Date of Award

Summer 1996

Degree Type

Thesis - Restricted

Degree Name

Master of Science (MS)

Department

Biology

Abstract

Bacteria can attach to many different kinds of surfaces. The process of bacterial adhesion to solid surfaces includes an initial reversible phase and a firmly bound irreversible phase. The marine bacteriumDeleya marina (ATCC 25374) was chosen as a model organism to study bacterial adhesion for the following reasons: 1) this bacterium possesses exopolysaccharides (EPS) and normally no other surface appendages; 2) the EPS have been indicated to be the mechanism by which this bacterium attaches to solid substrata; and 3) several mutants, both naturally occurring and transposon generated, that are deficient in EPS or have altered EPS characteristics are available for comparison. Cell surface hydrophobicity and charge are important in the initial process of bacterial adhesion and these characteristics are influenced by growth and nutrient conditions. These cell surface characteristics of Deleya marina were examined during growth and nutrient deprivation, and changes during growth were related to adhesion to different substrata. Hydrophobic interaction chromatography and electrostatic interaction chromatography showed changes in hydrophobicity and charge, respectively, during growth and starvation. Changes during growth correlated with cell adhesion to wettable and low-wettable substrata. It has been demonstrated that the EPS of Deleya marina is important in the adhesion of this bacterium. For the first time, the EPS of this bacterium was analyzed for monosaccharide composition after isolation and purification using gel and ion exchange chromatography. Two peaks (peak I and peak TI) which bad a molecular weight > 100,000 Daltons were isolated. Analysis with gas chromatography showed they were composed of ribose, glucose, mannose and galactose. Other measurements indicated the presence of uronic acids, hexosamines, pyruvate and some protein. It has been suggested that Deleya marina may use an EPS independent mechanism in the adhesion to hydrophobic (low-wettable) substrata. Pronase E was used to test the hypothesis that proteins may be involved in the adhesion to hydrophobic substrata. The enzyme had no effect on bacterial adhesion to hydrophilic (wettable) substrata but did appear to inhibit adhesion to the hydrophobic substratum. However, inactivated enzyme and bovine semm albumin had the same inhibitory effect on adhesion. The data indicated that a non-enzymatic adsorption effect was the cause of the inhibition. In addition, pronase E did not remove attached wild type cells from either hydrophilic or hydrophobic surfaces. However, it did remove EPS deficient mutant strains DMR and DMR-G from hydrophobic but not hydrophilic substrata. Addition of wild type EPS to cultures of the EPS deficient mutants did not allow them to recover their adhesion to levels similar to the wild type strain. Adhesion polymer footprints could be detected on both tissue-culture polystyrene (hydrophilic) and polystyrene (hydrophobic) substrata after cells were removed by sonication. It was demonstrated that footprints could be detected by direct staining with a lectin (Con A) conjugated to FITC after conditions for removal of the cells had been optimized. Although this bacterium appears to use separate mechanisms for adhesion to the two substrata no differences were detected in the footprints. It has been reported that the extracellular material from Deleya marina may influence the attachment of cypris larvae of the barnacle Balanua amphitrite. Experiments were conducted to examine the effects of purified EPS on barnacle attachment. The data indicate that when compared to controls EPS treatments of polystyrene surfaces were inhibitory to attachment. The data suggest that the purified EPS may contain inhibitory components.

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