A novel trans-envelope signalling complex in bacteria

Bacteria possess a cell envelope providing protection from harmful environmental stresses such as antibiotics. Gram-negative bacteria such as E. coli employ a cell envelope consisting of the outer membrane, the periplasm with the peptidoglycan layer, and the inner membrane. To maintain envelope function, bacteria must survey its integrity and activate genes for envelope synthesis and repair if necessary. Bacteria monitor their environments through two-component systems (TCSs). These systems sense environmental cues through histidine kinases in the cytoplasmic membrane, which activate cognate response regulators by phosphorylation. The response regulators in turn reprogram gene expression allowing for adaptation to the changing conditions. The TCS QseE/QseF, which is conserved in enteric bacteria, controls functions for cell envelope synthesis and repair. Moreover, QseE/QseF is required for virulence in pathogenic species. Recently, we discovered that QseE/QseF requires the third protein QseG for function. QseG is a lipoprotein attached to the inner leaflet of the outer membrane and undergoes a physical interaction with the periplasmic domain of kinase QseE, which according to current evidence stimulates phosphorylation of response regulator QseF. We hypothesize that QseG senses a signal in the outer membrane, presumably related to damage, and transduces this information into the cytoplasm to activate genes restoring envelope integrity. Genetic evidence suggests that QseG availability for interaction is regulated through its sequestration by an unknown factor in the periplasm, which shall be identified through ligand fishing and genetic screens. We speculate that QseG and QseE form a trans-envelope complex bridging the periplasm to control the phosphorylation state of QseF a possibility that shall be addressed through artificial alteration of the inner to outer membrane distance. To obtain mechanistic insight, we will determine the QseG structure and identify the interaction surfaces employed by QseG and QseE through mutational analysis and photo-crosslinking. Using transcriptome analysis and DNA binding assays we will assess whether the Qse system controls additional genes beyond the targets known so far. Finally, we will investigate the role of a second phosphorylation event in QseF, which possibly integrates additional information into this signaling pathway. Taken together, we propose to investigate a novel three-component signaling system, which transduces information across three different compartments to activate genes required for envelope homeostasis. Understanding how bacteria maintain and protect their cell envelope from damage is of prime importance to find novel strategies for fighting bacterial infections.

Lipoprotein QseG, kinase QseE and response regulator QseF form a three-component system that regulates expression of small RNA (sRNA) GlmY in Escherichia coli K-12. GlmY serves as decoy for the RNA-binding adapter protein RapZ. When not bound to GlmY, RapZ binds and presents the homologous sRNA GlmZ to endoribonuclease RNase E for degradation. GlmZ in turn regulates expression of enzyme GlmS, which synthesizes glucosamine-6-phosphate (GlcN6P), an essential metabolite required for cell envelope synthesis. We found that RapZ engages dynamically with QseG/QseE/QseF in response to GlcN6P availability to form a four-component signaling complex. When GlcN6P levels decrease, RapZ stimulates QseE/QseF phosphorylation by interaction, which increases glmY expression. As a result, GlmY sequesters RapZ into stable complexes, leaving GlmZ intact, which in turn activates glmS expression by base-pairing. Increased GlmS amounts replenish GlcN6P, which binds RapZ and displaces GlmY from the complex. Subsequently, GlmY gets rapidly degraded. Structural work yielded insight into the architecture of the RapZ:GlmZ complex and revealed overlapping GlcN6P and sRNA binding sites. Additional work suggests that RapZ moonlights and can catalyze de-phosphorylation of GlcN6P ensuring that a fraction of RapZ stays GlcN6P-free and can target GlmZ to decay when GlcN6P is replete. Elucidation of the structure of the RapZ:GlmZ:RNase E complex opened a perspective how other RNA-binding proteins may present substrates to RNase E. While RapZ is a modulator, QseG is essential for QseE/QseF phosphorylation. Screens revealed that QseG activates glmY expression via QseE/QseF in response to various envelope stresses. Interestingly, these higher GlmY levels do not necessarily result in activation of glmS expression by GlmZ, which is possibly due to concomitantly increased RapZ levels, which trigger GlmZ decay. QseG binds QseE by using a region close to the N-terminal lipidated residue anchoring QseG in the outer membrane. This observation contradicts the idea of a "trans-envelope complex" and argues for activation of QseE by QseG when the latter protein accumulates in the periplasm - presumably in response to stress. Time-resolved RNA-seq analysis showed that glmY is the only direct target gene regulated of QseF under standard growth conditions. However, a defined group of genes becomes up-regulated at later times, suggesting a regulatory mechanism distinct from binding to DNA. Finally, we employed RapZ and GlmZ to develop a tool that allows to release sRNAs of choice in a 5' monophosphorylated state in the cell through controlled processing from longer precursor RNAs. Using this tool, we could not observe a role of the 5' phosphorylation mark for target RNA decay, as previously proposed by other researchers. Instead, stability of a group of sRNAs is apparently determined by their 5' phosphorylation state.