30 research outputs found

    2023- The Twenty-seventh Annual Symposium of Student Scholars

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    The full program book from the Twenty-seventh Annual Symposium of Student Scholars, held on April 18-21, 2023. Includes abstracts from the presentations and posters.https://digitalcommons.kennesaw.edu/sssprograms/1027/thumbnail.jp

    Single-cell analysis of bacterial extracellular filament regulation and assembly

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    Im Laufe der Evolution haben Bakterienarten Ă€ußerst vielfĂ€ltige und ausgeklĂŒgelte extrazellulĂ€re Strukturen entwickelt, die es ihnen ermöglichen, Substrate in ihre Umgebung abzugeben oder Wirtszellen wĂ€hrend einer Invasion anzugreifen. Diese Sekretionssysteme sind an vielen bakteriellen Mechanismen wie Biofilmbildung, ZellmotilitĂ€t, Virulenz oder Gentransfer und Verbreitung von Antibiotikaresistenzen beteiligt. Das VerstĂ€ndnis des Aufbaus und der Regulierung dieser Strukturen ist angesichts der zunehmenden Entwicklung multiresistenter Bakterien von entscheidender Bedeutung. DarĂŒber hinaus geht der Aufbau solcher Strukturen unweigerlich auf Kosten wertvoller zellulĂ€rer Energieressourcen, was einen spannenden Parameter darstellt, um zu untersuchen, wie Bakterien den optimalen Mechanismus zum Ausgleich zwischen zellulĂ€ren Mechanismen und Energieverbrauch wĂ€hlen. Diese Arbeit konzentriert sich auf den Aufbau und die Regulierung bakterieller extrazellulĂ€rer Filamente, insbesondere des flagellaren Typ-III-Sekretionssystems (T3SS). Im ersten Kapitel werden Defekte in der Zellmorphologie aufgezeigt, die durch die Deletion des FlhE-Proteins wĂ€hrend des Zusammenbaus der Flagellen verursacht werden, was die Bedeutung der Regulierung des Membranpotentials verdeutlicht. Das zweite Kapitel zeigt, dass der Assemblierungsmechanismus der Flagellen-Filamente, welcher dem Injektions-Diffusions-Modell entspricht, im Vergleich zu anderen Sekretionssystemen schneller ist und fĂŒr die Energieerhaltung optimiert ist. Das dritte Kapitel untersucht die Rolle des Pilus beim Plasmid-Transfer, der mit einem Typ-IV-Sekretionssystem (T4SS) assoziiert ist, und liefert zusĂ€tzliche Hinweise darauf, dass der Pilus als Kanal fĂŒr den Plasmid-DNA-Transfer dienen kann. Im letzten Kapitel wird ein neuer Biosensor zur Messung des Gehalts an bis-(3'-5')-zyklischem Diguanosinmonophosphat (c-di-GMP) entwickelt, einem entscheidenden MolekĂŒl in bakteriellen Mechanismen, die ZellmotilitĂ€t und Biofilmbildung miteinander verbinden. Insgesamt bietet diese Arbeit Einblicke in die Regulation des flagellaren T3SS und des T4SS-Pilus, ein neues Werkzeug zur Untersuchung von c-di-GMP und Einblicke, wie Bakterien entscheidende Überlebensparameter und die Optimierung eines energiesparenden Aufbaus abwĂ€gen.Through evolution, bacterial species have developed highly diverse and sophisticated extracellular structures enabling them to secrete substrates in their environment or to target host cells during invasion. Those secretion systems are involved in many bacterial mechanisms such as biofilm formation, cell motility, virulence or gene transfer and antibiotic resistance dissemination. Understanding the assembly and regulation of these structures is crucial due to the increasing development of multi-drug resistant bacteria. Moreover, the assembly of such structures inevitably comes at the cost of valuable cellular energy resources, representing an exciting parameter to study how bacteria selected the optimal mechanism to balance cellular mechanisms and energy consumption. This thesis focuses on the assembly and regulation of bacterial extracellular filaments, notably the flagellar type III secretion system (T3SS) flagellum . The first chapter reveals cell morphology defects caused by the deletion of the FlhE protein during flagellum assembly, highlighting the importance of membrane potential regulation . Chapter two illustrates that the flagellar filament assembly mechanism, following the injection-diffusion model, is faster compared to other secretion systems and optimized for energy conservation. The third chapter investigates the role of the pilus in plasmid transfer, associated with a type IV secretion system (T4SS), and gives additional evidence that the pilus may also act as a channel for plasmid DNA transfer. The final chapter develops a new biosensor for measuring bis-(3’-5’)-cyclic diguanosine monophosphate (c-di-GMP) levels, a crucial molecule in bacterial mechanisms linking cell motility and biofilm formation. Overall, this thesis provides insights into the regulation of the flagellar T3SS and the T4SS pilus, a new tool to study c-di-GMP, and how bacteria balance crucial survival parameters and energy-conserving assembly optimization

