Large-scale snake genome analyses provide insights into vertebrate development.

Snakes are a remarkable squamate lineage with unique morphological adaptations, especially those related to the evolution of vertebrate skeletons, organs, and sensory systems. To clarify the genetic underpinnings of snake phenotypes, we assembled and analyzed 14 de novo genomes from 12 snake families. We also investigated the genetic basis of the morphological characteristics of snakes using functional experiments. We identified genes, regulatory elements, and structural variations that have potentially contributed to the evolution of limb loss, an elongated body plan, asymmetrical lungs, sensory systems, and digestive adaptations in snakes. We identified some of the genes and regulatory elements that might have shaped the evolution of vision, the skeletal system and diet in blind snakes, and thermoreception in infrared-sensitive snakes. Our study provides insights into the evolution and development of snakes and vertebrates.

Metabolic profiling identifies Qrich2 as a novel glutamine sensor that regulates microtubule glutamylation and mitochondrial function in mouse sperm.

In our prior investigation, we discerned loss-of-function variants within the gene encoding glutamine-rich protein 2 (QRICH2) in two consanguineous families, leading to various morphological abnormalities in sperm flagella and male infertility. The Qrich2 knockout (KO) in mice also exhibits multiple morphological abnormalities of the flagella (MMAF) phenotype with a significantly decreased sperm motility. However, how ORICH2 regulates the formation of sperm flagella remains unclear. Abnormal glutamylation levels of tubulin cause dysplastic microtubules and flagella, eventually resulting in the decline of sperm motility and male infertility. In the current study, by further analyzing the Qrich2 KO mouse sperm, we found a reduced glutamylation level and instability of tubulin in Qrich2 KO mouse sperm flagella. In addition, we found that the amino acid metabolism was dysregulated in both testes and sperm, leading to the accumulated glutamine (Gln) and reduced glutamate (Glu) concentrations, and disorderly expressed genes responsible for Gln/Glu metabolism. Interestingly, mice fed with diets devoid of Gln/Glu phenocopied the Qrich2 KO mice. Furthermore, we identified several mitochondrial marker proteins that could not be correctly localized in sperm flagella, which might be responsible for the reduced mitochondrial function contributing to the reduced sperm motility in Qrich2 KO mice. Our study reveals a crucial role of a normal Gln/Glu metabolism in maintaining the structural stability of the microtubules in sperm flagella by regulating the glutamylation levels of the tubulin and identifies Qrich2 as a possible novel Gln sensor that regulates microtubule glutamylation and mitochondrial function in mouse sperm.

A Chromosome-Level Genome Assembly of the Reef Stonefish (Synanceia verrucosa) Provides Novel Insights into Stonustoxin (sntx) Genes.

Reef stonefish (Synanceia verrucosa) is one of the most venomous fishes, but its biomedical study has been restricted to molecular cloning and purification of its toxins, instead of high-throughput genetic research on related toxin genes. In this study, we constructed a chromosome-level haplotypic genome assembly for the reef stonefish. The genome was assembled into 24 pseudo-chromosomes, and the length totaled 689.74 Mb, reaching a contig N50 of 11.97 Mb and containing 97.8% of complete BUSCOs. A total of 24,050 protein-coding genes were annotated, of which metalloproteinases, C-type lectins, and stonustoxins (sntx) were the most abundant putative toxin genes. Multitissue transcriptomic and venom proteomic data showed that sntx genes, especially those clustered within a 50-kb region on the chromosome 2, had higher transcription levels than other types of toxins as well as those sntx genes scatteringly distributed on other chromosomes. Further comparative genomic analysis predicted an expansion of sntx-like genes in the Percomorpha lineage including nonvenomous fishes, but Scorpaenoidei species experienced extra independent sntx duplication events, marking the clear-cut origin of authentic toxic stonustoxins. In summary, this high-quality genome assembly and related comparative analysis of toxin genes highlight valuable genetic differences for potential involvement in the evolution of venoms among Scorpaeniformes fishes.

