Fosfomycin is a frequently prescribed drug in the treatment of acute urinary tract infections. It enters the bacterial cytoplasm and inhibits the biosynthesis of peptidoglycans by targeting the MurA enzyme. Despite extensive pharmacological studies and clinical use, the permeability of fosfomycin across the bacterial outer membrane is largely unexplored. Here, we investigate the fosfomycin permeability across the outer membrane of Gram-negative bacteria by electrophysiology experiments as well as by all-atom molecular dynamics simulations including free-energy and applied-field techniques. Notably, in an electrophysiological zero-current assay as well as in the molecular simulations, we found that fosfomycin can rapidly permeate the abundant Escherichia coli porin OmpF. Furthermore, two triple mutants in the constriction region of the porin have been investigated. The permeation rates through these mutants are slightly lower than that of the wild type but fosfomycin can still permeate. Altogether, this work unravels molecular details of fosfomycin permeation through the outer membrane porin OmpF of E. coli and moreover provides hints for understanding the translocation of phosphonic acid antibiotics through other outer membrane pores.

Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfidebridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.

‡ Equally contributedThe complex cell envelope of Gram-negative bacteria comprises two membranes: the outer membrane (OM) and the cytoplasmic membrane. The two membranes delimit the periplasmic space of the bacterial cell and prevent the accumulation of toxic agents in the cytosol while regulating the access of nutrients vital for growth and cell function. The OM is the first barrier during compound uptake. It is composed of an asymmetric bilayer: an inner leaflet of phospholipids and an outer leaflet of lipopolysaccharides (LPS). Both OM leaflets combined prevent the efficient diffusion of hydrophilic as well as hydrophobic molecules. Porins, waterfilled channels spanning across the OM, enable passive diffusion of small, hydrophilic molecules into the periplasm. Substrate specificity is mainly defined by the constriction zone within the barrel structure of these porins, determining entry of molecules by factors such as size, shape, electric multipoles, and rigidity. 1,2,3 E. coli encodes multiple porins. The major porins OmpF and OmpC are highly abundant and both cation-selective, and it has been thought that they restrict the passage to compounds with a size-exclusion limit of about 600 Da. 4 However, it has recently been suggested that this limit should be redefined using other parameters. 5,6 The translocation of several classes of antibiotics, e.g. β-lactams and fluoroquinolones, through porins has been investigated extensively. Also, porin modification emerged as antibiotic resistance mechanism in clinical isolates, 7 based on specific changes in amino acid residues or decreased expression of wild-type porins. 8 Aminoglycosides (AGs) target the ribosome in the cytoplasm, thus they have to overcome both membranes in Gram-negative bacteria. Despite their frequent use as therapeutic agents, the mechanisms of their OM translocation remain incompletely understood. The self-promoted pathway is a proposed uptake mechanism. Here, divalent cations between LPS molecules are displaced by AGs, which leads to brief OM destabilization, thereby enabling OM translocation. 9

In the extremophile bacterium Deinococcus radiodurans, the outermost surface layer is tightly connected with the rest of the cell wall. This integrated organization provides a compact structure that shields the bacterium against environmental stresses. The fundamental unit of this surface layer (S-layer) is the S-layer deinoxanthin-binding complex (SDBC), which binds the carotenoid deinoxanthin and provides both, thermostability and UV radiation resistance. However, the structural organization of the SDBC awaits elucidation. Here, we report the isolation of the SDBC with a gentle procedure consisting of lysozyme treatment and solubilization with the nonionic detergent n-dodecyl-β-d-maltoside, which preserved both hydrophilic and hydrophobic components of the SDBC and allows the retention of several minor subunits. As observed by low-resolution single-particle analysis, we show that the complex possesses a porin-like structural organization, but is larger than other known porins. We also noted that the main SDBC component, the protein DR_2577, shares regions of similarity with known porins. Moreover, results from electrophysiological assays with membrane-reconstituted SDBC disclosed that it is a nonselective channel that has some peculiar gating properties, but also exhibits behavior typically observed in pore-forming proteins, such as porins and ionic transporters. The functional properties of this system and its porin-like organization provide information critical for understanding ion permeability through the outer cell surface of S-layer–carrying bacterial species.