Investigating the molecular mechanisms behind the transcriptional response in pressure adaptation.

Abstract

Despite 80% of Earth's microbial biomass existing in high pressure habitats in the Deep Ocean and within the continental crust, the mechanisms that allow for survival and growth in these thermodynamically unfavorable environments are poorly understood. Moreover, pressure resistance in pathogenic bacteria commonly found in food, such as Listeria monocytogenes and Escherichia coli, has emerged as more foods are sterilized by high pressure, rather than high temperature. To understand pressure adaptation, we have begun studying the molecular mechanisms of transcriptional adaptation to pressure. One important pathway in the pressure response is the heat shock pathway. To compare the heat shock response to pressure and temperature, we generated green fluorescent protein (GFP) promoter fusions of the central sigma factor genes’ transcriptional promoters and those of their downstream effectors. Activity of these promoter fusions was evaluated after heat shock and after pressure shock using scanning number and brightness (sN&B) fluorescence microscopy to determine the absolute number of GFP molecules produced in single cells and compared to the absolute number of GFP molecules produced in no shock conditions. Assuming transcriptional bursting at these promoters arises from release of supercoiling tension, we calculated the burst size and burst frequency from the distribution of the number of GFP molecules produced in each cell under each condition. These studies were carried out in the mesophilic E. coli MG1655 and in a moderately piezophilic (pressure-loving) strain adapted from MG1655, AN62. Studying both strains allowed us to quantify how the transcriptional response to changes in hydrostatic pressure occurs in both mesophiles and piezophiles, providing a more holistic model of the pressure response and adaptation phenomena.