Natural antibacterial clays, when hydrated and applied topically, kill human pathogens including antibiotic resistant strains proliferating worldwide. Only certain clays are bactericidal; those containing soluble reduced metals and expandable clay minerals that absorb cations, providing a capacity for extended metal release and production of toxic hydroxyl radicals. Here we show the critical antibacterial components are soluble Fe²⁺ and Al³⁺ that synergistically attack multiple cellular systems in pathogens normally growth-limited by Fe supply. This geochemical process is more effective than metal solutions alone and provides an alternative antibacterial strategy to traditional antibiotics. Advanced bioimaging methods and genetic show that Al³⁺ misfolds cell membrane proteins, while Fe²⁺ evokes membrane oxidation and enters the cytoplasm inflicting hydroxyl radical attack on intracellular proteins and DNA. The lethal reaction precipitates Fe³⁺-oxides as biomolecular damage proceeds. Discovery of this bactericidal mechanism demonstrated by natural clays should guide designs of new mineral-based antibacterial agents.

RseA sequesters RpoE (σ^E) to the inner membrane of Escherichia coli when envelope stress is low. Elevated envelope stress triggers RseA cleavage by the sequential action of two membrane proteases, DegS and RseP, releasing σ^E to activate an envelope stress reducing pathway. Revertants of a ΔdegP ΔbamB strain, which fails to grow at 37°C due to high envelope stress, harbored mutations in the rseA and rpoE genes. Null and missense rseA mutations constitutively hyper-activated the σ^E regulon and significantly reduced the major outer membrane protein (OMP) levels. In contrast, a novel rpoE allele, rpoE3, resulting from the partial duplication of the rpoE gene, increased σ^E levels greater than that seen in the rseA mutant background but did not reduce OMP levels. A σ^E-dependent RybB::LacZ construct showed only a weak activation of the σ^E pathway by rpoE3. Despite this, rpoE3 fully reversed the growth and envelope vesiculation phenotypes of ΔdegP. Interestingly, rpoE3 also brought down the modestly activated Cpx envelope stress pathway in the ΔdegP strain to the wild type level, showing the complementary nature of the σ^E and Cpx pathways. Through employing a labile mutant periplasmic protein, AcrA[subscript L222Q], it was determined that the rpoE3 mutation overcomes the ΔdegP phenotypes, in part, by activating a σ^E-dependent proteolytic pathway. Our data suggest that a reduction in the OMP levels is not intrinsic to the σ^E-mediated mechanism of lowering envelope stress. They also suggest that under extreme envelope stress, a tight homeostasis loop between RseA and σ^E may partly be responsible for cell death, and this loop can be broken by mutations that either lower RseA activity or increase σ^E levels.

Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.