Date of Award:

1985

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Nutrition, Dietetics, and Food Sciences

Advisor/Chair:

Daren P. Cornforth

Abstract

The effect of added iron on the growth of Clostridium botulinum type A in a chemically defined medium was studied. Growth of C. botulinum was supported by an iron level of 0.05 ug/ml with maximum growth observed at a level of 3 ug iron/ml.

Electron paramagnetic resonance (EPR) studies were conducted to detect the presence of iron-sulfur centers and iron-nitric oxide complexes in untreated and nitrite treated cell-free extracts of C. botulinum type A. Untreated extracts of C. botulinum exhibited EPR signals in the oxidized and reduced states characteristic of a "HiPiP-type" iron-sulfur center (g=2.02) in the oxidized state and a reduced signal at g=l.94, characteristic of a reduced iron-sulfur center. Extracts of C. botulinum treated with nitrite exhibited an EPR signal at g=2.035, characteristic of iron-nitrosyl complexes, with the simultaneous disappearance of the the signal at g=l.94. This indicates that nitrite reacts with the iron-sulfur centers in botulinal cells to form iron-nitrosyl complexes. Addition of ascorbate with nitrite intensified the EPR signal at g=2.035, probably by enhancing the reduction of nitrite to nitric oxide.

A cytochrome c reduction method was used for the determination of ferredoxin activity in untreated and nitrite treated cells of C. botulinum type A from which ferredoxin had been partially purified. Untreated extracts of C. botulinum reduced cytochrome c which demonstrates ferredoxin activity within the cells. Treatment of the cells with nitrite at a level of 1000 ppm for 45 min was found to inhibit ferredoxin activity by 90%. Boiling the partially purified ferredoxin from the untreated cells for 5 min inactivated the protein.

Pyruvate-ferredoxin oxidoreductase activity in partially purified extracts of nitrite treated and untreated cells of C. botulinum was determined by assaying for FAD reduction and acylhydroxamate formation. Nitrite treated cells exhibited an inhibition of 70% of FAD reducing activity and 80% inhibition of acylhydroxamate formation when compared to the untreated cells. Boiling inhibited the activity of partially purified oxidoreductase activity by more than 90% in both the assays.

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