The genome of the Wollemi pine, a critically endangered "living fossil" unchanged since the Cretaceous, reveals extensive ancient transposon activity.

We present the genome of the living fossil, Wollemia nobilis, a southern hemisphere conifer morphologically unchanged since the Cretaceous. Presumed extinct until rediscovery in 1994, the Wollemi pine is critically endangered with less than 60 wild adults threatened by intensifying bushfires in the Blue Mountains of Australia. The 12 Gb genome is among the most contiguous large plant genomes assembled, with extremely low heterozygosity and unusual abundance of DNA transposons. Reduced representation and genome re-sequencing of individuals confirms a relictual population since the last major glacial/drying period in Australia, 120 ky BP. Small RNA and methylome sequencing reveal conservation of ancient silencing mechanisms despite the presence of thousands of active and abundant transposons, including some transferred horizontally to conifers from arthropods in the Jurassic. A retrotransposon burst 8-6 my BP coincided with population decline, possibly as an adaptation enhancing epigenetic diversity. Wollemia, like other conifers, is susceptible to Phytophthora, and a suite of defense genes, similar to those in loblolly pine, are targeted for silencing by sRNAs in leaves. The genome provides insight into the earliest seed plants, while enabling conservation efforts.

Differences in the gut microbiome across typical ageing and in Parkinson's disease.

The microbiota-gut-brain axis' role in Parkinson's disease (PD) pathophysiology, and how this differs from typical ageing, is poorly understood. Presently, gut-bacterial diversity, taxonomic abundance and metabolic bacterial pathways were compared across healthy young (n = 22, 18-35 years), healthy older (n = 33, 50-80 years), and PD groups (n = 18, 50-80 years) using shotgun sequencing and compositional data analysis. Associations between the gut-microbiome and PD symptoms, and between lifestyle factors (fibre intake, physical activity, and sleep) and the gut-microbiome were conducted. Alpha-diversity did not differ between PD participants and older adults, whilst beta-diversity differed between these groups. Lower abundance of Butyricimonas synergistica, a butyrate-producer, was associated with worse PD non-motor symptoms in the PD group. Regarding typical ageing, Bifidobacterium bifidum, was greater in the younger compared to older group, with no difference between the older and PD group. Abundance of metabolic pathways related to butyrate production did not differ among the groups, while other metabolic pathways differed among the three groups. Sleep efficiency was positively associated with Roseburia inulinivorans in the older group. These results highlight the relevance of gut-microbiota to PD and that reduced butyrate-production may be involved with PD pathophysiology. Future studies should account for lifestyle factors when investigating gut-microbiomes across ageing and in PD. This article is part of the Special Issue on "Microbiome & the Brain: Mechanisms & Maladies".

Phenotypic Characterization and Comparative Genomic Analysis of Novel Salmonella Bacteriophages Isolated from a Tropical Rainforest.

Salmonella infections across the globe are becoming more challenging to control due to the emergence of multidrug-resistant (MDR) strains. Lytic phages may be suitable alternatives for treating these multidrug-resistant Salmonella infections. Most Salmonella phages to date were collected from human-impacted environments. To further explore the Salmonella phage space, and to potentially identify phages with novel characteristics, we characterized Salmonella-specific phages isolated from the Penang National Park, a conserved rainforest. Four phages with a broad lytic spectrum (kills >5 Salmonella serovars) were further characterized; they have isometric heads and cone-shaped tails, and genomes of ~39,900 bp, encoding 49 CDSs. As the genomes share a <95% sequence similarity to known genomes, the phages were classified as a new species within the genus Kayfunavirus. Interestingly, the phages displayed obvious differences in their lytic spectrum and pH stability, despite having a high sequence similarity (~99% ANI). Subsequent analysis revealed that the phages differed in the nucleotide sequence in the tail spike proteins, tail tubular proteins, and portal proteins, suggesting that the SNPs were responsible for their differing phenotypes. Our findings highlight the diversity of novel Salmonella bacteriophages from rainforest regions, which can be explored as an antimicrobial agent against MDR-Salmonella strains.

Complete Genome Sequence of Dickeya chrysanthemi Bacteriophage DchS19.

We characterized the complete genome of a lytic Dickeya chrysanthemi bacteriophage, DchS19, which was isolated from a soil sample in Sungai Petani, Kedah, Malaysia. The phage, from the Autographviridae family, has a 39,149-bp double-stranded DNA genome containing 49 protein-coding genes and shares 94.65% average nucleotide identity with Erwinia phage pEp_SNUABM_12.

