Journal Articles
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Item Isolation and characterization of Methanosphaera sp. ISO3-F5, a member of a novel and widespread species of rumen methanogens growing with methanol plus hydrogen(The Microbes, 2024-12-03) Jeyanathan J; Palevich N; Reilly K; Palevich FP; Maclean PH; Li D; Altermann E; Kim CC; van Scheepstal IM; Hoskin SO; Kelly WJ; Leahy SC; Attwood GT; Ronimus RS; Henderson G; Janssen PHRumen methanogens predominantly fall into two physiological groups: hydrogenotrophs which use hydrogen (H2) to reduce carbon dioxide (CO2) to methane (CH4), and methylotrophs which use H2 to reduce methanol and methylamines as substrates for methanogenesis. We used a dilution to extinction approach to isolate two hydrogenotrophic Methanocatella spp. and four cultures of methylotrophic methanogens from sheep rumen contents. Three of the methylotrophs were stable mixed cultures containing methanogens belonging to different lineages of the order Methanomassiliicoccales and one was a pure Methanosphaera culture. Methanosphaera sp. ISO3-F5 has a comparatively large genome (2.68 Mb) comprised of two replicons, a chromosome and a megaplasmid. The genome has an average G + C content of 30.5 % and encodes 2360 putative protein-coding genes. Cells of ISO3-F5 have a spherical shape, 0.6–1.2 µm in diameter, usually occurring in pairs or loose clumps, and have no flagellum. Cells stain Gram positive, have a single thick cell wall and divide by the formation of a cross wall. The optimum temperature for growth was 39°C to 42°C and the optimum pH was 6.7–6.8. Acetate was required for growth, but CH4 was not produced from acetate, formate, ethanol, methylamine, or isopropanol with or without H2/CO2. Volatile fatty acids and rumen fluid were also found to enhance the growth of ISO3-F5, while coenzyme M did not. ISO3-F5 produced CH4 from methanol in the presence of H2 and the genes encoding the necessary methanogenesis pathway have been identified. Based on morphological, physiological, and genomic characteristics, ISO3-F5 is a new species of the genus Methanosphaera. Our study shows that simple isolation methods allowed us to culture diverse and significant members of the rumen methanogen community.Item Genome Evolution and Introgression in the New Zealand mud Snails Potamopyrgus estuarinus and Potamopyrgus kaitunuparaoa(Oxford University Press on behalf of Society for Molecular Biology and Evolution, 2024-05-22) Fields PD; Jalinsky JR; Bankers L; McElroy KE; Sharbrough J; Higgins C; Morgan-Richards M; Boore JL; Neiman M; Logsdon JMWe have sequenced, assembled, and analyzed the nuclear and mitochondrial genomes and transcriptomes of Potamopyrgus estuarinus and Potamopyrgus kaitunuparaoa, two prosobranch snail species native to New Zealand that together span the continuum from estuary to freshwater. These two species are the closest known relatives of the freshwater species Potamopyrgus antipodarum-a model for studying the evolution of sex, host-parasite coevolution, and biological invasiveness-and thus provide key evolutionary context for understanding its unusual biology. The P. estuarinus and P. kaitunuparaoa genomes are very similar in size and overall gene content. Comparative analyses of genome content indicate that these two species harbor a near-identical set of genes involved in meiosis and sperm functions, including seven genes with meiosis-specific functions. These results are consistent with obligate sexual reproduction in these two species and provide a framework for future analyses of P. antipodarum-a species comprising both obligately sexual and obligately asexual lineages, each separately derived from a sexual ancestor. Genome-wide multigene phylogenetic analyses indicate that P. kaitunuparaoa is likely the closest relative to P. antipodarum. We nevertheless show that there has been considerable introgression between P. estuarinus and P. kaitunuparaoa. That introgression does not extend to the mitochondrial genome, which appears to serve as a barrier to hybridization between P. estuarinus and P. kaitunuparaoa. Nuclear-encoded genes whose products function in joint mitochondrial-nuclear enzyme complexes exhibit similar patterns of nonintrogression, indicating that incompatibilities between the mitochondrial and the nuclear genome may have prevented more extensive gene flow between these two species.Item High-coverage genomes to elucidate the evolution of penguins(Oxford University Press and BGI, 2019-09-18) Pan H; Cole TL; Bi X; Fang M; Zhou C; Yang Z; Ksepka DT; Hart T; Bouzat JL; Argilla LS; Bertelsen MF; Boersma PD; Bost C-A; Cherel Y; Dann P; Fiddaman SR; Howard P; Labuschagne K; Mattern T; Miller G; Parker P; Phillips RA; Quillfeldt P; Ryan PG; Taylor H; Thompson DR; Young MJ; Ellegaard MR; Gilbert MTP; Sinding M-HS; Pacheco G; Shepherd LD; Tennyson AJD; Grosser S; Kay E; Nupen LJ; Ellenberg U; Houston DM; Reeve