Reverse engineering: transaminase biocatalystdevelopment using ancestral sequencereconstruction

The development of new biocatalysts using ancestral sequence reconstruction is reported. When applied to an ω-transaminase, the ancestral proteins demonstrated novel and superior activities with eighty percent of the forty compounds tested compared to the modern day protein, and improvements in activity of up to twenty fold. These included a range of compounds pertinent as feedstocks in polyamide manufacture.MW and SK were supported by CSIRO Research Office Postdoctoral Fellowships

Genomic epidemiology reveals transmission patterns and dynamics of SARS-CoV-2 in Aotearoa New Zealand

New Zealand, a geographically remote Pacific island with easily sealable borders, implemented a nationwide 'lockdown' of all non-essential services to curb the spread of COVID-19. Here, we generate 649 SARS-CoV-2 genome sequences from infected patients in New Zealand with samples collected during the 'first wave', representing 56% of all confirmed cases in this time period. Despite its remoteness, the viruses imported into New Zealand represented nearly all of the genomic diversity sequenced from the global virus population. These data helped to quantify the effectiveness of public health interventions. For example, the effective reproductive number, Re of New Zealand's largest cluster decreased from 7 to 0.2 within the first week of lockdown. Similarly, only 19% of virus introductions into New Zealand resulted in ongoing transmission of more than one additional case. Overall, these results demonstrate the utility of genomic pathogen surveillance to inform public health and disease mitigation

TMFoldRec: a statistical potential-based transmembrane protein fold recognition tool.

BACKGROUND: Transmembrane proteins (TMPs) are the key components of signal transduction, cell-cell adhesion and energy and material transport into and out from the cells. For the deep understanding of these processes, structure determination of transmembrane proteins is indispensable. However, due to technical difficulties, only a few transmembrane protein structures have been determined experimentally. Large-scale genomic sequencing provides increasing amounts of sequence information on the proteins and whole proteomes of living organisms resulting in the challenge of bioinformatics; how the structural information should be gained from a sequence. RESULTS: Here, we present a novel method, TMFoldRec, for fold prediction of membrane segments in transmembrane proteins. TMFoldRec based on statistical potentials was tested on a benchmark set containing 124 TMP chains from the PDBTM database. Using a 10-fold jackknife method, the native folds were correctly identified in 77 % of the cases. This accuracy overcomes the state-of-the-art methods. In addition, a key feature of TMFoldRec algorithm is the ability to estimate the reliability of the prediction and to decide with an accuracy of 70 %, whether the obtained, lowest energy structure is the native one. CONCLUSION: These results imply that the membrane embedded parts of TMPs dictate the TM structures rather than the soluble parts. Moreover, predictions with reliability scores make in this way our algorithm applicable for proteome-wide analyses. AVAILABILITY: The program is available upon request for academic use

Whole-genome resequencing of Cucurbita pepo morphotypes to discover genomic variants associated with morphology and horticulturally valuable traits

