The studies reported herein indicate that, under the conditions commonly employed for cultivating Neisseria gonorrhoeae, colonial type T1 and T2 cultures of the organism dissociate to type T3 and T4 forms, and that this occurs both among populations of the organism grown in liquid media as well as in individual, well-isolated colonies grown on solid media.

A seliberia-like bacterium (SLO), isolated from reverse osmosis membranes was characterized by morphological, physiological and DNA studies. The helically twisted cells of this organism were often observed in star-shaped clusters. Depending on nutritional conditions, cells ranged from 0.5 to 21 um in length and possessed prosthecae. Small motile cells were produced by asymmetric fission or by a budding process. Ovoid "generative" cells were observed in mixed culture conditions or when the pure culture isolate was grown in the presence of humic acid. The SLO oxidatively utilized glucose, maltose, xylose, cellobiose, and several amino acids as sole carbon and energy sources. The organism is a strict aerobe and does not anaerobically respire. The moles percent guanine plus cytosine (mol% G + C) of the SLO DNA was 38% as compared with 63-67% for Seliberia stellata. Although the cellular morphology and physiology of the SLO closely resembles that of S. stellata, the SLO is considered to be a new species of Seliberia based on the presence of prosthecae and the mol% G + C.

Haemophilus influenzae, the primary causative factor in bacterial meningitis, displays a unique growth requirement for intact NAD. Selective inhibitors of the pyridine nucleotide-requiring enzymes from H. influenzae could have a pronounced effect on growth of the organism. Haemophilus malate dehydrogenase was purified 109-fold with a 26% recovery through a 4-step procedure involving salt fractionation, and hydrophobic and dye affinity chromatography. The purified enzyme was demonstrated to be a dimer of M,= 61,000. Initial velocity, product, and dead-end inhibition studies were consistent with an ordered bi-bi mechanism in which NAD is the first substrate bound to the enzyme. Several NAD analogs structurally altered in either the pyridine or purine moiety functioned as coenzymes in the reaction catalyzed. Selective interactions occurring at the coenzyme binding sites were investigated. Coenzyme-competitive inhibition by adenosine derivatives demonstrated important interactions of the pyrophosphate moiety of the coenzyme. Positive chain length effects in the coenzyme-competitive inhibition by aliphatic carboxylic acids indicated the presence of a hydrophobic region close to the pyrophosphate region at the coenzyme binding site. Several structural analogs of NAD and malate were evaluated as selective inhibitors of the enzyme. The enzyme was inactivated by incubation with diethylpyrocarbonate whereas no inactivation was observed with sulfhydryl reagents. Haemophilus influenzae 6-phosphogluconate dehydrogenase was purified 308-fold with a 16% recovery through a 4-step chromatographic procedure involving a PhenylSepharose hydrophobic column, and affinity chromatography on Matrex gel Green A, Matrex gel Red A, and 2',5’ADP-Sepharose resin. The purified enzyme was demonstrated to be a dimer of M,= 70,000. Initial velocity studies of 6-phosphogluconate oxidation indicated a sequential reaction mechanism. Although certain product and dead-end inhibition studies were consistent with an ordered mechanism, the direct binding of 6-phosphogluconate in protection experiments did not support a strictly ordered reaction sequence. Inhibition by adenosine derivatives indicated that the 2’-phosphate is important in binding to the coenzyme binding site of the enzyme. The 3-acetylpyridine analogs of NAD and NADP which support growth of H. influenzae were demonstrated to function as coenzymes with the two dehydrogenases studied. The most effective inhibitors of the purified malate dehydrogenase and 6-phosphogluconate dehydrogenase were observed to inhibit the growth of Haemophilus influenzae. However, the most potent inhibition of growth by 3-aminopyridine analogs of NAD and NADP could not be explained on the basis of interactions of these analogs with the two dehydrogenases studied. / Ph. D.

The value of the adenylate energy charge [(ATP)+1/2(ADP)/(ATP)+(ADP)+(AMP)] in Azotobacter vinelandii cells was monitored during growth and germination in flask cultures. The miximal value of 0.88 was attained during mid-log phase; this declined gradually to 0.50 by late stationary phase. When these cultures were transferred to encystment media, the adenylate energy charge decreased to an average value of 0.40 as the vegetative cells encysted and remained unchanged during the next 20 days. Encystment cultures wre composed of vegetative cells, encysting cells and mature cysts but the proportionate value of the energy charge could be assigned. Viability of the total population remained 95% or higher during the entire period studied. Azotobacter vinelandii cysts cultivated on phosphate-sufficient media. Although cell protein and nucleic acids were unaffected by phosphate deficiency, cell wall structures, oxygen uptake and sncystment were significantly affected. Phosphate-limited cysts contained much larger amounts of poly-beta-hydroxybutyric acid but had a lower adenylate energy charge than did control cysts. The ATP/ADP ratio was much lower in phosophate-deficient cysts than in the control cysts. The data indicate a "substrate saving" choice of three metabolic pathways available to cells of Azotobacter under different growth conditions.