1,720,987 research outputs found
Lytic Transglycosylases: Untangling Misconceptions in Bacterial Cell Wall Dynamics
No full text168 pagesThe bacterial cell wall comprises a strong, covalently closed network of peptidoglycan (PG) strands. While PG synthesis is generally essential for bacterial survival, the cell wall is also by necessity a dynamic structure and undergoes constant degradation and remodeling by “autolysins,” enzymes that break bonds within PG. One class of autolysin, the lytic transglycosylases (LTGs), cleaves the glycosidic linkages within PG strands. Despite LTGs having well-described biochemical properties, LTG redundancy and diversity have stymied understanding of their fundamental physiological roles. LTGs have been mostly assigned various non-essential, or poorly defined, pleiotropic functions and so there has been no clear evidence to explain why this extreme redundancy, usually indicating an essential function, is so widely conserved amongst diverse bacteria. The diarrheal pathogen Vibrio cholerae encodes eight known LTGs and inactivating single LTGs rarely generates a significant mutant phenotype from which to infer physiological importance. Therefore, rather than directly pursuing individual LTGs, we sought to explore the collective function of the entire enzymatic class by interrogating a mutant lacking all known LTGs. In doing so, we found that V. cholerae must retain at least one active LTG for survival and subsequently characterized the first truly essential role fulfilled by LTGs: clearance of PG debris from the periplasm which accumulates during normal cell wall expansion and remodeling, or during cell wall damage. Coincidentally, this addresses a fundamental question about how bacteria maintain the integrity of a dynamic cell wall through temporal separation of this LTG-mediated autolysis from synthesis, likely independent of previously hypothesized protein-protein interactions. By systematically re-introducing LTGs back into LTG-deficient mutants, we have also created a platform for empirically organizing diverse LTGs into functional families where previously they could only be categorized by their biochemistry. For example, one functional group includes LTGs that are specifically required for clearance of PG debris during septation and daughter cell separation. Another group likely contributes to the elusive, and now confirmed essential, function of releasing newly synthesized PG from the inner membrane. This platform is far from exhaustion and will continue to yield critical information about lytic transglycosylases and their relationship with cell wall homeostasis
Community Dynamics And Nutritional Benefits Of The Drosophila Gut Microbiota
Get PDFRecent advances in high throughput sequencing have provided important insights on the diversity and functional capabilities of gut microbiota in various animals. Despite tremendous sampling efforts in mammalian systems, the community dynamics and assembly patterns of gut microbiota are poorly understood, and experimental demonstration of their nutritional benefits remains largely absent. To address these issues, this study develops Drosophila as a model system to study: 1) the diversity of the gut microbiota, by characterizing the gut microbiota composition of laboratory Drosophila melanogaster and other Drosophila species across phylogeny using high-throughput sequencing of the 16S rRNA gene, 2) the nutritional benefits of gut microbiota under different dietary regimes, by comparing the performance and nutritional responses between conventional and axenic (i.e. microbefree) flies onto diets of systematically-varied nutrient (yeast-glucose) content. Results from this project demonstrate that Drosophila has a low-diversity gut bacterial community that is amenable for studying gut microbiota functions. The taxonomic composition appears to be inconstant, with no evidence for core taxa or co-evolution between the host and its microbiota. However, elimination of the gut microbiota results in prolonged host development and nutritional response to diet. The gut bacteria promote host health under conditions of nutritional stress resulting from unbalanced diet by increasing micronutrient (vitamins B) availability and/or reducing excessive dietary sugar. Future investigations will include examining the nutritional functions of individual gut bacteria via re-associations with axenic flies, and testing congruence between taxonomic and functional (microbiome) profiles of the gut microbiota in response to changing diet
INVESTIGATIONS INTO HOW BACTERIA INFLUENCE NUTRIENT AVAILABILITY IN THEIR ENVIRONMENT
Get PDFBacteria shape many of their interactions with other organisms through the manipulation of nutrient availability, whether through cooperation by providing nutrients, or through competition for nutrients. Thiamin is an essential vitamin necessary for all life, however, how bacteria shape the ecological interactions between organisms for this nutrient is not well understood. We employed a Drosophila melanogaster-microbiota model utilizing a chemically defined diet to understand the interaction between how the host is influenced by the microbiota’s interaction with the dietary component thiamin. We found that the Drosophila melanogaster microbiota provisions thiamin to its host in a low thiamin environment. This provision rescued development of Drosophila on a no thiamin diet, as axenic flies were unable to develop on this diet. Our study was a clear demonstration supporting the long standing hypothesis