    Structure and mechanism of two type III CRISPR defence nucleases activated by cyclic oligoadenylate

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    Prokaryotes have a wide range of antiviral strategies to defend against invading mobile genetic elements (MGEs). Type III CRISPR-Cas systems typically synthesise cyclic oligoadenylate (cOA) second messengers upon binding to cognate foreign RNA. These second messengers allosterically activate type III CRISPR ancillary proteins, potentiating a powerful immune response. Following the discovery of cOA signalling pathway, several ancillary proteins from Csx1/Csm6 family had been described. They sense cOA molecules with their CARF (CRISPR associated Rossman fold) domains and non-specifically cleave RNA with their effector domains. Here, we describe the structure and mechanism of two novel ancillary proteins Can1 and Can2. Can1 has a unique monomeric architecture that contains two CARF domains, a PD-(D/E)XK nuclease domain and a nuclease-like domain. It favours nicking scDNA in the presence of cyclic tetra-adenylate (cA₄) and metal ions. Can2 forms a canonical homodimer and each monomer contains a CARF domain and a PD-(D/E)XK nuclease domain. It exhibits both DNase and RNase activity in the presence of cA₄ and metal ions. It also provides effective immunity against plasmid and bacteriophage infection in a recombinant type III CRISPR-Cas system."This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/S000313/1 to M.F.W., BB/R008035/1 to T.M.G. and BB/T004789/1 to M.F.W. and T.M.G.); grants from Wellcome Trust Institutional Strategic Support Funding (204821/Z/16/Z to M.F.W. and T.M.G.); grants from China Scholarship Council (201703780015 to W.Z.). Funding for open access charge: RCUK block grant." -- Fundin

    Identification and characterisation of rumen bacteria with prominent roles in the ruminal metabolism of forages : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Microbiology and Genetics) at Massey University, Palmerston North, New Zealand

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    Foigures 1.2, 1.4, 1.5 & 1.6 are re-used with permission.This thesis documents the characterisation of two groups of rumen bacteria that are both prominent in forage-fed ruminants, with the aim to better understand their roles in ruminal metabolism. The first group, referred to as the R-7 group, has in recent years been shown to be one of the most abundant rumen bacterial groups, though the few isolated representative strains available were uncharacterised. Two strains of the group included in the Hungate1000 culture collection, R-7 and WTE2008, were selected for characterisation. To facilitate phylogenetic analyses of this group, the complete genomes of an additional three previously isolated R-7 group strains were sequenced. Genomic, phylogenetic and phenotypic characterisation of R-7 and WTE2008 demonstrated that despite their 16S rRNA gene sequences sharing 98.6-99.0% nucleotide identity, their genome-wide average nucleotide identity of 84% assigned them as separate species of a novel genus and family of the proposed order ‘Christensenellales’ using the Genome Taxonomy Database. Phenotypic characterisation showed that the strains were identical in morphology, and both possessed the ability to degrade plant cell wall polysaccharides xylan and pectin, but not cellulose. Acetate, ethanol, hydrogen and lactate were produced by both strains, though R-7 produced greater amounts of hydrogen than WTE2008, which instead produced more lactate. Based on these analyses, it is proposed that R-7 and WTE2008 belong to separate species (Aristaeella gen. nov. hokkaidonensis sp. nov. and Aristaeella lactis sp. nov., respectively) of a newly proposed family (Aristaeellaceae fam. nov.). The second bacterial group of interest, due to their dominant role in ruminal propionate production, was the Prevotella 1 group, following analyses of metatranscriptome datasets of rumen microbial communities of lucerne-fed sheep for dominant community members that express propionate pathway genes from succinate. Screening of 14 strains spanning the diversity of Prevotella 1 found that all except one P. brevis strain produced propionate in a cobalamin (vitamin B12)-dependent manner. To better understand the pathway and regulation of propionate production from succinate, a comparative multi-omics approach was used to test the hypothesis that propionate production is regulated by a cobalamin-binding riboswitch. Scanning of a completed genome assembly of Prevotella ruminicola KHP1 identified four ‘cobalamin’ family riboswitches. However, the riboswitches were not in close proximity to genes putatively involved in converting succinate to propionate, nor were these genes arranged in a single operon. Comparative genomics of the 14 screened strains found that all strains possessed all homologues of candidate propionate pathway genes identified in the KHP1 genome. However, the 13 propionate-producing strains possessed a putative transporter and three subunits encoding a putative methylmalonyl-CoA decarboxylase upstream but antisense to two genes encoding methylmalonyl-CoA mutase subunits, whereas the non-producing strain did not. Comparative transcriptomics and proteomics of KHP1 cultures in the presence and absence of cobalamin demonstrated that some gene candidates were upregulated by cobalamin at the transcriptome level, including co-located genes annotated as phosphate butyryltransferase and butyrate kinase, despite the strain not producing butyrate, suggesting that propionate production may occur via propionyl phosphate. However, only both subunits of methylmalonyl-CoA mutase showed greater transcript and protein abundances in the presence of cobalamin. These results show that while some propionate pathway candidate genes were differentially expressed between cobalamin treatments, they did not appear to be under direct control of a cobalamin-binding riboswitch. This study has contributed to our understanding of the roles of both Aristaeellaceae fam. nov. and Prevotella 1 in ruminal metabolism

