Other than more widely used methods, the use of styrene maleic acid copolymers allows the direct extraction of membrane proteins from the lipid bilayer into SMALPs keeping it in its native lipid surrounding.
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.
Under many conditions the killing of bacterial cells by antibiotics is potentiated by DNA damage induced by reactive oxygen species (ROS)1–3. A primary cause of ROS-induced cell death is the accumulation of DNA double-strand breaks (DSBs)1,4–6. DNA polymerase IV (pol IV), an error-prone DNA polymerase produced at elevated levels in cells experiencing DNA damage, has been implicated both in ROS-dependent killing and in DSBR7–15. Here, we show using single-molecule fluorescence microscopy that ROS-induced DSBs promote pol IV activity in two ways. First, exposure to the antibiotics ciprofloxacin and trimethoprim triggers an SOS-mediated increase in intracellular pol IV concentrations that is strongly dependent on both ROS and DSBR. Second, in cells that constitutively express pol IV, treatment with an ROS scavenger dramatically reduces the number of pol IV foci formed upon exposure to antibiotics, indicating a role for pol IV in the repair of ROS-induced DSBs.
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.
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