Thesis (M.A.)--Wake Forest University. Dept. of Religion, 2007. / Includes bibliographical references (leaves 76-79)

A group of Escherichia coli clones harbouring recombinant pBR322 plasmid, containing Cellulomonas fimi DNA inserts, that reacted with antiserum to C.fimi culture supernatant, was screened for their ability to hydrolyze carboxymethyl cellulose (CMC) and 4-methylumbeliferyll-β-D-cellobioside (MUC). A clone, pEC62, hydrolyzed MUC but did not hydrolyze CMC. The recombinant enzyme encoded by pEC62 was shown to be a β-glucosidase (cellobiase). C.fimii itself was shown to encode an extracellular β-glucosidase in C.fimi. This is the first report of an extracellular β-glucosidase from a bacterium. Deletion analysis localized the cloned gene (cbg)to the tet promoter proximal region of the 7.0 kilobase insert of pEC62. Further analysis and sequence data showed a highly active derivative of pEC62 contained a translational gene fusion between lacZ of pUC13 and cbg. From this data, a location for the cbg start site was proposed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Brain tissue samples were collected from individuals with histologically and biochemically confirmed Alzheimer's Disease (AD), as well as from a group of individuals without any signs of neurological disease (NNC). Ribonucleic acid (RNA) was extracted from these tissues, characterized by several chemical methods, and the yields were compared between AD and NNC groups. High molecular weight RNA could be effectively extracted from frozen postmortem human brain. In comparison to the NNC group, tissue RNA levels were reduced in the AD hippocampus, but not in the temporal cortex or substantia innominata (SI). No difference in the physical integrity of the RNA was apparent between AD and NNC groups. A high complexity complementary deoxyribonucleic acid (cDNA) library was prepared using RNA extracted from the NNC SI. Differential hybridization screening using a variety of cDNA probes was employed to identify mRNAs expressed differentially between AD and NNC tissue, and between SI and other human tissues. Many selected mRNAs were examined for specificity of expression in brain tissue and brain regions. The cDNA clone pSI3a-24 identified an mRNA, which, on Northern blot hybridization, was expressed in brain tissue but not in the other human tissues examined. The identified mRNA was unusually large, with a chain length estimated at 15,500 bases. Quantification of the brain tissue levels of this mRNA was carried out using a ribonuclease protection assay. Tissue levels were higher in the SI (40 pg/μg RNA) than in the temporal cortex (28.6 pg/μg), and were lowest in the cerebellum (11.2 pg/μ9). Levels of the mRNA in temporal cortex samples were increased 29% in the AD group, relative to NNC. No significant difference in the SI tissue levels was observed between AD and NNC groups. Hybridization analysis of human genomic DNA indicated that the mRNA was encoded by a single copy gene. Sequence analysis of the full 3 kilobases of cloned cDNA was completed. Computer database searches failed to identify any known nucleic acid sequence with homology to the cDNA. Examination of the cDNA sequence for potential polypeptide coding regions suggested that the corresponding mRNA has a 3' untranslated region of at least 3 kilobases. / Medicine, Faculty of / Graduate

