Microbiological Sciences Masters Theses

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Browsing Microbiological Sciences Masters Theses by browse.metadata.department "Veterinary and Microbiological Sciences"

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    Gene Regulation in Biofilms
    (North Dakota State University, 2011) Samanta, Priyankar
    Sessile bacterial communities which form on the solid surface or solid-liquid interface are known as biofilms. Both single species and multispecies biofilms are characterized by an extracellular matrix of polymeric substances which gives them several hundred times more antibiotic resistances than a planktonic bacterial culture. Though bacteria are the most common causative agent of various diseases, because of the high antibiotic resistance, biofilms cause complications of various diseases like cystic fibrosis, prosthetic valve endocarditis, chronic pulmonary diseases, catheter-associated urinary tract infections and several other diseases. From past studies, quorum sensing has been established as a novel target mechanism against biofilms; in this study, the two-component signal transduction systems (2CSTSs) have been focused. Once better understood, 2CSTSs can serve as a novel drug target and prevention mechanism for biofilm associated diseases. According to prior high-throughput experiments and phenotype microarray experiments by our lab, several 2CSTSs like OmpR-EnvZ, RcsCDB along with the global regulator FlhD/FlhC were hypothesized to have an important effect on various developmental stages of biofilm formation. From that past study, we postulated that acetate metabolism may be an important aspect for biofilm formation. In this study, we tested and confirmed this hypothesis. We observed biofilms formed by several mutants in 2CSTS, as well as mutants in acetate metabolism, using Scanning Electron Microscopy (SEM). We found quantitative and qualitative differences in the biofilm of the acetate mutants when compared to their isogenic parental Escherichia coli strain. An additional mutation in rcsB with acetate mutant strains forms less clumpy biofilms whereas an additional mutation in dcuR results in the formation of less biofilms. So the structural and the quantitative differences of acetate mutant biofilms depend on additional mutations in rcsB and dcuR. Though a number of studies have been done on the temporal gene expression within biofilms, spatial gene expression of the mature biofilm is a big gap of knowledge. The future aim of this study is to study the temporal as well as the spatial gene expression of different 2CSTSs in the biofilm. In my MS thesis, I have constructed selected promoter fused GFP /RFP plasmids and some other fusion plasmids were purchased from the promoter collections from Open Biosystems, lastly E. coli AJW678 bacterial strains were transformed with these GFP /RFP fused plasmids. A 96 well microtiter plate assay was performed to study the temporal expression from the promoters by quantifying the fluorescence intensity in the planktonic culture. According to this experiment, the highest expression of flhD was after 20 hours whereas, the expression of ompR increases up to 7 days, which indicates that the flhD expresses earlier than ompR. The decreasing phase of flhD expression was paralleled by the sharpest increase in ompR expression as phosphorylated OmpR is an inhibitor of flhD expression.
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    Glucose Uptake by the Cellulolytic Rumen Anaerobe Bacteroides Succinogenes
    (North Dakota State University, 1986) Franklund, Clifton Victor
    Glucose uptake by the cellulclytic rumen anaerobe, Bacteroides succinogenes S85, was measured under conditions that maintained anaerobiosis and osmotic stability. This organism was found to possess a highly specific, active transport mechanism for glucose. Evidence for a phosphoenol-pyruvate:g1ucose phosphotransferase system was not detected. Compounds that inhibit electron transport systems (non-heme iron chelators, and sulfhydryl reagents) were effective inhibitors of glucose uptake. The strongest inhibitors were compounds (proton and metal ionophores) that interfere with maintenance of the proton motive force. Compounds which interfere with ATP synthesis also inhibited glucose uptake, but a role for ATP in energizing uptake could not be inferred from these results. Oxygen prevented glucose uptake (75% inhibition), reflecting possible active sulfhydryl centers (above) or autooxidation of electron transport components. The results suggest the fumarate reductase-coupled electron transport system of B. succinogenes can generate a proton motive force that is used to energize glucose uptake. Na+ and Li+. but not K+, stimulated glucose uptake and may partly account for the growth requirement of B. succinogenes for Na+. However, the data were insufficient to conclude that glucose uptake occurs by a Na+ symport mechanism. Spheroplasts of B. succinogenes transported glucose as well as whole cells, indicating glucose uptake is not dependent on a periplasmic glucose binding protein. A variety of sugars including the nonmetabolizable analog, [inversely proportional symbol]-methylglucoside. did not inhibit glucose uptake. Only cellobiose and 2-deoxyglucose were active and neither behaved as a competitive inhibitor. Metabolism of both sugars was probably responsible for the inhibition. Cellobiose-grcwn B. succinogenes showed a reduced ability to transport glucose compared to glucose-grown cells. This may indicate regulation of synthesis of the glucose carrier protein by cellobiose through a mechanism other than catabolite repression. Differences in the ability to transport glucose were detected between transition cells (transition from lag to log phase of growth) and log-phase cells. However, the differences were not due to different glucose transport mechanisms. Alterations in the structural integrity of the cell envelope, as reflected by osmotic- and cold-sensitivity features of transition and log cells, may have affected the glucose uptake abilities in these cell types.