Transposon-Mediated Horizontal Transfer of the Host-Specific Virulence Protein ToxA between Three Fungal Wheat Pathogens

Most known examples of horizontal gene transfer (HGT) between eukaryotes are ancient. These events are identified primarily using phylogenetic methods on coding regions alone. Only rarely are there examples of HGT where noncoding DNA is also reported. The gene encoding the wheat virulence protein ToxA and the surrounding 14 kb is one of these rare examples. ToxA has been horizontally transferred between three fungal wheat pathogens (Parastagonospora nodorum, Pyrenophora tritici-repentis, and Bipolaris sorokiniana) as part of a conserved ∼14 kb element which contains coding and noncoding regions. Here we used long-read sequencing to define the extent of HGT between these three fungal species. Construction of near-chromosomal-level assemblies enabled identification of terminal inverted repeats on either end of the 14 kb region, typical of a type II DNA transposon. This is the first description of ToxA with complete transposon features, which we call ToxhAT. In all three species, ToxhAT resides in a large (140-to-250 kb) transposon-rich genomic island which is absent in isolates that do not carry the gene (annotated here as toxa−). We demonstrate that the horizontal transfer of ToxhAT between P. tritici-repentis and P. nodorum occurred as part of a large (∼80 kb) HGT which is now undergoing extensive decay. In B. sorokiniana, in contrast, ToxhAT and its resident genomic island are mobile within the genome. Together, these data provide insight into the noncoding regions that facilitate HGT between eukaryotes and into the genomic processes which mask the extent of HGT between these species.M.C.M. acknowledges The Sun Foundation’s Peer Prize for Women in Science for support to sequence additional ToxA isolates. E.H. acknowledges The Grains and Research Development Corporation (project UHS11002). M.C.M., A.M., S.S., and P.S.S. also acknowledge The Grains and Research Development Corporation for the collection of isolates (projects DAN00203 and DAN00177)

Genome-Based Analyses of Fitness Effects and Compensatory Changes Associated with Acquisition of blaCMY-, blaCTX-M-, and blaOXA-48/VIM-1-Containing Plasmids in Escherichia coli

Background: Resistance plasmids are under selective conditions beneficial for the bacterial host, but in the absence of selective pressure, this carriage may cause fitness costs. Compensation of this fitness burden is important to obtain competitive ability under antibiotic-free conditions. In this study, we investigated fitness effects after a conjugative transfer of plasmids containing various beta-lactamase genes transferred into Escherichia coli. (2) Methods: Fourteen beta-lactamase-encoding plasmids were transferred from clinical donor strains to E. coli J53. Growth rates were compared for all transconjugants and the recipient. Selected transconjugants were challenged in long-term growth experiments. Growth rates were assessed at different time points during growth for 500 generations. Whole-genome sequencing (WGS) of initial and evolved transconjugants was determined. Results: Most plasmid acquisitions resulted in growth differences, ranging from −4.5% to 7.2%. Transfer of a single blaCMY-16-carrying plasmid resulted in a growth burden and a growth benefit in independent mating. Long-term growth led to a compensation of fitness burdens and benefits. Analyzing WGS revealed genomic changes caused by Single Nucleotide Polymorphisms (SNPs) and insertion sequences over time. Conclusions: Fitness effects associated with plasmid acquisitions were variable. Potential compensatory mutations identified in transconjugants’ genomes after 500 generations give interesting insights into aspects of plasmid–host adaptationsPeer Reviewe

Metagenomic analysis facilitates ontogenetic investigations of microbiota composition and antimicrobial resistance in silver gull (Chroicocephalus novaehollandiae) chicks

