abstract / Biological Sciences / Doctoral / Doctor of Philosophy

Geok-yen Yeo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 225-233). / Title page --- p.i / Members of Thesis Advisory Committee --- p.ii / Abstract --- p.iii -iv / Acknowledgments --- p.v / Dedication --- p.vi / Table of Contents --- p.vii -xi / Chapter CHAPTER 1 --- INTRODUCTION --- p.1-31 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Fermentation --- p.1 / Chapter 1.3 --- Growth in Escherichia coli --- p.3 / Chapter 1.3.1 --- Aerobic growth in Escherichia coli --- p.3 / Chapter 1.3.2 --- The regulation of enzyme synthesis during cell metabolism --- p.7 / Chapter 1.3.3 --- Anaerobic growth in E. coli --- p.8 / Chapter 1.3.4 --- Anaerobic regulation by the transcriptional regulator Fnr --- p.12 / Chapter 1.3.5 --- "The case for ""Pasteur Control Proteins"" (PCP)" --- p.13 / Chapter 1.4 --- The family of alcohol dehydrogenases : An overview --- p.15 / Chapter 1.4.1 --- Molecular characteristics of alcohol dehydrogenases --- p.17 / Chapter 1.4.2 --- Residue conservation in alcohol dehydrogenases --- p.24 / Chapter 1.4.3 --- The effect of amino acid substitution on substrate specificity --- p.25 / Chapter 1.5 --- Alcohol dehydrogenases in bacteria --- p.28 / Chapter 1.5.1 --- Alcohol dehydrogenase in E. coli --- p.28 / Chapter 1.6 --- Aims of this study --- p.30 / Chapter CHAPTER 2 --- MATERIALS & METHODS --- p.32 -90 / Chapter 2.1 --- Bacterial strains --- p.32 / Chapter 2.2 --- Plasmids --- p.32 / Chapter 2.2.1 --- "Low copy number plasmid, pTJS75Km" --- p.32 / Chapter 2.2.2 --- "High copy number plasmid, pUC18" --- p.33 / Chapter 2.3 --- Bacterial culture media and solutions --- p.39 / Chapter 2.3.1 --- Luria Bertani (LB) medium --- p.39 / Chapter 2.3.2 --- L-Broth + MOPS --- p.39 / Chapter 2.3.3 --- "R medium, containing Triphenyltetrazolium chloride-ethanol (TTC-EtOH)" --- p.40 / Chapter 2.3.4 --- SOB and SOC media --- p.41 / Chapter 2.3.5 --- M9 Glucose medium --- p.42 / Chapter 2.3.6 --- Terrific Broth (TB) --- p.42 / Chapter 2.3.7 --- Rich Broth (RB) --- p.43 / Chapter 2.3.8 --- Antibiotic solutions --- p.43 / Chapter 2.4 --- Restriction endonucleases and other enzymes --- p.44 / Chapter 2.5 --- Isolation of chromosomal DNA --- p.45 / Chapter 2.5.1 --- Preparation of chromosomal DNA by spooling --- p.45 / Chapter 2.5.2 --- Preparation of chromosomal DNA by cesium chloride density gradient --- p.48 / Chapter 2.6 --- Isolation of plasmid DNA --- p.50 / Chapter 2.6.1 --- Large-scale preparation of plasmid by CsCl density gradient --- p.50 / Chapter 2.6.2 --- Small-scale preparation of plasmid DNA --- p.54 / Chapter 2.6.2. --- A Boiling method --- p.54 / Chapter 2.6.2. --- B Alkaline Lysis method --- p.55 / Chapter 2.6.3 --- Preparation of plasmid DNA by Qiagen columns --- p.56 / Chapter 2.7 --- Purification of DNA --- p.59 / Chapter 2.7.1 --- Ethanol precipitation --- p.59 / Chapter 2.7.2 --- Concentration and desalting using Centricon columns --- p.59 / Chapter 2.7.3 --- Purification of DNA by Geneclean procedure --- p.61 / Chapter 2.8 --- DNA cloning techniques --- p.63 / Chapter 2.8.1 --- Restriction endonuclease digestion --- p.63 / Chapter 2.8.2 --- Agarose-ethidium bromide gel electrophoresis --- p.65 / Chapter 2.8.2. --- A Gel loading buffer --- p.66 / Chapter 2.8.2. --- B Electro-elution of DNA --- p.67 / Chapter 2.8.3 --- Size fractionation --- p.68 / Chapter 2.8.3. --- A Salt gradient fractionation --- p.68 / Chapter 2.8.3. --- B Sucrose gradient --- p.70 / Chapter 2.8.4 --- Dephosphorylation of restriction-enzyme digested vector plasmid using calf intestinal phosphatase (CIP) --- p.71 / Chapter 2.8.5 --- Ligation of vector and insert --- p.72 / Chapter 