Rolf Jansen

Opening and Closing of the Bacterial RNA Polymerase Clamp

Clamping Down Crystal structures of RNA polymerase show that a “clamp” region which surrounds the DNA binding site can adopt conformations ranging from a closed to an open state. Chakraborty et al. (p. 591) used single-molecule fluorescence energy transfer experiments to detect the clamps conformational changes in solution during the transcription cycle. The results support a model in which a clamp opening allows DNA to be loaded into the active-center cleft and unwound. Direct interactions with DNA likely trigger clamp closure upon formation of a catalytically competent transcription initiation complex. Single-molecule fluorescence measurements define the clamp conformation during transcription initiation and elongation. Using single-molecule fluorescence resonance energy transfer, we have defined bacterial RNA polymerase (RNAP) clamp conformation at each step in transcription initiation and elongation. We find that the clamp predominantly is open in free RNAP and early intermediates in transcription initiation but closes upon formation of a catalytically competent transcription initiation complex and remains closed during initial transcription and transcription elongation. We show that four RNAP inhibitors interfere with clamp opening. We propose that clamp opening allows DNA to be loaded into and unwound in the RNAP active-center cleft, that DNA loading and unwinding trigger clamp closure, and that clamp closure accounts for the high stability of initiation complexes and the high stability and processivity of elongation complexes.

Structural, functional, and genetic analysis of sorangicin inhibition of bacterial RNA polymerase.

A combined structural, functional, and genetic approach was used to investigate inhibition of bacterial RNA polymerase (RNAP) by sorangicin (Sor), a macrolide polyether antibiotic. Sor lacks chemical and structural similarity to the ansamycin rifampicin (Rif), an RNAP inhibitor widely used to treat tuberculosis. Nevertheless, structural analysis revealed Sor binds in the same RNAP β subunit pocket as Rif, with almost complete overlap of RNAP binding determinants, and functional analysis revealed that both antibiotics inhibit transcription by directly blocking the path of the elongating transcript at a length of 2–3 nucleotides. Genetic analysis indicates that Rif binding is extremely sensitive to mutations expected to change the shape of the antibiotic binding pocket, while Sor is not. We suggest that conformational flexibility of Sor, in contrast to the rigid conformation of Rif, allows Sor to adapt to changes in the binding pocket. This has important implications for drug design against rapidly mutating targets.

Archazolid and apicularen: Novel specific V-ATPase inhibitors

BackgroundV-ATPases constitute a ubiquitous family of heteromultimeric, proton translocating proteins. According to their localization in a multitude of eukaryotic membranes, they energize many different transport processes. Since their malfunction is correlated with various diseases in humans, the elucidation of the properties of this enzyme for the development of selective inhibitors and drugs is one of the challenges in V-ATPase research.ResultsArchazolid A and B, two recently discovered cytotoxic macrolactones produced by the myxobacterium Archangium gephyra, and apicularen A and B, two novel benzolactone enamides produced by different species of the myxobacterium Chondromyces, exerted a similar inhibitory efficacy on a wide range of mammalian cell lines as the well established plecomacrolidic type V-ATPase inhibitors concanamycin and bafilomycin. Like the plecomacrolides both new macrolides also prevented the lysosomal acidification in cells and inhibited the V-ATPase purified from the midgut of the tobacco hornworm, Manduca sexta, with IC50 values of 20–60 nM. However, they did not influence the activity of mitochondrial F-ATPase or that of the Na+/K+-ATPase. To define the binding sites of these new inhibitors we used a semi-synthetic radioactively labelled derivative of concanamycin which exclusively binds to the membrane Vo subunit c. Whereas archazolid A prevented, like the plecomacrolides concanamycin A, bafilomycin A1 and B1, labelling of subunit c by the radioactive I-concanolide A, the benzolactone enamide apicularen A did not compete with the plecomacrolide derivative.ConclusionThe myxobacterial antibiotics archazolid and apicularen are highly efficient and specific novel inhibitors of V-ATPases. While archazolid at least partly shares a common binding site with the plecomacrolides bafilomycin and concanamycin, apicularen adheres to an independent binding site.

7-O-Malonyl Macrolactin A, a New Macrolactin Antibiotic from Bacillus subtilis Active against Methicillin-Resistant Staphylococcus aureus, Vancomycin-Resistant Enterococci, and a Small-Colony Variant of Burkholderia cepacia

ABSTRACT We report here the discovery, isolation, and chemical and preliminary biological characterization of a new antibiotic compound, 7-O-malonyl macrolactin A (MMA), produced by a Bacillus subtilis soil isolate. MMA is a bacteriostatic antibiotic that inhibits a number of multidrug-resistant gram-positive bacterial pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and a small-colony variant of Burkholderia cepacia. MMA-treated staphylococci and enterococci were pseudomulticellular and exhibited multiple asymmetric initiation points of septum formation, indicating that MMA may inhibit a cell division function.

