UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
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 given thesis/dissertation in DRUM.
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Item Using Recombinant PCR to Study Sequence Polymorphisms in a Family of Compact Albumin Binding Domains(2005-11-14) Rozak, David Anthony; Bryan, Philip N; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Sixteen homologs of a compact albumin binding domain were previously identified in six proteins and four bacterial species. These domains, which exhibit varied affinities for different albumins, have been shown to support bacterial growth in vitro, and may contribute to host specificity. This dissertation describes the development of a robust PCR-based recombination technique, which is applied to representatives of the albumin binding domain to identify and understand the impact of sequence polymorphisms on domain stability and function. Analysis of phage-selected recombinants highlights the potential impact of multiple mutations in stabilizing the selected domains and improving albumin binding through gains in hydrophilic surface area, direct modifications to the binding interface, and subtle changes in the position of the third helix. The most common mutant was encoded by three fourths of the selected phage and exhibited 5 and 10 fold increases in human and guinea pig albumin binding constants compared to the wild type streptococcal domain (G148-GA3). This study serves to validate further the application of in vitro recombination and phage display in the analysis of sequence polymorphisms. The recombination technique itself is shown to be well suited for producing multiple recombination events among compact heterologous domains and appears to offer several advantages over traditional DNA shuffling techniques.Item Patterns of reactivity of lantibiotics subtilin and nisin with molecular targets in Bacillus cereus and Bacillus subtilis 168(2005-01-21) Kuntumalla, Srilatha; Hansen, J Norman; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)ABSTRACT Title of Dissertation: PATTERNS OF REACTIVITY OF LANTIBIOTICS SUBTILIN AND NISIN WITH MOLECULAR TARGETS IN Bacillus cereus AND Bacillus subtilis 168 Srilatha Kuntumalla, Doctor of Philosophy, 2005 Dissertation Directed By: Professor J. Norman HansenDepartment of Chemistry and Biochemistry Subtilin and nisin belong to a unique class of antibiotics called lantibiotics that contain unusual dehydro and lanthionine amino acid residues. The gene-encoded antimicrobial peptides subtilin and nisin exhibit bactericidal effects against several Gram-positive bacteria and also inhibit bacterial spore outgrowth. Subtilin and nisin are structural analogs and possess similar mechanisms of antimicrobial action. Although nisin is very stable, subtilin previously isolated was highly unstable with loss of biological activity observed during storage. Subtilin isolated in this work using hydrophobic interaction chromatography was very stable, with biological activity retained for at least a few months after isolation. The possibility that specificity of subtilin and nisin towards sensitive Gram-positive bacteria is due to interaction of these lantibiotics with specific target proteins in susceptible bacteria was explored in this work. Phage display experiments performed to detect peptides interacting with subtilin identified a 12-mer peptide with a KTTLL motif found in ATP binding proteins such as ABC transporters and protein synthesis initiation factor IF-2 (~78 kDa). Binding of subtilin to specific ABC transporters in bacterial cell membrane would contribute to its specificity. Binding of subtilin to IF-2 would result in inhibition of protein synthesis suggesting an alternative mechanism of action for subtilin. Experiments performed to determine the nature of interaction of subtilin and nisin with bacterial cellular proteins detected both covalent and non-covalent interactions. The covalent interactions between bacterial proteins and subtilin or nisin were stable on boiling in SDS and analyzing by SDS-PAGE. These stable covalent adducts indicated that the electrophilic dehydro residues of subtilin and nisin were probably involved in covalent attachment with specific nucleophilic groups in bacterial protein targets. Covalent attachment of an antibiotic to its bacterial target has been previously observed with only a few antibiotics. Sites of nisin attachment to bacterial spores as visualized by electron microscopy showed nisin binds to highly localized regions on spore surfaces. Attempts to identify bacterial protein targets of subtilin and nisin using monomeric avidin and anti-FITC columns, respectively, resulted in isolation of proteins in ~70-80 kDa range. Further characterization of these proteins should help in understanding the specificity and antimicrobial mechanism of action of nisin and subtilin.