‡ 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

<p>Despite decades of therapeutic application of aminoglycosides, it is still a matter of debate if porins contribute to the translocation of the antibiotics across the bacterial outer membrane. Here, we quantified the uptake of kanamycin across the major porin channels OmpF and OmpC present in the outer membrane of <i>E. coli.</i> Our analysis revealed that, despite its relatively large size, about 10 - 20 kanamycin molecules per second permeate through OmpF and OmpC under a 10 mM concentration gradient, whereas OmpN does not allow the passage. Molecular simulations elucidate the uptake mechanism of kanamycin through these porins. Whole-cell studies with a decisive set of <i>E. coli</i> porin mutants provide evidence that translocation of kanamycin <i>via</i> porins is relevant for antibiotic potency.</p>

Negamycin is a natural pseudo-dipeptide antibiotic with promising activity against Gram-negative and Gram-positive bacteria, including Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus, and good efficacy in infection models. It binds to ribosomes with a novel binding mode, stimulating miscoding and inhibiting ribosome translocation. We were particularly interested in studying how the small, positively charged natural product reaches its cytoplasmic target in Escherichia coli. Negamycin crosses the cytoplasmic membrane by multiple routes depending on environmental conditions. In a peptide-free medium, negamycin uses endogenous peptide transporters for active translocation, preferentially the dipeptide permease Dpp. However, in the absence of functional Dpp or in the presence of outcompeting nutrient peptides, negamycin can still enter the cytoplasm. We observed a contribution of the DppA homologs SapA and OppA, as well as of DtpD, a proton-dependent oligopeptide transporter. Calcium strongly improves the activity of negamycin against both Gram-negative and Gram-positive bacteria, especially at concentrations around 2.5 mM, reflecting human blood levels. Calcium forms a complex with negamycin and facilitates its interaction with negatively charged phospholipids in bacterial membranes. Moreover, decreased activity at acidic pH and under anaerobic conditions point to a role of the membrane potential in negamycin uptake. Accordingly, improved activity at alkaline pH could be linked to increased uptake of [3H]negamycin. The diversity of options for membrane translocation is reflected by low resistance rates. The example of negamycin demonstrates that membrane passage of antibiotics can be multi-faceted and that for cytoplasmic anti-Gram-negative drugs, understanding of permeation and target interaction are equally important.

<p>Despite decades of therapeutic application of aminoglycosides, it is still a matter of debate if porins contribute to the translocation of the antibiotics across the bacterial outer membrane. Here, we quantified the uptake of kanamycin across the major porin channels OmpF and OmpC present in the outer membrane of <i>E. coli.</i> Our analysis revealed that, despite its relatively large size, about 10 - 20 kanamycin molecules per second permeate through OmpF and OmpC under a 10 mM concentration gradient, whereas OmpN does not allow the passage. Molecular simulations elucidate the uptake mechanism of kanamycin through these porins. Whole-cell studies with a decisive set of <i>E. coli</i> porin mutants provide evidence that translocation of kanamycin <i>via</i> porins is relevant for antibiotic potency.</p>