Molecular analysis of the anaerobic-inducible operon nrdDG from Salmonella typhimurium.

by Ng Wai-Leung.Thesis submitted in: August 1997.Thesis (M.Phil.)--Chinese University of Hong Kong, 1998.Includes bibliographical references (leaves 135-144).Title page --- p.iThesis Committee --- p.iiAbstract --- p.iiiAcknowledgments --- p.vAbbreviations --- p.viTable of contents --- p.viiList of figures --- p.xList of tables --- p.xiiiChapter Chapter 1. --- General introduction --- p.1Chapter Chapter 2. --- Literature reviewChapter 2.1 --- Biosynthesis of deoxyribonucleotide triphosphates --- p.3Chapter 2.2 --- Ribonucleotide reductase --- p.6Chapter 2.2.1 --- Class I ribonucleotide reductase --- p.6Chapter 2.2.2 --- Class II ribonucleotide reductase --- p.13Chapter 2.2.3 --- Class III ribonucleotide reductase --- p.14Chapter 2.3 --- Proposed mechanism for ribonucleotide reduction --- p.17Chapter 2.4 --- Allosteric control of ribonucleotide reductase --- p.21Chapter 2.4.1 --- Allosteric control of class I ribonucleotide reductase --- p.21Chapter 2.4.2 --- Allosteric control of class II and class III ribonucleotide reductases --- p.23Chapter 2.5 --- Evolution of ribonucleotide reductase --- p.25Chapter 2.6 --- Central metabolism pathways of enteric bacteria --- p.28Chapter 2.7 --- Regulation of gene expression by oxygen in enteric bacteria --- p.33Chapter 2.7.1 --- Regulation of gene expression by Fnr --- p.33Chapter 2.7.2 --- Regulation of gene expression by AcrAB --- p.39Chapter 2.7.3 --- Regulation of gene expression by NarXL and NarQP --- p.42Chapter 2.7.4 --- Other aspects in anaerobic gene expression --- p.45Chapter 2.7.5 --- Relationship between NrdD and anaerobic metabolism --- p.45Chapter 2.8 --- Objectives --- p.46Chapter Chapter 3. --- Molecular cloning and sequencing of nrdDG operon from Salmonella typhimuriumChapter 3.1 --- Introduction --- p.47Chapter 3.2 --- Material and methods --- p.48Chapter 3.2.1 --- Bacterial strains and bacteriophages --- p.48Chapter 3.2.2 --- Culture media --- p.48Chapter 3.2.3 --- Preparation of lambda lysate and phage DNA --- p.48Chapter 3.2.3.1 --- Plating out pf lambda phage and preparation of plate lysate --- p.48Chapter 3.2.3.2 --- Preparation of lambda lysate stock --- p.49Chapter 3.2.3.3 --- Preparation of lambda phage DNA --- p.50Chapter 3.2.4 --- Long distance polymerase chain reaction (LD-PCR) of nrdDG gene fragment --- p.51Chapter 3.2.5 --- Restriction enzyme digestion of LD-PCR products and subcloning of restriction fragments --- p.52Chapter 3.2.6 --- Confirmation of recombinants by colony-PCR --- p.53Chapter 3.2.7 --- Preparation of plasmid DNA by alkaline lysis using Wizard´ёØ Plus Miniprep DNA Purification System (Promega) --- p.54Chapter 3.2.8 --- DNA cycle sequencing by using dye-labeled dideoxy chain terminator and data collection --- p.55Chapter 3.2.9 --- Computer software for analyzing and manipulating DNA sequences --- p.57Chapter 3.3 --- Results --- p.59Chapter 3.3.1 --- Preparation of lambda DNA --- p.59Chapter 3.3.2 --- Long distance PCR amplification of nrdDG from lambda DNA --- p.59Chapter 3.3.3 --- Restriction digestion of LD-PCR products --- p.61Chapter 3.3.4 --- Subcloning of LD-PCR restriction fragments --- p.61Chapter 3.3.5 --- Miniprep of plasmid DNA from