Masters Theses

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Activation of acetate in acetate-grown Methanosarcina thermophila: purificationm and characterization of acetate kinase
Aceti, David John (Virginia Tech, 1980-04-05)
Extracts of acetate-grown Methanosarcina thermoghila were assayed for the presence of enzymes which might catalyze a proposed activation of acetate as the initial step in the pathway of methanogenesis from acetate by that organism. Acetate kinase and phosphate acetyltransferase activities of 4.9 and 49 μmoles of product/min/mg protein, respectively, were detected. Acetate kinase was purified 102- fold to a specific activity of 656 μmoles ADP formed/min/mg protein and was essentially homogeneous by denaturing gel electrophoresis. The native enzyme (Mr 94,000) was an α₂ homodimer with a subunit Mr of 53,000. Activity was optimal between pH 7.0 and 7.4 and was stable to heating at 70°C for 15 min. The apparent Km for acetate was 22 mM (Vmax = 668 μmoles ADP/min/mg protein) and 2.8 mM for ATP (Vmax = 777 pmoles ADP/min/ protein). The enzyme phosphorylated propionate at 602 of the rate with acetate but was unable to use formate. TTP, ITP, UTP, GTP, and CTP replaced ATP as the phosphoryl donor to acetate. One of several divalent cations was required for activity; the maximum rate was obtained with Mn2+.
Genetic analysis of nifF and nifA and site-directed mutagenesis of nifE in Azotobacter vinelandii
Bennett, Lisa Tracy (Virginia Tech, 1989-12-15)
Nitrogenase-catalyzed nitrogen fixation is a biochemically and genetically complex process requiring the participation of a number of different nif (nitrogen fixation) gene products. The nifF (electron transport), nifA (nif gene regulation) and nifE (FeMo-cofactor biosynthesis) genes from Azotobacter vinelandii were genetically analyzed. The nucleotide sequence of the nifF gene, which encodes a flavodoxin, was determined. Specific mutation strains indicated that in A vinelandii flavodoxin is not the unique physiological electron donor to nitrogenase. The nifF gene appears to be constitutively expressed but under nitrogen fixing conditions nifF gene expression is stimulated.
Mutagenesis of nifE and nifN from Azotobacter vinelandii
Wilson, Mark Steven Michael (Virginia Tech, 1988-12-15)
The products of nifE and nifN from Azotobacter vinelandii, which are involved in the biosynthesis of the iron-molybdenum cofactor (FeMo-co) co) from nitrogenase, have been analyzed using a variety of mutagenic techniques. NifE was the object of several site-specific, amino acid substitutions that were designed to elicit information regarding metal cluster ligands, subunit-subunit interactions, and the proposed transfer of FeMo-co.from a nifEN-products complex to the apo-MoFe protein. A model of metal cluster binding; regions within the nifEN-products is discussed insofar as it relates to the rationale for the targeting of particular amino acids for-substitution. A translational fusion between nifN and lacZ was constructed and used to study the regulation of nifEN. This gene fusion was regulated in the same manner as wild type nifN and produced a fusion protein which was enzymatically active with respect to substrates of Î²-galactosidase. Results from mutant strains which carry lesions in nifH or nifA in addition to the nifN
Role of polymorphonuclear leukocytes in the tumoricidal activity of Propionibacterium acnes
Murano, Elsa Alina (Virginia Polytechnic Institute and State University, 1987)
The mechanism responsible for the killing of tumor cells after injection of mice with a mixture of tumor cells and Propionibacterium acnes were investigated. Tumor cells were injected intramuscularly into Balb/c mice either alone or together with P. acnes vaccine. The tumor cells were then removed from the injection site 12 hours after injection, and transferred into fresh mice. Tumor cells from control animals given tumor cells only caused tumors when transferred into fresh mice 12 hours after injection whereas tumor cells from animals given both tumor cells and vaccine did not develop tumors in the fresh nice. ELISA tests were done to estimate the number of tumor cells in the lesions. In control animals given 10⁵ tumor cells the estimated