Mitogenome of the extinct Desert 'rat-kangaroo' times the adaptation to aridity in macropodoids.

The evolution of Australia's distinctive marsupial fauna has long been linked to the onset of continent-wide aridity. However, how this profound climate change event affected the diversification of extant lineages is still hotly debated. Here, we assemble a DNA sequence dataset of Macropodoidea-the clade comprising kangaroos and their relatives-that incorporates a complete mitogenome for the Desert 'rat-kangaroo', Caloprymnus campestris. This enigmatic species went extinct nearly 90 years ago and is known from a handful of museum specimens. Caloprymnus is significant because it was the only macropodoid restricted to extreme desert environments, and therefore calibrates the group's specialisation for increasingly arid conditions. Our robustly supported phylogenies nest Caloprymnus amongst the bettongs Aepyprymnus and Bettongia. Dated ancestral range estimations further reveal that the Caloprymnus-Bettongia lineage originated in nascent xeric settings during the middle to late Miocene, ~ 12 million years ago (Ma), but subsequently radiated into fragmenting mesic habitats after the Pliocene to mid-Pleistocene. This timeframe parallels the ancestral divergences of kangaroos in woodlands and forests, but predates their adaptive dispersal into proliferating dry shrublands and grasslands from the late Miocene to mid-Pleistocene, after ~ 7 Ma. We thus demonstrate that protracted changes in both climate and vegetation likely staged the emergence of modern arid zone macropodoids.

Generation and Characterization of a Zebrafish IL-2Rγc SCID Model.

The IL-2 family of cytokines act via receptor complexes that share the interleukin-2 receptor gamma common (IL-2Rγc) chain to play key roles in lymphopoiesis. Inactivating IL-2Rγc mutations results in severe combined immunodeficiency (SCID) in humans and other species. This study sought to generate an equivalent zebrafish SCID model. The zebrafish il2rga gene was targeted for genome editing using TALENs and presumed loss-of-function alleles analyzed with respect to immune cell development and impacts on intestinal microbiota and tumor immunity. Knockout of zebrafish Il-2rγc.a resulted in a SCID phenotype, including a significant reduction in T cells, with NK cells also impacted. This resulted in dysregulated intestinal microbiota and defective immunity to tumor xenotransplants. Collectively, this establishes a useful zebrafish SCID model.

Complete Genome Sequence of Salmonella enterica Bacteriophage PRF-SP1.

We characterized the complete genome sequence of the lytic Salmonella enterica bacteriophage PRF-SP1, isolated from Penang National Park, a conserved rainforest in northern Malaysia. The novel phage species from the Autographiviridae family has a 39,966-bp double-stranded DNA (dsDNA) genome containing 49 protein-encoding genes and shares 90.96% similarity with Escherichia phage DY1.

First Genome of Labyrinthula sp., an Opportunistic Seagrass Pathogen, Reveals Novel Insight into Marine Protist Phylogeny, Ecology and CAZyme Cell-Wall Degradation.

Labyrinthula spp. are saprobic, marine protists that also act as opportunistic pathogens and are the causative agents of seagrass wasting disease (SWD). Despite the threat of local- and large-scale SWD outbreaks, there are currently gaps in our understanding of the drivers of SWD, particularly surrounding Labyrinthula spp. virulence and ecology. Given these uncertainties, we investigated the Labyrinthula genus from a novel genomic perspective by presenting the first draft genome and predicted proteome of a pathogenic isolate Labyrinthula SR_Ha_C, generated from a hybrid assembly of Nanopore and Illumina sequences. Phylogenetic and cross-phyla comparisons revealed insights into the evolutionary history of Stramenopiles. Genome annotation showed evidence of glideosome-type machinery and an apicoplast protein typically found in protist pathogens and parasites. Proteins involved in Labyrinthula SR_Ha_C's actin-myosin mode of transport, as well as carbohydrate degradation were also prevalent. Further, CAZyme functional predictions revealed a repertoire of enzymes involved in breakdown of cell-wall and carbohydrate storage compounds common to seagrasses. The relatively low number of CAZymes annotated from the genome of Labyrinthula SR_Ha_C compared to other Labyrinthulea species may reflect the conservative annotation parameters, a specialized substrate affinity and the scarcity of characterized protist enzymes. Inherently, there is high probability for finding both unique and novel enzymes from Labyrinthula spp. This study provides resources for further exploration of Labyrinthula spp. ecology and evolution, and will hopefully be the catalyst for new hypothesis-driven SWD research revealing more details of molecular interactions between the Labyrinthula genus and its host substrate.

