SignificanceThe outer membrane of Gram-negative bacteria is a highly impermeable barrier to a range of toxic chemicals and is responsible for the resistance of these bacteria to important classes of antibiotics. In this work, we show that plant pathogenic Pectobacterium spp. acquire iron from the small, stable, and abundant iron-containing plant protein ferredoxin by transporting ferredoxin across the outer membrane for intracellular processing by a highly specific protease, which induces iron release. The presence of homologous uptake and processing proteins in a range of important animal and plant pathogens suggests an exploitable route through which large molecules can penetrate the outer membrane of Gram-negative bacteria.

SummaryIn a commonly accepted model, in response to stimuli, bacterial histidine kinases undergo a conformational transition between an active and inactive form. Structural information on histidine kinases is limited. By using ion mobility-mass spectrometry (IM-MS), we demonstrate an exchange between two conformational populations of histidine kinase ExsG that are linked to different levels of kinase activity. ExsG is an atypical signaling protein that incorporates an uncommon histidine kinase catalytic core at the C terminus preceded by an N-terminal “receiver domain” that is normally associated with the response regulator proteins in two-component signal transduction systems. IM-MS analysis and enzymatic assays indicate that phosphorylation of the ExsG receiver domain stabilizes the “compact” form of the protein and inhibits kinase core activity; in contrast, nucleotide binding required for kinase activity is associated with the more open conformation of ExsG.

Genome homologies led to the identification of Streptomyces aureorectus DSM 41692 and Streptomyces virens DSM 41465 as producers of the antibiotic nucleocidin, and also 4’-fluoroadenosine.

RAS is a major anticancer drug target which requires membrane localization to activate downstream signal transduction. The direct inhibition of RAS has proven to be challenging. Here, we present a novel strategy for targeting RAS by stabilizing its interaction with the prenyl-binding protein PDE6D and disrupting its localization. Using rationally designed RAS point mutations, we were able to stabilize the RAS:PDE6D complex by increasing the affinity of RAS for PDE6D, which resulted in the redirection of RAS to the cytoplasm and the primary cilium and inhibition of oncogenic RAS/ERK signaling. We developed an SPR fragment screening and identified fragments that bind at the KRAS:PDE6D interface, as shown through cocrystal structures. Finally, we show that the stoichiometric ratios of KRAS:PDE6D vary in different cell lines, suggesting that the impact of this strategy might be cell-type-dependent. This study forms the foundation from which a potential anticancer small-molecule RAS:PDE6D complex stabilizer could be developed.