Herbert Irschik

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.

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.

Myxobacteria: a source of new antibiotics

Abstract Myxobacteria form highly colored macroscopic fruiting bodies on rotting wood and other substrates. The organisms can move by gliding or creeping, for example, across glass and agar surfaces. They also produce a large number of unusual secondary metabolites some of which have considerable potential as antibiotics. The large-scale cultivation of myxobacteria has also, therefore, become of great interest.

Production of the Tubulin Destabilizer Disorazol in Sorangium cellulosum: Biosynthetic Machinery and Regulatory Genes

Myxobacteria show a high potential for the production of natural compounds that exhibit a wide variety of antibiotic, antifungal, and cytotoxic activities. 1 ,  2 The genus Sorangium is of special biotechnological interest because it produces almost half of the secondary metabolites isolated from these microorganisms. We describe a transposon‐mutagenesis approach to identifying the disorazol biosynthetic gene cluster in Sorangium cellulosum So ce12, a producer of multiple natural products. In addition to the highly effective disorazol‐type tubulin destabilizers, 3 – 5 S. cellulosum So ce12 produces sorangicins, potent eubacterial RNA polymerase inhibitors, 6 bactericidal sorangiolides, and the antifungal chivosazoles. 7 ,  8 To obtain a transposon library of sufficient size suitable for the identification of the presumed biosynthetic gene clusters, an efficient transformation method was developed. We present here the first electroporation protocol for a strain of the genus Sorangium. The transposon library was screened for disorazol‐negative mutants. This approach led to the identification of the corresponding trans‐acyltransferase core biosynthetic gene cluster together with a region in the chromosome that is likely to be involved in disorazol biosynthesis. A third region in the genome harbors another gene that is presumed to be involved in the regulation of disorazol production. A detailed analysis of the biosynthetic and regulatory genes is presented in this paper.

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.

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.

Analysis of the Sorangicin Gene Cluster Reinforces the Utility of a Combined Phylogenetic/Retrobiosynthetic Analysis for Deciphering Natural Product Assembly by trans‐AT PKS

The sorangicins are a group of polyketide antibiotics produced by several strains of the myxobacterium Sorangium cellulosum, whose activity is derived from inhibition of eubacterial RNA polymerase. The core structure of sorangicins A and B, the metabolites of highest abundance, comprises a 31-membered lactone that exhibits many unusual features, including a tetrasubstituted terahydropyran, a trisubstituted dihydropyran, and a signature C31–C36 bicyclic ether moiety; sorangicin B lacks a hydroxyl group at C22 relative to sorangicin A (Scheme 1). The sorangicins are the most potent myxobacterial antibiotics identified to date, exhibiting activity against both Gram-positive and Gram-negative species. The corresponding glycosides are only poorly active, however, suggesting that this modification might serve to protect S. cellulosum from its own antibiotics. Mode-of-action studies have demonstrated that sorangicin A inhibits the eubacterial RNA polymerase (RNAP). 5, 6] Binding occurs at the same site targeted by the polyketide rifampicin, an antibiotic that is used in combination therapy for the treatment of tuberculosis. However, the finding that clinical isolates of rifampicin-resistant RNAP show partial cross-resistance to sorangicin A 7] has discouraged further attempts to develop the sorangicins for therapeutic use. Nonetheless, recent studies on the mechanism of resistance have revealed that sorangicin A exhibits significantly greater tolerance to mutation-induced changes in the shape of the RNAP binding pocket relative to rifampicin, which is likely due its greater conformational flexibility. This finding suggests that structural analogues of the sorangicins could have utility in tackling strains of tuberculosis that are rifampicin resistant. We aimed to facilitate the generation of such sorangicin variants by using genetic engineering, by providing a detailed understanding of sorangicin biosynthesis in S. cellulosum. We previously reported the identification of a partial sorangicin gene cluster in S. cellulosum So ce12. The cluster was initially located by a mariner-based transposition approach within So ce12, as described previously. A mutant library consisting of 300 clones was screened by bioassay with the sorangicin-sensitive indicator strain Staphylococcus aureus, yielding four sorangicin-negative phenotypes. Disruption of sorangicin biosynthesis in each case was confirmed by analysis of culture extracts of the mutants by HPLC (Supporting Information). The transposition site in each mutant was then identified by “transposon recovery” (Supporting Information). Briefly, genomic DNA was digested with a restriction enzyme that did not cut within the transposable element, and then the DNA was liga ted and transformed into E. coli, yielding plasmids harboring the transposition site and flanking chromosomal elements. Sequencing of these regions revealed genes encoding polyketide synthases (PKSs) that are consistent with the polyketide origin of the sorangicins. Following the generation of a BAC library of Scheme 1. Structures of sorangicins A, B, and C1. The gray boxes indicate structural differences between sorangicins B and C1 and sorangicin A.

Damage of Streptococcus mutans biofilms by carolacton, a secondary metabolite from the myxobacterium Sorangium cellulosum

BackgroundStreptococcus mutans is a major pathogen in human dental caries. One of its important virulence properties is the ability to form biofilms (dental plaque) on tooth surfaces. Eradication of such biofilms is extremely difficult. We therefore screened a library of secondary metabolites from myxobacteria for their ability to damage biofilms of S. mutans.ResultsHere we show that carolacton, a secondary metabolite isolated from Sorangium cellulosum, has high antibacterial activity against biofilms of S. mutans. Planktonic growth of bacteria was only slightly impaired and no acute cytotoxicity against mouse fibroblasts could be observed. Carolacton caused death of S. mutans biofilm cells, elongation of cell chains, and changes in cell morphology. At a concentration of 10 nM carolacton, biofilm damage was already at 35% under anaerobic conditions. A knock-out mutant for comD, encoding a histidine kinase specific for the competence stimulating peptide (CSP), was slightly less sensitive to carolacton than the wildtype. Expression of the competence related alternate sigma factor ComX was strongly reduced by carolacton, as determined by a pcomX luciferase reporter strain.ConclusionsCarolacton possibly interferes with the density dependent signalling systems in S. mutans and may represent a novel approach for the prevention of dental caries.

Biosynthesis of Thuggacins in Myxobacteria: Comparative Cluster Analysis Reveals Basis for Natural Product Structural Diversity

The thuggacins are macrolide antibiotics that are active against Mycobacterium tuberculosis, the causative agent of tuberculosis. Distinct variants of these structures are produced by the myxobacteria Sorangium cellulosum So ce895 and Chondromyces crocatus Cm c5, which differ in side chain structure and modification by hydroxylation. We report here a comparative analysis of the biosynthetic gene clusters in these strains, which reveals the mechanistic basis for this architectural diversity. Although the polyketide-nonribosomal peptide cores of the molecules are highly similar, the underlying biosynthetic machineries exhibit an unexpected degree of divergence. Furthermore, the S. cellulosum gene cluster contains a crotonyl-CoA reductase (CCR) homolog not present in C. crocatus, which likely participates in assembling the unusual hexyl side chain of the So ce895 thuggacins, whereas the distinct hydroxylation pattern may result from variable action of a conserved FMN-dependent monooxygenase. Indeed, inactivation of the monooxygenase gene in C. crocatus resulted in production of both mono- and di-deshydroxy thuggacin derivatives, providing direct evidence for the role of this enzyme in the pathway. Finally, integration of a Tn5-derived npt promotor upstream of the thuggacin cluster in C. crocatus led to a significant increase in thuggacin production.