Date of Award

2002

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Botany and Plant Pathology

Advisor

John D. Tjepkema

Second Committee Member

Robert E. Cashon

Third Committee Member

Christa R. Schwintzer

Abstract

Hemoglobins have been identified in root nodules of many actinorhizal plants. When cultured in vitro, the actinomycete Frankia strain CcI3 produces hemoglobin when grown with or without supplied nitrogen. The cyanobacterium, Nostoc commune, also produces hemoglobin in vitro, although only under nitrogen-fixing, microaerobic conditions, and in less than one fifth of the explored strainslspecies. The objectives of this study were to determine if Frankia strains EANlpec, ArI3, EUNlf, CcI.17, and Cc13, members of diverse genogroups, are capable of producing hemoglobin in vitro, to characterize the oxygen kinetics of the hemoglobin, and to determine the effect of nitrogen, oxygen, and carbon dioxide on the amount of hemoglobin produced. Frankia cells were disrupted under an atmosphere of carbon monoxide and the carbonmonoxy absorption spectrum of the crude extract was used to calculate the hemoglobin concentration in the cells. Hemoglobin was present in all five strains when grown on nitrogen-free (-N) or nitrogen-supplied (+N) medium. Thus it is likely that hemoglobins are produced by most Frankia strains. In four of the five strains, the hemoglobin concentrations were similar in -N and +N culture. This does not support an association between nitrogen fixation and hemoglobin expression. Hemoglobin in crude extracts from -N cultures of EANlPec was partially purified by ion exchange chromatography and then subjected to size exclusion chromatography. The molecular mass, 13.4 f 0.2 kDa (mean * SE, n = 3), is consistent with that of truncated hemoglobins, such as the Nostoc hemoglobin. The hemoglobin in other EANlPec ion exchange fractions was further purified using a CO-pressurized concentration cell with a 10 kDa exclusion membrane. The absorption spectra obtained from this sample showed carbonmonoxy and oxyhemoglobin absorption peaks typical of a hemoglobin. This same sample was also used to determine the hff value for oxygen; 131.2 f 5.8 s-' (mean * SE, n = 6) and 166 + 8.2 s" (mean * SE, n = 7) for the hemoglobin from -N and +N cultures, respectively. Cultures of EANlp grown at 2% and 20% 0 2 showed no effect of 0 2 on hemoglobin concentration in the +N treatments @ = 0.632). The -N treatments showed less growth than the +N treatments and the hemoglobin concentration was greater at 2% than 20% 0 2 . -Cultures of Frankia strain EANlPec and CcI3 grew more efficiently when supplied with 0.2% C02 when compared to 0.0% C02. This suggests that addition of C02 to cultures could assist in Frankia growth at low initial densities, such as isolation from root nodules. The very rapid oxygen dissociation rate of EANlp hemoglobin is two to threefold faster than that of Frankia strain CcI3 and Nostoc hemoglobin, which have been proposed to function in facilitated diffusion of oxygen. If hemoglobin is localized within the small volume of the vesicle or periphery of the cells, the concentrations in the immediate region in which it is found might approach those necessary for facilitated oxygen transport.

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