AH; Johnson K; Masello JF; Stracke T; McKinlay B; Borboroglu PG; Zhang D-X; Zhang GBACKGROUND: Penguins (Sphenisciformes) are a remarkable order of flightless wing-propelled diving seabirds distributed widely across the southern hemisphere. They share a volant common ancestor with Procellariiformes close to the Cretaceous-Paleogene boundary (66 million years ago) and subsequently lost the ability to fly but enhanced their diving capabilities. With ∼20 species among 6 genera, penguins range from the tropical Galápagos Islands to the oceanic temperate forests of New Zealand, the rocky coastlines of the sub-Antarctic islands, and the sea ice around Antarctica. To inhabit such diverse and extreme environments, penguins evolved many physiological and morphological adaptations. However, they are also highly sensitive to climate change. Therefore, penguins provide an exciting target system for understanding the evolutionary processes of speciation, adaptation, and demography. Genomic data are an emerging resource for addressing questions about such processes. RESULTS: Here we present a novel dataset of 19 high-coverage genomes that, together with 2 previously published genomes, encompass all extant penguin species. We also present a well-supported phylogeny to clarify the relationships among penguins. In contrast to recent studies, our results demonstrate that the genus Aptenodytes is basal and sister to all other extant penguin genera, providing intriguing new insights into the adaptation of penguins to Antarctica. As such, our dataset provides a novel resource for understanding the evolutionary history of penguins as a clade, as well as the fine-scale relationships of individual penguin lineages. Against this background, we introduce a major consortium of international scientists dedicated to studying these genomes. Moreover, we highlight emerging issues regarding ensuring legal and respectful indigenous consultation, particularly for genomic data originating from New Zealand Taonga species. CONCLUSIONS: We believe that our dataset and project will be important for understanding evolution, increasing cultural heritage and guiding the conservation of this iconic southern hemisphere species assemblage.Item Genome sequence of the entomopathogenic Serratia entomophila isolate 626 and characterisation of the species specific itaconate degradation pathway(BioMed Central Ltd, 2022-12) Vaughan AL; Altermann E; Glare TR; Hurst MRHBACKGROUND: Isolates of Serratia entomophila and S. proteamaculans (Yersiniaceae) cause disease specific to the endemic New Zealand pasture pest, Costelytra giveni (Coleoptera: Scarabaeidae). Previous genomic profiling has shown that S. entomophila isolates appear to have conserved genomes and, where present, conserved plasmids. In the absence of C. giveni larvae, S. entomophila prevalence reduces in the soil over time, suggesting that S. entomophila has formed a host-specific relationship with C. giveni. To help define potential genetic mechanisms driving retention of the chronic disease of S. entomophila, the genome of the isolate 626 was sequenced, enabling the identification of unique chromosomal properties, and defining the gain/loss of accessory virulence factors relevant to pathogenicity to C. giveni larvae. RESULTS: We report the complete sequence of S. entomophila isolate 626, a causal agent of amber disease in C. giveni larvae. The genome of S. entomophila 626 is 5,046,461 bp, with 59.1% G + C content and encoding 4,695 predicted CDS. Comparative analysis with five previously sequenced Serratia species, S. proteamaculans 336X, S. marcescens Db11, S. nematodiphila DH-S01, S. grimesii BXF1, and S. ficaria NBRC 102596, revealed a core of 1,165 genes shared. Further comparisons between S. entomophila 626 and S. proteamaculans 336X revealed fewer predicted phage-like regions and genomic islands in 626, suggesting less horizontally acquired genetic material. Genomic analyses revealed the presence of a four-gene itaconate operon, sharing a similar gene order as the Yersinia pestis ripABC complex. Assessment of a constructed 626::RipC mutant revealed that the operon confer a possible metabolic advantage to S. entomophila in the initial stages of C. giveni infection. CONCLUSIONS: Evidence is presented where, relative to S. proteamaculans 336X, S. entomophila 626 encodes fewer genomic islands and phages, alluding to limited horizontal gene transfer in S. entomophila. Bioassay assessments of a S. entomophila-mutant with a targeted mutation of the itaconate degradation region unique to this species, found the mutant to have a reduced capacity to replicate post challenge of the C. giveni larval host, implicating the itaconate operon in establishment within the host.Item The genomes of precision edited cloned calves show no evidence for off-target events or increased de novo mutagenesis(BioMed Central Ltd, 2021-06-17) Jivanji S; Harland C; Cole S; Brophy