    RNA, the Epicenter of Genetic Information

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    The origin story and emergence of molecular biology is muddled. The early triumphs in bacterial genetics and the complexity of animal and plant genomes complicate an intricate history. This book documents the many advances, as well as the prejudices and founder fallacies. It highlights the premature relegation of RNA to simply an intermediate between gene and protein, the underestimation of the amount of information required to program the development of multicellular organisms, and the dawning realization that RNA is the cornerstone of cell biology, development, brain function and probably evolution itself. Key personalities, their hubris as well as prescient predictions are richly illustrated with quotes, archival material, photographs, diagrams and references to bring the people, ideas and discoveries to life, from the conceptual cradles of molecular biology to the current revolution in the understanding of genetic information. Key Features Documents the confused early history of DNA, RNA and proteins - a transformative history of molecular biology like no other. Integrates the influences of biochemistry and genetics on the landscape of molecular biology. Chronicles the important discoveries, preconceptions and misconceptions that retarded or misdirected progress. Highlights major pioneers and contributors to molecular biology, with a focus on RNA and noncoding DNA. Summarizes the mounting evidence for the central roles of non-protein-coding RNA in cell and developmental biology. Provides a thought-provoking retrospective and forward-looking perspective for advanced students and professional researchers

    White Paper 2: Origins, (Co)Evolution, Diversity & Synthesis Of Life

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    Publicado en Madrid, 185 p. ; 17 cm.How life appeared on Earth and how then it diversified into the different and currently existing forms of life are the unanswered questions that will be discussed this volume. These questions delve into the deep past of our planet, where biology intermingles with geology and chemistry, to explore the origin of life and understand its evolution, since “nothing makes sense in biology except in the light of evolution” (Dobzhansky, 1964). The eight challenges that compose this volume summarize our current knowledge and future research directions touching different aspects of the study of evolution, which can be considered a fundamental discipline of Life Science. The volume discusses recent theories on how the first molecules arouse, became organized and acquired their structure, enabling the first forms of life. It also attempts to explain how this life has changed over time, giving rise, from very similar molecular bases, to an immense biological diversity, and to understand what is the hylogenetic relationship among all the different life forms. The volume further analyzes human evolution, its relationship with the environment and its implications on human health and society. Closing the circle, the volume discusses the possibility of designing new biological machines, thus creating a cell prototype from its components and whether this knowledge can be applied to improve our ecosystem. With an effective coordination among its three main areas of knowledge, the CSIC can become an international benchmark for research in this field

    Heads or tails? An insight into the nature of antibacterial structures of an entomopathogenic bacterium Brevibacillus laterosporus : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