Stereochemical Determination and Complex Biosynthetic Assembly of Etnangien, a Highly Potent RNA Polymerase Inhibitor from the Myxobacterium Sorangium cellulosum

A potent novel analogue of the natural macrolide antibiotic etnangien, a structurally unique RNA polymerase inhibitor from myxobacteria, is reported. It may be readily obtained from fermentation broths of Sorangium cellulosum and shows high antibiotic activity, comparable to that of etnangien. However, it is much more readily available than the notoriously labile authentic natural product itself. Importantly, it is stable under neutral conditions, allowing for elaborate NMR measurements for assignment of the 12 hydroxyl- and methyl-bearing stereogenic centers. The full absolute and relative stereochemistries of these complex polyketides were determined by a combination of extensive high-field NMR studies, including J-based configuration analysis, molecular modeling, and synthetic derivatization in combination with an innovative method based on biosynthetic studies of this polyketide which is also presented here. A first look into the solution conformation and 3D structure of these promising macrolide antibiotics is reported. Finally, the complete biosynthetic gene cluster was analyzed in detail, revealing a highly unusual and complex trans-AT type polyketide biosynthesis, which does not follow colinearity rules, most likely performs programmed iteration as well as module skipping, and exhibits HMG-CoA box-directed methylation.

Streptococcus mutans inhibits Candida albicans hyphal formation by the fatty acid signaling molecule trans-2-decenoic acid (SDSF).

In the human mouth, fungi and several hundred species of bacteria coexist. Here we report a case of interkingdom signaling in the oral cavity: A compound excreted by the caries bacterium Streptococcus mutans inhibits the morphological transition from yeast to hyphae, an important virulence trait, in the opportunistic fungus Candida albicans. The compound excreted by S. mutans was originally studied because it inhibited signaling by the universal bacterial signal autoinducer‐2 (AI‐2), determined by the luminescence of a Vibrio harveyi sensor strain. The inhibitor was purified from cell‐free culture supernatants of S. mutans guided by its activity. Its chemical structure was elucidated by using NMR spectroscopy and GC‐MS and proved to be trans‐2‐decenoic acid. We show that trans‐2‐decenoic acid does not inhibit AI‐2‐specific signaling, but rather the luciferase reaction used for its detection. A potential biological role of trans‐2‐decenoic acid was then discovered. It is able to suppress the transition from yeast to hyphal morphology in the opportunistic human pathogen Candida albicans at concentrations that do not affect growth. The expression of HWP1, a hyphal‐specific signature gene of C. albicans, is abolished by trans‐2‐decenoic acid. trans‐2‐Decenoic acid is structurally similar to the diffusible signal factor (DSF) family of interkingdom‐signaling molecules and is the first member of this family from a Gram‐positive organism (Streptococcus DSF, SDSF). SDSF activity was also found in S. mitis, S. oralis, and S. sanguinis, but not in other oral bacteria. SDSF could be relevant in shaping multispecies Candida bacteria biofilms in the human body.

Isolation and spectroscopic structure elucidation of sorangicin a, a new type of macrolide-polyether antibiotic from gliding bacteria - XXX.

Abstract The broad spectrum antibiotic sorangicin A (1) has been isolated from Sorangium cellulosum , and its structure was determined spectroscopically as a 31-membered macrocyclic hydroxy-lactone carboxylic acid, containing four ether rings.

Marinoquinolines A-F, pyrroloquinolines from Ohtaekwangia kribbensis (Bacteroidetes).

Marinoquinoline A (1) was isolated from the gliding bacterium Ohtaekwangia kribbensis together with the novel marinoquinolines B-F (2-6). Their structures were elucidated from NMR and HRESIMS data. The pyrroloquinolines showed weak antibacterial and antifungal activities and moderate cytotoxicity against four growing mammalian cell lines with IC(50) values ranging from 0.3 to 8.0 μg/mL. In a screening against tropical parasites marinoquinolines A-F (1-6) showed activity against Plasmodium falciparum K1 with IC(50) values between 1.7 and 15 μM.

Rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNA polymerase active center

Rifamycin antibacterial agents inhibit bacterial RNA polymerase (RNAP) by binding to a site adjacent to the RNAP active center and preventing synthesis of RNA products >2–3 nt in length. Recently, Artsimovitch et al. [(2005) Cell 122:351–363] proposed that rifamycins function by allosteric modulation of binding of Mg2+ to the RNAP active center and presented three lines of biochemical evidence consistent with this proposal. Here, we show that rifamycins do not affect the affinity of binding of Mg2+ to the RNAP active center, and we reassess the three lines of biochemical evidence, obtaining results not supportive of the proposal. We conclude that rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNAP active center.

Apicularen A and B, Cytotoxic 10-Membered Lactones with a Novel Mechanism of Action fromChondromyces Species (Myxobacteria): Isolation, Structure Elucidation, and Biosynthesis

A novel highly cytotoxic metabolite, apicularen A (1), was isolated in a screening of the myxobacterial genus Chondromyces. The structure of 1 is characterized by a salicylic acid residue as part of a 10-membered lactone, which bears an acylenamine side chain. Compound 1 is an inhibitor of the proliferation of human cancer cell lines and induces apoptosis. Apicularen A (1) is present in nearly every strain of C.apiculatus, C. pediculatus, C. lanuginosus and C. robustus. Habitually 1 is accompanied by different amounts of a more polar variant, apicularen B (2), which was identified as 11-O-(2-N-acetamido-2-deoxy-β-D-glucopyranosyl)apicularen. According to feeding experiments with 13C-labeled acetates, glycine, and methionine, apicularen A (1) is an acetate-derived polyketide containing a glycine residue as precursor of the enamine. Uncommonly, the C3 starter unit is not assembled from propionate but from acetate and methionine.