B; Garrick D; Snell R; Littlejohn M; Laible GBACKGROUND: Animal health and welfare are at the forefront of public concern and the agricultural sector is responding by prioritising the selection of welfare-relevant traits in their breeding schemes. In some cases, welfare-enhancing traits such as horn-status (i.e., polled) or diluted coat colour, which could enhance heat tolerance, may not segregate in breeds of primary interest, highlighting gene-editing tools such as the CRISPR-Cas9 technology as an approach to rapidly introduce variation into these populations. A major limitation preventing the acceptance of CRISPR-Cas9 mediated gene-editing, however, is the potential for off-target mutagenesis, which has raised concerns about the safety and ultimate applicability of this technology. Here, we present a clone-based study design that has allowed a detailed investigation of off-target and de novo mutagenesis in a cattle line bearing edits in the PMEL gene for diluted coat-colour. RESULTS: No off-target events were detected from high depth whole genome sequencing performed in precursor cell-lines and resultant calves cloned from those edited and non-edited cell lines. Long molecule sequencing at the edited site and plasmid-specific PCRs did not reveal structural variations and/or plasmid integration events in edited samples. Furthermore, an in-depth analysis of de novo mutations across the edited and non-edited cloned calves revealed that the mutation frequency and spectra were unaffected by editing status. Cells in culture, however, appeared to have a distinct mutation signature where de novo mutations were predominantly C > A mutations, and in cloned calves they were predominantly T > G mutations, deviating from the expected excess of C > T mutations. CONCLUSIONS: We found no detectable CRISPR-Cas9 associated off-target mutations in the gene-edited cells or calves derived from the gene-edited cell line. Comparison of de novo mutation in two gene-edited calves and three non-edited control calves did not reveal a higher mutation load in any one group, gene-edited or control, beyond those anticipated from spontaneous mutagenesis. Cell culture and somatic cell nuclear transfer cloning processes contributed the major source of contrast in mutational profile between samples.Item Leveraging an existing whole-genome resequencing population data set to characterize toll-like receptor gene diversity in a threatened bird(John Wiley and Sons, Ltd, 2022-10) Magid M; Wold JR; Moraga R; Cubrinovska I; Houston DM; Gartrell BD; Steeves TE; Hohenlohe PASpecies recovery programs are increasingly using genomic data to measure neutral genetic diversity and calculate metrics like relatedness. While these measures can inform conservation management, determining the mechanisms underlying inbreeding depression requires information about functional genes associated with adaptive or maladaptive traits. Toll-like receptors (TLRs) are one family of functional genes, which play a crucial role in recognition of pathogens and activation of the immune system. Previously, these genes have been analysed using species-specific primers and PCR. Here, we leverage an existing short-read reference genome, whole-genome resequencing population data set, and bioinformatic tools to characterize TLR gene diversity in captive and wild tchūriwat'/tūturuatu/shore plover (Thinornis novaeseelandiae), a threatened bird endemic to Aotearoa New Zealand. Our results show that TLR gene diversity in tchūriwat'/tūturuatu is low, and forms two distinct captive and wild genetic clusters. The bioinformatic approach presented here has broad applicability to other threatened species with existing genomic resources in Aotearoa New Zealand and beyond.Item Mining the 99 Lives Cat Genome Sequencing Consortium database implicates genes and variants for the Ticked locus in domestic cats (Felis catus)(John Wiley and Sons Ltd on behalf of Stichting International Foundation for Animal Genetics, 2021-06) Lyons LA; Buckley RM; Harvey RJ; 99 Lives Cat Genome ConsortiumTabby patterns of fur coats are defining characteristics in wild and domestic felids. Historically, three autosomal alleles at one locus (Tabby): Abyssinian (Ta ; a.k.a. ticked), mackerel (Tm ; a.k.a. striped) and blotched (tb ; a.k.a. classic, blotched) were thought to control these patterns in domestic cats and their breeds. Currently, at least three loci influence cat tabby markings, two of which are designated Tabby and Ticked. The Tabby locus is laeverin (LVRN) and affects the mackerel and blotched patterns. The unidentified gene for the Ticked locus on cat chromosome B1 was suggested to control the presence or absence of the ticked pattern (Tabby - Abyssinian (Ta ; a.k.a. ticked). The cat reference genome (Cinnamon, the Abyssinian) has the ticked phenotype and the variant dataset and coat phenotypes from the 99 Lives Cat Genome Consortium (195 cats) were used to