Microbial Physiology Commons^™

Investigating The Mechanisms Of Microbial Interactions Using A Model Cystic Fibrosis Polymicrobial Community, Bassam El Hafi

Dartmouth College Ph.D Dissertations

Cystic fibrosis (CF) airway microbial communities are complex ecosystems where interspecies interactions influence pathogen survival and disease progression. We leverage a CF-relevant in vitro four-species model composed of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica, alongside a microbiome dataset with over 4,000 respiratory samples from publicly available sequencing data of CF patient samples, to explore the dynamics of microbial interactions in CF. On a broader scale, we performed microbial network analysis to examine how the latest modulator therapy, elexacaftor/tezacaftor/ivacaftor (ETI), might influence CF microbial communities. Our analysis revealed a reduction in node connections and …

Metabolite Analysis To Understand Factors Limiting Microbial Fermentation At High Substrate Concentrations, Bishal D. Sharma

Dartmouth College Ph.D Dissertations

Clostridium thermocellum, a thermophilic anaerobic bacterium, holds promise as a biocatalyst for conversion of lignocellulosic biomass into biofuels. This microbe has been subjected to various metabolic engineering strategies, including the deletion of genes encoding secondary fermentation products such as H₂, formate, acetate, and lactate, as well as the expression of heterologous genes to redirect carbon flux toward ethanol production. Despite these efforts, achieving economically viable ethanol titers remains a challenge. We hypothesized that studying how intracellular metabolite concentrations change as fermentation stops could reveal metabolic bottlenecks or regulatory mechanisms limiting ethanol production at high titers. However, metabolomics studies at …

Biochemical And Molecular Analysis Of Anr-Dependent Microoxic Fitness Factors In Pseudomonas Aeruginosa, Stacie E. Balsam

Dartmouth College Ph.D Dissertations

Pseudomonas aeruginosa is a prominent opportunistic pathogen that is often found in microoxic environments like sites of infection and colony biofilms. P. aeruginosa can grow at oxygen (O2) at concentrations as little as 3 μM and also grows anaerobically by generating energy using nitrate as an alternate electron acceptor. An important way P. aeruginosa adapts to low O2 is through Anr, a transcription factor that upregulates many genes important for microoxic and anoxic fitness.

Specifically, Anr regulates mhr which encodes a hemerythrin protein (Mhr) that binds O2 with affinities relevant to microoxia and is also necessary for full fitness of …

Causes And Consequences Of Lasr Mutant Selection In Pseudomonas Aeruginosa Populations, Dallas L. Mould

Dartmouth College Ph.D Dissertations

Change is the only constant in life, and these changes, though random in nature, can have consequences. Quorum sensing is heterogeneous in phenotype and prone to negative selection. In P. aeruginosa, the regulator LasR is frequently non-functional in phylogenetically diverse isolates. Through repeated experimental evolution and mathematical modeling, we show that differences in growth enable lasR mutant evolutionary success and this requires a system enabling metabolic choices, known as carbon catabolite repression (or catabolite repression). The differences in catabolite repression between wild type and lasR mutants enable altered metabolite preferences, and the resulting differences in metabolic states enable intraspecies …

Development Of A Core Clostridium Thermocellum Kinetic Metabolic Model Consistent With Multiple Genetic Perturbations, Satyakam Dash, Ali Khodayari, Jilai Zhou, Evert K. Holwerda, Daniel Olson, Lee Lynd, Costas Maranas

Dartmouth Scholarship

Clostridium thermocellum is a Gram-positive anaerobe with the ability to hydrolyze and metabolize cellulose into biofuels such as ethanol, making it an attractive candidate for consolidated bioprocessing (CBP). At present, metabolic engineering in C. thermocellum is hindered due to the incomplete description of its metabolic repertoire and regulation within a predictive metabolic model. Genome-scale metabolic (GSM) models augmented with kinetic models of metabolism have been shown to be effective at recapitulating perturbed metabolic phenotypes.

In this effort, we first update a second-generation genome-scale metabolic model (iCth446) for C. thermocellum by correcting cofactor dependencies, restoring elemental and charge balances, …

The Armadillo Repeat Protein Pf16 Is Essential For Flagellar Structure And Function In Plasmodium Male Gametes, Ursula Straschil, Arthur M. Talman, David J. P. Ferguson, Karen A. Bunting, Zhengyao Xu, Elizabeth Bailes, Robert E. Sinden, Anthony A. Holder, Elizabeth F. Smith

Dartmouth Scholarship

Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the world's population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasite's flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that …

Parasites Alter Community Structure, Chelsea L. Wood, James E. Byers, Kathryn L. Cottingham, Irit Altman

Dartmouth Scholarship

Parasites often play an important role in modifying the physiology and behavior of their hosts and may, consequently, mediate the influence hosts have on other components of an ecological community. Along the northern Atlantic coast of North America, the dominant herbivorous snail Littorina littorea structures rocky intertidal communities through strong grazing pressure and is frequently parasitized by the digenean trematode Cryptocotyle lingua. We hypothesized that the effects of parasitism on host physiology would induce behavioral changes in L. littorea, which in turn would modulate L. littorea's influence on intertidal community composition. Specifically, we hypothesized that C. lingua …

Conservation Of The Pho Regulon In Pseudomonas Fluorescens Pf0-1, Russell D. Monds, Peter D. Newell, Julia A. Schwartzman, George A. O'Toole

Dartmouth Scholarship

The Pho regulon integrates the sensing of environmental inorganic phosphate (Pi) availability with coregulation of gene expression, mediating an adaptive response to Pi limitation. Many aspects of the Pho regulon have been addressed in studies of Escherichia coli; however, it is unclear how transferable this knowledge is to other bacterial systems. Here, we report work to discern the conservation of the Pho regulon in Pseudomonas fluorescens Pf0-1. We demonstrate by mutational studies that PhoB/PhoR and the Pst system have conserved functions in the regulation of Pi-induced phosphatase activities, as well as expression of other Pi-regulated genes. A genetic screen was …

Susceptibility Of Biofilms To Bdellovibrio Bacteriovorus Attack, Daniel Kadouri, George A. O'Toole

Dartmouth Scholarship

Biofilms are communities of microorganisms attached to a surface, and the growth of these surface attached communities is thought to provide microorganisms with protection against a range of biotic and abiotic agents. The capability of the gram-negative predatory bacterium Bdellovibrio bacteriovorus to control and reduce an existing Escherichia coli biofilm was evaluated in a static assay. A reduction in biofilm biomass was observed as early as 3 h after exposure to the predator, and an 87% reduction in crystal violet staining corresponding to a 4-log reduction in biofilm cell viability was seen after a 24-h exposure period. We observed that …

Protoporphyrinogen Oxidation, A Step In Heme Synthesis In Soybean Root Nodules And Free-Living Rhizobia., Nicholas J. Jacobs, Susan E. Borotz, Mary Lou Guerinot

Dartmouth Scholarship

Extracts of the crude bacteroid fraction of symbiotically grown Bradyrhizobium japonicum were much more active in oxidizing protoporphyrinogen to protoporphyrin than were extracts of cells grown under free-living conditions, especially when assayed in atmospheres containing only traces of oxygen. This correlates with the higher heme content of the microaerophilic nodules. Furthermore, the high level of oxidative activity in the crude bacteroid fraction was associated with an uncharacterized membrane fraction, probably of plant origin, that was separable from the bacteroids by Percoll gradient centrifugation.