Glucose dehydrogenase-deficient (Gcd$\sp-$) mutants of Pseudomonas cepacia 249 accumulated copious amounts of exopolymer on solid media containing 0.2% yeast extract supplemented with excess glucose. The majority of P. cepacia strains isolated from the pulmonary tracts of patients with cystic fibrosis were Gcd$\sp+$ indicating there was no correlation between glucose dehydrogenase-deficiency and the capacity of this bacterium to colonize the respiratory tract. Activities of glucokinase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, and enzymes of the Entner-Doudoroff pathway were significantly lower in Gcd$\sp+$ than in Gcd$\sp-$ bacteria. The decrease in the levels of these enzymes in the Gcd$\sp+$ strains was associated with a dramatic drop in the pH of the culture medium due to conversion of glucose to gluconic and 2-ketogluconic acids. Both Gcd$\sp+$ and Gcd$\sp-$ strains accumulated polymer when gluconate, mannitol or fructose were substituted for glucose. Strain 249 and most other P. cepacia strains examined failed to form exopolymer in liquid medium. However, P. cepacia 389, a Gcd$\sp-$ clinical isolate accumulated large amounts of exopolymer in liquid and on solid media. When grown on media containing 0.2% yeast extract, 1% gluconate and 0.02% calcofluor white, this strain formed an exopolymer which reacted with the dye and fluoresced bright-green under UV-light. The exopolymers produced by strains 249 and 389 were distinct from alginic acid formed by Pseudomonas aeruginosa. Exopolymer of P. cepacia 249 contained galactose, glucose, rhamnose, mannose and uronic acid. However, strain 389 formed an exopolymer composed of glucose and galactose. When strain 389 was grown with excess gluconate the polymer also contained succinate. Thus the fluorescence phenotype of this strain in the presence of calcofluor white appeared to depend upon succinylation of its polymer. Synthesis of the exopolymer presumably involved the conversion of glucose-6-phosphate to glucose-1-phosphate, UDP-glucose and UDP-galactose by the enzymes phosphoglucomutase, UDP-glucose pyrophosphorylase and UDP-glucose-4-epimerase. Extracts of P. cepacia strains 389 and 249 contained significant levels of these enzymes. Mutants of strain 389 that either overproduced or failed to produce exopolymer were isolated. One Exo$\sp-$ mutant had only 10% of the normal level of phosphoglucomutase suggesting that exopolymer formation depended upon conversion of G6P to G1P.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-8079 |
| Date | 01 January 1991 |
| Creators | Sage, Andrew Erwin |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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