[EN] Cucurbita pepo contains two cultivated subspecies, each of which encompasses four fruit-shape morphotypes (cultivar groups). The Pumpkin, Vegetable Marrow, Cocozelle, and Zucchini Groups are of subsp. pepo and the Acorn, Crookneck, Scallop, and Straightneck Groups are of subsp. ovifera. Recently, a de novo assembly of the C. pepo subsp. pepo Zucchini genome was published, providing insights into its evolution. To expand our knowledge of evolutionary processes within C. pepo and to identify variants associated with particular morphotypes, we performed whole-genome resequencing of seven of these eight C. pepo morphotypes. We report for the first time whole-genome resequencing of the four subsp. pepo (Pumpkin, Vegetable Marrow, Cocozelle, green Zucchini, and yellow Zucchini) morphotypes and three of the subsp. ovifera (Acorn, Crookneck, and Scallop) morphotypes. A high-depth resequencing approach was followed, using the BGISEQ-500 platform that enables the identification of rare variants, with an average of 33.5X. Approximately 94.5% of the clean reads were mapped against the reference Zucchini genome. In total, 3,823,977 high confidence single-nucleotide polymorphisms (SNPs) were identified. Within each accession, SNPs varied from 636,918 in green Zucchini to 2,656,513 in Crookneck, and were distributed homogeneously along the chromosomes. Clear differences between subspecies pepo and ovifera in genetic variation and linkage disequilibrium are highlighted. In fact, comparison between subspecies pepo and ovifera indicated 5710 genes (22.5%) with Fst > 0.80 and 1059 genes (4.1%) with Fst = 1.00 as potential candidate genes that were fixed during the independent evolution and domestication of the two subspecies. Linkage disequilibrium was greater in subsp. ovifera than in subsp. pepo, perhaps reflective of the earlier differentiation of morphotypes within subsp. ovifera. Some morphotype-specific genes have been localized. Our results offer new clues that may provide an improved understanding of the underlying genomic regions involved in the independent evolution and domestication of the two subspecies. Comparisons among SNPs unique to particular subspecies or morphotypes may provide candidate genes responsible for traits of high economic importance.This work has been supported by Hellenic Agricultural Organization (ELGO) Demeter. Furthermore, we thank the Conselleria de Educacio, Investigacio, Cultura i Esport (Generalitat Valenciana) for funding Project Prometeo 2017/078 "Seleccion de Variedades Tradicionales y Desarrollo de Nuevas Variedades de Cucurbitaceas Adaptadas a la Produccion Ecologica". Also, this work was supported by Chiang Mai University.Xanthopoulou, A.; Montero-Pau, J.; Mellidou, I.; Kissoudis, C.; Blanca Postigo, JM.; Picó Sirvent, MB.; Tsaballa, A.... (2019). Whole-genome resequencing of Cucurbita pepo morphotypes to discover genomic variants associated with morphology and horticulturally valuable traits. Horticulture Research. 6:1-17. https://doi.org/10.1038/s41438-019-0176-9S1176Maynard, D. & Paris, H. in The Encyclopedia of Fruits & Nuts (eds Paull, R. E. & Janick, J.) 276–313 (CABI, New Jersey, U.S.A., 2018).Paris, H. S. in Genetics and Genomics of Cucurbitaceae, Grumet, Rebecca, Katzir, Nurit, Garcia-Mas, Jordi (Eds.) 111–154 (Springer, New York, U.S.A., 2016).Whitaker, T. W. & Davis, G. N. Cucurbits (Leonard Hill (Books) Ltd., London, and Interscience Publishers Inc., New York, 1962).Paris, H. S. History of the cultivar-groups of Cucurbita pepo. Hortic. Rev. 25, 71–170 (2001).Paris, H. S. A proposed subspecific classifiaction for Cucurbita pepo. Phytologia (USA) 61, 133–138 (1986).Lira, R., Andres, T. C. & Nee, M. in Systematic and Ecogeographic Studies on Crop Genepools, Vol. 9, 1–115 (International Plant Genetic Resources Institute, Roma, Italia, 1995).Castellanos-Morales, G. Historical biogeography and phylogeny of Cucurbita: insights from ancestral area reconstruction and niche evolution. Mol. Phylogenet. Evol. 128, 38–54 (2018).Paris, H. S., Lebeda, A., Křistkova, E., Andres, T. C. & Nee, M. H. Parallel evolution under domestication and phenotypic differentiation of the cultivated subspecies of Cucurbita pepo (Cucurbitaceae). Econ. Bot. 66, 71–90 (2012).Dong, W., Wu, D., Li, G., Wu, D. & Wang, Z. Next-generation sequencing from bulked segregant analysis identifies a dwarfism gene in watermelon. Sci. Rep. 8, 2908 (2018).Galpaz, N. et al. Deciphering genetic factors that determine melon fruit‐quality traits using RNA‐Seq‐based high‐resolution QTL and eQTL mapping. Plant J. 94, 169–191 (2018).Gur, A. et al. Genome-wide linkage-disequilibrium mapping to the candidate gene level in melon (Cucumis melo). Sci. Rep. 7, 9770 (2017).Blanca, J. et al. Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae). BMC Genom. 12, 104 (2011).Esteras, C. et al. High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping. BMC Genom. 13, 80 (2012).Montero-Pau, J. et al. An SNP-based saturated genetic map and QTL analysis of fruit-related traits in Zucchini using genotyping-by-sequencing. BMC Genom. 18, 94 (2017).Vicente-Dólera, N. et al. First TILLING platform in Cucurbita pepo: a new mutant resource for gene function and crop improvement. PLoS ONE 9, e112743 (2014).Wyatt, L. E., Strickler, S. R., Mueller, L. A. & Mazourek, M. An acorn squash (Cucurbita pepo ssp. ovifera) fruit and seed transcriptome as a resource for the study of fruit traits in Cucurbita. Hortic. Res. 2, 14070 (2015).Xanthopoulou, A. et al. De novo comparative transcriptome analysis of genes involved in fruit morphology of pumpkin cultivars with extreme size difference and development of EST-SSR markers. Gene 622, 50–66 (2017).Montero‐Pau, J. et al. De novo assembly of the zucchini genome reveals a whole‐genome duplication associated with the origin of the Cucurbita genus. Plant Biotechnol. J. 16, 1161–1171 (2018).Garcia-Mas, J. et al. Cloning and mapping of resistance gene homologues in melon. Plant Sci. 161, 165–172 (2001).Xanthopoulou, A. et al. Comparative analysis of genetic diversity in Greek Genebank collection of summer squash (‘Cucurbita pepo’) landraces using start codon targeted (SCoT) polymorphism and ISSR markers. Aust. J. Crop Sci. 9, 14 (2015).Huang, J. et al. A reference human genome dataset of the BGISEQ-500 sequencer. Gigascience 6, gix024 (2017).Natarajan, K. N. et al. Comparative analysis of sequencing technologies for single-cell transcriptomics. Genome Biol. 20, 70 (2019).Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).Tian, L. et al. Transcript and proteomic analysis of developing white lupin (Lupinus albus L.) roots. BMC Plant Biol. 9, 1 (2009).Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).Bradbury, P. J. et al. TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23, 2633–2635 (2007).Chang, C. C. et al. Second-generation PLINK: rising to the challenge of larger and richer datasets. Gigascience 4, 7 (2015).Team, R. C. (2015). http://www.r-project.org/ .Krzywinski, M. I. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).Kosman, E. & Leonard, K. J. Similarity coefficients for molecular markers in studies of genetic relationships between individuals for haploid, diploid, and polyploid species. Mol. Ecol. 14, 415–424 (2005).Huson, D. H. & Bryant, D. Estimating Phylogenetic Trees and Networks Using SplitsTree 4. www.splitstree.org (2005).Danecek, P. et al. The variant call format and VCFtools. Bioinformatics 27, 2156–2158 (2011).Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strainw1118; iso-2; iso-3. Fly 6, 80–92 (2012).Wu, S. et al. A common genetic mechanism underlies morphological diversity in fruits and other plant organs. Nat. Commun. 9, 4734 (2018).Drevensek, S. et al. The Arabidopsis TRM1–TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes. Plant Cell 24, 178–191 (2012).Sievers, F. et al. Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2014).Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587 (2017).Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q. & Vinh, L. S. UFBoot2: improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518–522 (2017).Bailey, T. L. et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202–W208 (2009).Leida, C. et al. Variability of candidate genes, genetic structure and association with sugar accumulation and climacteric behavior in a broad germplasm collection of melon (Cucumis melo L.). BMC Genet. 16, 28 (2015).Esteras, C. et al. SNP genotyping in melons: genetic variation, population structure, and linkage disequilibrium. Theor. Appl. Genet. 126, 1285–1303 (2013).Maria José Gonzalo et al. Re-evaluation of the role of Indian germplasm as center of melon diversification based on genotyping-by-sequencing analysis. BMC Genom. 20, p. 448 (2019).Nimmakayala, P. et al. Single nucleotide polymorphisms generated by genotyping by sequencing to characterize genome-wide diversity, linkage disequilibrium, and selective sweeps in cultivated watermelon. BMC Genom. 15, 767 (2014).Gonzalo, M. J. & Monforte, A. J. in Genetics and Genomics of Cucurbitaceae, Grumet, Rebecca, Katzir, Nurit, Garcia-Mas, Jordi (Eds.) 269–290 (Springer, New York, U.S.A., 2016).Pomares-Viciana, T. et al. First RNA-seq approach to study fruit set and parthenocarpy in zucchini (Cucurbita pepo L.). BMC Plant Biol. 19, 61 (2019).Lu, S. et al. The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of β-carotene accumulation. Plant Cell 18, 3594–3605 (2006).Jin, B., Kim, J., Jung, J., Kim, D. & Park, Y. Characterization of IQ domain gene homologs as common candidate genes for elongated fruit shape in cucurbits. Hortic. Sci. Technol. 36, 85–97 (2018).van der Knaap, E. et al. What lies beyond the eye: the molecular mechanisms regulating tomato fruit weight and shape. Front. Plant Sci. 5, 227 (2014).Xiao, H., Jiang, N., Schaffner, E., Stockinger, E. J. & Van Der Knaap, E. A retrotransposon-mediated gene duplication underlies morphological variation of tomato fruit. Science 319, 1527–1530 (2008).Dou, J. et al. Genetic mapping reveals a candidate gene (ClFS1) for fruit shape in watermelon (Citrullus lanatus L.). Theor. Appl. Genet. 131, 947–958 (2018).Pan, Y. et al. Round fruit shape in WI7239 cucumber is controlled by two interacting quantitative trait loci with one putatively encoding a tomato SUN homolog. Theor. Appl. Genet. 130, 573–586 (2017).Liu, J. et al. Banana Ovate family protein MaOFP1 and MADS-box protein MuMADS1 antagonistically regulated banana fruit ripening. PLoS ONE 10, e0123870 (2015).Liu, J. et al. Mu MADS 1 and Ma OFP 1 regulate fruit quality in a tomato ovate mutant. Plant Biotechnol. J. 16, 989–1001 (2018).Cong, B., Barrero, L. S. & Tanksley, S. D. Regulatory change in YABBY-like transcription factor led to evolution of extreme fruit size during tomato domestication. Nat. Genet. 40, 800 (2008).Huang, Z., Van Houten, J., Gonzalez, G., Xiao, H. & van der Knaap, E. Genome-wide identification, phylogeny and expression analysis of SUN, OFP and YABBY gene family in tomato. Mol. Genet. Genom. 288, 111–129 (2013).Bowman, J. L. The YABBY gene family and abaxial cell fate. Curr. Opin. Plant Biol. 3, 17–22 (2000).Liu, J., Van Eck, J., Cong, B. & Tanksley, S. D. A new class of regulatory genes underlying the cause of pear-shaped tomato fruit. Proc. Natl Acad. Sci. USA 99, 13302–13306 (2002).Tsaballa, A., Pasentsis, K., Darzentas, N. & Tsaftaris, A. S. Multiple evidence for the role of an Ovate-like gene in determining fruit shape in pepper. BMC Plant Biol. 11, 46 (2011).Wang, S., Chang, Y., Guo, J. & Chen, J. G. Arabidopsis Ovate family protein 1 is a transcriptional repressor that suppresses cell elongation. Plant J. 50, 858–872 (2007).Lazzaro, M. D., Wu, S., Snouffer, A., Wang, Y. & Van Der Knaap, E. Plant organ shapes are regulated by protein interactions and associations with microtubules. Front. Plant Sci. 9, 1766 (2018)