Chitinophaga extrema sp. nov., isolated from subsurface soil and leaf litter in a tropical peat swamp forest.

A Gram-negative, filamentous aerobic bacterium designated as strain Mgbs1T was isolated on 12 April 2017 from the subsurface soil and leaf litter substrate at the base of a Koompassia malaccensis tree in a tropical peat swamp forest in the northern regions of the state of Selangor, Malaysia (3° 39' 04.7' N 101° 17' 43.7'' E). Phylogenetic analyses based on the full 16S rRNA sequence revealed that strain Mgbs1T belongs to the genus Chitinophaga with the greatest sequence similarity to Chitinophaga terrae KP01T (97.65 %), Chitinophaga jiangningensis DSM27406T (97.58 %), and Chitinophaga dinghuensis DHOC24T (97.17 %). The major fatty acids of strain Mgbs1T (>10 %) are iso-C15 : 0, C16 : 1 ω5c and iso-C17 : 0 3-OH while the predominant respiratory quinone is menaquinone-7. Strain Mgbs1T has a complete genome size of 8.03 Mb, with a G+C content of 48.5 mol%. The DNA-DNA hybridization (DDH) score between strain Mgbs1T and C. jiangningensis DSM27406T was 15.9 %, while in silico DDH values of strain Mgbs1T against C. dinghuensis DHOC24T and C. terrae KP01T were 20.0 and 19.10% respectively. Concurrently, Average Nucleotide Identity (ANI) scores between strain Mgbs1T against all three reference strains are 73.2 %. Based on the phenotypic, chemotaxonomic, and phylogenetic consensus, strain Mgbs1T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga extrema sp. nov. is proposed (=DSM 108835T=JCM 33276T).

Ultrahigh-Resolution Mass Spectrometry Method for Resolving 13C-Enrichment Patterns in a Microalgal Lipidome.

The analysis of 13C-labeled lipids by mass spectrometry is challenging due to the complexity from labeling the large number of carbon atoms in lipids. To further add to the complexity, different adducts can be produced during electrospray ionization and in-source fragmentation, which can create complex overlapping isotope patterns that can only be resolved using high-resolution mass spectrometry. Co-elution of lipids even after chromatographic separation also adds to the potential for overlapping mass spectra. Here, we describe a procedure that enables full 13C-labeled patterns to be resolved in complex microalgal lipid extracts as well a procedure that provides structural labeling information. Mass resolving powers of 240000 full width half-maximum (fwhm) and fast targeted MS/MS allowed the differentiation of isotopologues, adducts, and unresolved lipid species after chromatographic separation. This enabled the percentage of 13C enrichment to be calculated for each individual lipid species over a time series in the microalgal lipidome. The application of tandem mass spectrometry (MS/MS) also allowed the degree of labeling within the headgroup vs acyl chains to be determined, further adding to the detail of information collected. This information is particularly useful for studying lipid synthesis and remodeling processes and can be extended to other biological systems.

Complete genome sequence of Sphingomonas paucimobilis AIMST S2, a xenobiotic-degrading bacterium.

Complete genomes of xenobiotic-degrading microorganisms provide valuable resources for researchers to understand molecular mechanisms involved in bioremediation. Despite the well-known ability of Sphingomonas paucimobilis to degrade persistent xenobiotic compounds, a complete genome sequencing is lacking for this organism. In line with this, we report the first complete genome sequence of Sphingomonas paucimobilis (strain AIMST S2), an organophosphate and hydrocarbon-degrading bacterium isolated from oil-polluted soil at Kedah, Malaysia. The genome was derived from a hybrid assembly of short and long reads generated by Illumina HiSeq and MinION, respectively. The assembly resulted in a single contig of 4,005,505 bases which consisted of 3,612 CDS and 56 tRNAs. An array of genes involved in xenobiotic degradation and plant-growth promoters were identified, suggesting its' potential role as an effective microorganism in bioremediation and agriculture. Having reported the first complete genome of the species, this study will serve as a stepping stone for comparative genome analysis of Sphingomonas strains and other xenobiotic-degrading microorganisms as well as gene expression studies in organophosphate biodegradation.

