The role of DnaK as an indicator of growth state and starvation in Escherichia coli

The Escherichia coli molecular chaperone DnaK has been implicated as a critical protein in the normal functioning of the cell. During growth, damaged or defective proteins are replaced by newly synthesized protein or diluted by division. However during starvation, there is insufficient energy and no cell division to remove or repair this damage. DnaK might represent one mechanism to minimize damage during starvation. The first section of this thesis attempts to better understand the role of DnaK during starvation. Two methods were used, an examination of the physiological consequences of DnaK deficiency and of DnaK excess. In addition, a new role was found for DnaK in regulating sigma factor abundance during starvation. dnaK deletion mutants were found to exhibit two starvation-related phenotypes, one of which was mediated through RpoS and the second acted in an rpoS-independent manner. These results indicate that DnaK plays a role in gene expression as well as being a molecular chaperone for the repair of denatured proteins. Stationary phase overproduction of DnaK was previously shown to be toxic and this toxicity likely results from depletion of DnaK\u27s transcription factor, [special characters omitted]. The next phase of this thesis concerned efforts to better understand DnaK toxicity through the isolation and characterization of multicopy plasmid suppressors. The results of the preceding studies led to the identification of a set of proteins, DnaK, Dps, and Fis, ideally suited for the determination of single cell growth state. Dps abundance is inversely correlated with growth rate and was used as an indicator of starvation (stationary phase). Fis plays a critical role coordinating rRNA synthesis with growth and was used as an indicator of growth. DnaK was used as a control for probe access. An immunofluorescent technique targeting these proteins was then developed and applied to studies of coliform bacteria physiology in municipal wastewater