Genomic evidence reveals intraspecific divergence of the hot-spring snake (Thermophis baileyi), an endangered reptile endemic to the Qinghai-Tibet plateau.

Understanding how and why species evolve requires knowledge on intraspecific divergence. In this study, we examined intraspecific divergence in the endangered hot-spring snake (Thermophis baileyi), an endemic species on the Qinghai-Tibet Plateau (QTP). Whole-genome resequencing of 58 sampled individuals from 15 populations was performed to identify the drivers of intraspecific divergence and explore the potential roles of genes under selection. Our analyses resolved three groups, with major intergroup admixture occurring in regions of group contact. Divergence probably occurred during the Pleistocene as a result of glacial climatic oscillations, Yadong-Gulu rift, and geothermal fields differentiation, while complex gene flow between group pairs reflected a unique intraspecific divergence pattern on the QTP. Intergroup fixed loci involved selected genes functionally related to divergence and local adaptation, especially adaptation to hot spring microenvironments in different geothermal fields. Analysis of structural variants, genetic diversity, inbreeding, and genetic load indicated that the hot-spring snake population has declined to a low level with decreased diversity, which is important for the conservation management of this endangered species. Our study demonstrated that the integration of demographic history, gene flow, genomic divergence genes, and other information is necessary to distinguish the evolutionary processes involved in speciation.

Molecular characterization of a teleost-specific toll-like receptor 22 (tlr22) gene from yellow catfish (Pelteobagrus fulvidraco) and its transcriptional change in response to poly I:C and Aeromonas hydrophila stimuli.

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) that can recognize pathogen-associated molecular patterns (PMPs) and play important roles in the innate immune system in vertebrates. In this study, we identified a teleost-specific tlr22 gene from yellow catfish (Pelteobagrus fulvidraco) and its immune roles in response to different pathogens were also determined. The open reading frame (ORF) of the tlr22 was 2892 bp in length, encoding a protein of 963 amino acids. Multiple protein sequences alignment, secondary and three-dimensional structure analyses revealed that TLR22 is highly conserved among different fish species. Phylogenetic analysis showed that the phylogenetic topology was divided into six families of TLR1, TLR3, TLR4, TLR5, TLR7 and TLR11, and TLR22 subfamily was clustered into TLR11 family. Meanwhile, synteny and gene structure comparisons revealed functional and evolutionary conservation of the tlr22 gene in teleosts. Furthermore, tlr22 gene was shown to be widely expressed in detected tissues except barbel and eye, with highest expression level in liver. The transcription of tlr22 was significantly increased in spleen, kidney, liver and gill tissues at different timepoints after Poly I:C infection, suggesting TLR22 plays critical roles in defensing virus invasion. Similarly, the transcription of tlr22 was also dramatically up-regulated in spleen, kidney and gill tissues with different patterns after Aeromonas hydrophila infection, indicating that TLR22 is also involved in resisting bacteria invasion. Our findings will provide a solid basis for the investigation the immune functions of tlr22 gene in teleosts, as well as provide useful information for disease control and treatment for yellow catfish.

The American Paddlefish Genome Provides Novel Insights into Chromosomal Evolution and Bone Mineralization in Early Vertebrates.

Sturgeons and paddlefishes (Acipenseriformes) occupy the basal position of ray-finned fishes, although they have cartilaginous skeletons as in Chondrichthyes. This evolutionary status and their morphological specializations make them a research focus, but their complex genomes (polyploidy and the presence of microchromosomes) bring obstacles and challenges to molecular studies. Here, we generated the first high-quality genome assembly of the American paddlefish (Polyodon spathula) at a chromosome level. Comparative genomic analyses revealed a recent species-specific whole-genome duplication event, and extensive chromosomal changes, including head-to-head fusions of pairs of intact, large ancestral chromosomes within the paddlefish. We also provide an overview of the paddlefish SCPP (secretory calcium-binding phosphoprotein) repertoire that is responsible for tissue mineralization, demonstrating that the earliest flourishing of SCPP members occurred at least before the split between Acipenseriformes and teleosts. In summary, this genome assembly provides a genetic resource for understanding chromosomal evolution in polyploid nonteleost fishes and bone mineralization in early vertebrates.

