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- Creators: Chang, Yung
- Creators: Harrison, Jon F.
Description
Protein folding is essential in all cells, and misfolded proteins cause many diseases. In the Gram-negative bacterium Escherichia coli, protein folding must be carefully controlled during envelope biogenesis to maintain an effective permeability barrier between the cell and its environment. This study explores the relationship between envelope biogenesis and cell stress, and the return to homeostasis during envelope stress. A major player in envelope biogenesis and stress response is the periplasmic protease DegP. Work presented here explores the growth phenotypes of cells lacking degP, including temperature sensitivity and lowered cell viability. Intriguingly, these cells also accumulate novel cytosolic proteins in their envelope not present in wild-type. Association of novel proteins was found to be growth time- and temperature-dependent, and was reversible, suggesting a dynamic nature of the envelope stress response. Two-dimensional gel electrophoresis of envelopes followed by mass spectrometry identified numerous cytoplasmic proteins, including the elongation factor/chaperone TufA, illuminating a novel cytoplasmic response to envelope stress. A suppressor of temperature sensitivity was characterized which corrects the defect caused by the lack of degP. Through random Tn10 insertion analysis, aribitrarily-primed polymerase chain reaction and three-factor cross, the suppressor was identified as a novel duplication-truncation of rpoE, here called rpoE'. rpoE' serves to subtly increase RpoE levels in the cell, resulting in a slight elevation of the SigmaE stress response. It does so without significantly affecting steady-state levels of outer membrane proteins, but rather by increasing proteolysis in the envelope independently of DegP. A multicopy suppressor of temperature sensitivity in strains lacking degP and expressing mutant OmpC proteins, yfgC, was characterized. Bioinformatics suggests that YfgC is a metalloprotease, and mutation of conserved domains resulted in mislocalization of the protein. yfgC-null mutants displayed additive antibiotic sensitivity and growth defects when combined with null mutation in another periplasmic chaperone, surA, suggesting that the two act in separate pathways during envelope biogenesis. Overexpression of YfgC6his altered steady-state levels of mutant OmpC in the envelope, showing a direct relationship between it and a major constituent of the envelope. Curiously, purified YfgC6his showed an increased propensity for crosslinking in mutant, but not in a wild-type, OmpC background.
ContributorsLeiser, Owen Paul (Author) / Misra, Rajeev (Thesis advisor) / Jacobs, Bertram (Committee member) / Chang, Yung (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2010
Description
Anoxia tolerance is strongly correlated with tolerance to heat, desiccation, hyperosmotic shock, freezing, and other general stressors, suggesting that anoxia tolerance is broadly related to stress tolerance. Age affects the capacity of many animals to survive anoxia, but the basis to this ontogenic variation is poorly understood. We exposed adult Drosophila, 1, 3, 5, 7, 9, and 12 days past eclosion, to six hours of anoxia and assessed survival 24-hours post-treatment. Survival of anoxia declined strongly with age (from 80% survival for one-day-old flies to 10% survival for 12 day-old-flies), a surprising result since adult fly senescence in Drosophila is usually observed much later. In anoxia, adenosine triphosphate (ATP) levels declined rapidly (< 30 min) to near-zero levels in both 1 and 12-day old adults; thus the higher anoxia-tolerance of young adults is not due to a better capacity to keep ATP elevated. Relatively few physiological parameters are reported to change over this age range in D. melanogaster, but gut bacterial content increases strongly. As a partial test for a causal link between bacterial load and anoxia tolerance, we replaced food daily, every third day, or every sixth day, and assayed survival of six hours of anoxia and bacterial load at 12 days of age. Anoxia tolerance for 12-day old flies was improved by more food changes and was strongly and negatively affected by bacterial load. These data suggest that increasing bacterial load may play an important role in the age-related decline of anoxia tolerance in Drosophila.
ContributorsSargent, James (Author) / Harrison, Jon F. (Thesis advisor) / Haydel, Shelly (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2020