Date of Award

1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

School of Nutrition and Food Sciences

First Advisor

Donal F. Day

Abstract

The plant pathogen responsible for halo blight in legumes, Pseudomonas syringae pathovar phaseolicola, produces an alginate exopolysaccharide which prevents desiccation and enhances the "water soaking" effect associated with pathogenesis. These bacteria demonstrated elevated extracellular alginate lyase activity during log growth and stationary phases in batch cultures. An analysis of the substrate specificity indicated enzymatic activity on both acetylated and non-acetylated alginate. The release of the enzyme(s) in log growth phase and the subsequent decrease in production in stationary phase have not been previously reported. Extracellular activity during rapid cell growth was confirmed by continuous culture in both nutrient broth and a glucose-simple salts media. The activity levels in both media peaked at 60% of the washout rate, though the nutrient broth grown cells produced twice the activity per mg cell. An analysis of media components in batch growth indicated both glutamate and iron concentrations affected the level of alginate lyase activity in log growth phase. This analysis also suggested a functional relationship between growth rate and activity level, as slow growing batch cultures displayed higher activity per mg cell than fast growing cultures. Batch-grown Pseudomonas syringae cells at a fixed cell density adhered to glass linearly over time. The corresponding rates of adhesion increased logarithmically with increasing cell density. An adhesion rate analysis of continuous culture cells displayed an absence of adhesion except at very low dilution rates, suggesting only stationary phase cells adhered. Effluent from a micro-ecosystem which contained starved, sessile cells was analyzed for cell number, cell adhesion, and extracellular alginate lyase activity upon nutrient addition. The addition of nutrient to these cells initiated an increase in cell number and exopolysaccharide in the biofilm within the microecosystem. This biofilm was disrupted after three hours of nutrient addition by alginate lyase(s), resulting in increased cell numbers in the effluent and the release of putative stationary phase cells which adhered downstream. These observations suggest a role for alginate lyase(s) in P. syringae as part of a release mechanism for viable cells from biofilms.

ISBN

9780591904833

Pages

119

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