Genome of Laudakia sacra Provides New Insights into High-Altitude Adaptation of Ectotherms.

Anan's rock agama (Laudakia sacra) is a lizard species endemic to the harsh high-altitude environment of the Qinghai-Tibet Plateau, a region characterized by low oxygen tension and high ultraviolet (UV) radiation. To better understand the genetic mechanisms underlying highland adaptation of ectotherms, we assembled a 1.80-Gb L. sacra genome, which contained 284 contigs with an N50 of 20.19 Mb and a BUSCO score of 93.54%. Comparative genomic analysis indicated that mutations in certain genes, including HIF1A, TIE2, and NFAT family members and genes in the respiratory chain, may be common adaptations to hypoxia among high-altitude animals. Compared with lowland reptiles, MLIP showed a convergent mutation in L. sacra and the Tibetan hot-spring snake (Thermophis baileyi), which may affect their hypoxia adaptation. In L. sacra, several genes related to cardiovascular remodeling, erythropoiesis, oxidative phosphorylation, and DNA repair may also be tailored for adaptation to UV radiation and hypoxia. Of note, ERCC6 and MSH2, two genes associated with adaptation to UV radiation in T. baileyi, exhibited L. sacra-specific mutations that may affect peptide function. Thus, this study provides new insights into the potential mechanisms underpinning high-altitude adaptation in ectotherms and reveals certain genetic generalities for animals' survival on the plateau.

Morphometric and population genomic evidence for species divergence in the Chimarrichthys fish complex of the Tibetan Plateau.

The uplift of the Tibetan Plateau altered the environmental conditions of the local area substantially. Here, we conducted a comprehensive investigation based on morphometrics, population genomics, and climatic factors to evaluate phenotypic and genome-level variations in a radiation of Chimarrichthys catfish endemic to the Plateau. Discriminant function analysis showed phenotypic differences of Chimarrichthys between rivers with respect to elevation. Genetic structure analysis based on 6606 single nucleotide polymorphisms (SNPs) deduced genetic differences between rivers, and species delimitation indicated that the Chimarrichthys fish complex could be divided into three species. Restriction site-associated DNA tags were mapped to the gene sets of Glyptosternon maculatum, and matches were searched against databases for Gene Ontology annotation. Genomic regions exhibiting marked differences among localities represented a range of biological functions, including growth (gdf11), bone development (bmp8a), cellular response to light stimulus (opn3), regulation of the rhodopsin-mediated signalling pathway (grk1), immune response (rag1 and ung), reproductive process (antxr2), and regulation of intracellular iron levels (ireb2). The tag44126, where gene gdf11 is located, was identified as an outlier exhibiting divergence between rivers with altitude differences, and the SNP is thymine (T) in Dadu and Yalong River (~2700 m), but guanine (G) in Jinsha and Qingyi rivers (~2200 and ~ 684 m), suggesting a possible effect of altitude on its differentiation.

Diurnal rhythm expression of transcription factor NF-κB subunit p65 in Pelteobagrus vachellii.

