Kathrin Schneider

Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42.

Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Its 3,918-kb genome, containing an estimated 3,693 protein-coding sequences, lacks extended phage insertions, which occur ubiquitously in the closely related Bacillus subtilis 168 genome. The B. amyloliquefaciens FZB42 genome reveals an unexpected potential to produce secondary metabolites, including the polyketides bacillaene and difficidin. More than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores by pathways not involving ribosomes. Besides five gene clusters, known from B. subtilis to mediate nonribosomal synthesis of secondary metabolites, we identified four giant gene clusters absent in B. subtilis 168. The pks2 gene cluster encodes the components to synthesize the macrolactin core skeleton.

Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens

The genome of plant-associated Bacillus amyloliquefaciens FZB42 harbors an array of giant gene clusters involved in synthesis of lipopeptides and polyketides with antifungal, antibacterial and nematocidal activity. Five gene clusters, srf, bmy, fen, nrs, dhb, covering altogether 137 kb, were shown to direct synthesis of the cyclic lipopeptides surfactin, bacillomycin, fengycin, an unknown peptide, and the iron-siderophore bacillibactin. In addition, one gene cluster encoding enzymes involved in synthesis and export of the antibacterial dipeptide bacilysin is also functional in FZB42. Three gene clusters, mln, bae, and dfn, with a total size of 199 kb were shown to direct synthesis of the antibacterial acting polyketides macrolactin, bacillaene, and difficidin. In total, FZB42 dedicates about 340 kb, corresponding to 8.5% of its total genetic capacity, to synthesis of secondary metabolites. On the contrary, genes involved in ribosome-dependent synthesis of lantibiotics and other peptides are scarce. Apart from two incomplete gene clusters directing immunity against mersacidin and subtilin, only one peptide-like compound has been detected in the culture fluid that inhibits the growth of B. subtilis lacking the alternative sigma factor W.

Structural and Functional Characterization of Three Polyketide Synthase Gene Clusters in Bacillus amyloliquefaciens FZB 42

Although bacterial polyketides are of considerable biomedical interest, the molecular biology of polyketide biosynthesis in Bacillus spp., one of the richest bacterial sources of bioactive natural products, remains largely unexplored. Here we assign for the first time complete polyketide synthase (PKS) gene clusters to Bacillus antibiotics. Three giant modular PKS systems of the trans-acyltransferase type were identified in Bacillus amyloliquefaciens FZB 42. One of them, pks1, is an ortholog of the pksX operon with a previously unknown function in the sequenced model strain Bacillus subtilis 168, while the pks2 and pks3 clusters are novel gene clusters. Cassette mutagenesis combined with advanced mass spectrometric techniques such as matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization mass spectrometry revealed that the pks1 (bae) and pks3 (dif) gene clusters encode the biosynthesis of the polyene antibiotics bacillaene and difficidin or oxydifficidin, respectively. In addition, B. subtilis OKB105 (pheA sfp(0)), a transformant of the B. subtilis 168 derivative JH642, was shown to produce bacillaene, demonstrating that the pksX gene cluster directs the synthesis of that polyketide. The GenBank accession numbers for gene clusters pks1(bae), pks2, and pks3(dif) are AJ 634060.2, AJ 6340601.2, and AJ 6340602.2, respectively.

