Theses and Dissertations

Defense Date

1-1996

Document Type

Dissertation

Degree Name

Ph.D. Oceanography/Marine Biology

Department

Oceanographic Center

First Advisor

Curtis M. Burney

Second Advisor

Richard E. Dodge

Abstract

The following is a study of the regulation of production of a catabolic enzyme, beta-glucosidase, by isolated strains of marine bacteria. Catabolic enzymes transform organic matter to monosaccharides which are utilized as an energy source for growth by bacteria. The bacterial strains were isolated from the Gulf Stream off the coast of Florida, as well as from particulate matter collected from waters adjacent to the Florida coast.

The first section describes the preparation of a liquid medium using sterile saltwater supplemented with inorganic nutrients and a carbohydrate component. This medium allowed growth of marine bacteria under carbohydrate-limiting conditions. A solid agar version of the media was also prepared, which allowed isolation of individual colonies of marine bacteria under carbohydrate-limiting conditions.

The second section describes analyses of the regulation of beta-glucosidase production by five isolated bacterial strains using methylumbelliferyl-glucopyranoside (MUF-glu) as a model substrate. The beta-glucosidase hydrolysis of MUF-glu to glucose and a highly fluorescing product, methylumbelliferon (MUF ), allowed a measurement of enzyme activity in laboratory cultures.

The experiments showed that four of the five bacterial strains isolated could regulate production of beta-glucosidase. When cellobiose, in particular, was the only carbohydrate present, the four strains showing regulatory ability produced elevated levels of enzyme activity. This elevated enzyme activity was not observed when glucose was provided as the only carbohydrate source. The fifth strain showed only low-level enzyme activity in the presence of cellobiose or glucose. This is the first evidence of the regulation of beta-glucosidase activity in particular strains of marine bacteria.

Authenticity of beta-glucosidase activity was confirmed with known inhibitors of beta-glucosidase, gluconic acid, and glucose. The enzyme activities of all the isolated strains, measured by hydrolysis of MUF-glu to fluorescent MUF, showed sensitivity to both enzyme inhibitors. The sensitivity was observed as lower MUF production compared to control assay samples with no inhibitor added.

The first isolated bacterial strain, from Gulf Stream waters, also showed an ability to repress the production of beta-glucosidase in the presence of glucose. This strain was tested with cylic AMP, known to neutralize glucose repression of beta-galactosidase in E.coli. Cyclic AMP, however, did not neutralize the effect· of glucose on repressing beta-glucosidase activity in the isolated marine bacterium.

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