Ancestral function and diversification of a horizontally acquired oomycete carboxylic acid transporter

This is the author accepted manuscript. The final version is available from OUP via the DOI in this recordHorizontal gene transfer (HGT) can equip organisms with novel genes, expanding the repertoire of genetic material available for evolutionary innovation and allowing recipient lineages to colonise new environments. However, few studies have characterised the functions of HGT genes experimentally or examined post-acquisition functional divergence. Here we report the use of ancestral sequence reconstruction and heterologous expression in Saccharomyces cerevisiae to examine the evolutionary history of an oomycete transporter gene family that was horizontally acquired from fungi. We demonstrate that the inferred ancestral oomycete HGT transporter proteins and their extant descendants are predominantly localised to the plasma membrane when expressed in yeast, and that they transport dicarboxylic acids which are intermediates of the tricarboxylic acid cycle. The substrate specificity profile of the most ancestral protein has largely been retained throughout the radiation of oomycetes, including in both plant and animal pathogens and in a free-living saprotroph, indicating that the ancestral HGT transporter function has been maintained by selection across a range of different lifestyles. No evidence of neofunctionalization in terms of substrate specificity was detected for different HGT transporter paralogues which have different patterns of temporal expression. However, a striking expansion of substrate range was observed for one plant pathogenic oomycete, with a HGT derived paralogue from Pythium aphanidermatum encoding a protein that enables tricarboxylic acid uptake in addition to dicarboxylic acid uptake. This demonstrates that HGT acquisitions can provide functional additions to the recipient proteome as well as the foundation material for the evolution of new protein functions.This work was supported by a Philip Leverhulme Award to T. A. R. We acknowledge related project funding from the Gordon and Betty Moore Foundation (GBMF5514). T.A.R. is supported by a Royal Society University Research Fellowshi

ORGANIZATION AND REGULATION OF PROTEINS REQUIRED FOR INITIATION OF CELL DIVISION IN ESCHERICHIA COLI

The process of bacterial cell division relies on the assembly of multiple proteins to form the cell division machine, or “divisome.” FtsZ, a bacterial homolog of eukaryotic tubulin, forms a ring-like structure, the “Z ring,” at midcell and serves as a scaffold for divisome assembly and potential force generator during synthesis of the division septum. Both FtsA, a bacterial homolog of eukaryotic actin, and ZipA, a single-pass transmembrane protein, anchor the Z ring to the inner membrane, are required for the localization of other divisome proteins to midcell, and likely influence the assembly state of FtsZ. Positioning of the Z ring at midcell must be precise in order to ensure efficient cell division, and the MinCDE proteins have an important role in restricting the Z ring to midcell. I found that an artificial fusion of the C-terminus of MinC with MinD, instead of inhibiting Z ring formation as expected, restricted FtsA localization to only part of an intact Z ring. This resulted in many cells with division septa mostly on the side where FtsA localized, causing jackknifing of dividing cells and supporting the importance of a symmetrical FtsA ring for normal cell division. I then investigated localization patterns for FtsZ, FtsA, and ZipA in normal cells using 3D-SIM super-resolution microscopy, which showed that they generally colocalize in puncta instead of a uniformly continuous ring. Finally, I investigated the assembly of purified FtsA and FtsA mutant proteins by electron microscopy and assessed their ability to bind membranes. These studies have refined our view of the organization of the proto-ring proteins FtsZ, FtsA, and ZipA

Molecular insights into the mating system of the marine diatom Pseudo-nitzschia multistriata using genetic and genomic approaches

Sexual reproduction is a fundamental phase in the life cycle of diatoms, linked to the production of genotypic diversity and the formation of large-sized initial cells that ensure population persistence. It occurs only within cells below a certain threshold size and, in heterothallic diatoms, only between strains of opposite mating types. We aim at identifying genes involved in mating type determination in the marine planktonic diatom Pseudo-nitzschia multistriata. This species is recorded in coastal waters worldwide and produces the neurotoxin domoic acid. A reference genome has been generated and transcriptomes have been produced for strains of opposite mating type (MT + and MT-). Differential expression analysis provided a list of candidate MT-biased genes validated with qPCR. Four MT-biased genes were identified, two in MT+ and two in MT-. The expression pattern of the candidate genes was followed in a 24 hours' time course experiment to verify whether they were regulated in dependence of light or cell cycle phases. Experimental evidences demonstrated their involvement during mating recognition in early stages of sexual reproduction while preliminary genetic analyses excluded that they could be the master gene responsible for mating type determination. The description of the four genes was improved through computational characterization to understand their role in the chemical communication occurring between opposite mating types. A further step towards the identification of the MT locus will include a Bulked Segregant Analysis applied to a library of 30 MT+ and 30 MT- F1 strains obtained through DNA deep sequencing. Elucidating the molecular and genetic basis of MT determination and sexual reproduction in diatoms will contribute to a better understanding of the regulation and evolution of their life cycles and reproductive strategies. Results from this study could also provide molecular markers to trace the distribution of MT + and MT - cells in environmental samples

