Cyclic di-AMP traps proton-coupled K+ transporters of the KUP family in an inward-occluded conformation.

Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K+/H+ symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating potassium ion uptake. However, at elevated intracellular K+ concentrations, further K+ accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K+ binding site and therefore traps KimA in an inward-occluded conformation.

Molecular Mechanisms for Bacterial Potassium Homeostasis.

Potassium ion homeostasis is essential for bacterial survival, playing roles in osmoregulation, pH homeostasis, regulation of protein synthesis, enzyme activation, membrane potential adjustment and electrical signaling. To accomplish such diverse physiological tasks, it is not surprising that a single bacterium typically encodes several potassium uptake and release systems. To understand the role each individual protein fulfills and how these proteins work in concert, it is important to identify the molecular details of their function. One needs to understand whether the systems transport ions actively or passively, and what mechanisms or ligands lead to the activation or inactivation of individual systems. Combining mechanistic information with knowledge about the physiology under different stress situations, such as osmostress, pH stress or nutrient limitation, one can identify the task of each system and deduce how they are coordinated with each other. By reviewing the general principles of bacterial membrane physiology and describing the molecular architecture and function of several bacterial K+-transporting systems, we aim to provide a framework for microbiologists studying bacterial potassium homeostasis and the many K+-translocating systems that are still poorly understood.

Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair.

Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.

Native mass spectrometry goes more native: investigation of membrane protein complexes directly from SMALPs.

Other than more widely used methods, the use of styrene maleic acid allows the direct extraction of membrane proteins from the lipid bilayer into SMALPs keeping it in its native lipid surrounding. Here we present the combined use of SMALPs and LILBID-MS, allowing determination of oligomeric states of membrane proteins of different functionality directly from the native nanodiscs.