The pleiotropic transcription factor nuclear factor-kappa B (NF-κB) has important functions in viral resistance. In the present study, we isolated a p65 subunit of NF-κB cDNA from Pelteobagrus vachellii (termed Pvp65) and characterized it. The full-length p65 cDNA comprised 3651 bp, including a 148-bp 5'-untranslated region (UTR), a 106-bp 3'-UTR, and an open reading frame encoding a 1067-amino acid putative protein. The protein sequence comprised a DNA binding motif, a Rel-homology domain, a Rel protein signature, a putative transcription activation domain, a nuclear localization signal, and a transcription initiation factor IIA domain. The expression of Pvp65 displayed a daily rhythm, with an acrophase at approximately at 15:32 h in the liver, 11:34 h in the spleen, and 16:45 h in the head kidney. In addition, infection with Aeromonas hydrophila caused Pvp65 expression to increase significantly (P < 0.05), and peaking at 12 h post infection in the spleen, at 24 in the head kidney, and at 12 h in the liver. Thus, NF-κB expression might be under light/dark cycle control in P. vachellii, and may be involved in the immune response to A. hydrophila.

High throughput screening of small immune peptides and antimicrobial peptides from the Fish-T1K database.

Analyses of transcriptomic datasets have the potential to reveal genetic markers underlying ecological adaptations. In the present study, we leverage the expanding dataset generated by the Fish-T1K Project (Transcriptomes of 1000 Fishes) to characterize small peptides that may be implicated in the immune system of fishes. We focused our analyses on sequences smaller than 360 bp obtained from gill transcriptomes of 87 ray-finned fishes (Actinopterygii). Functional annotation of short transcripts revealed that the number of small immune peptides varied significantly among the studied species. High-throughput screening of antimicrobial peptides (AMPs) with homologous searches was used to characterize the composition of innate immune defense factors present in fishes. We analyzed the putative effects of habitat, climatic zone and genetic system on the distribution of small peptides among species. Our results highlight the utility of large transcriptomic datasets such as Fish-T1K to explore patterns of variation at macroevolutionary scales and to discover novel peptides that may be used for further investigation and drug development.

Genome and Transcriptome Sequencing of casper and roy Zebrafish Mutants Provides Novel Genetic Clues for Iridophore Loss.

casper has been a widely used transparent mutant of zebrafish. It possesses a combined loss of reflective iridophores and light-absorbing melanophores, which gives rise to its almost transparent trunk throughout larval and adult stages. Nevertheless, genomic causal mutations of this transparent phenotype are poorly defined. To identify the potential genetic basis of this fascinating morphological phenotype, we constructed genome maps by performing genome sequencing of 28 zebrafish individuals including wild-type AB strain, roy orbison (roy), and casper mutants. A total of 4.3 million high-quality and high-confidence homozygous single nucleotide polymorphisms (SNPs) were detected in the present study. We also identified a 6.0-Mb linkage disequilibrium block specifically in both roy and casper that was composed of 39 functional genes, of which the mpv17 gene was potentially involved in the regulation of iridophore formation and maintenance. This is the first report of high-confidence genomic mutations in the mpv17 gene of roy and casper that potentially leads to defective splicing as one major molecular clue for the iridophore loss. Additionally, comparative transcriptomic analyses of skin tissues from the AB, roy and casper groups revealed detailed transcriptional changes of several core genes that may be involved in melanophore and iridophore degeneration. In summary, our updated genome and transcriptome sequencing of the casper and roy mutants provides novel genetic clues for the iridophore loss. These new genomic variation maps will offer a solid genetic basis for expanding the zebrafish mutant database and in-depth investigation into pigmentation of animals.

Whole Genome Sequencing of the Blue Tilapia (Oreochromis aureus) Provides a Valuable Genetic Resource for Biomedical Research on Tilapias.

Blue tilapia (Oreochromis aureus) has been an economically important fish in Asian countries. It can grow and reproduce in both freshwater and brackish water conditions, whereas it is also considered as a significant invasive species around the world. This species has been widely used as the hybridization parent(s) for tilapia breeding with a major aim to produce novel strains. However, available genomic resources are still limited for this important tilapia species. Here, we for the first time sequenced and assembled a draft genome for a seawater cultured blue tilapia (0.92 Gb), with 97.8% completeness and a scaffold N50 of 1.1 Mb, which suggests a relatively high quality of this genome assembly. We also predicted 23,117 protein-coding genes in the blue tilapia genome. Comparisons of predicted antimicrobial peptides between the blue tilapia and its close relative Nile tilapia proved that these immunological genes are highly similar with a genome-wide scattering distribution. As a valuable genetic resource, our blue tilapia genome assembly will benefit for biomedical researches and practical molecular breeding for high resistance to various diseases, which have been a critical problem in the aquaculture of tilapias.