2.8.6 --- Preparation of competent cells --- p.73 / Chapter 2.8.7 --- DNA transformation --- p.75 / Chapter 2.8.7.A --- By heat shock --- p.75 / Chapter 2.8.7.B --- By electroporation --- p.75 / Chapter 2.9 --- Screening for adhC transformants --- p.78 / Chapter 2.9.1 --- Screening for adhC clones --- p.78 / Chapter 2.9.2 --- Screening for pUC18 transformants --- p.79 / Chapter 2.10 --- Confirmation of adhC clones --- p.80 / Chapter 2.10.1 --- Reproduction of red colonies on R plates and antibiotic resistance --- p.80 / Chapter 2.10.2 --- T7 phage test for E. coli strains --- p.80 / Chapter 2.10.3 --- Plasmid size determination --- p.82 / Chapter 2.10.4 --- Re-transformation into E. coli host strains --- p.82 / Chapter 2.10.5 --- Physiological study of adhC clones --- p.83 / Chapter 2.10.6 --- Alcohol dehydrogenase assay --- p.84 / Chapter 2.11 --- The dye-binding method of protein determination --- p.87 / Chapter 2.12 --- Special procedures --- p.88 / Chapter 2.12.1 --- Generation of adh clones with deletions --- p.88 / Chapter 2.12.2 --- Sequencing reactions --- p.89 / Chapter CHAPTER 3 --- RESULTS: PART I Cloning and Restriction Mapping of the adhC mutation in a low copy number plasmid vector --- p.91 -122 / Chapter 3.1 --- Introduction: Cloning strategy --- p.91 / Chapter 3.2 --- Cloning of the adh mutation from strain CC2807B (an ADH overproducing mutant strain) in pTJS75Km --- p.93 / Chapter 3.2.1 --- Construction of the 'HK' clones --- p.93 / Chapter 3.3 --- Restriction mapping of the adh clones --- p.101 / Chapter 3.4 --- Subcloning the adhC insert --- p.110 / Chapter 3.4.1 --- Construction of plasmid pHK14 --- p.110 / Chapter 3.4.2 --- Construction of plasmid pHK15 --- p.115 / Chapter 3.4.3 --- Construction of plasmid pSS22 --- p.121 / Chapter 3.5 --- Remarks concerning the clones --- p.121 / Chapter CHAPTER 4 --- RESULTS:PART II Cloning and Sequencing of the adhC mutation in a high copy number plasmid vector --- p.123 -148 / Chapter 4.1 --- Introduction --- p.123 / Chapter 4.1.1 --- Choice of sequencing strategy --- p.123 / Chapter 4.1.2 --- An attempt to eliminate clone instability --- p.124 / Chapter 4.2 --- Subcloning of adh insert in pUC18 --- p.125 / Chapter 4.2.1 --- Study of adh clone EPR --- p.125 / Chapter 4.2.2 --- Re-construction of plasmid pEPR ( = pEE5) --- p.126 / Chapter 4.2.3 --- Construction of plasmids pEH2 and pEH3 --- p.127 / Chapter 4.2.4 --- Construction of a nested deletion library --- p.138 / Chapter CHAPTER 5 --- RESULTS : PART III Sequencing of the Mutation --- p.149 -177 / Chapter 5.1 --- Nucleotide sequencing --- p.149 / Chapter 5.2 --- Sequencing of the cloned adhC gene insert --- p.150 / Chapter 5.3 --- Analysis of the sequenced DNA by DNASIS computer software --- p.151 / Chapter 5.3.1 --- Search for codons associated with initiation and termination of transcription using the open reading frame (ORF) search --- p.151 / Chapter 5.3.2 --- Translation of the nucleotide sequence at the open reading frame (start 223 - end 2896) --- p.152 / Chapter 5.4 --- Search for DNA sequence homology with known DNA sequences --- p.152 / Chapter 5.4.1 --- Sequence homology of the structural gene (nucleotide # 223- #28%) : Two nucleotide changes revealed in DNA sequence of the structural gene adhE of Escherichia coli --- p.153 / Chapter 5.4.2 --- adhC mutation is due to changes in two amino acids --- p.153 / Chapter 5.4.3 --- The DNA sequence 5' of the mutated structural gene (upstream sequence) --- p.155 / Chapter 5.4.4 --- The DNA sequence 3' of the mutated structural gene (downstream sequence) --- p.156 / Chapter 5.5 --- Comparisons between the computer-predicted properties of the mutant and wild-type protein --- p.156 / Chapter 5.5.1 --- Prediction of the alcohol dehydrogenase protein secondary structure by the Robson Method --- p.156 / Chapter 5.5.2 --- Isoelectric point prediction --- p.156 / Chapter CHAPTER 6 --- RESULTS : PART IV Comparative Studies of Alcohol Dehydrogenase Expressionin adhC Strains and Clones --- p.178 -203 / Chapter 6.1 --- Introduction --- p.178 / Chapter 6.1.1 --- Basis for the alcohol dehydrogenase assay --- p.178 / Chapter 6.1.2 --- Choice of assay method --- p.179 / Chapter 6.1.3 --- Points to consider for ADH assay --- p.179 / Chapter 6.2 --- General growth characteristics of bacterial strains --- p.181 / Chapter 6.2.1 --- Plate cultures --- p.181 / Chapter 6.2.2 --- Overnight liquid cultures --- p.183 / Chapter 6.2.3 --- Batch liquid cultures --- p.183 / Chapter 6.2.4 --- ADH activity of strain CC2807B --- p.190 / Chapter 6.2.5 --- Comparison of ADH activity --- p.192 / Chapter 6.3 --- Investigating the mutated ADH enzyme --- p.197 / Chapter 6.3.1 --- Oxygen inactivation of the mutated enzyme --- p.197 / Chapter 6.3.2 --- Thermostability of the mutated enzyme --- p.201 / Chapter CHAPTER 7 --- DISCUSSION --- p.204 -220 / Chapter 7.1 --- Cloning of the adhC mutation --- p.204 / Chapter 7.1.1 --- Instability of clones in plasmid vector pUC18 --- p.204 / Chapter 7.1.2 --- Eliminating 'toxic' genes adjacent to adh locus --- p.207 / Chapter 7.1.3 --- Cloning in pTJS75Km low copy number vector --- p.208 / Chapter 7.2 --- DNA sequence of the adhC clones --- p.211 / Chapter 7.2.1 --- The basis for sequencing pUC 18-derived clones --- p.211 / Chapter 7.2.2 --- Homology to known alcohol dehydrogenases (ADH) sequences --- p.213 / Chapter 7.3 --- Findings concerning the adhC mutation --- p.217 / Chapter 7.3.1 --- How amino acid substitutions may affect an enzyme --- p.217 / Chapter 7.3.2 --- Physiological aspects of the bacterial cell due to the mutated enzyme --- p.218 / Chapter 7.4 --- Conclusions --- p.220 / APPENDICES --- p.221 -224 / REFERENCES --- p.225 -233

The glpF gene, which encodes a cytoplasmic membrane protein that facilitates the diffusion of glycerol into the cell, and the glpK gene, which encodes glycerol kinase, map near minute 88 on the linkage map of Escherichia coli K-12. In the present work, the nucleotide sequence of the 843 base Pair glpF gene, 430 base pairs of the glipK gene, and the intervening sequence between the two genes were determined. The control region for the glpFK operon was identified and sequenced. The gipK gene product was purified to near homogeneity by streptomycin sulfate and ammonium sulfate fractionation with subsequent DEAE Sephadex chromatography. N-terminal amino acid analysis identified the startpoint of translation for the gipK gene. The transcription start site was identified 71 base pairs upstream from the proposed translation start codon for glpF. Preceding the transcription start site were -10 and -35 sequences Similar to the consensus sequences for gram bacterial promoter elements. DNase I footprinting was used to identify two binding sites for the CAMP-cAMP receptor protein (CRP) complex upstream from and overlapping the putative -35 sequence. Four binding sites for the glp repressor were located sequentially along the DNA extending from -89 (relative to the start point of transcription) to within the -10 region. Two additional repressor binding sites were identified within the glpK coding region. Interaction of these operator sites with those in the control region was identified. The affinity of the gilp repressor for the control regions of the glpD, glpACB-gipTQ, and glpFK operons was compared by titration studies using a strain harboring a glpT:lacZ fusion and a glpRⁿ mutation. / Ph. D.