The oprF gene encoding porin protein F from Pseudomonas aeruginosa was cloned onto a cosmid vector into Escherichia coli. Protein F was expressed in large amounts in E. coli and retained its heat- and reduction-modifiable and immunological characteristics. The cloned oprF gene product was purified from E. coli and characterized with respect to pore-forming ability in black lipid bilayers. Small channels, with an average single channel conductance of approximately 0.4 nS, were observed. A similar small channel size was observed for native protein F. The oprF sequences were used as a DNA-DNA hybridization probe with chromosomal DNA from the 17 IATS (International Antigen Typing Scheme) strains of P. aeruginosa, 52 clinical isolates and the non-aeruginosa Pseudomonads. Conservation of oprF sequences was observed among all the P. aeruginosa strains and to a lesser extent among the non-aeruginosa strains of the P. fluorescens rRNA homology group. Insertion mutations in the oprF gene were created in vivo by Tn1mutagenesis of the cloned gene in E. coli and in vitro by insertion of the streptomycin-encoding Ω fragment into the cloned gene, followed by transfer of the mutated protein F gene back into P. aeruginosa and homologous recombination with the chromosome. The oprF mutants were characterized by gel electrophoresis and immunoblotting, and it was shown that the mutants had lost protein F. The P. aeruginosa oprF mutants were characterized with respect to growth rates, antibiotic permeability and cell surface hydrophobicity. The results of these studies indicated that major alterations in the cell surface had occurred and that the cells were unable to grow in a non-defined liquid medium without added electrolytes. Marginal differences were observed in MICs (minimum inhibitory concentrations) of hydrophilic antibiotics for the oprF mutants compared with their protein F-sufficient parents. The putative roles of protein F in antibiotic permeability and general outer membrane permeability are discussed. Evidence for extensive homologies between protein F, the OmpA protein of E. coli and PHIII of Neisseria gonorrhoeae are presented. A role for protein F in prophylactic anti-Pseudomonas therapy, as a target for vaccine development, is proposed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The β-glucosidase (Abg) from ATCC 21400, an Agrobacterium species, was purified to homogeneity. The protein was cleaved with cyanogen bromide and the peptides were purified by reversed phase high pressure liquid chromatography. The partial amino-acid sequences for three CNBr peptides, CNBr1, CNBr2 and CNBr3, were determined by automated Edman degradation. A sequence from CNBr2 was used to synthesize a mixture of oligonucleotides which was used as a hybridization probe to identify a recombinant DNA clone carrying the gene for β-glucosidase. A single clone was isolated which expressed an enzymatic activity that hydrolyzed several β-glucosides. The enzymatic activity produced by this clone could be adsorbed by rabbit antiserum raised against the Agrobacterium enzyme. The direction of transcription of the β-glucosidase gene was determined by verifying the DNA sequence 3' to the oligonucleotide probe binding site. After subcloning the gene a high level of expression was obtained in the plasmid vector pUC18 using the lacZ gene promoter. The nucleotide sequence of the 1599 bp insert in pABG5 was determined using the chain terminator method. The start of the protein coding region was determined by aligning the amino terminal sequence of the protein with the predicted amino acid sequence of the cloned gene. The open reading frame was 1387 nucleotides and contained 458 codons. The molecular weight calculated from the deduced amino acid sequence agreed with that observed from both the native and recombinant enzymes. The predicted amino acid composition from the open reading frame matched with that determined for the native β-glucosidase. The stop codon of this coding region was followed by a potential stem loop structure which may be the transcriptional terminator. There was a region of the deduced Abg sequence which had homology to a region from two other β-glucosidase sequences. This region of homology contained a putative active site by analogy with the active site of hen egg white lysozyme. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Pathogenic bacteria colonise their host animals by means of a complex set of interactions. The host defensive mechanisms attack foreign microorganisms and attempt to rid the animal of the invaders, while the bacteria express a variety of functions to ensure their survival under adverse conditions, some of which damage the host and cause the clinical symptoms of disease. Adhesins are the bacterial structures which mediate adherence to specific host tissues and therefore permit the colonisation of areas from which the bacteria would normally be removed. T4. I have genetically cloned and analysed an adhesin from a pathogenic strain of Escherichia coli isolated from a child with enteritis. The genetic information was transferred to laboratory strains of E. coli and was expressed under similar conditions as in the parent strain, generating material with the same adherence and antigenic properties. Thus, the cloned genes enabled laboratory strains to adhere to human colon, but not to duodenum, in the same manner as the parent. This probably accounts in large part for the tissue specificity of the pathogen which caused dysentery-like symptoms consistent with colonisation of, and damage to, the colon. The cloned genes encoded the synthesis of the adhesin as fine fibrils ('fimbriae') on the bacterial surface, approximately 2 nm in diameter. The 14,000 dalton protein subunits were assembled into very high molecular weight aggregates and were purified by size fractionation. The genetic determinant occupied about 6,000 basepairs of DNA, indicating a system of genes for the synthesis, export and assembly of functional adhesin. The genetic map was very similar to those of adhesins from another enteritis isolate and a urinary tract pathogen, suggesting an evolutionary relationship between these E. coli strains. However, the protein subunits of the three adhesins appear to differ, indicating a degree of divergence.

Resistance to aminoglycosides in the organisms that produce them is often ascribed to the well characterised and clinically important antibiotic modifying enzymes. However, at least three aminoglycoside producing actinomycetes, namely Micromonospora purpurea, Streptomyces tenjimariensis, and Streptomyces tenebrarius possess ribosomes that are refractory to some members of this class of drugs. In these cases, resistance is due to methylation of rRNA of the small ribosomal subunit. This study supports the possibility that this mechanism might be more widespread than hitherto suspected. Two of the methylase genes have been analysed at the nucleotide level and their transcripts mapped. The gentamicin resistance methylase gene (kgmA) from M. purpurea codes for a 36 kDa protein consisting of 249 amino acids. Like most actinomycete genes, kgmA is not expressed in E. coli from its own promoter, although the determinant was expressed in this Gram-negative host as a result of DNA rearrangement. Sequence analysis of the mutated plasmid suggested that the methylase was expressed as a translational fusion with the lacZ' gene of pUC18, a view that was later confirmed. Transcript mapping revealed that kgmA is probably read from a single promoter but that it might be part of a polycistron. The second gene examined confers resistance to kanamycin and apramycin, and originated in S. tenjimariensis. This determinant (kamA) was shown to encode a predicted protein of 155 amino acids with a molecular weight of 19 kDa. Unlike kgmA, this gene could not be expressed as either a transcriptional or translational fusion in E. coli. Transcription of kamA is directed by tandem promoters and is a monocistron since the the transcript terminates only 160 bp downstream of the stop codon.

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