Whole Genome Sequencing of Chinese White Dolphin (Sousa chinensis) for High-Throughput Screening of Antihypertensive Peptides.

Chinese white dolphin (Sousa chinensis), also known as the Indo-Pacific humpback dolphin, has been classified as "Vulnerable" on the IUCN Red List of Threatened Species. It is a special cetacean species that lives in tropical and subtropical nearshore waters, with significant differences from other cetaceans. Here, we sequenced and assembled a draft genome of the Chinese white dolphin with a total length of 2.3 Gb and annotation of 18,387 protein-coding genes. Genes from certain expanded families are potentially involved in DNA replication and repairing, suggesting that they may be related to adaptation of this marine mammal to nearshore environments. We also discovered that its historical population had undergone a remarkable bottleneck incident before the Mindel glaciation. In addition, a comparative genomic survey on antihypertensive peptides (AHTPs) among five representative mammals with various residential habitats (such as remarkable differences in exogenous ion concentrations and sea depth) revealed that these small bioactive peptides were highly conserved among these examined mammals, and they had the most abundant hits in collagen subunit proteins, especially for two putative AHTP peptides Gly-Leu-Pro (GLP) and Leu-Gly-Pro (LGP). Our genome assembly will be a valuable resource for further genetic researches on adaptive ecology and conservation biology of cetaceans, and for in-depth investigations into bioactive peptides in aquatic and terrestrial mammals for development of peptide-based drugs to treat various human cardiovascular diseases.

Genome Sequencing of the Japanese Eel (Anguilla japonica) for Comparative Genomic Studies on tbx4 and a tbx4 Gene Cluster in Teleost Fishes.

Limbs originated from paired fish fins are an important innovation in Gnathostomata. Many studies have focused on limb development-related genes, of which the T-box transcription factor 4 gene (tbx4) has been considered as one of the most essential factors in the regulation of the hindlimb development. We previously confirmed pelvic fin loss in tbx4-knockout zebrafish. Here, we report a high-quality genome assembly of the Japanese eel (Anguilla japonica), which is an economically important fish without pelvic fins. The assembled genome is 1.13 Gb in size, with a scaffold N50 of 1.03 Mb. In addition, we collected 24 tbx4 sequences from 22 teleost fishes to explore the correlation between tbx4 and pelvic fin evolution. However, we observed complete exon structures of tbx4 in several pelvic-fin-loss species such as Ocean sunfish (Mola mola) and ricefield eel (Monopterus albus). More interestingly, an inversion of a special tbx4 gene cluster (brip1-tbx4-tbx2b- bcas3) occurred twice independently, which coincides with the presence of fin spines. A nonsynonymous mutation (M82L) was identified in the nuclear localization sequence (NLS) of the Japanese eel tbx4. We also examined variation and loss of hindlimb enhancer B (HLEB), which may account for pelvic fin loss in Tetraodontidae and Diodontidae. In summary, we generated a genome assembly of the Japanese eel, which provides a valuable genomic resource to study the evolution of fish tbx4 and helps elucidate the mechanism of pelvic fin loss in teleost fishes. Our comparative genomic studies, revealed for the first time a potential correlation between the tbx4 gene cluster and the evolutionary development of toxic fin spines. Because fin spines in teleosts are usually venoms, this tbx4 gene cluster may facilitate the genetic engineering of toxin-related marine drugs.

