Browsing by Author "Samanta, Priyankar"

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Gene Expression and Evolution in Escherichia Coli Biofilm
(North Dakota State University, 2014) Samanta, Priyankar
Biofilms can be defined as a complex aggregation of bacterial communities that involves many gene regulatory mechanisms, as well as evolutionary processes to increase biodiversity. Specific Aim 1 used a gene regulation approach to identity novel targets for the development of biofilm prevention and treatment techniques. The goal was to determine genes that get expressed early in biofilm development (prevention targets) and genes that get expressed late and in the outer layer of the biofilm (treatment targets). Biofilm formation is regulated by numerous regulators, including the two-component osmoregulator system EnvZ/OmpR, the colanic acid activator rcsCDB and the global regulator FlhD/FlhC. In this study, we determined the temporal and spatial expression of flhD, ompR and rcsB in E. coli k-12 AJW678 biofilm, as well as the gene expression of flhD in isogenic ompR and rcsB mutants. Results indicated that flhD was expressed early, and in the outer layer of the mature biofilm. We concluded that FlhD/FlhC would be the first target for novel prevention and treatment technique. One mechanism to increase biodiversity in biofilm is the insertion of transposon elements, which was investigated as Specific Aim 2. Insertion of IS elements into the flhD promoter resulted in increased motility in numerous E. coli K-12 strains has been shown in previous study. In this study, we recovered isolates from biofilm, where IS1 had inserted in the flhD promoter further downstream than in previously described strains. These isolates showed reduced motility. We also wanted to determine the effect of an IS element insertion on regulation of flhD expression by OmpR and RcsB in biofilm. Temporal and spatial gene expression of three different GFP-tagged flhD promoters was measured. The results indicated that IS5 insertion in the flhD promoter at the published hotspot did not have any effect on regulation of flhD expression by OmpR and RcsB in biofilm.
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.