Browsing by Subject "Bacterial genetics"
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Item Bioenergetics of DNA Transport in Bacillus Subtilis(Texas Tech University, 1971-12) LaRue, Michael KingNot Available.Item Cloning of genes necessary for survival of Erwinia chrysanthemi in plant tissue(Texas Tech University, 1997-12) Oner, FusunErwinia chrysanthemi is an important plant pathogenic bacterium that causes soft-rot disease in many plants. The pectin-degrading enzymes secreted by the bacterium appear to be the pathogenic agents. These enzymes degrade the pectinaceous component of the plant cell wall into monomers, saturated and unsaturated dimers, trimers and oligomers of galacturonic acid that are taken into the bacterial cell to be used as carbon and energy sources and for pectolytic enzyme induction. This indicates that the transport of these compounds is a vital step in the pathogenicity of the bacterium. The uptake system for the dimer molecuIe,digalacturonic acid (dGA), in E. chrysanthemi EC 16 was studied and shown to be a separate system from the galacturonic acid (GA) uptake system. Two dGA uptake mutants, T5 and T9, were constructed from E. chrysanthemi EC 16 by transposon mutagenesis. Inverse Polymerase Chain Reaction (IPCR) was used to isolate the genes disrupted by the transposon Tn5 insertion. The 1-kb IPCR fragment obtained was sequenced after digesting with HaelU to remove the primer from one end, and the sequence was searched in GenBank and found to be 53% homologous to H. influenzae citrate lyase beta chain based on the deduced amino acid sequence. The 5' end of exwr was used to probe the EC 16 genomic library to identify genes vrith common regulatory sequences to exuT and presumably dgaT. A nucleotide sequence of 735 bp was identified which contained the partial sequence for both exuT and uxaCA operons. This region contained upstream sequences of exuT from E. chrysanthemi EC 16 that have not been previously identified. Additionally, a highly conserved cAMPCRP binding site is also present in this region.Item In vitro characterization of cAMP receptor protein mutated at position 127(Texas Tech University, 1996-12) Leu, Sew-FenThe cyclic adenosine 3',5' monophosphate (cAMP) receptor protein (CRP) complexed with cAMP binds a region upstream of the lactose operon promoter (lacF) to facilitate RNA polymerase (RNAP) recognition of lacF. The DNA binding and lacF activation characteristics of four mutant forms of CRP were investigated. These proteins contained a single amino acid substitution (either cysteine [C], glycine [G], isoleucine [I] or serine [S]) for threonine (T) at position 127. All but the T127G CRP mediated lacF activation in the presence of cAMP. CRPicAMP-mediated lacF activity differed for the T127C, T127I and T127S forms of CRP. The level of lacF activation observed for T127C CRP was comparable to wild-type (WT) CRP. The levels mediated by the T127I CRP or the T127S CRP were about one-half that of WT CRP. Transcription reaction mixtures that contained each of these forms of CRP differed in the rate of formation of the /ACPIRNAP open complex. DNase I footprint analysis of complexes formed between lacF DNA and the position 127 CRP mutants showed the following. First, stable DNAiCRP: cAMP complexes formed only in reactions that contained WT CRP or T127C CRP. Second, stable DNA:CRP:cAMP:RNAP complexes formed in reactions that contained the WT, T127C, T127G, T127I or T127S CI. Protection of the lacF CRP-binding site by the T127G, T127I or T127S CRP was less complete than for WT or T127C CRP. Third, stable, non-specific DNAiCRP complexes formed at high CRP concentrations (in the absence of cAMP) in reactions that contained the T127C or the T127G CRP; cAMP addition produced site-specific DNA binding for T127C CRP but not for T127G CRP. Non-specific DNA binding was not observed for the WT, T127I or T127S forms of CRP. The results of this study show that position 127 amino acid substitutions in CRP differentially affect cAMP-mediated changes in CRP structure that: (1) minimize non-specific DNA affinity and introduce high affinity site-specific DNA binding in the absence of RNAP and (2) establish the productivity of CRPiRNAP interactions at lacF. Importantly, these data also show that these position 127 amino acid substitutions have little effect on CRP:RNAP:/flcP binding cooperativity.Item Probing the structure and reactivity of the Bacillus cereus 5/B/6 metallo-Beta-lactamase through site-directed mutagenesis and mechanism-based inactivation(Texas Tech University, 1997-12) Yi, XiaomingThe discovery of peniciUin by Sir Alexander Fleming in 1929 was accidental but revolutionary. In 1938, Chain, Foley and their associates purifíed the active agent from a culture filtrate of Penicilium notatum. The subsequent introduction of penicillin into clinical use revolutionized the treatment of many infectious diseases and started a new era of chemotherapy in the history of medicine (Abraham, 1981).