The First Transcriptome Assembly of Yenyuan Stream Salamander (Batrachuperus yenyuanensis) Provides Novel Insights into Its Molecular Evolution.

The Yenyuan stream salamander (Batrachuperus yenyuanensis) has been previously evaluated with regards to phylogeny, population genetics, and hematology, but genomic information is sparse due to the giant genome size of salamanders which contain highly repetitive sequences, thus resulting in the lack of a complete reference genome. This study evaluates the encoding genetic sequences and provides the first transcriptome assembly of Yenyuan stream salamander based on mixed samples from the liver, spermary, muscle and spleen tissues. Using this transcriptome assembly and available encoding sequences from other vertebrates, the gene families, phylogenetic status, and species divergence time were compared or estimated. A total of 13,750 encoding sequences were successfully obtained from the transcriptome assembly of Yenyuan stream salamander, estimated to contain 40.1% of the unigenes represented in tetrapod databases. A total of 88.79% of these genes could be annotated to a biological function by current databases. Through gene family clustering, we found multiple possible isoforms of the Scribble gene-whose function is related to regeneration-based on sequence similarity. Meanwhile, we constructed a robust phylogenetic tree based on 56 single-copy orthologues, which indicates that based on phylogenetic position, the Yenyuan stream salamander presents the closest relationship with the Chinese giant salamander (Andrias davidianus) of the investigated vertebrates. Based on the fossil-calibrated phylogeny, we estimated that the lineage divergence between the ancestral Yenyuan stream salamander and the Chinese giant salamander may have occurred during the Cretaceous period (~78.4 million years ago). In conclusion, this study not only provides a candidate gene that is valuable for exploring the remarkable capacity of regeneration in the future, but also gives an interesting insight into the understanding of Yenyuan stream salamander by this first transcriptome assembly.

A comprehensive phylogeny of the genus Kurixalus (Rhacophoridae, Anura) sheds light on the geographical range evolution of frilled swamp treefrogs.

Currently, the genus Kurixalus comprises 14 species distributed in Southern, Southeast and East Asia. Because of their relatively low dispersal capability and intolerance of seawater, this group is ideal for the study of terrestrial range evolution, especially that portion of its range that extends into the island archipelagos of Southern Asia. We assembled a large dataset of mitochondrial and nuclear genes, and estimated phylogeny by maximum likelihood and Bayesian methods, and we explored the history of each species via divergence-time estimation based on fossil-calibrations. A variety of ancestral-area reconstruction strategies were employed to estimate past changes of the species' geographical range, and to evaluate the impact of different abiotic barriers on range evolution. We found that frilled swamp treefrogs probably originated in Taiwan or South Vietnam in the Oligocene. Alternatively, the lineage leading to Kurixalus appendiculatus strongly supports a hypothesis of terrestrial connection between the Indian and Asian continents in the Oligocene. The outcome of both our divergence-time estimates and ancestral-area reconstruction suggests that the divergence between species from Indochina and Taiwan can probably be attributed to the opening of the South China Sea, approximately 33 million years ago. We could not find evidence for dispersal between mainland China and Taiwan Island. Formation of both Mekong and Red River valleys did not have any impact on Kurixalus species diversification. However, coincidence in timing of climate change and availability of plausible dispersal routes from the Oligocene to the middle Miocene, plausibly implied that Kurixalus diversification in Asia resulted from contemporaneous, climate-induced environmental upheaval (Late Oligocene Warming at 29 Ma; Mi-1 glaciation since 24.4-21.5 Ma; Mid-Miocene Climatic Optimum at 14 Ma), which alternatively opened and closed dispersal routes.

High Throughput Identification of Antihypertensive Peptides from Fish Proteome Datasets.

