Gene Expression in Response to Desiccation Stress

Non-Technical Summary: Desiccation sensitivity and desiccation tolerance can impact the survival of microbial as well as animal and plant cells. Even though some organisms are considered to be desiccation-tolerant, it is not known how long cells can survive desiccation conditions, and all of the mechanisms that cells use to protect themselves against drying are not understood. When bacterial cells experience a significant removal of water and become metabolically inactive, they may no longer be able to form colonies, but may be viable, and enter a viable-but-non-culturable (VBNC) state. These cells can no longer be detected by traditional culture methods, but are still alive and able to divide once favorable growth conditions are encountered. Therefore, entry into and recovery from the VBNC state for organisms, particularly pathogens, poses a significant challenge to the food packaging industry, the clinical lab and to human health. The recovery of E. coli strain JM109 from desiccation stress will be examined as an example of microbes important to the food industry and human health. Understanding the mechanisms of desiccation tolerance will be critical to devising and implementing strategies for detecting and eliminating contaminating microbes<P> Approach: To determine the effects of desiccation stress on viability of E. coli K-12, cultures of E. coli K-12 strain JM109 will be grown, harvested, and resuspended in buffer. The cells will be aliquoted into microtiter dishes or erlenmeyer flasks and placed into humidity-controlled chambers for drying for various lengths of time from six hours to fourteen days. The dried cell samples will be resuspended in pyruvate buffer and incubated at 95% RH for up to seven days. During the rehydration time, triplicate cell samples will be removed periodically from the microtiter dish and used for dilution plating to determine viable cell number, optical density measurement at OD650, and total cell counts using phase contrast microscopy. These results will be compared to the number of cells in each sample prior to drying and rehydration to calculate the percent recovery. These drying experiments will be extended to include different growth, drying and rehydration conditions. Since desiccation and subsequent rehydration can impact the integrity and fluidity of cell membranes, a potential mechanism for desiccation tolerance may involve changes in the phospholipid composition of the membrane. Fatty acid analysis of cell samples will determine the types and relative amounts of fatty acids present in non-dried cells, dried cells, and cells during rehydration. Changes in membrane fluidity in response to desiccation and during rehydration will be assessed by Fourier Transform Infrared (FTIR) spectroscopy, which can be used to measure the membrane's phase transition temperature (Tm). The Tm of cell samples will be measured by FTIR in collaboration with Dr. Al Aksan in the Department of Mechanical Engineering at the University of Minnesota. As time and resources permit, proteomic and genomic approaches will be used to identify changes in gene expression in response to desiccation stress. Samples of cells that had been dried and rehydrated will be analyzed for changes in protein profiles when compared to cells that had not been subjected to desiccation using two-dimensional gel electrophoresis. Polypeptide spots that appear to be at least two-fold more intense in the experimental sample when compared to the control will be picked from the gel and analyzed by mass spectrometry to quickly identify proteins of interest. Since membranes are critical to survival and are affected by desiccation, we will be particularly interested in membrane proteins or proteins involved in lipid biosynthesis that may be up-regulated in response to drying conditions. Another approach for detecting changes in gene expression in response to desiccation stress will be to analyze gene transcription profiles using microarray analysis. As for the proteomic experiments, samples of cells that had been dried and rehydrated for various lengths of time will be harvested, and total RNA will be isolated to prepare biotin-labeled probes that will be hybridized to an array of genomic DNA fragments. Genes expressed at least two-fold higher in cells that had been exposed to desiccation stress when compared to control cells will be identified by data base searches.