Investigation of the EnvZ/OmpR Bacterial Signaling System using Single Particle Tracking and Single Molecule Force Spectroscopy

Abstract

The EnvZ/OmpR two-component regulatory system in bacteria regulates the porins OmpF and OmpC in response to changes in osmolality of the environment. The cytoplasmic portion of the sensor kinase EnvZ (EnvZc) has been shown to be the osmo-sensor (Wang et al., EMBO J, 2012). At high osmolality, the four-helix bundle domain of the EnvZc dimer is relaxed, increasing the autophosphorylation of EnvZ. On the other hand, at low osmolality, the four-helix bundle domain is stretched, leading to lower autophosphorylation. After EnvZ is autophosphorylated, the phosphoryl group is transferred to the DNA binding protein OmpR. OmpR∼P binds to the ompF and ompC promoters to regulate their expression. Since the osmo-sensing capability of EnvZ is associated with stretch-relaxation dynamics, we wanted to understand and characterize the mechanical stability of EnvZ in the absence and presence of ATP- or OmpR/OmpR∼P-binding. In our initial experiments, we used magnetic tweezers to stretch a single EnvZc molecule. This gives two unfolding events from the dimerization and ATP binding domains. Although there is a wealth of in vitro biochemical studies of EnvZ and OmpR, an understanding of the signal transduction from EnvZ to OmpR in vivo is poorly understood. We used single-particle tracking of an OmpR-PAmcherry fusion to examine the relative abundance of DNA-bound/free OmpR obtained from the mobility of the OmpR-PAmCherry tracks. This was then used to determine how the fraction of bound/free OmpR changed in response to environmental stress. Our preliminary results indicate that the bound fraction of OmpR is localized at certain cellular positions, which could be the location of the genes regulated by OmpR. Supported by Mechanobiology RCE from the Ministry of Education, Singapore and VA-IO1BX000372 to LJK.