The proliferation of antimicrobial resistance (AMR) in recent decades has been fuelled by the development, overconsumption and misuse of antibiotic therapeutics and non-medical antimicrobials. The evolution of AMR-bacteria is predominantly underpinned by the horizontal transfer of antimicrobial resistance genes (ARGs), which allows AMR to be transferred between bacterial species and accumulate within bacteria, even in the absence of antimicrobial selection pressures. It has been speculated that avian wildlife, in particular waterbird species such as gulls, act as reservoirs of AMR, facilitating the propagation of AMR-bacteria that can then be transmitted into humans. The current thesis, therefore, investigated whether silver gull (Chroiocephalus novaehollandiae) chicks may play a role in the evolution and proliferation of AMR-bacteria. Firstly, numerous DNA extraction protocols were trialled to optimise the extraction of DNA from multi-species microbiota samples. Metagenomic sequencing and analysis were then conducted for 60 temporal multi-species microbiota samples from 23 silver gull chicks, which had been subcultured for Enterobacteriaceae, a subpopulation of the microbiome enriched in AMR-associated bacteria. The data were examined in order to characterise bacterial and AMR composition and to analyse ontogenetic shifts in the microbiome to assess if the silver gull chick microbiome has the ability to accumulate and maintain AMR. Metagenomic analysis revealed a diverse Gammaproteobacterial community of 125 species and 30 genera, and the majority of species were of a genus containing pathogenic and AMR-associated species. Additionally, metagenomic analysis detected a large ARG pool, with 115 ARGs identified across all samples. These ARGs were dominated by Beta-lactams and Aminoglycoside resistance phenotypes, which are the most commonly used antibiotics in Australia. Ontogenetic analyses further showed that both bacterial and AMR profiles were in constant flux and varied between samples and within individual chicks. Additionally, both richness and composition measures for bacterial species and ARGs were significantly correlated, such that the ARG profile within a sample was correlated to the bacterial profile. The high diversity of AMR-associated bacteria and ARGs found in these samples indicates that the silver gull microbiome may act as an ‘ecological sponge’ of AMR-bacterial contamination in the environment, which transiently colonise the gut. Moreover, these findings indicate that ARGs are not accumulated and do not persist over time but are brought in with the transient bacteria they are associated with. The current thesis, therefore, concludes that silver gull chicks do not contribute to the proliferation and evolution of AMR-bacteria, and further, do not act as a 6 reservoir to support the propagation of AMR-bacteria. Moreover, this thesis presents the first longitudinal metagenomic study to examine the dynamic nature of the microbiome and AMR in a wildlife host. Future studies should investigate potential sources of the AMR contamination reflected in this silver gull population, and explore potential reservoirs of AMR, including wildlife populations and the non-human environment

Horizontal gene transfer and the unusual genomic architecture of bdelloid rotifers

Bdelloid rotifers are microscopic aquatic animals, notable for their ancient asexuality and their extreme desiccation tolerance. In the absence of sexual reproduction, bdelloids have persisted for over 40 million years, diverging into >450 morphologically distinct species. Despite the two-fold cost of sex, asexual lineages tend to be short-lived and species poor. Many theories exist to explain the success of sexual reproduction, and in the light of these, ancient asexual lineages are an evolutionary paradox. Understanding the persistence and speciation of ancient asexuals may provide clues to factors underlying the success of sexual reproduction. Bdelloid rotifers have unusual genomic features that may have provided some compensation for their long-term absence of sexual reproduction. Here I focus on two: multiple gene copies and horizontal gene transfer (HGT). Bdelloids have multiple copies of many genes, and are considered degenerate tetraploids. In genomes influenced by the opposing forces of gene conversion and divergence of former alleles, I examine the relationships between, and biochemical implications of divergence of a multi-gene family of alpha tubulin. Horizontally acquired genes were initially identified in sub-telomeric regions of two species of bdelloid rotifer. In order to understand what role foreign genes might have played in bdelloid evolution we need to examine the extent, frequency and mechanism of HGT. Here I develop a bioinformatics pipeline for identifying horizontally acquired genes in transcriptomes. By comparing HGT in a number of bdelloid species I demonstrate that the majority of transcribed foreign genes were acquired before the divergence of extant bdelloid species, but the presence of more recently acquired genes implies that HGT is ongoing. By comparing the extent of HGT in closely related species with different desiccation frequencies I provide initial support for the hypothesis that bdelloid HGT is facilitated by DNA breakage and repair during cycles of desiccation and rehydration.Open Acces

EVOLUTION OF THE CIRCADIAN CLOCK IN EXTREME ENVIRONMENT: LESSONS FROM CAVEFISH.

Evolution has been strongly influenced by the daily cycles of temperature and light imposed by the rotation of the Earth. Fascinating demonstrations of this are seen in extreme environments such as caves where some animals have remained completely isolated from the day-night cycle for millions of years. Most of these species show convergent evolution, sharing a range of striking physical properties such as eye loss. One fundamental issue is whether “hypogean” species retain a functional circadian clock. This highly conserved, physiological timing mechanism allows organisms to anticipate daily environmental changes and is synchronized primarily by light. The Somalian cavefish, Phreatichthys andruzzii does possess a circadian clock that is entrained by a daily regular feeding time but strikingly, not by light. Under constant conditions the P. andruzzii clock oscillates with an extremely long period and also lacks normal temperature compensation. We document multiple mutations affecting a light-induced clock gene, Period2 as well as the genes encoding the extra-retinal photoreceptors Melanopsin (Opn4m2) and TMT-opsin. Remarkably, we show that ectopic expression of zebrafish homologs of these opsins rescues light induced clock gene expression in P. andruzzii cells. Thus, by studying this natural mutant we provide direct evidence for a peripheral light-sensing function of extra-retinal opsins in vertebrates. Furthermore, the properties of this cavefish illustrate that evolution in constant darkness leads not only to anatomical changes but also to loss of gene function linked with the detection and anticipation of the day-night cycle

Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism.

Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells

Técnicas avançadas de biologia molecular e transcritómica aplicadas a actinomycetes

Nowadays, the resistance to antibiotics is turning to be one of the biggest concerns of this century. It is risking our public health and might be, in the future, one of the deadliest health issues. Therefore, a solution is needed. Streptomyces is known as a genus that is responsible for many antibiotics. However, in the latest years, the number of antibiotics that reached the clinical use diminished, due to various reasons. The secondary metabolism is composed by a number of processes that, even though aren’t of extreme importance to the cell survival, can give it several advantages. Most of those advantages come as secondary metabolites, and some are known as antibiotics. On the other hand, phosphorus is one of the most important elements to any organism, and, therefore, a mechanism is needed to make sure that this element is regulated. One of the systems that does that depends on PhoR-PhoP. With the discovery of a new type of RNAs, the sRNAs (which have between 50 and 400 nucleotides) the investigation of new compounds with pharmaceutical and industrial importance may continue to go forward, since some of those molecules may function as regulators of the secondary metabolism and, therefore, be related to antibiotics. The aim of this work is to identify sRNAs that are implicated in the phosphate regulation, which is the main assimilation form of phosphorus. My objective in this study was to experience what was like to work in a laboratory, by learning some molecular biology and transcriptomic techniques, such as DNA introduction into cells and nucleic acid extraction, needed for this study.Hoje em dia, a resistência a antibióticos está a tornar-se numa das maiores preocupações do século. Esta problemática está a arriscar a nossa saúde pública e pode vir a tornar-se, no futuro, num dos problemas de saúde mais letais. Portanto, uma solução é necessária. Streptomyces é conhecido como o género que é responsável por imensos antibióticos. Porém, nestes últimos anos, o número de antibióticos que chegaram a uso clínico diminuiu, devido a várias razões. Metabolismo secundário é composto de vários processos que, ainda que não sejam fundamentais para a sobrevivência da célula, dão-lhe imensas vantagens. A maior parte destas vantagens originam-se a partir dos metabolitos secundários, alguns deles conhecidos como antibióticos. Por outro lado, fósforo é um dos elementos mais importantes para qualquer organismo e, por isso, necessita de um mecanismo que se assegure que ele está sempre regulado. Um destes sistemas depende de PhoR-PhoP. Com a descoberta de um novo tipo de RNAs, os sRNAs (que têm entre 50 a 400 nucleótidos), a investigação de novos compostos com importância farmacêutica e industrial pode continuar a avançar, dado que algumas destas moléculas podem funcionar como reguladoras do metabolismo secundário e, por isso, estar relacionadas com antibióticos. O objetivo deste trabalho é identificar sRNAs que estão implicados na regulação de fosfato, a principal forma de assimilação de fósforo. A minha função era experienciar o que era trabalhar num laboratório, ao aprender algumas técnicas de biologia molecular e transcritómica, como introdução de DNA em células e extração de ácidos nucleicos, necessárias para este projeto.Mestrado em Biologia Molecular e Celula

Collateral sensitivity in clinical Escherichia coli isolates

Background At present time, antimicrobial resistance is emerging more rapidly than the development of novel antimicrobials, presenting a serious threat to how we prevent and treat infectious diseases. Several treatment strategies to counteract this development have been proposed, among these is the use of collateral sensitivity in clinical treatment. The ability to predict collateral sensitivity and cross-resistance effects is essential to exploiting this concept. In this study, we aimed to investigate the patterns of collateral sensitivity and cross-resistance in ciprofloxacin resistant isolates carrying gyrA and parC mutations. Method Ciprofloxacin resistant isolates were evolved from three clinical E. coli strain using static and dynamic selection methods. Isolates were selected based on identified mutations and level of ciprofloxacin resistance measured with diffusion gradient strips. DNA sequencing was used to detect mutations in gyrA and parC. Resistant isolates carrying at least one gyrA and parC mutation were characterized by IC90 assays with micro-broth dilutions of six unrelated antimicrobial agents. The observed collateral sensitivity and cross-resistance effects were displayed in a heat map. Results Various non-synonymous point mutations in gyrA and parC were identified in several of the generated ciprofloxacin resistant isolates. These mutants displayed collateral sensitivity and cross-resistance to several unrelated antimicrobials. Collateral sensitivity to gentamicin and trimethoprim was observed in the majority isolates. Cross-resistance effects were found in several mutants, specifically to ceftazidime, chloramphenicol and colistin. Conclusion Our findings suggest that ciprofloxacin resistant mutants with gyrA and parC mutations display a clear tendency of collateral sensitivity to gentamicin, an effect which potentially can be exploited in future treatment. However, we propose further investigation into specific point mutations within these genes, to better understand the observed variations in collateral sensitivity and cross-resistance

