Date of Award

1989

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

John M. Larkin

Abstract

Many strains of Beggiatoa, Vitreoscilla, and Thiothrix were found to be capable of nitrogen fixation and to harbor plasmid DNA of various sizes, including large plasmids, and several plasmids in the 12 to 15 Mdal size range. Nitrogenase induction and activity in Beggiatoa alba were studied with respect to the effects of oxygen, pH, temperature, and nitrogenous compounds. Induction required microaerobic conditions. Nitrate and nitrite repressed induction, but glutamine did not. A pH optimum of 6.5 to 8.0 and a temperature optimum of 29$\sp\circ$C were observed. Nitrogenase activity was immediately and incompletely inhibited by ammonium, but was not inhibited by nitrate, nitrite, and amino acids. The effects of different concentrations of methionine sulfoximine, an inhibitor of bacterial ammonium assimilation and transport, on ammonium inhibition of nitrogenase suggested that ammonium transport rather than assimilation was involved. Thallium inhibited activity similarly to ammonium, which, along with other experimental observations, implies that inhibition by ammonium is caused by the ionic effect that ammonium transport may have on the proton motive force. An effort was made to clone Beggiatoa alba nif genes. Recombinant DNA molecules containing B. alba DNA and pBR322 were used to transform E. coli. However, the Klebsiella pneumoniae nifHDK probe that was to be used for screening recombinants showed weak hybridization with B. alba DNA. Unsuccessful attempts were made to optimize hybridization. During this portion of the investigation, it was found that B. alba B18LD DNA is resistant to digestion by several restriction endonucleases, which suggests that the DNA contains modified bases. A technique was developed for purifying DNA from agarose gels. The procedure is an improvement over others because it is rapid, easily reproducible, and gives good yields of DNA.

Pages

183

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