The heat shock response is a highly conserved genetic mechanism which is induced by a wide range of environmental stimuli. Although intensively studied in both prokaryotes and eukaryotes, no regulatory mechanism has been identified by which the environmental stimuli affect expression of the heat shock genes. In addition, although many inducers of the heat shock response are known to cause DNA damage, the role of heat shock in repair of DNA damage remains unclear. Mutants of Escherichia coli defective in the recA, uvrA, and xthA genes are more sensitive to heat than wild type. However, these mutants are able to develop thermotolerance, suggesting that thermotolerance is an inducible response capable of repairing heat-induced DNA damage independent of recA, uvrA, and xthA. Thermotolerance itself is shown to depend on the dnaK gene, directly linking the E. coli heat shock response to thermotolerance. In addition, the dnaK mutant is sensitive to heat and H₂O₂, but not to UV suggesting that the DnaK protein may function to protect cells from the specific DNA damage caused by heat and H₂O₂. An E. coli grpE mutant was found to be substantially more resistant to 50°C heat treatment than wild type. However, grpE⁻ cells have the same H₂O₂ and UV sensitivity as wild type. This implies that the conditions, for which a grpE mutation is beneficial, are unique to heat exposure and are not caused by H₂O₂ or UV exposure. Furthermore, heat shock protein synthesis occurs sooner in the grpE mutant than in wild type, indicating that the grpE gene product of E. coli may act as a negative regulator of the heat shock response. An adenyl cyclase deletion mutant of E. coli (cya) failed to exhibit a heat shock response even after 30 min. at 42°C. Furthermore, a presumptive cyclic AMP receptor protein (CRP) binding site exists within the promoter region of the E. coli htpR gene. Together, these results suggest that the cya gene may regulate the heat shock response, through cyclic AMP, by directly affecting the level of expression of the heat shock sigma factor.

In this dissertation I explore the ecology of bacteriophage T4, a virus of Escherichia coli. In particular, I argue that the life history of bacteriophage T4 can be divided into the growth and survival of T4 phages in three distinct environments. I argue that these environments are distinguished by at least two T4 phage sensory systems. These include (i) the sensing of secondary adsorption by infecting phages and (ii) the determination of the concentration of monovalent cations and free tryptophan in solution about free T4 phage particles. The first environment consists of high concentrations of uninfected, logarithmic phase E. coli cells. These concentrations are approximately 10⁶ E. coli cells/ml and greater. This environment occurs in the prefecal colonic lumen of animals. Here T4 phages exhibit unimpeded logarithmic growth. The second environment contains high concentrations of infected E. coli cells, low concentrations of uninfected E. coli cells, and high concentrations of free T4 phage particles. This second environment also occurs in the prefecal colonic lumen of animals and represents the maturation of environments supporting logarithmic T4 phage population growth. Such phage phenotypes as secondary exclusion and lysis inhibition characterize T4 phage growth in this environment. The third environment consists of extra-colonic waters. Here T4 phages avoid infecting E. coli cells and exhibit strategies that maximize their stability. These strategies in extra-colonic waters increase the potential of T4 phages to disseminate successfully from colon to colon. I employ this enhanced understanding of T4 phage ecology, outlined above, in an exploration of the ecology of the repair of DNA damage by T4 phages.

