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Item Phenotypic and Genotypic Effects of FlhC Mediated Gene Regulation in Escherichia Coli O157:H7(North Dakota State University, 2011) Sule, PreetiEscherichia coli (E.coli) 0157:H7, a pathogen belonging to the enterohemorrhagic group of E.coli, has long been a concern to human health. The pathogen causes a myriad of symptoms in humans, ranging from diarrhea and malaise to renal failure. Human infection with the spread of the pathogen is mainly attributed to consumption of contaminated food material such as meat. Decontamination of meat via sprays have to date been the most commonly practiced method to reduce contamination, which now has little relevance in the face of developing resistance by the pathogen. In the following study we investigated FlhC mediated gene regulation in E. coli 0157:H7 on the surface of meat, in an attempt to recognize FlhC regulated targets, which may ultimately serve as targets for the development of novel decontaminating sprays. Microarray experiments were conducted to compare gene expression levels between a parental E. coli 0157:H7 strain and its isogenic flhC mutant, both grown on meat. Putative FlhC targets were then grouped based on their function. Realtime PCR experiment was done to confirm the regulation. Additionally, experiments were done to investigate the phenotypic effects of the regulation. To test the effect of FlhC on biofilm formation, an ATP based assay was first developed in E.coli K-12, which has been detailed in the following dissertation. This assay was used to quantify biofilm biomass in E. coli 0157. Microarray experiments revealed 287 genes as being down regulated by FlhC. These genes belonged to functions relating to cell division, metabolism, biofilm formation and pathogenicity. Real-time PCR confirmed the regulation of 87% of the tested genes. An additional 13 genes were tested with real-time PCR. These belonged to the same functional groups, but were either not spotted on the microarray chips or had missing data points and were hence not included in the analysis. All 13 of these genes appeared to be regulated by FlhC. The phenotypic experiments performed elucidated that the FlhC mutants divided to 20 times higher cell densities, formed five times more biofilm biomass and were twice as pathogenic in a chicken embryo lethality assay, when compared to the parental strain. The following dissertation also reports the development of a combination assay for the quantification of biofilm that takes advantage of the previously mentioned ATP assay and the PhenotypeMicroarray TM (PM) system. The assay was developed using the parental E. coli strain AJW678 and later applied to its isogenic flhD mutant to elaborate on the differences in nutritional requirements between the two strains during biofilm formation. Metabolic modeling and statistical testing was also applied to the data obtained. This assay will be used in the future to study biofilm formation by the parental strain E. coli 0157:H7 strain and its isogenic FlhC mutants on single carbon sources, hence identifying potential metabolites which differentially support biofilm formation in the parental and the mutant strain.Item Gene Regulation in Biofilms(North Dakota State University, 2011) Samanta, PriyankarSessile 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.