Stress Response by Alternative σ-factor, RpoH, and Analysis of Posttranslational Modification of the Heat Shock Protein, Dnak, in Escherichia coli
Description: Bacteria have developed specialized responses that involve the expression of particular genes present in a given regulon. Sigma factors provide regulatory mechanisms to respond to stress by acting as transcriptional initiation factors. This work focuses on σ32 during oxidative stress in Escherichia coli. The differential response of key heat shock (HS) genes was investigated during HS and oxidative stress using qPCR techniques. While groEL and dnaJ experienced increases in transcriptional response to H2O2 (10 mM), HS (42°C), and paraquat (50 mM) exposure, the abundance of dnaK over the co-chaperones was apparent. It was hypothesized that DnaK undergoes oxidative modification by reactive carbonyls at its Lys-rich C-terminus, accounting for the differential response during oxidative stress. A σ32-mediated β-galactosidase reporter was devised to detect the activity of wild-type DnaK and DnaKV634X modified to lack the Lys-rich C-terminus. Under unstressed conditions and HS, σ32 was bound at the same rate in both strains. When subjected to H2O2, the WT DnaK strain produced significantly higher β-galactosidase than DnaKV634X (one-tailed Student’s t test p=0.000002, α=0.05) and approached the same level of output as the lacZ positive control. The β-galactosidase assay indicates that DnaK undergoes Lys modification in the WT strain, preventing the protein from binding σ32, increasing the activity of σ32, and resulting in higher β-galactosidase activity than the DnaKV634X strain. In the DnaKV634X strain DnaK continues to bind σ32 so that σ32 could not promote the production of β-galactosidase. These findings demonstrate how DnaK is oxidatively modified, hindering the interaction with σ32 in manner distinct from HS.
Date: May 2015
Creator: Martinez, Sarah N.