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    Bacterium eating viruses” (phages) are one of the biotic factors that can disrupt the mass production of bacteria through the lysis of the cells during growth. Brevibacillus laterosporus (Laubach) is an entomopathogenic, gram-positive, and spore-forming bacterium found around the world, but individual strains can differ greatly in their virulence and hosts. Since 2012, three B. laterosporus strains (Bl 1821L, Bl 1951, Bl Rsp) have been discovered and characterised in New Zealand. All the strains exhibited pathogenicity against diamondback moth larvae and mosquitoes. However, during culturing, the cultures often lost virulence, which was perceived to be due to growth issues and the presence of potential bacteriophages. Though isolates Bl 1821L and Bl 1951 are under development as a biopesticide, the lack of consistent production has hindered their development. Based on these issues this research project commenced to isolate and characterise the suspected bacteriophages, cure the bacteria of the invading phage particles, compare the virulence of phage cured and uncured strains against diamondback moth, and finally develop a production protocol that would allow a biopesticide to be developed from this insect pathogenic bacterium free of phages. However, initial work involving classical phage isolation and enumeration assays (plaque and serial dilutions) and an electron microscopic examination could not substantiate the presence of putative phage particles, despite bioinformatic predictions indicating the presence of intact phages in Bl 1821L, Bl 1951, and Bl Rsp genomes. Assessment of electron micrographs of mitomycin C induced cultures (Bl 1821L & Bl 1951) and bioactivity tests of polyethylene glycol precipitated cultures validated the presence of incomplete phage particles with hexagonal or phage head (encapsulating) and contractile tail-sheath like structures. Subsequent N-terminal sequencing of a prominent ~48 kD protein of SDS-PAGE after mitomycin C induction led to the discovery of putative antibacterial phage tail-like bacteriocins (PTLBs) in the Bl 1821L genome. The putative Bl 1821L PTLB displayed a broader spectrum of activity than the Bl 1951 PTLB. N-terminal sequencing of purified ~48 kD protein of Bl 1821L identified a phage-like element PBSX protein XkdK in the Bl 1821L genome. BLASTp analysis of Bl 1821L 48 kD identified protein accessions with >90% amino acid similarity to the phage tail-sheath protein of Bl LMG 15441. Using the same methodology an XkdK homolog was identified in the Bl 1951 genome. Although the translated product of the Bl 1821L xkdK gene encoding region exhibited amino acid identity to the analogous region of Bl 1951, the bioinformatic analysis revealed some differences in the operon surrounding the gene, a region which corresponded to the PBSX region in Bacillus subtilis. Bioinformatically, the PBSX-like region in Bl 1951 encodes imperfect repeats of glycine rich proteins (1700 bp long) while a putative phage region resides in the analogous Bl 1821L region. A second putative antibacterial protein (bacteriocin) of ~30 kD was also found in SDS PAGE analysis of purified Bl 1821L and Bl 1951 putative antibacterial protein. N-terminal sequencing of purified ~30 kD protein identified matches to both a 25 kD hypothetical and a 30 kD putative encapsulating protein homologs residing in each of the genomes. Various purification methods employed in this study enabled the purification of one putative antibacterial protein (~30 kD) of Bl 1951 and two putative antibacterial proteins (~30 kD & ~48 kD) of Bl 1821L. Subsequent TEM examination of purified antibacterial Bl 1821L proteins revealed the presence of phage head-like encapsulin (~30 kD) and polysheath-like (~48 kD) structures. Although only the ~30 kD protein was purified from Bl 1951, both the phage head-like encapsulin and polysheath-like structures were observed under an electron microscope. SDS-PAGE analysis of spontaneously produced putative antibacterial proteins of Bl 1821L and Bl 1951 upon purification also yielded two prominent bands of ~30 kD and ~48 kD. Assaying size exclusion chromatographic fractions of Bl 1951 harbouring the 30 kD against Bl 1821L resulted in the presence of small Bl 1821L cells that may be indicative of persister cell formation in the population of Bl 1821L. BLASTp analysis of the homologs Bl 1821L and Bl 1951 30 kD amino acid sequences identified >97% amino acid identity to the Linocin M18 bacteriocin family protein of Bl LMG 15441 and Bl GI-9 which are known as encapsulating proteins. Using the bioinformatic tools AMPA and CellPPD motifs relating to the bactericidal activity and cell penetrating peptides were identified. Antibacterial activity of the identified 25 kD hypothetical and 30 kD putative encapsulating proteins of Bl 1821L was further