Cell envelope dynamics and solvent tolerance in Pseudomonas putida

Author

Holly Chubb Pinkart

Date of Award

12-1996

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

David C. White

Committee Members

Thomas Montie, Cynthia Peterson, Gary Sayler

Abstract

The role of the cell envelope in the solvent tolerance mechanisms of Pseudomonas putida was investigated. The responses of a solvent tolerant strain, P. putida Idaho, and a solvent sensitive strain, P. putida MW1200 were examined in terms of phospholipid and fatty acid content and composition, phospholipid biosynthetic rate, and alteration of lipopolysaccharide following exposure to a non-metabolizable solvent, o-xylene. Following o-xylene exposure, P. putida MW1200 exhibited a decrease in total phospholipid content. In contrast, P. putida Idaho demonstrated an increase in phospholipid content 1-6 hours following o-xylene exposure. Both strains produced trans-unsaturated fatty acids in response within 5 minutes of xylene exposure. The Idaho strain exhibited an increase in saturated fatty acids, a response not seen in P. putida MW1200. These alterations serve to compensate for increased membrane fluidity induced by intercalation of o-xylene within the phospholipids. Analysis of phospholipid biosynthesis showed P. putida Idaho to have a higher basal rate of phospholipid synthesis than MW1200. This rate increased significantly following exposure to xylene, and was independent of xylene concentration. Both strains showed little significant turnover of phospholipid in the absence of xylene. In the presence of xylene, both strains showed increased phospholipid turnover. The rate of turnover was significantly greater in the P. putida Idaho strain than in the MW1200 strain. These results suggest that P. putida Idaho has a greater ability than the solvent sensitive MW1200 strain to repair damaged membranes through efficient turnover and increased phospholipid biosynthesis. Surface structure of the two strains was also quite different, as evidenced by scanning electron microscopy, PAGE analysis, and chemical composition of LPS isolated from each strain. In the presence of xylene, the Idaho strain altered its LPS to include increased amounts of hydroxy fatty acids and charged sugar residues. These changes maintained a hydrophilic cell surface less permeable than the MW1200 strain to difloxacin and 1-N-phenylnaphthylamine. A two-phase model for solvent tolerance was proposed. The first phase includes the fatty acid alterations, which allow for short term (1-2 hours) survival of high concentrations of solvent. The second phase, which includes increased phospholipid biosynthesis and cell surface alteration, allows for long term survival.