An integrative approach to discovering cryptic species within the Bemisia tabaci whitefly species complex

Bemisia tabaci is a cryptic whitefly-species complex that includes some of the most damaging pests and plant-virus vectors of a diverse range of food and fibre crops worldwide. We combine experimental evidence of: (i) differences in reproductive compatibility, (ii) hybrid verification using a specific nuclear DNA marker and hybrid fertility confirmation and (iii) high-throughput sequencing-derived mitogenomes, to show that the “Mediterranean” (MED) B. tabaci comprises at least two distinct biological species; the globally invasive MED from the Mediterranean Basin and the “African silver-leafing” (ASL) from sub-Saharan Africa, which has no associated invasion records. We demonstrate that, contrary to its common name, the “ASL” does not induce squash silver-leafing symptoms and show that species delimitation based on the widely applied 3.5% partial mtCOI gene sequence divergence threshold produces discordant results, depending on the mtCOI region selected. Of the 292 published mtCOI sequences from MED/ASL groups, 158 (54%) are low quality and/or potential pseudogenes. We demonstrate fundamental deficiencies in delimiting cryptic B. tabaci species, based solely on partial sequences of a mitochondrial barcoding gene. We advocate an integrative approach to reveal the true species richness within cryptic species complexes, which is integral to the deployment of effective pest and disease management strategies