validated through gene expression in a gram-positive bacterium Bacillus subtilis WB800N and the subsequent assay tests and SDS-PAGE analysis of purified proteins from the recombinants. A preliminary assessment of the expressed 25 kD hypothetical gene (pHT01-hypo) exhibited an increased effect against Bl 1821L and Bl 1951 compared to the 30 kD putative encapsulating gene (pHT01-encap). The putative antibacterial proteins (bacteriocins) of Bl 1821L and Bl 1951 sustained the antagonistic activity against B. laterosporus over a wide range of pHs and temperatures. A loss in the antagonistic activity of putative bacteriocins of Bl 1821L and Bl 1951 after treatment with proteolytic enzymes authenticated their proteinaceous nature. However, catalase treatment could not abrogate the inhibitory action of the putative antibacterial proteins of Bl 1821L and Bl 1951, which showed that growth inhibition was not due to hydrogen peroxide production. This study describes the first examples of the spontaneous induction of high molecular weight (HMW) bacteriocins of Bl 1821L and Bl 1951 from the genus Brevibacillus. Spontaneously induced HMW bacteriocins of Bl 1951 affected the growth of bacterium by causing a significant decline in the number of viable cells after 18 hours of culture inoculation that corresponded to the highest antagonistic activity against Bl 1951 and Bl 1821L. Likewise, though not at a significant level spontaneously induced HMW bacteriocins of Bl 1821L decreased the number of viable cells of Bl 1821L after 18 hours of growth. Antagonistic activity of putative antibacterial proteins in growth assays not only varied between the different proteins but also with the state of the proteins (crude or purified). Crude lysate of Bl 1821L and Bl 1951 harbouring both the putative encapsulating (30 kD) and phage tail-like (48 kD) proteins prominently caused autocidal activity with a decrease of 30.1% and 48.4% in the number of viable cells. The purified ~30 kD putative encapsulating protein of Bl 1821L and Bl 1951 exhibited bactericidal activity against both strains. Numerous plausible killing mechanisms of 30 kD putative encapsulating protein of Bl 1821L and Bl 1951 are proposed. These include the activation of stress relevant transcriptional regulator family proteins (PadR & MarR) or YtxJ protein under some unknown stresses, iron malnutrition, failure of ferritin protein to detoxify iron, and cell penetrating peptides activity. The purified ~48 kD putative phage tail-sheath protein of the PBSX-like region of Bl 1821L was active against Bl 1951. Bactericidal activity of ~48 kD purified phage tail-sheath protein of Bl 1821L was likely due to the contractile injection system by forming pores in susceptible cells. Overall, this research identified and characterised two bactericidal proteins of ~30 kD (encapsulating) and ~48 kD (phage tail-sheath) in Bl 1821L and one ~30 kD encapsulating protein in Bl 1951 that were found to be implicated in the growth issues of the insect pathogenic strains Bl 1821L and Bl 1951. A hypothetical protein (25 kD) with more potent putative antibacterial activity in Bl 1821L and Bl 1951 was also identified and expressed late in the project but requires further investigation. The findings provided a wealth of knowledge that will be useful in the future development of a biopesticide from this beneficial bacterium

    YOUMARES 9 - The Oceans: Our Research, Our Future

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    This open access book summarizes peer-reviewed articles and the abstracts of oral and poster presentations given during the YOUMARES 9 conference which took place in Oldenburg, Germany, in September 2018. The aims of this book are to summarize state-of-the-art knowledge in marine sciences and to inspire scientists of all career stages in the development of further research. These conferences are organized by and for young marine researchers. Qualified early-career researchers, who moderated topical sessions during the conference, contributed literature reviews on specific topics within their research field

    YOUMARES 9 - The Oceans: Our Research, Our Future

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    This open access book summarizes peer-reviewed articles and the abstracts of oral and poster presentations given during the YOUMARES 9 conference which took place in Oldenburg, Germany, in September 2018. The aims of this book are to summarize state-of-the-art knowledge in marine sciences and to inspire scientists of all career stages in the development of further research. These conferences are organized by and for young marine researchers. Qualified early-career researchers, who moderated topical sessions during the conference, contributed literature reviews on specific topics within their research field
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