Characterizing Outer Membrane Biogenesis and the Stress Responses that Maintain the Cell Envelope in Escherichia coli

Abstract

The outer membrane of Escherichia coli serves as selective permeability barrier allowing for the influx of nutrients and efflux of waste, while preventing the uptake of toxic molecules from the environment. The outer membrane is composed of integral β-barrel proteins (OMPs), lipoproteins and an asymmetrical lipid bilayer with lipopolysaccharide in the outer leaflet and phospholipids in the inner. This thesis focuses on the role of the Bam complex and periplasmic chaperones in the assembly of various classes of OMPs as well as the Cpx stress response and its role in maintaining the integrity of the cell envelope.

Recent controversial studies suggested that bactericidal antibiotics including aminoglycosides (AGAs), and toxic small molecules, such as hydroxyurea (HU), kill bacteria the same way; namely, by generating reactive oxygen species (ROS) via a process requiring activation of the Cpx stress response. To determine if yet to be identified Cpx regulon members might be involved in generating ROS we undertook high throughput analysis of the Cpx regulon via microarray. This high throughput analysis expanded known members of the Cpx regulon and demonstrated a role for the Cpx response in modulating peptidoglycan. However none of the putative Cpx regulon members identified were involved in the generation of ROS. We suggest an opposite, protective role for Cpx. We show that a dominant cpxA* mutation that constitutively activates the Cpx stress response confers a high level of resistance to both HU and AGAs in a CpxR-dependent manner. However the Cpx response does not increase resistance to all classes of bactericidal antibiotics, thus it is unlikely that all bactericidal antibiotics kill by the same mechanism.

This work also examines the role of the Bam complex and periplasmic chaperones in OMP assembly. Although the periplasmic chaperone Skp was known to function in the backup chaperone pathway for OMPs, no direct role for Skp in OMP assembly had been demonstrated. We demonstrate a role for Skp in the biogenesis of LptD and FhuA that was masked by redundancy with FkpA.

The OMPs affected by skp fkpA form one class of substrates, while substrates affected by null mutations in bamB such as the porins, form another class. To investigate these classes of substrates more closely, we examined the effect of mutations in bamB in synthetic genetic backgrounds and the effect of lowering the amount of functional Bam complex by manipulating essential members of the Bam complex. Results from the latter set of experiments suggest that lowering the amount of functional Bam complex has a strong, preferential effect on assembly of multimers. Taken together, the results presented here raise intriguing questions about how multimers are assembled in the non-fluid OM environment.