recombinants and verification of nrdDG identities --- p.64Chapter 3.3.6 --- Nucleotide sequence of nrdDG --- p.66Chapter 3.4 --- Discussions --- p.72Chapter 3.4.1 --- Sequence analysis of S. typhimurium nrdDG --- p.72Chapter 3.4.2 --- Experimental design --- p.79Chapter Chapter 4. --- Transcriptional regulation of anaerobic ribonucleotide reductase in Salmonella typhimurium in aerobic and anaerobic environmentsChapter 4.1 --- Introduction --- p.84Chapter 4.2 --- Materials and methods --- p.86Chapter 4.2.1 --- Bacteria and bacteriophages strainsChapter 4.2.2 --- Culture media --- p.86Chapter 4.2.3 --- Construction and characterization of oxrA mutant --- p.87Chapter 4.2.3.1 --- Preparation of P22 lysate of TN2336 --- p.87Chapter 4.2.3.2 --- P22 transduction for construction of oxrA mutant --- p.87Chapter 4.2.3.3 --- Characterization of oxrA mutant --- p.87Chapter 4.2.4 --- Extraction of bacterial RNA by hot phenol method --- p.88Chapter 4.2.5 --- Formaldehyde gel electrophoresis of RNA --- p.88Chapter 4.2.6 --- Reverse transcriptase polymerase chain reaction (RT-PCR) of nrdD transcript --- p.89Chapter 4.2.7 --- Transfer of DNA/RNA to solid support --- p.90Chapter 4.2.7.1 --- Transfer of DNA to solid support by Southern blotting --- p.90Chapter 4.2.7.2 --- Transfer of RNA to solid support by Northern blotting --- p.91Chapter 4.2.7.3 --- RNA Dot blot --- p.91Chapter 4.2.8 --- Preparation of radioactive-labeled probes for hybridization --- p.92Chapter 4.2.8.1 --- Synthesis of radioactive-labeled probes by labeling --- p.92Chapter 4.2.8.2 --- Preparation of RNA probe by in vitro transcription --- p.93Chapter 4.2.9 --- Hybridization and membrane washing conditions --- p.95Chapter 4.2.10 --- Normalization of samples by 16S ribosomal RNA (rRNA) --- p.95Chapter 4.3 --- Results --- p.97Chapter 4.3.1 --- Preparation of RNA --- p.97Chapter 4.3.2 --- RT-PCR of nrdD transcript --- p.97Chapter 4.3.3 --- Northern blot analysis of nrdD transcript --- p.103Chapter 4.3.4 --- Dot blot hybridization analysis of nrdD expression in an oxrA mutant --- p.103Chapter 4.4 --- Discussions --- p.107Chapter 4.4.1 --- Expression of nrdD of S. typhimurium in aerobic and anaerobic environments --- p.107Chapter 4.4.2 --- Experimental design --- p.110Chapter Chapter 5. --- Characterization of nrdD::Tn10 mutant of S. typhimuriumChapter 5.1 --- Introduction --- p.112Chapter 5.2 --- Materials and methods --- p.112Chapter 5.2.1 --- Bacteria and bacteriophages strains --- p.113Chapter 5.2.2 --- Transduction of zzz-3875::Tn10 to S. typhimurium --- p.113Chapter 5.2.3 --- Characterization of zzz-3875::Tn10 by Southern hybridization --- p.113Chapter 5.2.3.1 --- Preparation of genomic DNA from S. typhimurium --- p.113Chapter 5.2.3.2 --- Restriction enzyme digestion of genomic DNA and Southern hybridization --- p.114Chapter 5.2.4 --- Characterization of growth pattern of nrdD::Tn10 mutant --- p.115Chapter 5.3 --- Results --- p.116Chapter 5.3.1 --- Characterization of zzz-3 875: :Tn7 0 in S. typhimurium --- p.116Chapter 5.3.2 --- Characterization of growth pattern of nrdD mutant --- p.120Chapter 5.4 --- Discussions --- p.125Chapter Chapter 6. --- General DiscussionsChapter 6.1 --- General discussions --- p.131Chapter 6.2 --- Further studies --- p.134References --- p.13