Heterologous Expression and Characterization of Hybrid Two-Component Systems from Gut Bacteria

Two component systems (TCSs) are the primary signal transduction modality by which bacteria sense and respond to the environment. Classical TCSs are comprised of an inner membrane bound sensor histidine kinase (SK) and a cytoplasmic transcription factor known as a response regulator (RR). In the presence of an extracellular input signal, the SK phosphorylates the RR, which then binds to a promoter, activating transcription. Recently, a new TCS subfamily called the hybrid TCSs (HTCSs) has been identified wherein the SK and RR are fused into a single membrane-bound protein. While the functional implications of this architecture are poorly understood, HTCSs are found extensively throughout the genus Bacteroides, which comprise approximately 50% of the human gut microbiome.  Additionally, HTCSs have been shown to sense diverse host- and diet-derived carbohydrates, which are linked to host physiology and disease. Hindering their further characterization and use for synthetic biology, no full-length HTCS has been expressed or characterized outside the native organism. Here, we demonstrate that a HTCS can be expressed and function in E. coli, though with considerable toxicity and a weaker transcriptional response than is observed in the native organism. Through systematic expression of truncation variants of several human gut-derived HTCSs, we have identified an N-terminal predicted signal peptide targeting the Sec translocon as the major source of toxicity. Complete removal of this domain results in a dramatic reduction in toxicity of the overexpressed protein but is accompanied by a loss of ligand sensitivity in the fructose sensor BT1754. We are currently working to optimize protein expression and enhance promoter output through protein and promoter engineering as well as identify the ligands sensed by HTCSs without known inputs. This work has implications in understanding the dynamics of the human gut microbiome, understanding the functional implications of the HTCS architecture, and engineered novel diagnostics and therapeutic agents for the human gut.