Phylogenetic relationships in southern African Bryde's whales inferred from mitochondrial DNA : further support for subspecies delineation between the two allopatric populations

Bryde’s whales (Balaenoptera edeni) are medium-sized balaenopterids with tropical and subtropical distribution. There is confusion about the number of species, subspecies and populations of Bryde’s whale found globally. Two eco-types occur off South Africa, the inshore and offshore forms, but with unknown relationship between them. Using the mtDNA control region we investigated the phylogenetic relationship of these populations to each other and other Bryde’s whale populations. Skin, baleen and bone samples were collected from biopsy-sampled individuals, strandings and museum collections. 97 sequences of 674 bp (bp) length were compared with published sequences of Bryde’s whales (n = 6) and two similar species, Omura’s (B. omurai) and sei (B. borealis) whales (n = 3). We found eight haplotypes from the study samples: H1–H4 formed a distinct, sister clade to pelagic populations of Bryde’s whales (B. brydei) from the South Pacific, North Pacific and Eastern Indian Ocean. H5–H8 were included in the pelagic clade. H1–H4 represented samples from within the distributional range of the inshore form. Pairwise comparisons of the percentage of nucleotide differences between sequences revealed that inshore haplotypes differed from published sequences of B. edeni by 4.7–5.5% and from B. brydei by 1.8–2.1%. Ten fixed differences between inshore and offshore sequences supported 100% diagnosability as subspecies. Phylogenetic analyses grouped the South African populations within the Bryde’s-sei whale clade and excluded B. edeni. Our data, combined with morphological and ecological evidence from previous studies, support subspecific classification of both South African forms under B. brydei and complete separation from B. edeni.PostprintPeer reviewe

Genomic epidemiology of syphilis reveals independent emergence of macrolide resistance across multiple circulating lineages.

Syphilis is a sexually transmitted infection caused by Treponema pallidum subspecies pallidum and may lead to severe complications. Recent years have seen striking increases in syphilis in many countries. Previous analyses have suggested one lineage of syphilis, SS14, may have expanded recently, indicating emergence of a single pandemic azithromycin-resistant cluster. Here we use direct sequencing of T. pallidum combined with phylogenomic analyses to show that both SS14- and Nichols-lineages are simultaneously circulating in clinically relevant populations in multiple countries. We correlate the appearance of genotypic macrolide resistance with multiple independently evolved SS14 sub-lineages and show that genotypically resistant and sensitive sub-lineages are spreading contemporaneously. These findings inform our understanding of the current syphilis epidemic by demonstrating how macrolide resistance evolves in Treponema subspecies and provide a warning on broader issues of antimicrobial resistance

Uncoupled activation and cyclization in catmint reductive terpenoid biosynthesis

Terpene synthases typically form complex molecular scaffolds by concerted activation and cyclization of linear starting materials in a single enzyme active site. Here we show that iridoid synthase, an atypical reductive terpene synthase, catalyzes the activation of its substrate 8-oxogeranial into a reactive enol intermediate, but does not catalyze the subsequent cyclization into nepetalactol. This discovery led us to identify a class of nepetalactol-related short-chain dehydrogenase enzymes (NEPS) from catmint (Nepeta mussinii) that capture this reactive intermediate and catalyze the stereoselective cyclisation into distinct nepetalactol stereoisomers. Subsequent oxidation of nepetalactols by NEPS1 provides nepetalactones, metabolites that are well known for both insect-repellent activity and euphoric effect in cats. Structural characterization of the NEPS3 cyclase reveals that it binds to NAD+ yet does not utilize it chemically for a non-oxidoreductive formal [4 + 2] cyclization. These discoveries will complement metabolic reconstructions of iridoid and monoterpene indole alkaloid biosynthesis