Bacterial metabolite sensing
Overview: Bacteria have an enormous impact on humans. For example, infectious diseases cause a third of deaths worldwide. Furthermore, bacteria-based biotechnologies are central to developing a more sustainable economy. Advancing these areas requires uncovering new aspects of bacterial physiology. One such frontier involves deciphering how bacteria communicate using chemical messages, in the form of metabolites, to adapt to their environment. In the King lab, we explore how bacteria sense reactive metabolites. Reactive metabolites can form unique covalent adducts with protein residues that can serve as a switch to regulate protein function. We study modifications that can spontaneously revert, making their abundance on proteins responsive to local metabolite levels - an important requirement for sensing. An often overlooked class of metabolites that exemplify this behaviour are the essential metabolic gases, O₂ and CO₂, which trigger adaptive biological responses within diverse bacteria. We focus on two main areas: 1) protein carboxylation in CO₂ sensing, 2) reversible protein oxidation in redox sensing. Through exploring these areas, we aim to contribute knowledge that enables creating new antibiotics and bolsters development of sustainable biotechnologies.
Area 1: Protein carboxylation as a CO₂ sensing mechanism
CO₂ is an essential biological gas that triggers adaptive responses within diverse bacteria. For example, high CO₂ levels within humans triggers virulence factor expression within certain pathogens. Other capnophiles (CO₂-loving bacteria), fix CO₂ as a carbon source to fuel metabolism. Understanding how bacteria sense CO₂ levels and adjust their metabolism could provide exciting opportunities for carbon capture and sustainable production of industrial products. However, the basic biochemical mechanisms through which bacteria sense CO₂ are unknown. Though little explored, one direct way that CO₂ can impact proteins is through modification of the primary amine of lysine residues to form a carbamate (Lys-CO₂). This modification completely changes the structure and charge state at these sites in a reversible manner. In the King lab, we are developing chemoproteomic methods to uncover Lys-CO₂ sites at the proteome-wide level and exploring its potential role as a biochemical regulatory mechanism of CO₂ sensing.
Proteome-wide identification of Lys-CO₂
We describe the first quantitative proteomic method to identify Lys-CO₂ sites in proteins. Using this method, we identify 20 high-confidence Lys-CO₂ sites. From among our panel of modified proteins, we characterize a novel Lys-CO₂ site within the effector pocket of metabolic sensor PII from cyanobacteria. We uncover a unique negative biochemical control mechanism whereby Lys-CO₂ formation antagonizes binding of PII to its regulatory effector ligand ATP over a physiological range of CO₂. To our knowledge, this represents the first example of CO₂ acting as a negative biochemical regulator of proteins.
Area 2: Reversible protein oxidation in redox sensing
Oxidative respiration generates reactive oxygen species (ROS) that damage biomolecules. To protect against oxidative damage, bacteria evolved an intricate cellular redox network to detoxify ROS and reverse oxidative damage on biomolecules. Importantly, ROS are elevated during exposure to bactericidal antibiotics and contribute toward killing by these antibiotics. Therefore, blocking redox sensing pathways provides an enticing potential avenue to sensitize bacteria to current antibiotics. However, effective therapeutic targeting of redox sensing will require uncovering the detailed molecular mechanisms that regulate the redox network. In the King lab, we study new forms of reversible oxidation in proteins. Our goal is to uncover insights into the bacterial redox network that can be targeted therapeutically.