Intrinsically Disordered Protein Regions in Human Evolution and as Therapeutic Targets

Intrinsically disordered proteins (IDPs) and IDP regions (IDPRs) fail to form stable structures but have important biological functions via interacting with various molecular partners (proteins, DNA, RNA, glycosaminoglycans). We hypothesized that IDPRs are potential targets for therapeutics development because they are reservoirs of evolutionary innovation, and they play crucial roles in adaptation to pathogens. We first studied the evolution of IDPRs in the human proteome and compared it with the proteome of non-human primates. We have found that evolutionary young protein-coding genes have included low conserved regions in the N-terminal part of proteins, and such regions are linked to high disorder scores. Although humans share high genomic similarities with their closest relatives such as chimpanzees and other non-human primates, we are more susceptible to diseases. It has been shown that humans respond differently to infectious diseases when compared to primates, the study of structural differences in evolutionary young human proteins could potentially help to discover new therapeutic targets that are linked to human-specific pathologies. In addition, we propose that disordered regions are important targets in vaccine development. Pathogens incorporate disordered regions on their proteomes to interact more efficiently with their host. We chose several IDPRs of the spike protein of the SARS CoV-2 virus to demonstrate in vivo that these regions are antigenic. Our Chimeric Qβ virus-like particles displaying disordered segments of SARS CoV-2 spike protein-induced robust antibody responses in BALB/c mice. We demonstrate evidence that IDPRs can be efficient epitopes for the development of efficacious vaccines

Manipulation of the levels of pyruvate decarboxylase and alcohol dehydrogenase for submergence tolerance in rice

Rice plants are partially or completely submerged when fields are flooded. During submergence, rice plants encounter anaerobic conditions, and suffer severe injury, often death, leading to major crop losses in countries affected by monsoonal flooding. Pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) catalyse thanol fermentation (EF), the major energy-producing pathway under conditions of low oxygen and are among the anaerobic polypeptides induced under such conditions. The importance of EF is emphasised by the reduced survival and germination of the ADH null mutants of maize, barley, rice and Arabidopsis under anaerobic conditions. The research described in this thesis has taken a transgenic approach to manipulate the levels of PDC and ADH to determine whether altering the EF pathway can affect anaerobic tolerance in rice. It was found that one antisense ADHl (rice gene)line had decreased levels of both ADHl and ADH2 polypeptides, and greatly reduced ADH activity of 6% of that of wild type (WT- untransformed Taipei). This antisense ADHI line showed reduced ethanol production and coleoptile growth under anoxia, and mature plants exhibited reduced survival when submerged in anaerobic water, suggesting ADH plays a role in seed germination and plant survival under anoxia. One sense ADH2 (cotton gene) line had significantly increased levels of ADH activity compared to WT and a flooding tolerant rice variety FR13A in air and under hypoxia. No significant increase in ethanol production was observed in the line which overproduced ADH by 439% of WT. Similar levels of anoxia tolerance were found in mature plants of the line which over-produced ADH and WT whereas in the flooding tolerant variety, anoxia tolerance was much higher. This suggests that over-production of ADH increases neither ethanol production nor anaerobic survival. Three independently transformed lines of the rice PDCl driven by an anaerobically inducible promoter contained an increased level of PDC1 polypeptides. A moderate increase in PDC activity and ethanol production compared to WT was also observed in these lines under anaerobic conditions. Effects of anoxia on seed germination were assayed in these lines over-producing PDC and neither retardation nor acceleration of germination was observed. However, mature plants showed decreased survival under anaerobiosis. On the contrary, hybrid plants over-expressing both PDC and ADH were found to have better anaerobic tolerance than plants over-producing PDC alone. These results indicate that overproducing PDC plants suffered from some kind of toxicity which was counterbalanced and/or neutralised in plants over-producing ADH along with PDC. Acetaldehyde levels were appreciably higher in the plants over-producing PDC compared to WT and hybrid plants over-producing both PDC and ADH indicating that acetaldehyde might cause early senescence in plants over-producing PDC alone under anaerobic conditions. No transformed lines with either over-producing PDC, ADH or both PDC and ADH had increased submergence tolerance relative to the WT, however lines often had different metabolic rates and demonstrated the versatility of a molecular approach to evaluating metabolic controls affecting plant growth and survival. A second objective of this research was to study the expression of the rice PDCl and PDC3 promoters in various tissues of rice by GUS histochemical analysis. Translational fusion of the PDCl promoter-GUS gave positive blue staining in embryos, endosperm, shoots, and roots and showed strong anaerobic induction in shoots and roots. GUS staining was found in anthers but absent in pollen. In immunoblotting analysis using an antibody raised against the rice PDC1 polypeptide, the PDC band corresponding to PDCl was also absent in pollen of untransformed Taipei, suggesting that the rice PDCl gene is not expressed in pollen. Nine independently transformed lines of the rice PDC3 promoter-GUS fusion (translational) did not express GUS in any vegetative tissues even under anaerobic conditions. GUS staining was seen in the pollen of three independently transformed lines of PDC3-GUS. A novel PDC band with a MWt of approx. 62 kDa was found in immunoblots of pollen of untransformed Taipei using an antibody generated against the rice PDCl, indicating that the rice PDC3 has pollen specific expression. Expression of PDC3 was seen after the first mitosis of microspores and increased with maturation, implying that it may have a role during pollen germination. The final objective was to study the cis-acting regulatory elements required for anaerobic induction in the rice PDCl promoter. GUS histochemical analysis of transcriptional fusions of the various lengths of 5' truncated PDCl promoter-GUS revealed that the regulation of the GUS reporter gene did not mirror that of the endogenous PDCl and the PDCl promoter acted in a constitutive manner