numbers dropped to 10³ cells at 24 hours, but thereafter rose steadily. Palpable tumors were present 7-10 days later. In animals given 10⁵ tumor cells + 500 ug of P. acnes vaccine, estimated tumor cell numbers fell steadily, and could not be detected after 2 days. Palpable tumors never developed in these animals. These results indicate that tumor cells are killed, or rendered nontumorigenic, during the first 12 hours after co-injection into mice with P. acnes Histological studies showed that injection of P. acnes vaccine, with or without tumor cells, induced large numbers of polymorphonuclear leukocytes (PMNs) at 12 hours. To determine the role of PMNs in the killing of tumor cells, tumor cells were incubated with supernatant obtained from the phagocytosis mixture of PHNs and P. genes. After a 2- hr. incubation, the tumor cells were washed and injected into fresh mice. No tumors developed, indicating that a product of the phagocytosis of P. acnes by PMNs played a role in the killing of tumor cells. Bacterial vaccines such as P. freudenreichii, which are poorly protective against tuner cells, produced phagocytosis supernatants which were unable to kill tumor cells. Various oxygen radical scavengers/inhibitors were used to test their effect on the toxicity of the supernatant on tumor cells and chinese hamster ovary cells. Both azide and catalase rendered the supernatant nontoxigenic, suggesting that H₂O₂, produced by PHNs during phagocytosis of P. acnes, is responsible for the killing of tumor cells. However, the addition of catalase 30 minutes after the start of phagocytosis had no effect on the toxicity of the supernatant, suggesting that H₂O₂ is converted to other toxic radicals during the course of phagocytosis of P. acnes by PMNs. The oxygen consumption levels of PHNs during phagocytosis of P. acnes or other bacterial vaccines was measured and found to be similar regardless of the antitumor ability of the vaccine used. This suggests that the difference in the ability of various vaccines to protect mice against tumors may be in the production of a particular oxygen radical by PMNs during phagocytosis, and not in the production of different quantities of the same radicals.
Site-directed mutagenesis of the nitrogenase MoFe protein from Azotobacter vinelandii
Setterquist, Robert Alan (Virginia Polytechnic Institute and State University, 1989)
A model describing the potential amino acid ligands to the four 4Fe-4S centers (P-clusters) within the Azotobacter vinelandii nitrogenase MoFe protein is presented. Based on interspecies and intersubunit amino acid comparisons of the α- and ß-subunits of the MoFe protein, and the FeMoco biosynthetic proteins, NifE and NifN, four conserved residues (Cys62, His83, Cys88, Cys154 all proposed P-cluster ligands) within the α- subunit were targeted for site-directed mutagencsis studies. In order to define a range of acceptable substitutions, 35 specific site-mutants have been constructed, each with a different amino acid replacement at one of the four targeted positions. Previous studies indicated that these residues were important for MoFe activity, and may act as metallocenter ligands. Unusual redox and spectroscopic properties of the Fe-S centers suggest the involvement of ligands other than the four typical cysteines, though extrusion requirements indicate that some thiol ligands are likely. Surprisingly, mutants with an Asp, Gly, Thr, or Ser substituted for Cys88 are still capable of diazotrophic growth (Nif+), though whole cell and crude extract acetylene reduction activity is lowered. Several substitutions (Cys, Asp, Phe, Asn, Met, Tyr, Leu) are tolerated at the His83 position, these Nif+ mutant strains also have varying acetylene reduction rates and growth rates. All mutants with substitutions at positions 62, 154, resulted in complete loss of diazotrophic growth. The results could be interpreted by the following explanations: 1) Our proposed model for the P-cluster ligation within the MoFe protein is incorrect. 2) Some substitutions permit P-cluster rearrangement to a semi-functional state. 3) Either, P-clusters are not absolutely essential for diazotrophic growth, or the enzyme can function with a reduced number of these metal centers.