Labyrinthopeptins: A New Class of Carbacyclic Lantibiotics

Lantibiotics are peptides that are ribosomally synthesized from bacteria such as staphylococci, lactobacilli, and actinomycetes. The common structural characteristic of lantibiotics is the noncanonical amino acid lanthionine (Lan, 1; Figure 1), which confers conformational stability to the peptide. The most prominent representative is nisin, which is a lipid II binder, and has been known for its use as an antimicrobial food preservative for over 40 years. The majority of studies on molecular targets and bioactivities are focused on potential applications of lantibiotics as anti-infectives. Duramycin (Moli1901) is in phase II clinical trials for the treatment of cystic fibrosis because of its ability to increase chloride transport in airway epithelium. Biosurfactant function in the life cycle of streptomycetes has been elucidated for some members such as SapB. Herein, we present the structures, the biosynthesis gene cluster, and the bioactivities of labyrinthopeptins, which are lantibiotics that contain labionin, an unprecedented carbacyclic, posttranslationally modified amino acid. The culture extracts of the novel actinomycete Actinomadura namibiensis DSM 6313 attracted our attention because of their activity against the Herpes simplex virus. Active fractions of the extracts contained a peptide that was isolated by chromatographic methods. The high-resolution ESI-FTICR mass spectrum showed a mass of 984.3333 Da for the doubly charged sodium adduct of the compound, corresponding to a neutral monoisotopic mass of 1922.6872 Da and the molecular formula C85H110N20O24S4 (Dm/m= 0.7 ppm). Amino acid analysis revealed Gly and the l-enantiomers of Ala, Thr, Leu, Asx, Cys, Phe, Glx, Trp (ratio 1:1:1:2:1:2:1:1:2). However, the total molecular mass of the detected amino acids indicated a considerable mass difference, which could not be correlated with known peptidic or lantibiotic posttranslational modifications. Resolution of the structure by H NMR spectroscopy was impeded by broad signals in parts of the spectrum. The X-ray structure at 1.0 resolution (Figure 1) enabled interpretation of the analytical data and displayed several unique structural features. In view of its labyrinthine structure, the compound was named labyrinthopeptin A2 (2). Labyrinthopeptin A2 has a globular structure that consists primarily of hydrophobic amino acids. Formally, the structure can be dissected into two nonapeptides. Each peptide bears a C-terminal Cys residue that forms a disulfide bond, which is a comparatively rare modification in lantibiotics, but is found for sublancin 168 from B. subtilis. Each nonapeptide contains a tetrapeptide (ring A) and a pentapeptide (ring B) that share a quaternary aC atom; labyrinthopeptin A rings are formed by a methylene group between the aC atoms of Lab1/ Lab10 and Lab4/Lab13 (Figure 1). A carbacyclic side-chain linkage is unprecedented in peptides and proteins. We propose the name labionin (Lab) for the corresponding amino acid (Figure 1). Labionin 3 represents an aC quaternary substituted amino acid with a subtle structural resemblance to a-aminoisobutyric acid (Aib) or isovaline (Iva), which are incorporated in fungal peptaibol-type antibiotics. The stereocenters of 3 can be assigned to (2S,4S,8R)-labionin (Lab), which is consistent with the configuration of (2S,6R)lanthionine of other lantibiotics. The formation of the 11membered ring that involves 3 forces the peptide backbone into a conformation with cis-amide bonds between Asp2– Trp3 and Thr11–Gly12, respectively (Figure 1). The presence of cis-amide bonds and the absence of a hydrogen bond between Lab1–Lab4 and Lab10–Lab13, respectively, show that the turn motif in 2 is clearly different from a b-turn motif. Subsequent identification of the biosynthetic gene cluster was performed from a cosmid library of A. namibiensis by means of degenerated primer probes, followed by sequencing [*] Dr. T. Schmiederer, Dr. K. Schneider, Dr. A. Reicke, Dr. D. Butz, Dr. S. Keller, Prof. Dr. R. D. S ssmuth Technische Universit t Berlin, Fakult t II—Institut f r Chemie Strasse des 17. Juni 124, 10623 Berlin (Germany) Fax: (+49)30-314-24205 E-mail: suessmuth@chem.tu-berlin.de Homepage: http://www2.tu-berlin.de/fb5/Suessmuth/ contact.html Dr. K. Meindl, Prof. Dr. G. M. Sheldrick Universit t G ttingen (Germany)

Caboxamycin, a new antibiotic of the benzoxazole family produced by the deep-sea strain Streptomyces sp. NTK 937*

Caboxamycin, a new benzoxazole antibiotic, was detected by HPLC-diode array screening in extracts of the marine strain Streptomyces sp. NTK 937, which was isolated from deep-sea sediment collected in the Canary Basin. The structure of caboxamycin was determined by mass spectrometry, NMR experiments and X-ray analysis. It showed inhibitory activity against Gram-positive bacteria, selected human tumor cell lines and the enzyme phosphodiesterase.

Proximicin A,B and C, Novel Aminofuran Antibiotic and Anticancer Compounds Isolated from Marine Strains of the Actinomycete Verrucosispora

A family of three novel aminofuran antibiotics named as proximicins was isolated from the marine Verrucosispora strain MG-37. Proximicin A was detected in parallel in the marine abyssomicin producer “Verrucosispora maris” AB-18-032. The characteristic structural element of proximicins is 4-amino-furan-2-carboxylic acid, a hitherto unknown γ-amino acid. Proximicins show a weak antibacterial activity but a strong cytostatic effect to various human tumor cell lines.

