Characterization of an Ancient Lepidopteran Lateral Gene Transfer

<div><p>Bacteria to eukaryote lateral gene transfers (LGT) are an important potential source of material for the evolution of novel genetic traits. The explosion in the number of newly sequenced genomes provides opportunities to identify and characterize examples of these lateral gene transfer events, and to assess their role in the evolution of new genes. In this paper, we describe an ancient lepidopteran LGT of a glycosyl hydrolase family 31 gene (GH31) from an <i>Enterococcus</i> bacteria. PCR amplification between the LGT and a flanking insect gene confirmed that the GH31 was integrated into the <i>Bombyx mori</i> genome and was not a result of an assembly error. Database searches in combination with degenerate PCR on a panel of 7 lepidopteran families confirmed that the GH31 LGT event occurred deep within the Order approximately 65–145 million years ago. The most basal species in which the LGT was found is <i>Plutella xylostella</i> (superfamily: Yponomeutoidea). Array data from <i>Bombyx mori</i> shows that GH31 is expressed, and low dN/dS ratios indicates the LGT coding sequence is under strong stabilizing selection. These findings provide further support for the proposition that bacterial LGTs are relatively common in insects and likely to be an underappreciated source of adaptive genetic material.</p> </div

A branching diagram showing the hypothesized relationships of lepidopteran families.

<p>PCR with sequencing, and bioinformatic searches were used to identify GH31 LGTs in the lepidopteran species from families highlighted by the blue boxes. The only family in which the LGT was not confirmed by degenerate PCR is Lymantriidae (red box). The most basal species in which the LGT was discovered is <i>P. xylostella</i> (Yponomeutidae). This places the timescale of LGT acquisition (red arrow) to the Cretaceous (145-66 mya), as this is when early radiations of ditrysian superfamilies likely occurred <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059262#pone.0059262-Grimaldi1" target="_blank">[26]</a>. The branch arrangements are based on data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059262#pone.0059262-Regier1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059262#pone.0059262-Cho1" target="_blank">[36]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059262#pone.0059262-Kristensen1" target="_blank">[37]</a>.</p

Microsynteny exists in the region surrounding DpGH31, BmGH31 and PxGH31.

<p>Microsynteny of genes surrounding the GH31 LGT regions in <i>B. mori</i>, <i>D. plexippus</i> and <i>P. xylostella</i>. The genes’ direction of transcription is indicated by arrows with LGTs shown in red. Genes that are one to one blast orthologs between <i>B. mori, D. plexippus,</i> and <i>P. xylostella</i> are indicated by matching colors and connecting gray lines. The PCR primers used to amplify between BmGH31 and the neighboring gene (BGIBMGA013897) on scaffold 3099 are indicated by small blue arrows on BmGH31 and BGIBMGA013897.</p

Molecular evidence supports that BmGH31 is part of the <i>B. mori</i> genome.

<p>PCR was used to amplify across the boundary between the BmGH31 LGT and a flanking insect gene called <i>BGIBMGA013897</i> that is conserved in divergent insect species. The sequence boundary between BmGH31 (red) and <i>BGIBMGA013897</i> (blue) was obtained by sequencing the PCR product. The scaffold positions along scaffold3899 are shown above the gene models with the region amplified by the PCR is highlighted by the box. Note that the coding region of BGIBMGA013897 is located on the opposite strand.</p

The GH31 LGT is ancient in origin.

<p>A.) Putative homologs of DpGH31 identified using degenerate PCR on genomic DNA from a panel of diverse lepidoptera species. Phylogenetic relationships of the families are taken from Cho et al 2011 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059262#pone.0059262-Cho1" target="_blank">[36]</a>. <i>Bombxy mori</i> genomic DNA was used as the positive control (+). Faint bands could be observed for <i>P. populi</i> and <i>A. velutinana</i> on the original gel. B.) A Bayesian phylogenetic tree derived from GH31-LGT conceptually translated DNA sequences. DNA sequences used were obtained either by degenerate PCR or searches of available lepidopteran genome sequences. Well supported clades are resolved for members of the Noctuoidea and Geometroidea. The phylogenetic relationships of species from the Bombycoid complex and the butterflies could not be resolved in this analysis (posterior probability values less than 90%).</p

Bayesian phylogeny of DpGH31 and BmGH31 sequences.

<p>Unrooted Bayesian phylogenetic tree of GH31-like protein sequences from prokaryote and eukaryote species. The clade grouping the <i>D. Plexippus</i> and <i>B. mori</i> LGTs (DpGH31 and BmGH31 shown in red) with the <i>E. faecalis</i> GH31 sequence is supported by a 100% posterior probability. The division of the sequences into Prokaryote and Eukaryote clades, indicted by the curved lines, is supported by a >98% posterior probabilities, with the Lepidopteran LGTs clearly within the bacterial clade. Only posterior probability values at key nodes are not shown for clarity. The tentative protein annotations were made based on the information available at NCBI using the following key: hypothetical (HYP), glycosyl hydrolase family 31 (GH31), alpha-glucosidase (AG), neutral alpha-glucosidase (NAG), glucosidase (GLUC). Gene identifier numbers for all sequences in the phylogeny are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059262#pone.0059262.s001" target="_blank">Table S1</a>.</p

Activation of intestinal tuft cell-expressed Sucnr1 triggers type 2 immunity in the mouse small intestine.

Until recently, little is known about whether succinate, a key metabolite in the Krebs cycle and microbial propionate synthesis, has any physiological role in the gut. De Vadder et al. (1) showed that dietary succinate or microbiota-produced succinate improves glucose homeostasis via intestinal gluconeogenesis. Given the expression of the succinate receptor Sucnr1 in the intestine, we asked if dietary succinate has additional effects in the intestine. In this report, we find that Sucnr1 is specifically expressed in intestinal tuft cells but not in other types of intestinal epithelial cells. Exogenous succinate induces tuft and goblet cell hyperplasia in wild-type mice, hallmark features of mucosal type 2 immunity in response to parasitic infections. In contrast, monomethyl succinate, a structural analog of succinate but not a ligand for Sucnr1, fails to induce tuft cell hyperplasia. Succinate-induced type 2 responses are mediated by Sucnr1, and tuft cell-expressed chemosensory signaling elements gustducin and Trpm5. Mice deficient for Sucnr1, gustducin or Trpm5 show neither tuft nor goblet cell expansion in response to succinate feeding. Perturbation of microbiota by antibiotics (e.g., streptomycin) or chemically-induced intestinal motility disturbance (e.g., PEG-3350) can lead to an elevated level of succinate in the perturbed intestine. Tuft cells expand in the distal ileum juxtaposed to the cecum in the Sucnr1 heterozygous but not homozygous mice, suggesting that microbiota-produced succinate is sufficient to trigger type 2 immunity. Altogether, our data suggest that dietary and microbiota-produced succinate activates intestinal tuft cell-expressed Sucnr1 to triggers type 2 immunity