A study of treponemal antigens
Burke, Margaret-Ann (Virginia Polytechnic Institute and State University, 1987)
The antigenic relationship between T. denticola, T. socranskii subsp. Socranskii, T. socranskii subsp. Buccale, T. socranskii subsp. paredis, Treponeme D, T. vincentii, T. phagedenis biotype Reiter, T. minutum, and T. refringens was studied using fluorescent antibody staining and those organisms were found to contain a group of common antigens. The cross-reactivity was removed when the antisera was absorbed with Reiter cells. There was a second group of common antigens that were shared by the subspecies of T. socranskii, Treponeme D, and T. pectinvorum. Triton-extracts from different treponemal species were found to contain very few cross-reacting antigens as demonstrated by immunodiffusion, crossed immunoelectrophoresis, ELISA, and Western blots. An immunodiffusion test using treponemal Triton-extracts and antisera against five treponeme species was developed for the routine identification of oral treponemes that were in either pure or mixed cultures. T. denticola cells were found to bind to fibronectin-coated microscope slides. Proteins, that bind fibronectin, in the Triton-extracts of T. denticola, were detected in a fibroectin-captured ELISA. The Triton-extracts also reacted with rabbit fibronectin antiserum and rabbit albumin antiserum in immunodiffusion and ELISA assays. Three proteins with molecular weight of 60K, 47K, and 21K, were eluted from a agarose-gelatin-fibronectin column and reacted in Wester blots with T. denticola antiserum.
Treponema hyodysenteriae: growth and production of hemolysin
Russell, Laura René (Virginia Tech, 1988-03-08)
Treponema hyodysenteriae is the causative agent of swine dysentery, a mucohemorrhagic disease of the intestines. T. hyodysenteriae requires phospholipids and cholesterol (or cholestanol) for growth, and it produces a a-hemolysin (TH) which is induced (300 fold) by the addition of RNA core to cultures. TH is bound to the RNA core which acts as a carrier or stabilizer. I\lyobjectives were to (i) obtain successful continuous growth of T. hyodysenteriae; (ii) study the production of the hemolysin produced by T. hyodysenteriae; (Ui) study the effects of different oligonucleotide carriers on the induction of hemolysin, (iv) examine various purification procedures for nipid, efficient partial purification of the hemolysin, (v) separate the hemolysin from the RNA core carrier, and (vi) to detennme whether T. hyodysenteriae can use coprostanol, the most common intestinal sterol, to satisfy its sterol requirement. I found that optimal growth of T. hyodysenteriae could be achieved by using BHI- glucose broth supplemented with calf serum (I O~/o). serum replacement (100/0), or phosphatidylcholine liposomes which contained cholesterol or cholestanol. Optimal growth required 1 % 02 and stirring of the culture. l\laximal hemolytic titers were obtained during late-log to early-stationary phase by cultures grown in the presence of RNA core. Hemolysin production was induced as soon as 5 minutes after addition of RNA core to cultures. This production was inhibited by chloramphenicol. Polyguanylic acid and RNase treated RNA core did not significantly increase hemolytic titers of cultures grown in their presence. Partial purification of hemolysin was achieved by acetic acid clarification followed by ammonium sulfate precipitation (65% saturation). With these procedures > 90% of the hemolytic activity was recovered. Although, hydroxylapatite adsorption and polyethyleneimine precipitation completely adsorbed or precipitated hemolytic activity I I was unable to efficiently recover the activity. Partially purified hemolysin was c}10toxic to CHO cells, and caused lysis rather than the rounding effect caused by many cytotoxins.
The use of page and gas chromatography of cellular fatty for the rapid identification of Fusobacteria and Capnocytophaga
Litz, Julie S. (Virginia Polytechnic Institute and State University, 1987)
The use of PAGE of soluble cellular proteins and gas chromatography of cellular fatty acids was studied to determine the possible use of both methods for rapid identification of anaerobic bacteria. Species of Fusobacterium and Capnocytophaga were analyzed to determine the identification accuracy of each system. The electrophoretic patterns of soluble cellular proteins and the cellular fatty acid patterns determined by gas chromatography were found to remain relatively constant within the species examined. This similarity allowed for the development of automated systems using computer programs to analyze the patterns, and compare them to the patterns of known species. Procedures for gas chromatography of cellular fatty acids were developed by Myron Sasser, University of Delaware, and Microbial Identification Systems (Suite 115 Barksdale Professional Center, Newark, Delaware 19711), in cooperation with Hewlett Packard. From this study it was determined that the accuracy of identification of the PAGE analyses was not high, and therefore this method would have limited use in a clinical laboratory. Gas chromatography of cellular fatty acids had a relatively high accuracy, which is still being improved.

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Browsing Masters Theses by Department "Anaerobic Microbiology"

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