Theses and Dissertations from UMD

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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM

More information is available at Theses and Dissertations at University of Maryland Libraries.

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    Characterization of a novel Escherichia coli exopolysaccharide and its biosynthesis by NfrB
    (2024) Fernando, Sashika Hansini Lakmali; Poulin, Myles B; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Biofilms are made from an association of bacterial cells and extracellular products dominated by a plethora of exopolysaccharides. Accumulating evidence have demonstrated that the bacterial second messenger cyclic-di-guanosine monophosphate (c-di-GMP) promotes the synthesis of these exopolysaccharides through direct allosteric activation of glycosyltransferase enzymes. The Escherichia coli inner membrane protein NfrB, which together with the outer membrane protein NfrA acts as a receptor system for phage N4, contains a N-terminal glycosyltransferase domain and C-terminal c-di-GMP binding domain. Recent research revealed that NfrB is a novel, c-di- GMP controlled glycosyltransferase that is proposed to synthesize a N-acetylmannosamine containing polysaccharide product, though the exact structure and function of this remains unknown. Nfr polysaccharide production impedes bacterial motility, which suggests a possible role of the Nfr proteins in bacterial biofilm formation. Here, we carry out in-vivo synthesis of novelNfr polysaccharide followed by its structural characterization. Preliminary data from MALDI- TOF mass spectrometry and Solid State 13C NMR spectroscopy indicated that the Nfr polysaccharide is mainly a homo polymer of poly-?-(1®4)-N-acetylmannosamine, bound to an aglycone. In addition, we report efforts to develop of a Nfr polysaccharide binding and detection tool, through the mutation of YbcH, a putative Nfr polysaccharide hydrolase enzyme. These studies advance the understanding of Nfr polysaccharide biosynthesis and could offer potential new targets for the development of antibiofilm and antibacterial therapies.
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    Food Safety Implications of Biofilms Formed by Resident Bacteria in Fresh-cut Processing Environments
    (2013) Liu, Tong; Lo, Yangming M.; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Biofilms on equipment surfaces can be vectors for cross-contaminations in food processing facilities. A particular problem is that biofilms can protect pathogenic bacteria from daily cleaning and disinfection operations. In the present study, microflora were recovered from fresh-cut processing facilities, identified, and tested for biofilm forming potential. Subsequently, dual-species biofilms of selected isolates and Escherichia coli O157:H7 were investigated. Approximately 30% of the isolates were potent biofilm formers, producing large amounts of biomass. A hundred and seventeen tested isolates were identified into 23 genera, including plant related bacteria and coliforms with some opportunistic pathogens. Dual-species biofilms formed by Burkholderia caryophylli or Ralstonia insidiosa and E. coli O157:H7 manifested increased biomass in comparison to their monocultures. Additionally, about a one-log unit increase of E. coli O157:H7 cell counts were observed for both dual-species biofilms. To test the effects of environmental factors on growth of R. insidiosa and E. coli O157:H7 in dual-species biofilms, factors tested included low temperature (10 oC), media with different composition of nutrient sources (10% TSB, M9, 1.25% Cantaloupe Juice) and a continuous culture system with limited nutrients. E. coli O157:H7 cell counts increased for all tested conditions. To examine bacterial localization within biofilms, confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) were used. Images showed distinct spatial distributions with E. coli O157:H7 commonly located at the bottom and also interspersed among R. insidiosa cells. To test the interactions with other pathogens, R. insidiosa was co-cultured with Listeria monocytogenes, Salmonella spp., and Shiga toxin-producing E. coli. Cells counts for 7 out of 9 tested pathogenic bacteria strains were increased (0.36-1.60 CFU log units). It is notable that the biomass formed by R. insidiosa and L. monocytogenes was much greater than those produced by other tested combinations. These results indicate that R. insidiosa could be a food safety risk in fresh-cut processing environments by providing protective habitats for pathogenic bacteria.
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    TOWARD A BETTER UNDERSTANDING OF THE CELLULAR, MOLECULAR AND GENETIC BASES OF THE RUGOSE MORPHOLOGY OF SALMONELLA TYPHIMURIUM
    (2005-08-05) Anriany, Yuda Adha; Joseph, Sam W; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The origin of rugose (wrinkled) colony morphology of Salmonella Typhimurium, which is formed only at stationary phase, low temperature, and under low osmolarity, is attributed to the production of an extracellular matrix and is associated with biofilm and pellicle formation, likely for survival strategies. The regulator CsgD is required for the synthesis of both matrix components: curli, encoded by the csgBAC operon, and cellulose, encoded by the bcs operon. Transcription of csgD, in turn, depends upon a number of transcriptional regulators such as SigmaS, OmpR and HNS. Using random mutagenesis, two groups of mutants with altered rugose phenotype were identified. The two mutations in the first group caused retardation of rugosity and altered waaG and ddhC, which are required for synthesis of the core and O antigen of lipopolysaccharide synthesis, respectively. Both mutants exhibited lack of motility, decreased levels of curli, and, especially in the waaG mutant, increased cellulose production. In media containing high osmolarity, both mutants produced more biofilms. Non-polar gene knockout and complementation performed on the waaG further confirmed these phenotypes in this transposon mutant. Thus, alteration in the LPS seemed to influence both curli and cellulose in opposing manners, and appeared to direct cells toward alternative pathways to produce biofilm matrix. The regulatory mutation in the second group affected hfq, and produced only minimal amounts of cellulose and curli protein. This phenotype was confirmed in an hfq deletion mutant. Transcriptional fusion between the csgB or csgD promoter and lacZ showed a drastic reduction in activity of both promoters in an hfq mutant compared to that in the wt. These were surprising results given the known function of Hfq as a post-transcriptional regulator, including in the regulation of SigmaS-encoding gene rpoS. However, when the promoter activity was measured in an rpoS hns background, where transcription continues under Sigma70, significant reduction was still shown in the hfq mutant. Deletion of the gene that codes for DsrA, a sRNA which, together with Hfq, is required for translation of rpoS at low temperatures, had minimal effect in both promoters. These results indicate that Hfq may regulate both promoters independent of SigmaS.