Antihypertensive peptides (AHTPs) are a group of small peptides with the main role to block key enzymes or receptors in the angiotensin genesis pathway. A great number of AHTPs have been isolated or digested from natural food resources; however, comprehensive studies on comparisons of AHTPs in various species from the perspective of big data are rare. Here, we established a simplified local AHTP database, and performed in situ mapping for high throughput identification of AHTPs with high antihypertensive activity from high-quality whole proteome datasets of 18 fish species. In the 35 identified AHTPs with reported high activity, we observed that Gly-Leu-Pro, Leu-Pro-Gly, and Val-Ser-Val are the major components of fish proteins, and AHTP hit numbers in various species demonstrated a similar distributing pattern. Interestingly, Atlantic salmon (Salmo salar) is in possession of far more abundant AHTPs compared with other fish species. In addition, collagen subunit protein is the largest group with more matching AHTPs. Further exploration of two collagen subunits (col4a5 and col8a1) in more fish species suggested that the hit pattern of these conserved proteins among teleost is almost the same, and their phylogeny is consistent with the evolution of these fish species. In summary, our present study provides basic information for the relationship of AHTPs with fish proteins, which sheds light on rapid discovery of marine drugs or food additives from fish protein hydrolysates to alleviate hypertension.

A Genomic Survey of Angiotensin-Converting Enzymes Provides Novel Insights into Their Molecular Evolution in Vertebrates.

Angiotensin-converting enzymes, ACE and ACE2, are two main elements in the renin⁻angiotensin system, with a crucial role in the regulation of blood pressure in vertebrates. Previous studies paid much attention to their physiological functions in model organisms, whereas the studies on other animals and related evolution have been sparse. Our present study performed a comprehensive genomic investigation on ace and ace2 genes in vertebrates. We successfully extracted the nucleotide sequences of ace and ace2 genes from high-quality genome assemblies of 36 representative vertebrates. After construction of their evolutionary tree, we observed that most of the phylogenetic positions are consistent with the species tree; however, certain differences appear in coelacanths and frogs, which may suggest a very slow evolutionary rate in the initial evolution of ace and ace2 in vertebrates. We further compared evolutionary rates within the entire sequences of ace and ace2, and determined that ace2 evolved slightly faster than ace. Meanwhile, we counted that the exon numbers of ace and ace2 in vertebrates are usually 25 and 18 respectively, while certain species may occur exon fusion or disruption to decrease or increase their exon numbers. Interestingly, we found three homologous regions between ace and ace2, suggesting existence of gene duplication during their evolutionary process. In summary, this report provides novel insights into vertebrate ace and ace2 genes through a series of genomic and molecular comparisons.

A Comprehensive Genome Survey Provides Novel Insights into Bile Salt Hydrolase (BSH) in Lactobacillaceae.

Bile salt hydrolase (BSH) is a well-known enzyme that has been commonly characterized in probiotic bacteria, as it has cholesterol-lowering effects. However, its molecular investigations are scarce. Here, we build a local database of BSH sequences from Lactobacillaceae (BSH⁻SDL), and phylogenetic analysis and homology searches were employed to elucidate their comparability and distinctiveness among species. Evolutionary study demonstrates that BSH sequences in BSH⁻SDL are divided into five groups, named BSH A, B, C, D and E here, which can be the genetic basis for BSH classification and nomenclature. Sequence analysis suggests the differences between BSH-active and BSH-inactive proteins clearly, especially on site 82. In addition, a total of 551 BSHs from 107 species are identified from 451 genomes of 158 Lactobacillaceae species. Interestingly, those bacteria carrying various copies of BSH A or B can be predicted to be potential cholesterol-lowering probiotics, based on the results of phylogenetic analysis and the subtypes that those previously reported BSH-active probiotics possess. In summary, this study elaborates the molecular basis of BSH in Lactobacillaceae systematically, and provides a novel methodology as well as a consistent standard for the identification of the BSH subtype. We believe that high-throughput screening can be efficiently applied to the selection of promising candidate BSH-active probiotics, which will advance the development of healthcare products in cholesterol metabolism.