preparation, compared to matched tissues from worm tails. The expression site of representative highlighted genes was then validated using whole munt in situ hybridisation (WISH). Mapping of transcript reads to the S. mansoni genome assembly using Cufflinks identified ~90 genes that were differentially expressed >fourfold in the head preparation; ~50 novel transcripts were also identified by de novo assembly using Trinity. The largest subset (27) of secreted proteins was encoded by microexon genes (MEGs), the most intense focus identified to date. Expression of three (MEGs 12, 16, 17) was confirmed in the anterior gland and five (MEGs 8.1, 9, 11, 15 and 22) in the posterior gland. The other major subset comprised nine lysosomal hydrolases (aspartyl proteases, phospholipases and palmitoyl thioesterase), again localised to the glands. Conclusions A proportion of the MEG-encoded secretory proteins can be classified by their primary structure. We have suggested testable hypotheses about how they might function, in conjunction with the lysosomal hydrolases, to mediate the biological processes that occur in the esophagus lumen. Antibodies bind to the esophageal secretions in both permissive and self-curing hosts, suggesting that the proteins represent a novel panel of untested vaccine candidates. A second major task is to identify which of them can serve as immune targets.

A large genomic island allows Neisseria meningitidis to utilize propionic acid, with implications for colonization of the human nasopharynx.

Neisseria meningitidis is an important human pathogen that is capable of killing within hours of infection. Its normal habitat is the nasopharynx of adult humans. Here we identify a genomic island (the prp gene cluster) in N. meningitidis that enables this species to utilize propionic acid as a supplementary carbon source during growth, particularly under nutrient poor growth conditions. The prp gene cluster encodes enzymes for a methylcitrate cycle. Novel aspects of the methylcitrate cycle in N. meningitidis include a propionate kinase which was purified and characterized, and a putative propionate transporter. This genomic island is absent from the close relative of N. meningitidis, the commensal Neisseria lactamica, which chiefly colonizes infants not adults. We reason that the possession of the prp genes provides a metabolic advantage to N. meningitidis in the adult oral cavity, which is rich in propionic acid‐generating bacteria. Data from classical microbiological and sequence‐based microbiome studies provide several lines of supporting evidence that N. meningitidis colonization is correlated with propionic acid generating bacteria, with a strong correlation between prp‐containing Neisseria and propionic acid generating bacteria from the genus Porphyromonas, and that this may explain adolescent/adult colonization by N. meningitidis.

A different path: revealing the function of staphylococcal proteins in biofilm formation.

Staphylococcus aureus and Staphylococcus epidermidis cause dangerous and difficult to treat medical device‐related infections through their ability to form biofilms. Extracellular poly‐N‐acetylglucosamine (PNAG) facilitates biofilm formation and is a vaccination target, yet details of its biosynthesis by the icaADBC gene products is limited. IcaC is the proposed transporter for PNAG export, however a comparison of the Ica proteins to homologous exo‐polysaccharide synthases suggests that the common IcaAD protein components both synthesise and transport the PNAG. The limited distribution of icaC to the Staphylococcaceae and its membership of a family of membrane‐bound acyltransferases, leads us to suggest that IcaC is responsible for the known O‐succinylation of PNAG that occurs in staphylococci, identifying a potentially new therapeutic target specific for these bacteria.