In the yeast Saccharomyces cerevisiae the production of respiratory competent mitochondria requires the activity of several hundred proteins, the vast majority of which are encoded by nuclear genes and imported into mitochondria. A subset of these nuclear gene products are required for the expression of individual mitochondrial genes. Expression of the mitochondrial gene cob, encoding cytochrome b, requires the activity of at least seven nuclear gene products, including CBP1, CBS1 and CBS2. CBP1 is required for the stability of cob transcripts, while CBS1 and CBS2 are required for translation of cob mRNA. Previous analyses of mitochondrial rearrangements that suppress cbp1, cbs1, or cbs2 mutations have led to the hypothesis that CBP1, CBS1 and CBS2 interact with the 5' untranslated leader (UTR) of cob transcripts. To further define cob sequence important for stability or translation of cob transcripts, the expression of mutant cob genes, in which portions of the cob coding sequence or 5' UTR have been deleted, has been analyzed in vivo. Quantitation of steady-state levels of cob transcripts in wild-type CBP1 and mutant cbp1 strains carrying deletion mitochondrial genomes leads to the conclusion that a 63 nucleotide sequence is sufficient for the CBP1-dependent stability of cob transcripts. This sequence encompasses the site of cleavage that produces the mature 5' end of cob mRNA from precursor transcripts. Determination of the 5' ends of mature cob mRNAs in the deletion strains indicated that this sequence is also sufficient for correct positioning of the cleavage. The data suggest that the cleavage is CBP1-dependent and occurs at a specific distance 5' of a recognition site located within the defined 63 nucleotides. In addition, the data suggest that the stability of the mature cob mRNA produced by the cleavage is dependent on sequence at its 5' end. Lastly, an analysis of the structure of a high molecular weight cob transcript present in a respiratory incompetent deletion strain leads to the hypothesis that 5' UTR sequence between -200 and -4 is required for interaction with factors, such as CBS1 or CBS2, required for translation of cob transcripts.

A small lipid-containing bacteriophage PRD1 encodes a DNA polymerase that utilizes a protein primer for the initiation of DNA replication. The purification of PRD1 DNA polymerase has been hampered by the insolubility of the overexpressed enzyme in E. coli cells. A simple and rapid procedure for purification of the overexpressed PRD1 DNA polymerase has been developed. This method is based on guanidine hydrochloride denaturation and renaturation of the insoluble PRD1 DNA polymerase overexpressed in E. coli containing recombinant plasmid pEJG. The purified DNA polymerase was extensively characterized and found to be indistinguishable from the normal soluble PRD1 DNA polymerase as judged by enzymatic properties. These properties include: protein-primed initiation of PRD1 DNA replication, strand-displacement DNA synthesis, DNA polymerase processivity, 3' to 5' exonuclease activity and filling-in repair type DNA synthesis. Furthermore, the kinetic parameters determined for dNTPs and primer-terminus were of the same order of magnitude. The availability of a simple purification procedure for PRD1 DNA polymerase should permit detailed structure-function analysis of this enzyme. All known family B DNA polymerases contain a conserved region of amino acids, KX₆₋₇YG, which appears to be correspond to the "finger" alpha helix O of the Klenow fragment of E. coli DNA polymerase I, a family A DNA polymerase. A site-directed mutagenesis study has been applied to access the functional role of the invariant amino acid lysine-340 of PRD1 DNA polymerase. Mutant DNA polymerases were overexpressed and purified to near homogeneity. The results showed that the modification of the lysine-340 of PRD1 DNA polymerase abolishes the polymerase activity without affecting the 3' to 5' exonuclease activity. Site-directed mutagenesis studies revealed that residues important for the 3' to 5' exonuclease activity, particularly metal binding ligands for the Klenow fragment, are all conserved in PRD1 DNA polymerase as well. Although PRD1 DNA polymerase has a smaller 3' to 5' exonuclease domain, active sites appear to be very similar to those of the Klenow fragment. Moreover, the metal binding ligands were also found to be important for the strand-displacement activity, a unique feature of PRD1 DNA polymerase.

Uric acid is a nitrogenous compound that is produced as an end product of the deamination of amino acids. Many insects and all reptiles and birds excrete the majority of excess nitrogen as solid uric acid granules that are suspended in the urine. However, the solid uric acid is not in crystalline form, but is packaged into small, spherical structures that are usually 3 - 15 μm in diameter. Further, the uric acid that is dissolved in the urine exceeds its aqueous solubility limit and the solubility limits of the potassium and sodium urate salts. Thus, a factor(s) in the urine is facilitating urate in exceeding its solubility limits and in causing urate to form spheres rather than crystals. I have reported that avian urine contains unusually high amounts of protein (1-3 mg/ml), and that the uric acid spheres also contain protein. Linear regression analysis shows that the amount of uric acid and protein in urine are positively correlated (r = 0.80, p<0.005), suggesting a functional relationship between the two. Equilibrium dialysis of urine before and after its treatment with protease shows the urinary protein capable of binding urate (and calcium). After isolating and purifying a sphere protein, I was able to generate sheep anti-sphere protein antibodies. By using these and commercially obtained anti-chicken serum albumin antibodies in a series of western blot analyses, I have identified serum albumin as the sphere protein and as a major urinary protein in birds. In summary, my data show serum albumin to playa significant role in the excretion of uric acid in birds. Because urine from all uricotelic species I have examined contains similar spheres, I am suggesting that all uricoteles may use like, or analogous, proteins to facilitate the excretion of urates.