Horizontal transfer, selection and maintenance of antibiotic resistance determinants

Scientific abstract Antibiotic resistance, especially in Gram-negative pathogens, represents a substantial clinical and financial burden to our society. The presented work investigated mechanisms and evolutionary dynamics that promote the emergence and maintenance of resistance in bacteria. In the first study, conserved collateral susceptibility changes were identified across resistant uropathogenic Escherichia coli, which included also changes towards increased sensitivity of these isolates to certain antibiotics. This so-called collateral sensitivity potentiated the effect of antibiotics and prevented the selection of resistant isolates compared to wildtype strains. The mechanism and fitness cost of resistance were important predictors of collateral responses in resistant bacteria, and their rapid clinical identification could inform future evolution-based infection treatment. The second study demonstrated the potential of a transposable element (Tn1) to spread antibiotic resistance during natural transformation of Acinetobacter baylyi. In the course of this horizontal gene transfer mechanism, Tn1-containing DNA entered the bacterial cell, and specific host, as well as transposon proteins, facilitated Tn1-insertion into the recipient chromosome. A mechanistic model of transposition-mediated natural transformation from a circular, cytoplasmic intermediate is presented. In the third study, uropathogenic E. coli improved its permissiveness towards two unrelated multidrug resistance plasmids while adapting to a new environmental niche. Mutations in the CCR and ArcAB regulatory systems resulted in transcriptional downregulation of plasmid genes and explained plasmid cost reduction. The presented evolutionary dynamics improve our understanding of successful associations between bacterial pathogens and resistance plasmids and provide a novel solution to the so-called ‘plasmid paradox’. In a broader perspective, the findings of this thesis advanced our knowledge on the selection, spread and maintenance of antibiotic resistance in bacteria, which is important to counteract its evolution

Role of DNA methylation in WNT5A Promoter B expression in Osteosarcoma (SaOS-2) cells.

WNT5A, a member of the WNT family of secreted proteins, activates the non-canonical WNT pathway, regulates developmental events and is involved in tissue homeostasis. Misregulation of WNT5A has been associated with various types of cancer including pancreatic, colorectal, breast, lung and osteosarcoma. Recent studies have shown that WNT5A expression in cancer cells involves non-genetic (epigenetic) changes. The WNT5A gene has two similar transcription start sites (termed promoter A and promoter B in this study) and transcription from these sites give rise to two different messages that encode different protein isoforms. Epigenetic studies to date have focused on the WNT5A promoter A, however our preliminary analysis found that promoter B transcripts are nearly absent in osteosarcoma cells, but present in normal osteoblasts. The goal of this study was to determine if the decrease in promoter B transcripts in osteosarcoma was due to DNA methylation. We identified 6 CpG islands in the WNT5A intron 1 region which contains promoter B and exon 1B, and compared them with data available in NCBI epigenomics website. The NCBI database was also screened for data on the methylation status of these islands in various other cell lines and cancer types. The database analysis shows that there is little or no methylation of Regions 1 - 6 in cells, that normally express WNT5A (fibroblast, chondrocytes, and mesenchymal stem cells). However, in some colorectal tumor tissue there is extensive methylation within intron 1. In adjacent normal colon mucosa tissue, Regions 3 and 4 showed some degree of methylation. Methylation status of the CpG islands in normal osteoblasts and osteosarcoma (SaOS-2) was determined using Sodium bisulfite sequencing. The sequencing data showed that CpG Regions 1 and 2 are unmethylated. Regions 3, 4 and 5 are completely methylated and Region 6 is partially methylated. In normal osteoblasts all 6 regions were unmethylated. A demethylation experiment using 5-Aza-cytidine was performed to determine if decrease in methylation would increase promoter B transcripts. Treatment of SaOS-2 cells with 1µM 5-Aza-cytidine resulted in a 120 fold increase of promoter B transcripts. The methylation data of SaOS-2 cells treated with 1µM 5-Aza-cytidine shows that CpG Region 6 is more prone to demethylation when compared to CpG islands 3, 4, 5. Two clones of Region 6 showed that 4 out of 5 CpG's analyzed were demethylated. 5 clones of Region 3 and 4 showed that only 1-2 CpG's were demethylated out of 24 CpG's in Region 3 and 27 CpG's in Region 4. Overall these results suggest that promoter B transcription is down regulated by DNA methylation of CpG Region 6 and Region 6 is more prone to demethylation compared to Regions 3, 4 and 5