Insights into microbial community structure and diversity in oil palm waste compost.

Empty fruit bunch (EFB) and palm oil mill effluent (POME) are the major wastes generated by the oil palm industry in Malaysia. The practice of EFB and POME digester sludge co-composting has shown positive results, both in mitigating otherwise environmentally damaging waste streams and producing a useful product (compost) from these streams. In this study, the bacterial ecosystems of 12-week-old EFB-POME co-compost and POME biogas sludge from Felda Maokil, Johor were analysed using 16S metagenome sequencing. Over ten phyla were detected, with Chloroflexi being the predominant phylum, representing approximately 53% of compost and 23% of the POME microbiome reads. The main bacterial lineage found in the compost and POME was Anaerolinaceae (Chloroflexi) with 30% and 18% of the total gene fragments, respectively. The significant differences between compost and POME communities were abundances of Syntrophobacter, Sulfuricurvum and Coprococcus. No methanogens were identified due to the bias in general 16S primers to eubacteria. The preponderance of anaerobic species in the compost and high abundance of secondary metabolite fermenting bacteria is due to an extended composting time, with anaerobic collapse of the pile due to the tropical heat. Predictive functional profiles of the metagenomes using 16S rRNA marker genes suggest that the presence of enzymes involved in degradation of polysaccharides such as glucoamylase, endoglucanase and arabinofuranosidase, all of which were strongly active in POME. Eubacterial species associated with cellulytic methanogenesis were present in both samples.

Cryopreservation methods are effective for long-term storage of Labyrinthula cultures.

Marine heterotrophic protists of the Labyrinthulomycota are of interest for their biotechnological (e.g. thraustochytrid production of lipids) and ecological (e.g. wasting disease and rapid blight by pathogens of the genus Labyrinthula) applications; culture-based laboratory studies are a central technique of this research. However, maintaining such microorganism cultures can be labour- and cost-intensive, with a high risk of culture contamination and die-off over time. Deep-freeze storage, or cryopreservation, can be used to maintain culture back-ups, as well as to preserve the genetic and phenotypic properties of the microorganisms; however, this method has not been tested for the ubiquitous marine protists Labyrinthula spp. In this study, we trialled 12 cryopreservation protocols on 3 Labyrinthula sp. isolates of varying colony morphological traits. After 6 mo at -80°C storage, the DMSO and glycerol protocols were the most effective cryoprotectants compared to methanol (up to 90% success vs. 50% success, respectively). The addition of 30% horse serum to the cryoprotectant solution increased Labyrinthula sp. growth success by 20-30%. We expect that these protocols will provide extra security for culture-based studies, as well as opportunities for long-term research on key Labyrinthula sp. isolates.

A Simple Method for Measuring Carbon-13 Fatty Acid Enrichment in the Major Lipid Classes of Microalgae Using GC-MS.

A simple method for tracing carbon fixation and lipid synthesis in microalgae was developed using a combination of solid-phase extraction (SPE) and negative ion chemical ionisation gas chromatography mass spectrometry (NCI-GC-MS). NCI-GC-MS is an extremely sensitive technique that can produce an unfragmented molecular ion making this technique particularly useful for stable isotope enrichment studies. Derivatisation of fatty acids using pentafluorobenzyl bromide (PFBBr) allows the coupling of the high separation efficiency of GC and the measurement of unfragmented molecular ions for each of the fatty acids by single quadrupole MS. The key is that isotope spectra can be measured without interference from co-eluting fatty acids or other molecules. Pre-fractionation of lipid extracts by SPE allows the measurement of 13C isotope incorporation into the three main lipid classes (phospholipids, glycolipids, neutral lipids) in microalgae thus allowing the study of complex lipid biochemistry using relatively straightforward analytical technology. The high selectivity of GC is necessary as it allows the collection of mass spectra for individual fatty acids, including cis/trans isomers, of the PFB-derivatised fatty acids. The combination of solid-phase extraction and GC-MS enables the accurate determination of 13C incorporation into each lipid pool. Three solvent extraction protocols that are commonly used in lipidomics were also evaluated and are described here with regard to extraction efficiencies for lipid analysis in microalgae.