that animal microbiotas function to provision thiamin and other vitamins to their host. A small subset of bacteria produce the enzyme thiaminase I, which degrades thiamin to its two moieties. The biological function of this enzyme is not understood. We used a genomic approach to investigate a potential function of this enzyme and found that it is located in a conserved operon in three thiaminase I producing Paenibacillus species, with other genes involved in thiamin salvage and production of the thiamin antimetabolite bacimethrin, suggesting it may play a role in thiamin salvage and competition for this nutrient. We further investigated the biological role of thiaminase I using Burkholderia thailandensis, where we generated thiamin auxotrophs. We found that the enzyme plays a role in thiamin salvage as it allows for auxotrophic strains to grow in media conditions when strains lacking thiaminase I cannot, as it recycles precursors from thiamin and certain analogs. Using a genomic approach, we also investigated the biosynthetic and metabolic potential of the unique, giant bacterial intestinal symbiont ‘Candidatus Epulopiscium viviparous’ of the surgeonfish Naso tonganus. We found that this bacterium’s genome is enriched for carbohydrate metabolism, as it has the potential to degrade a vast array of carbohydrates present in its host’s diet, allowing them to supply assimilable nutrients, vitamins, and protein to their host
Unraveling The Nest Microbiome: Characterizing Avian-Associated Bacterial Communities And Their Influences On Host Transgenerational Immune Investment
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Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
ESSENTIAL PATHWAYS AND GENE REGULATION FOR CELL WALL HOMEOSTASIS IN BACILLUS SUBTILIS
Get PDFThe cell wall is an essential component of most bacterial cells, and cell wall targeting antibiotics have greatly improved human health and life span. Despite intense research over the last 50 years, we still lack a complete understanding of how bacteria maintain cell wall homeostasis. Here we attempt to integrate new data from the last few years with some established ideas in the field, and we propose a new model of cell wall biogenesis in rod-shaped bacteria (Chapter 1). The lipid II cycle is central to peptidoglycan (PG) synthesis. A common C55 lipid carrier, undecaprenyl-pyrophosphate (UPP) is used for the synthesis of both peptidoglycan and wall teichoic acids to ferry precursors across the cytoplasmic membrane. Here we demonstrate that B. subtilis requires either one of two UPP phosphatases, UppP or BcrC, for the recycling of this essential molecule for continuous synthesis of cell wall (Chapter 2). Peptidoglycan synthesis relies on intermediates derived from central metabolism. We found that an aspB mutant is auxotrophic for aspartate and lyses when grown on Difco sporulation medium due to limitation of the peptidoglycan precursor meso-2,6-diaminopimelate (mDAP), a downstream metabolic intermediate of aspartate. Interestingly, we found that when bacteria experience a shortage of aspartate, the first breakpoint is not protein but peptidoglycan synthesis, which predisposes them to cell wall weakness and sensitizes them to antibiotics targeting late steps of PG synthesis. This work highlights the ability of perturbations of central metabolism to sensitize cells to peptidoglycan synthesis inhibitors (Chapter 3). Bacillus subtilis uses alternative sigma factors to regulate gene transcription upon stresses. Being a powerful double-edged sword, it is essential to keep this regulatory network under check. Here we show that the absence of its anti-sigma factor(s) leads to dysregulation of SigM, which drives a positive feedback loop for its own synthesis and SigM accumulates to a toxic level. High SigM activity overproduces membrane proteins and causes protein secretion stress, which lead to cell morphology and severe growth defects (Chapter 4). Collectively, the work in this dissertation provides additional insights of essential pathways and gene regulation for cell wall homeostasis in Bacillus subtilis
Development Of Micro And Nanoparticle Substrates For Sers Detection Of Pesticides And Delivery Of Chemotherapeutic Drugs And Pilot Scale Process Development And Cgmp Production Of Cancer Testis Antigen Melan-A
Get PDFDetection or delivery of a diverse range of biomolecules in field portable devices is an important area of research in the fields of environmental pollution, homeland security, and medicine. The demand for hand-held and low cost detection in these fields has led to the development of novel analytical systems such as Surface Enhanced Raman Spectroscopy (SERS) and Molecularly Imprinted Polymers (MIP). SERS is rapidly emerging as a tool for biological assays, chemical sensing, and electrochemistry. Whereas, Molecularly Imprinted Polymers have been developed as systems for solid phase extraction, chemical separation, and controlled release. In this dissertation, several systems were developed using nanotechnology and polymer synthesi s to develop chemical sensors and a drug delivery system. A substrate for pesticide detection was developed by conjugating oligonucleotides specific for the pesticide malathion to the surface of a SERS substrate that detected the organophosphorus pesticide malathion down to the micromolar level. A SERS substrate was also developed by grafting a molecularly imprinted polymer of methacrylic acid and ethylene glycol dimethacrylate to surface of a gold coated silica microparticle that was able to capture and detect the pesticide thiabendazole. Molecularly ii imprinted polymers were investigated as a drug delivery system for the chemotherapeutic doxorubicin which showed strong imprinting and temperature sensitive controlled release of drug. Lastly, a cGMP pilot scale process was developed to produce gram levels of the cancer vaccine Melan-A for phase I clinical trials. ii
