Growing resistance of pathogenic bacteria and shortage of antibiotic discovery platforms challenge the use of antibiotics in the clinic. This threat calls for exploration of unconventional sources of antibiotics and identification of inhibitors able to eradicate resistant bacteria. Here we describe a different class of antibiotics, odilorhabdins (ODLs), produced by the enzymes of the non-ribosomal peptide synthetase gene cluster of the nematode-symbiotic bacterium Xenorhabdus nematophila. ODLs show activity against Gram-positive and Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae, and can eradicate infections in animal models. We demonstrate that the bactericidal ODLs interfere with protein synthesis. Genetic and structural analyses reveal that ODLs bind to the small ribosomal subunit at a site not exploited by current antibiotics. ODLs induce miscoding and promote hungry codon readthrough, amino acid misincorporation, and premature stop codon bypass. We propose that ODLs' miscoding activity reflects their ability to increase the affinity of non-cognate aminoacyl-tRNAs to the ribosome.

Entomopathogenic bacteria of the genus Xenorhabdus are known to be symbiotically associated with soil dwelling nematodes of the Steinernematidae family. These bacteria are transported by their nematode hosts into the hemocoel of the insect larvae, where they proliferate and produce insecticidal proteins, inhibitors of the insect immune system and antimicrobial molecules. In this study, we describe the discovery of a new family (PAX) of five antimicrobial compounds produced by fermentation of the Xenorhabdus nematophila F1 strain and purified by cation exchange chromatography and reversed phase chromatography. The chemical structure of PAX 3, a lysine-rich cyclolipopetide, was obtained from the analysis of homo and heteronuclear 2D NMR and confirmed by MS-MS experiments. The five members of the PAX family showed significant activity against plants and human fungal pathogens and moderate activity against few bacteria and yeast. No cytotoxicity was observed on CHO or insect cells.

Worldwide spreading of drug-resistant pathogens makes mechanistic understanding of antibiotic action an urgent task. The macrocyclic antibiotic lipiarmycin (Lpm), which is under development for clinical use, inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Using genetic and biochemical approaches, we show that Lpm targets the sigma(70) subunit region 3.2 and the RNAP beta' subunit switch-2 element, which controls the clamping of promoter DNA in the RNAP active-site cleft. Lpm abolishes isomerization of the 'closed'-promoter complex to the transcriptionally competent 'open' complex and blocks sigma(70)-stimulated RNA synthesis on promoter-less DNA templates. Lpm activity decreases when the template DNA strand is stabilized at the active site through the interaction of RNAP with the nascent RNA chain. Template DNA-strand fitting into the RNAP active-site cleft directed by the beta' subunit switch-2 element and the sigma(70) subunit region 3.2 is essential for promoter melting and for de novo initiation of RNA synthesis, and our results suggest that Lpm impedes this process.

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The dramatic rise of antibiotic-resistant bacteria over the past two decades has stressed the need for completely novel classes of antibacterial agents. Accordingly, recent advances in the study of prokaryotic transcription open new opportunities for such molecules. This paper reports the structure-activity relationships of a series of phenyl-furanyl-rhodanines (PFRs) as antibacterial inhibitors of RNA polymerase (RNAP). The molecules have been evaluated for their ability to inhibit transcription and affect growth of bacteria living in suspension or in a biofilm and for their propensity to interact with serum albumin, a critical parameter for antibacterial drug discovery. The most active of these molecules inhibit Escherichia coli RNAP transcription at concentrations of

This is, to our knowledge, the first example of a small molecule inhibitor of this interaction.

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