Oxidative folding, the process by which folding and disulfide oxidation occur in concert, is a critical step in the production of many extracellular proteins and is therefore centrally linked to a vast multitude of important physiological functions. The primary focus of this dissertation is the remarkable disulfide oxidoreductase DsbA, the sole catalyst of oxidative folding in Escherichia coli. DsbA was the first oxidative folding catalyst to be discovered, and remains the strongest known oxidant among the thioredoxin superfamily of disulfide oxidoreductases due to unique biochemical and biophysical properties. Through the activity of its substrate repertoire, which includes adhesion structures and toxins, DsbA is an essential component of many pathogenic processes and therefore is an active target for the development of novel antibiotics. Though DsbA has been analyzed through a host of biochemical, genetic, and cellular experiments over the quarter-century since its identification, the elucidation of certain mechanistic details of its catalytic process have proven elusive to conventional techniques. This primarily results from the experimental difficulties in independently monitoring the progress of folding and oxidation during oxidative folding that arise with conventional, ensemble-averaged approaches. In this work, single molecule force spectroscopy methods are applied to investigate the process of oxidative folding as catalyzed by DsbA. Through observing single substrate molecules as they undergo DsbA-catalyzed oxidative folding, a precise kinetic analysis of the enzyme is constructed. DsbA is demonstrated to be a highly effective catalyst of oxidative folding, outperforming its eukaryotic counterpart by substantial margins in every metric considered. This efficacy complements the strong preference for simpler disulfide connectivity patterns in the Escherichia coli proteome, which in conjunction likely represent a strategy for navigating the physiological demands that are imposed by the inherent speed of prokaryotic life, in which a generation can be as short as twenty minutes.

Rapid degradation of certain short lived "timing" proteins is an effective mechanism for cells to control important regulatory pathways. The mechanisms by which regulatory proteases recognize their substrates are not well understood. Escherichia coli Lon, an energy dependent protease highly conserved in many prokaryotes and eukaryotes provides a model system to study protease/substrate interactions. RcsA, a regulator of capsule synthesis, when present in levels high enough to saturate Lon, cannot protect SulA, a cell division inhibitor, from being degraded. These observations suggest Lon recognizes its different substrates with different affinities. The different affinities of these substrates might relate to the role these substrates play in the cell: stabilization of RcsA leads to a nonlethal phenotype (capsule), while stabilization of SulA leads to lethal filamentation. To further examine protease/substrate interactions, targeted mutagenesis was employed to select for mutations in rcsA which give rise to mutant RcsA protein no longer degraded by Lon protease. Two mutants with an increased half-life in the presence of Lon were identified. Their mutations fall into the C-terminal region of RcsA, supporting the hypothesis that this region is involved in the interaction of RcsA with Lon. Stabilization of RcsA was dependent on its partner RcsB; the interaction of RcsA with RcsB is believed to protect RcsA from Lon dependent degradation. However, it was shown that rcsA expression is enhanced in the presence of RcsB, and RcsA protein cannot be detected in strains mutant for RcsB in the presence or absence of Lon. Furthermore, rcsA expression was shown to be activated by RcsA itself: rcsA::lacZ expression is low in the absence of RcsA. A conserved 25 by motif, designated "RcsA-Box" was identified in the promoter region of the rcsA and capsule (cps) genes. This motif was shown to be a likely candidate for RcsA binding: high level expression of both cps::lacZ and rcsA::lacZ fusions was shown to be dependent on the presence of the "RcsA-Box". These studies expand the understanding of the specific interactions between regulatory proteases and their targets, specifically as they relates to complex regulatory networks. / Graduation date: 1998

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Mn-salophen complex with superoxide-scavenging activity was prepared from manganese(III) acetate dihydrate and salophen in ethanol. Visible absorption spectrum of the red-brown solution exhibited a broad absorption band at 430 - 450 nm with two shoulders between 500 and 600 nm which were absent with either salophen or manganic acetate alone. Titration of salophen with manganese(III) was consistent with a 1:1 Mn to salophen stoichiometry of the complex based on changes in the absorbance at 500 nm or of superoxide scavenging activity. The SOD-like activity of the complex in the xanthine-xanthine oxidase/cytochrome <i>c</i> assay was 1450 units/mg salophen. The SOD activity of the complex was suppressed 50% in the presence of EDTA (1 mM), but was not altered in the presence of bovine serum albumin (1 mg/ml) or crude protein extract of <i>E. coli</i> QC779 <i>sodA sodB</i> (1 mg/ml). <i>E. coli</i> QC779 <i>sodA sodB</i> grew scantily after an 8 hour lag phase in aerobic M63 glucose minimal medium. / Ph. D.