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<p>Our purpose was to gain knowledge of the bacterial demographic using recently isolated strains causing uncomplicated UTI; we also sought to understand how capsular type has changed over time. Using multiplex PCR, data show Group 2 encapsulated strains have significantly increased in uncomplicated UTI in the past twenty years, suggesting an expansion of previously under-represented capsule types. Additionally, we find K1 encapsulated extra-intestinal pathogenic <italic>Escherichia coli</italic> (ExPEC) are significantly associated with symptomatic UTI.</p><p>K1 ExPEC are able to catabolize sialic acid for energy (glycolysis), and membrane production (LPS and peptidoglycan), as well as synthesize sialic acid for capsule expression. NanR is an ExPEC global regulator of known virulence factors, however regulation via NanR is controlled by sialic acid. Using an established murine model, fluorescent microscopy, high performance liquid chromatography, and tissue culture methods, we find K1 ExPEC use sialic acid as a signaling molecule to determine location within the host.</p><p>Capsule and Type-1 pili (T1P) are important for ExPEC pathogenesis, both in the urinary tract and in more disseminated disease; coordinated regulation of these two virulence factors has been implicated. Using a tissue culture model, Western blot analysis, T1P agglutination and reporter assays, we found capsule and T1P are counter-regulated, and that this mechanism is conserved in non-pathogenic <italic>E. coli</italic>.</p> / Dissertation

Ocr is unusual among proteins in its ability to form a transparent gel at high ammonium sulphate concentrations. This transition was investigated using a combination of spectroscopic, microscopic and rheological techniques. It occurs sharply at a concentration of 3.2M ammonium sulphate and is not observed with other types of salt. Rheological measurements showed that rather than precipitating under such conditions, ocr forms a weak viscoelastic gel. Far UV circular dichroism spectra reveal that ocr does not denature in the gel phase, while near UV CD spectra suggest the formation of long, helical structures. Well resolved fibrils were observed using atomic force microscopy. They were over 1µm in length and varied between 2.6nm to 10.4nm in height, corresponding to the thickness and length of the ocr dimer. Ocr is a highly charged protein (-56e at pH 8) and is shaped like a banana. We argue that it is stabilized in specifically aggregated structures at large salt concentrations by these physical properties. Electrostatic repulsions between proteins are screened by salts, allowing proteins to approach close enough to aggregate. The charge on ocr is high enough to resist such precipitation. However, at 3.2M ammonium sulphate we suggest that the salt molecules bridge neighbouring ocr dimers via hydrogen bonds, connecting amino acid carboxyl groups with the ammonium groups of the salt. The banana-shaped dimers stack on top of each other, forming long helical fibrils that intertwine into a semi flexible network.

<p>Bacterial colonization of the upper respiratory tract is the first step in the pathogenesis of nontypeable Haemophilus influenzae (NTHi) disease. Examination of the determinants of NTHi colonization process has been hampered by the lack of an appropriate animal model. To address this, we have developed a model of NTHi colonization in adult rhesus macaques that involves intranasal inoculation of 1x105 CFU and results in persistent colonization of the upper respiratory tract for at least three weeks with no signs of disease, mimicking asymptomatic colonization of humans. Using this model, we assessed the contributions to colonization of the HMW1 and HMW2 adhesive proteins. In competition experiments, the parent strain expressing both HMW1 and HMW2 was able to efficiently out-compete an isogenic mutant strain expressing neither HMW1 nor HMW2. In experiments involving inoculation of single isogenic derivatives of NTHi strain 12, the strains expressing HMW1 or HMW2 or both were able to colonize efficiently, while the strain expressing neither HMW1 nor HMW2 colonized inefficiently. Furthermore, colonization resulted in antibody production against HMW1 and HMW2 in one-third of the animals, demonstrating that colonization can be an immunizing event. In conclusion, we have established that NTHi is capable of colonizing the upper respiratory tract of rhesus macaques, in some cases associated with stimulation of an immune response. The HMW1 and HMW2 adhesive proteins play a major role in the process of colonization.</p><p>After establishing that the HMW1 and HMW2 proteins are colonization factors we further investigated the determinants of HMW1 function. HMW1 is encoded in the same genetic locus as two other proteins, HMW1B and HMW1C, with which HMW1 must interact in order to be functional. Interaction with HMW1C in the cytoplasm results in the glycosylation of HMW1. By employing homologues of HMW1C that glycosylate HMW1 in slightly different patterns we show that the pattern of modification is critical to HMW1 function. Structural analysis showed a change in protein structure when the pattern of HMW1 modification differed. We also identified two specific sites which must be glycosylated for HMW1 to function properly. These point mutations did not have a significant effect on protein structure, suggesting that glycosylation at those specific sites is instead necessary for interaction of HMW1 with its receptor. HMW1B is an outer membrane pore through which HMW1 is transported to reach the bacterial cell surface. We observed that HMW1 isolated from the cytoplasm has a different structure than HMW1 isolated from the bacterial cell surface. By forcing HMW1 to be secreted in a non-HMW1B dependent manner, we show that secretion alone is not sufficient for HMW1 to obtain a functional structure. This leads us to hypothesize that there is something specific in the interaction between HMW1 and HMW1B that aids in proper HMW1 folding.</p><p>The NTHi HMW1C glycosyltransferase mediates unconventional N-linked glycosylation of HMW1. In this system, HMW1 is modified in the cytoplasm by sequential transfer of hexose residues. To determine if this mechanism of N-linked glycosylation is employed by species other than NTHi, we examined Kingella kingae and Aggregatibacter aphrophilus homologues of HMW1C. We found both homologues to be functional glycosyltransferases and identified their substrates as the K. kingae Knh and the A. aphrophilus EmaA trimeric autotransporter proteins. LC-MS/MS analysis revealed multiple sites of N-linked glycosylation on Knh and EmaA. Without glycosylation, Knh and EmaA failed to facilitate wild type levels of bacterial autoaggregation or adherence to human epithelial cells, establishing that glycosylation is essential for proper protein function.</p> / Dissertation