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Production of unique metabolites by the marine dinoflagellate Prorocentrum minimum Trick, Charles Gordon


Marine phytoplankton produce extracellular metabolites which may be important in controlling the interactions among species or the competition for a limiting nutrient. While the absolute amount of these metabolites may be small compared to the primary organics released by the phytoplankton cell, the control of the production of these unique metabolites may be an important factor in the ecology of the producing species. These compounds have not been extensively studied due to the difficulty in isolating these minute quantities from seawater. In this thesis, two externally produced metabolites have been investigated. The concentration of 1 -(2,6,6-trimethyl-4-hydroxycyclohexenyl)- 1,3-butanedione, a nor-caroteniod commonly referred to as the β-diketone, was quantitatively determined during the exponential and senescent stages of growth of Prorocentrum mi n imum in P-, N-, and iron-deficient batch cultures. The β-diketone was released extracellularly in a single 'pulse' during the stationary stage of growth. Several factors such as temperature, irradiance, type of nutrient-deficiency (N, P, or Fe), and the ambient nitrate concentration were important in establishing the amount of the β-diketone produced. The environmental factors did not influence the temporal pattern of production, only the absolute amount of the β-diketone produced. The limits of the range of production of the β-diketone were narrower than the range of maximum growth for any environmental influence. The inhibition of growth and the heterotrophic uptake of glucose by marine bacteria, demonstrated the antibacterial properties of the β-diketone. The second extracellular organic examined was prorocentrin. Prorocentrin is the extracellular siderophore produced by Prorocentrum minimum, P. mariae-lebouriae, and P.gracile. Functionally similar compounds are produced by Thalassiosira pseudonana and Dunaliella tertiolecta. This study is first to characterize this type of high-affinity iron (111)-transport system in marine eukaryotic phytoplankton. The pattern of siderophore production by all species is the same, although the absolute amount of the material produced is species specific. There is no intracellular or extracellular siderophore production under iron-sufficient culture conditions. When iron was deficient there was a short period of rapid extracellular siderophore production during the stationary stage of growth. The intracellular prorocentrin concentration was very low which suggests that de novo synthesis of the prorocentrin occurs just prior to extracellular release. The persistence of the extracellular siderophore in the culture medium was brief. There was an increase in the in vivo fluorescence following the loss of the siderophore from the medium. The increase in in vivo fluorescence was not accompanied by an increase in cell concentration. An hypothesis concerning the mechanism of the iron-uptake system is proposed. Procedures for the isolation and characterization of prorocentrin are presented. Prorocentrin appears to be a tri-hydroxamate siderophore with a molecular weight between 560 and 590 daltons. The iron-prorocentrin complex is stable over a wide pH range.

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