Evolutionary Stability Of Fungal-Bacterial Endosymbioses
Get PDFMany eukaryotes interact with heritable endobacteria to satisfy diverse metabolic needs. Of the characterized fungal-bacterial symbioses, endobacterial associations with the Gigasporaceae (Glomeromycota) and Rhizopus microsporus (Mucoromycotina) are the best described. Both fungal hosts associate with closely related bacterial endosymbionts from the Burkholderia lineage of [beta]-proteobacteria. Through investigating patterns of co-divergence between partners, we have shown that the Glomeribacter-Glomeromycota symbiosis is at least 400 million years old, while still remaining non-essential for the host. To further explore what adaptations have taken place to allow for the persistence of this association, we created a computational pipeline which utilizes patterns of adaptation to infer microbial lifestyle. We show that this pipeline is effective at inferring microbial lifestyle, and that genes involved in DNA regulation, energy metabolism, and pathogenicity are likely important for survival of Ca. Glomeribacter within their fungal hosts. Additionally, we identified that non-essential endosymbionts are as effective at purging slightly deleterious mutations from their genomes as free-living organisms. Unlike Glomeribacter, Burkholderia rhizoxinica, the endosymbiont of Rhizopus microsporus is capable of free living yet is simultaneously of great importance to host survival. Our work has revealed that endosymbionts are required for sexual reproduction of the fungal host. Through phenotypic observation and transcriptome profiling, we found that endosymbionts control fungal reproduction through hijacking of host reproductive machinery. Specifically, bacteria control expression levels of Ras2, a signaling protein important for reproductive development as well as filamentous growth. We also exploited endosymbiont control over reproduction to explore conservation of sexually relevant genes across Fungi, including the Mucoromycotina. This approach identified several genes that appear core to all fungal reproduction, as well as reproduction related genes which are specific to members of the Mucoromycotina. In particular, we found two candidate class C seven transmembrane G-protein coupled receptors (GPCRs), TriR1 and TriR2, which may be responsible for perception of trisporic acid during mating in Mucoromycotina. These receptors are closely related to the retinoic acid GPCRs present in animal systems
EXAMINING THE ROLE OF HOST DEPENDENCE ON THE ECOLOGY AND EVOLUTION OF EPULOSCIUM SPP. AND THEIR RELATIVES
No full textStudies of the gut microbiome have enlightened our perspective on the contributions of microbes to animal health. Commensal bacteria have evolved strategies to maintain associations with their host and gain a foothold in this competitive environment through colonization, maintenance and transmission. Here I studied how dominant commensals of herbivorous surgeonfish have evolved strategies to overcome the obstacles to host association and as a result of this interaction, further explain the unusual biology of these commensals. Epulopiscium spp. and relatives (known as epulos) are morphologically diverse and form a monophyletic clade within the Lachnospiraceae XIVb cluster (Order: Clostridiales). They are renowned for their large size, some reaching lengths up to 0.6 mm, and their extreme polyploidy, containing 100,000s of copies of a ~3.2 Mb genome. The reproductive cycle of epulos follows a predictable diurnal pattern. Establishing a single-cell sequencing technique, I explored questions that were once unattainable for this uncultured bacterium. First, I conducted a population analysis of the mutually exclusive interaction between Epulopiscium sp. type B and Naso tonganus in the Great Barrier Reef. I found evidence of allopatric speciation, governed by host migratory preferences between reef and island habitats. Maintaining genomic diversity for the symbiont depends on horizontal transmission by the host and rampant recombination to overcome genome purification in this polyploid bacteria. Next, I explored the distribution and diversity of co-resident symbiont populations across three Acanthurus species. Genomic insights into their metabolic potential confirmed that the two cohabitating epulos convert ammonia/urea to amino acids. However, differences among these epulo lineages for the ability to degrade dietary complex polysaccharides and diverse strategies for conserving energy suggest niche differentiation in this competitive environment. Lastly, by examining the transcriptomic profiles of an endospore-forming epulo in Naso unicornis, I have shown that the metabolic and reproductive life cycle of this epulo population is synchronized with the feeding/fasting cycles of the host. In conclusion, I suggest that the evolution of epulos is largely influenced by their dependency on surgeonfish hosts. A possible reason for successful transmission and maintenance of epulos in herbivorous surgeonfish worldwide is their ability to anticipate the circadian cycles of their host
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