Mating interactions of the harmful dinoflagellate alexandrium tamarense from UK coastal waters

The Alexandrium tamarense species complex is a group of economically and ecologically important marine dinoflagellates. The complex is comprised of three morphospecies A. tamarense, A. fundyense and A. catenella grouped according to ribosomal DNA or ‘ribotype’. There are five ribotype type groups (I-V) each consisting entirely of toxic or non-toxic isolates. Toxic isolates are associated with harmful algal blooms (HAB’s) due to their ability to produce powerful neurotoxins, which are responsible for outbreaks of paralytic shellfish poisoning in areas of shellfish production. Sexual reproduction in A. tamarense has important implications for the initiation and termination of HAB’s associated with these species. Resistant, long-lived hypnozygotes are formed during blooms through gamete fusion and deposited in sediments. These hypnozygotes provide the source of inoculum of motile vegetative A. tamarense cells in temperate zones during subsequent spring/summer blooms. This study provides further insight into the mating interactions between toxic Group I and non-toxic Group III isolates primarily from UK coastal waters. Study of the effect of temperature on the mating interactions of A. tamarense Group I and Group III in culture showed that temperature had a significant effect on both groups. Co-cultures of compatible Group III isolates showed a significant decrease (p<0.05) in hypnozygote yield at 15°C, compared to 20°C. In contrast the mating compatibility of co-cultures of Group I isolates showed significant increase (p<0.05) at 15°C, compared to 20°C. Similar to other studies, compatible Group I and Group III isolates formed non-viable hybrid hypnozygotes in co-culture. Comparison of the average vigour of inbred Group I crosses and outbred Group I/III crosses suggest that Group I isolates are more likely to out-breed with a compatible Group III isolate. A finding that may have significance in areas where the two groups co-occur. Preliminary data suggesting the presence of both Group I and Group III ribotypes in some isolates has been generated from a nested single cell PCR/qPCR protocol using group specific primers. These data were compared to a dual probe whole cell fluorescent in situ hybridisation (whole cell FISH) assay of isolates. Whole cell FISH showed no dual expression of ribosomal RNA. This suggests that some A. tamarense Group I and Group III isolates may have rDNA pseudogenes corresponding to different ribotypes. If correct this could have implications for the overestimation of A. tamarense group diversity in natural populations when using rDNA sequences for identification

DNA Methylation in Rectal Cancer: Validating Findings of an Epigenome-Wide Association Study