identify candidate genes and variants associated with the Ticked locus. Two strategies were used to find the Ticked allele(s), one considered Cinnamon with the reference allele or heterozygous (Strategy A) and the other considered Cinnamon as having the variant allele or heterozygous (Strategy B). For Strategy A, two variants in Dickkopf Wnt Signaling Pathway Inhibitor 4 (DKK4), a p.Cys63Tyr (B1:41621481, c.188G>A) and a less common p.Ala18Val (B1:42620835, c.53C>T) variant are suggested as two alleles influencing the Ticked phenotype. Bioinformatic and molecular modeling analysis suggests that these changes disrupt a key disulfide bond in the Dkk4 cysteine-rich domain 1 or Dkk4 signal peptide cleavage respectively. All coding variants were excluded as Ticked alleles using Strategy B.Item Genomic insights into the secondary aquatic transition of penguins(Springer Nature Limited, 2022-07-19) Cole TL; Zhou C; Fang M; Pan H; Ksepka DT; Fiddaman SR; Emerling CA; Thomas DB; Bi X; Fang Q; Ellegaard MR; Feng S; Smith AL; Heath TA; Tennyson AJD; Borboroglu PG; Wood JR; Hadden PW; Grosser S; Bost C-A; Cherel Y; Mattern T; Hart T; Sinding M-HS; Shepherd LD; Phillips RA; Quillfeldt P; Masello JF; Bouzat JL; Ryan PG; Thompson DR; Ellenberg U; Dann P; Miller G; Dee Boersma P; Zhao R; Gilbert MTP; Yang H; Zhang D-X; Zhang GPenguins lost the ability to fly more than 60 million years ago, subsequently evolving a hyper-specialized marine body plan. Within the framework of a genome-scale, fossil-inclusive phylogeny, we identify key geological events that shaped penguin diversification and genomic signatures consistent with widespread refugia/recolonization during major climate oscillations. We further identify a suite of genes potentially underpinning adaptations related to thermoregulation, oxygenation, diving, vision, diet, immunity and body size, which might have facilitated their remarkable secondary transition to an aquatic ecology. Our analyses indicate that penguins and their sister group (Procellariiformes) have the lowest evolutionary rates yet detected in birds. Together, these findings help improve our understanding of how penguins have transitioned to the marine environment, successfully colonizing some of the most extreme environments on Earth.Item Whole-genome sequencing and ad hoc shared genome analysis of Staphylococcus aureus isolates from a New Zealand primary school(Springer Nature Limited, 2021-10-13) Scott P; Zhang J; Anderson T; Priest PC; Chambers S; Smith H; Murdoch DR; French N; Biggs PJEpidemiological studies of communicable diseases increasingly use large whole-genome sequencing (WGS) datasets to explore the transmission of pathogens. It is important to obtain an initial overview of datasets and identify closely related isolates, but this can be challenging with large numbers of isolates and imperfect sequencing. We used an ad hoc whole-genome multi locus sequence typing method to summarise data from a longitudinal study of Staphylococcus aureus in a primary school in New Zealand. Each pair of isolates was compared and the number of genes where alleles differed between isolates was tallied to produce a matrix of "allelic differences". We plotted histograms of the number of allelic differences between isolates for: all isolate pairs; pairs of isolates from different individuals; and pairs of isolates from the same individual. 340 sequenced isolates were included, and the ad hoc shared genome contained 445 genes. There were between 0 and 420 allelic differences between isolate pairs and the majority of pairs had more than 260 allelic differences. We found many genetically closely related S. aureus isolates from single individuals and a smaller number of closely-related isolates from separate individuals. Multiple S. aureus isolates from the same individual were usually very closely related or identical over the ad hoc shared genome. Siblings carried genetically similar, but not identical isolates. An ad hoc shared genome approach to WGS analysis can accommodate imperfect sequencing of the included isolates, and can provide insights into relationships between isolates in epidemiological studies with large WGS datasets containing diverse isolates.Item Genomic and genetic analyses of diversity and plant interactions of Pseudomonas fluorescens(BIOMED CENTRAL LTD, 2009) Silby MW; Cerdeno-Tarraga AM; Vernikos GS; Giddens SR; Jackson RW; Preston GM; Zhang XX; Moon CD; Gehrig SM; Godfrey SAC; Knight CG; Malone JG; Robinson Z; Spiers AJ; Harris S; Challis GL; Yaxley AM; Harris D; Seeger K; Murphy L; Rutter S; Squares R; Quail MA; Saunders E; Mavromatis K; Brettin TS; Bentley SD; Hothersall J; Stephens E; Thomas CM; Parkhill J; Levy SB; Rainey PB; Thomson NRBACKGROUND: Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species. RESULTS: Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome. CONCLUSIONS: P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.