Gene family expansion and functional diversification of chitinase and chitin synthase genes in Atlantic salmon (Salmo salar).

Chitin is one of the most abundant polysaccharides in nature, forming important structures in insects, crustaceans, and fungal cell walls. Vertebrates on the other hand are generally considered "nonchitinous" organisms, despite having highly conserved chitin metabolism-associated genes. Recent work has revealed that the largest group of vertebrates, the teleosts, have the potential to both synthesize and degrade endogenous chitin. Yet, little is known about the genes and proteins responsible for these dynamic processes. Here, we used comparative genomics, transcriptomics, and chromatin accessibility data to characterize the repertoire, evolution, and regulation of genes involved in chitin metabolism in teleosts, with a particular focus on Atlantic salmon. Reconstruction of gene family phylogenies provides evidence for an expansion of teleost and salmonid chitinase and chitin synthase genes after multiple whole-genome duplications. Analyses of multi-tissue gene expression data demonstrated a strong bias of gastrointestinal tract expression for chitin metabolism genes, but with different spatial and temporal tissue specificities. Finally, we integrated transcriptomes from a developmental time series of the gastrointestinal tract with chromatin accessibility data to identify putative transcription factors responsible for regulating chitin metabolism gene expression (CDX1 and CDX2) as well as tissue-specific divergence in the regulation of gene duplicates (FOXJ2). The findings presented here support the hypothesis that chitin metabolism genes in teleosts play a role in developing and maintaining a chitin-based barrier in the teleost gut and provide a basis for further investigations into the molecular basis of this barrier.

Comparative regulomics supports pervasive selection on gene dosage following whole genome duplication.

BACKGROUND: Whole genome duplication (WGD) events have played a major role in eukaryotic genome evolution, but the consequence of these extreme events in adaptive genome evolution is still not well understood. To address this knowledge gap, we used a comparative phylogenetic model and transcriptomic data from seven species to infer selection on gene expression in duplicated genes (ohnologs) following the salmonid WGD 80-100 million years ago. RESULTS: We find rare cases of tissue-specific expression evolution but pervasive expression evolution affecting many tissues, reflecting strong selection on maintenance of genome stability following genome doubling. Ohnolog expression levels have evolved mostly asymmetrically, by diverting one ohnolog copy down a path towards lower expression and possible pseudogenization. Loss of expression in one ohnolog is significantly associated with transposable element insertions in promoters and likely driven by selection on gene dosage including selection on stoichiometric balance. We also find symmetric expression shifts, and these are associated with genes under strong evolutionary constraints such as ribosome subunit genes. This possibly reflects selection operating to achieve a gene dose reduction while avoiding accumulation of "toxic mutations". Mechanistically, ohnolog regulatory divergence is dictated by the number of bound transcription factors in promoters, with transposable elements being one likely source of novel binding sites driving tissue-specific gains in expression. CONCLUSIONS: Our results imply pervasive adaptive expression evolution following WGD to overcome the immediate challenges posed by genome doubling and to exploit the long-term genetic opportunities for novel phenotype evolution.

Aromatic-Mediated Carbohydrate Recognition in Processive Serratia marcescens Chitinases.

Microorganisms use a host of enzymes, including processive glycoside hydrolases, to deconstruct recalcitrant polysaccharides to sugars. Processive glycoside hydrolases closely associate with polymer chains and repeatedly cleave glycosidic linkages without dissociating from the crystalline surface after each hydrolytic step; they are typically the most abundant enzymes in both natural secretomes and industrial cocktails by virtue of their significant hydrolytic potential. The ubiquity of aromatic residues lining the enzyme catalytic tunnels and clefts is a notable feature of processive glycoside hydrolases. We hypothesized that these aromatic residues have uniquely defined roles, such as substrate chain acquisition and binding in the catalytic tunnel, that are defined by their local environment and position relative to the substrate and the catalytic center. Here, we investigated this hypothesis with variants of Serratia marcescens family 18 processive chitinases ChiA and ChiB. We applied molecular simulation and free energy calculations to assess active site dynamics and ligand binding free energies. Isothermal titration calorimetry provided further insight into enthalpic and entropic contributions to ligand binding free energy. Thus, the roles of six aromatic residues, Trp-167, Trp-275, and Phe-396 in ChiA, and Trp-97, Trp-220, and Phe-190 in ChiB, have been examined. We observed that point mutation of the tryptophan residues to alanine results in unfavorable changes in the free energy of binding relative to wild-type. The most drastic effects were observed for residues positioned at the "entrances" of the deep substrate-binding clefts and known to be important for processivity. Interestingly, phenylalanine mutations in ChiA and ChiB had little to no effect on chito-oligomer binding, in accordance with the limited effects of their removal on chitinase functionality.

Thermodynamic Relationships with Processivity in Serratia marcescens Family 18 Chitinases.

The enzymatic degradation of recalcitrant polysaccharides is accomplished by synergistic enzyme cocktails of glycoside hydrolases (GHs) and accessory enzymes. Many GHs are processive which means that they remain attached to the substrate in between subsequent hydrolytic reactions. Chitinases are GHs that catalyze the hydrolysis of chitin (ß-1,4-linked N-acetylglucosamine). Previously, a relationship between active site topology and processivity has been suggested while recent computational efforts have suggested a link between the degree of processivity and ligand binding free energy. We have investigated these relationships by employing computational (molecular dynamics (MD)) and experimental (isothermal titration calorimetry (ITC)) approaches to gain insight into the thermodynamics of substrate binding to Serratia marcescens chitinases ChiA, ChiB, and ChiC. We show that increased processive ability indeed corresponds to more favorable binding free energy and that this likely is a general feature of GHs. Moreover, ligand binding in ChiB is entropically driven; in ChiC it is enthalpically driven, and the enthalpic and entropic contributions to ligand binding in ChiA are equal. Furthermore, water is shown to be especially important in ChiA-binding. This work provides new insight into oligosaccharide binding, getting us one step closer to understand how GHs efficiently degrade recalcitrant polysaccharides.