Background: Preliminary studies conducted by our group utilised the Illumina Infinium Human Methylation 450k Beadchip array to perform an epigenome-wide association study (EWAS) of 15 matched rectal tumour (RT) and adjacent mucosa (AM) samples. 176 differentially methylated probes (DMPs) were identified (P<0.00001). RT was also characterised by significantly reduced global methylation in comparison to AM. Aims: This study aimed to validate specific and global DNA methylation differences identified by our preliminary work. We then sought to replicate the findings in additional samples. Finally, we attempted to identify correlations between DNA methylation differences and clinicopathological tumour features. Materials and Methods: Polymerase chain reaction (PCR) and bisulphite pyrosequencing assays were designed and optimised to quantify DNA methylation at nine DMPs nominated by our EWAS. Pearson’s test was used to calculate the correlation between 450k and pyrosequencing methylation values. Replication was performed in an additional cohort of 68 matched colorectal tumour and AM pairs. Global DNA methylation of the discovery cohort was quantified using the luminometric methylation assay (LUMA). Potential relationships between tumour features and differential methylation were investigated using univariate (t-tests or ANOVA) and multivariate analyses (logistic regression). Results: All DMPs selected for validation showed strong correlations between bisulphite pyrosequencing and Illumina 450k methylation values (r= 0.87-0.97). Global hypomethylation was observed in RT (54.6%) when compared to AM (63.5%, P = 0.021). All probes displayed significant levels of differential methylation in the replication cohort (P = <2.2e-16). No significant associations were observed between DNA methylation and clinicopathological tumour features, however this may reflect the relatively small number of samples assessed. Conclusions: Our studies have identified and validated a novel methylomic signature of rectal cancer. Although no clinicopathological correlations were observed with the DMPs investigated, others may represent potential targets in the diagnosis, monitoring and risk stratification of rectal cancer.Northcott Medical FoundationThe Wolfson FoundationThe Royal College of Surgeons of EnglandBowel Cancer Wes

Black seabream, spondyliosoma cantharus : stock structure in the eastern atlantic and characterisation of the biology and fishery in the portuguese coast

Tese de doutoramento, Ciências do Mar, Universidade de Lisboa, Faculdade de Ciências, 2018In this thesis the stock structure of the black seabream, Spondyliosoma cantharus, along the Eastern Atlantic is explored using 4 different methodologies, body morphometry, otolith shape and stable isotopes ratio and genetics (mitochondrial and nuclear markers). Samples were gathered for five European areas (English Channel, Bay of Biscay, Galicia, west Portuguese coast – Peniche, and south Portuguese coast – Algarve), and two areas from African coast (Canary Islands and Angola). Results from morphometric analyses support the existence of different phenotypic stocks in each sampled area, while stable isotope ratios show fuzzier results with only 3 areas clearly distinguished: Angola, Canary Islands and Bay of Biscay. Genetic analyses were enhanced with samples from Mediterranean Sea and Cape Verde. A clear structuration is present between regions: North east Atlantic, Mediterranean Sea, Cape Verde and Angola, not being detected any population structuring within each region; however high levels of private haplotypes for all populations were observed, which can indicate that not enough dispersal/gene flow exists to homogenize more recent mutations at the ecological timescale. Integrating results from all methods, in a holistic stock structure analyses, the scenario with 6 different stock units in the North-eastern Atlantic is the most probable. Life history parameters for the species were also evaluated for the west Portuguese coast, since these are key parameters for a conscientious stock assessment. Maximum age of 17 years was assigned for a specimen with 38 cm. Age at growth was best described with the L00 hyperbolic modification of von Bertalanffy curve. Under this model, a change in growth occurs around 8 years, which corresponds roughly to the average age for sex reversal in the species. The estimated exploitation rate was relatively high (0.43 – 0.62), indicating that although the species is not the main target of the fisheries, its management needs careful attention. Considering the reproductive strategy of the species, half of the females’ population was mature at 18.41 cm and changed sex at 25.62 cm. Sex change takes only a brief period of time, since transitional individuals were scarce and most of them showed oocytes regressing into cystic structures. The species presents a clear indeterminate fecundity type with massive atresia happening at the end of the spawning season. Mean values of 203 oocytes and 5431 oocytes by gram of eviscerated female were estimated for relative batch fecundity and relative annual fecundity, respectively.Fundação para a Ciência e a Tecnologia (FCT), SFRH/BD/92769/201