UCSF

The outer membrane (OM) is the first line of defense for Gram-negative bacteria against the exterior environment, as it forms an impermeable lipid barrier to protect the cell from antibiotics and other stresses. This impermeability is due to the fact that the OM is an asymmetric lipid bilayer, consisting of outer leaflet lipopolysaccharides and inner leaflet phospholipids. Additionally, there are many proteins unique to the OM, mainly the Outer Membrane Porins (OMPs) and OM lipoproteins. The cell utilizes dedicated machines to assemble each of the proteins and lipids into this complex folding environment. As OM integrity is vital, the cell needs to have complex surveillance systems to monitor folding and assembly of all OM components. In E. coli and related bacteria, homeostasis of the OM is monitored by the essential transcription factor, σ^E, which regulates OM assembly by transcribing a suite of ~100 protein encoding genes and two small, regulatory RNAs (sRNAs).

σ^E activity is controlled by the degradation rate of its anti–σ, RseA, which holds σ^E inactive in the inner membrane. σ^E is further regulated by RseB, which binds to RseA and protects RseA from proteolytic cleavage. While mechanisms that can cause RseA degradation are known, the in vivo signal that relieves the inhibitory effect of RseB was not understood.

In this thesis, I set out to understand the role of RseB in the σ^E signaling system, and to identify the signal that inactivated RseB in vivo. I found that the presence of RseB significantly inhibited the dynamic range of σ^E activation, such that σ^E is activated only when signals to activate RseA cleavage and inhibit RseB were both present in the cell. I next discovered that RseB is inactivated by lipopolysaccharde, thus only in the presence of two concomittant sources of envelope stress is σ^E maximally activated. As the σ^E, RseA, and RseB operon is conserved in genomes with σ^E, we hoped that our findings regarding the role of RseB in Escherichia coli would be a broadly applicable paradigm for how Gram-negative bacteria regulate OM homeostasis.

I found it curious that the assembly machines for the lipoproteins of the OM were not represented in the σ^E regulon, even though this regulon encodes genes involved in every step of lipopolysaccharide and OMP transport and assembly. As lipoproteins are the major proteins of the OM, I set out to determine whether σ^E senses lipoprotein folding stresses and whether σ^E had additional mechanisms to regulate lipoprotein folding. I discovered a new σ^E–dependent sRNA (MicL) that decreases translation of the lipoprotein Lpp, the most abundant protein in the cell. By regulating Lpp translation through MicL, σ^E is able to regulate a major fraction of cellular translation and control the majority of protein flux destined to the OM. Additionally, misaccumulation of Lpp induces the σ^E response and proper regulation of Lpp plays an important role in envelope homeostasis.