Date of Award

1990

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

First Advisor

Taylor, Norman F.,

Keywords

Chemistry, Biochemistry.

Rights

CC BY-NC-ND 4.0

Abstract

The outer membrane of Pseudomonas putida is prepared using both lysozyme digestion and sucrose density gradient separation of French pressed whole cell suspensions of the bacteria. The sucrose density gradient preparation gives greater yields and is more suited to large scale preparations of the outer membrane of P. putida. Examination of the outer membrane preparations by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that it contains 9 major protein bands. These proteins are similar but not identical to the outer membrane proteins of P. aeruginosa. One particular protein, Band E (42.9 kDa), is induced by growth of P. putida on glucose, maltose and lactose but repressed by growth on organic acids including gluconic acid and the citric acid cycle intermediates citrate, malate and succinate. Cell surface protein labelling using $\sp{125}$I autoradiography shows that most outer membrane proteins including band E are expressed at the cell surface. The defluorination of 4-deoxy-4-fluoro-D-glucose by whole cells P. putida, was induced by growth of cells on glucose and repressed by growth on succinate. However, when cells are grown on gluconate defluorination is still induced. This suggests that band E is not responsible for the observed defluorinating activity observed in outer membrane preparations of P. putida. The inhibition of the defluorinating activity of outer membranes by antibiotics and the concommitant inhibition of whole cell contamination after 24 hours of incubation suggests that the previously observed defluorination of 4FG by outer membranes of P. putida is caused by contaminating whole cells. Band E is extracted from outer membrane preparations using ionic and non-ionic detergents. Extraction of band E with non-ionic detergents requires the use of EDTA. Band E is purified from a Lubrol PX-EDTA extract using DEAE Sephacel ion-exchange chromatography. By an identical procedure a glucose inducible protein (D1) is isolated from the outer membrane of P. aeruginosa. A comparative amino acid analysis indicates that proteins D1 and E are almost homologous with respect to amino acid content. Circular dichroism spectroscopy indicated that band E is very high in $\beta$-sheet content. A purified band E is used to immunize rabbits for the production of antisera against band E. Western blot analysis shows that the antisera obtained is specific not to band E but to lipopolysaccharide.Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1990 .S278. Source: Dissertation Abstracts International, Volume: 52-11, Section: B, page: 5807. Supervisor: Norman F. Taylor. Thesis (Ph.D.)--University of Windsor (Canada), 1990.

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