A Genomic Screening Approach to the Structure-Guided Identification of Drug Candidates from Natural Sources

The potential of actinomycetes to produce natural products has been exploited for decades. Recent genomic sequence analyses have revealed a previously unrecognized biosynthetic potential and diversity. In order to rationally exploit this potential, we have developed a sequence‐guided genetic screening strategy. In this “genome mining” approach, genes that encode tailoring enzymes from natural product biosyntheses pathways serve as indicator genes for the identification of strains that have the genetic potential to produce natural products of interest. We chose halogenases, which are known to be involved in the synthesis of halometabolites as representative examples. From PCR screening of 550 randomly selected actinomycetes strains, we identified 103 novel putative halogenase genes. A phylogenetic analysis of the corresponding putative halogenases, and the determination of their sequential context with mass spectrometric analysis of cultures filtrates revealed a distinct correlation between the sequence and secondary metabolite class of the halometabolite. The described screening strategy allows rapid access to novel natural products with predetermined structural properties.

The Final Steps of Bacillaene Biosynthesis in Bacillus amyloliquefaciens FZB42: Direct Evidence for β,γ Dehydration by a trans-Acyltransferase Polyketide Synthase†

The antibiotic bacillaene (1; Scheme 1) is the prototype of a growing class of polyketides synthesized by a family of polyketide synthases (PKSs) termed trans-acyltransferase (AT) PKSs. 3] These poorly studied enzymes are giant multimodular proteins that have evolved independently from the textbook (cis-AT) PKSs involved in, for example, erythromycin biosynthesis. Since they are rare in actinomycetes, the first model organisms used for studies on polyketide biosynthesis, trans-AT PKSs have long been overlooked. However, they are now known to be widespread among many other rich natural product sources and responsible for the production of several pharmacologically relevant polyketides, including antibiotics of the mupirocin and streptogramin groups and the antitumor drug candidate bryostatin 1. trans-AT PKSs have also attracted considerable ecological interest, since they occur in bacteria with unusual lifestyles, such as symbionts, pathogens, and anaerobes. 6] Another peculiarity that sets them apart from cisAT enzymes and raises fundamental questions about how polyketides are assembled by these proteins is the high frequency of biosynthetic features that do not conform with classical PKS rules. 3,7] During functional studies with 1 as a model, we discovered that deletion of the thioesterase (TE) domain, which releases the assembled polyketide chain from the PKS, results in the production of virtually all intermediates. This unusual phenomenon enabled us to unravel a large part of bacillaene biosynthesis and, in combination with phylogenetic analyses, to establish a set of colinearity rules that can be applied to the prediction and discovery of natural products from trans-AT PKS sequences. Several questions, however, remained unanswered after the studies on 1. The triene system in the C3–C8 region exhibits rare b,g-type unsaturation instead of the standard a,b pattern; the resulting enamide moiety is known in few other polyketides. Since 1 and its advanced precursors are highly unstable, our previous structural investigations to characterize the bacillaene assembly had relied on HPLC coupled to high-resolution MS (HRMS). This technique, however, does not distinguish between constitutional isomers and thus leaves unclear when and how trans-AT PKSs catalyze olefinic shifts (Scheme 1). We also noticed that in late cultures of the bacillaene producer Bacillus amyloliquefaciens FZB42, 1 was almost entirely replaced by a new compound of unknown structure (see Figure S1 in the Supporting Information). This result raised the question of whether 1 is really the true biosynthetic end product. Finally, the bacillaene (bae) PKS terminates with two noncanonical modules (modules 16 and 17, Scheme 1), the ketosynthase (KS) domains of which were predicted by bioinformatic analysis to be unable to catalyze chain-elongation reactions. The role of such seemingly superfluous terminal modules, which are ubiquitous in trans-AT PKSs, is presently unknown. Another architectural oddity is a dehydratase (DH) domain in module 16 which could not be attributed to any dehydration reaction. To examine the function of modules 16 and 17, we genetically engineered the PKS mutants JM54-2, with a deletion of module 17 + TE, and JM122, in which module 17 plus the TE domain are fused directly onto module 15. HPLC–HRMS analysis showed that both strains produced a series of prematurely released intermediates, the two most advanced of which had the molecular formulae C34H48N2O6 [*] J. Moldenhauer, Dr. M. Engeser, Prof. Dr. J. Piel Kekul Institut f r Organische Chemie und Biochemie Universit t Bonn Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany) Fax: (+ 49)228-739-712 E-mail: joern.piel@uni-bonn.de Homepage: http:/www.chemie.uni-bonn.de/oc/ak_piel

Proximicins A, B, and C—Antitumor Furan Analogues of Netropsin from the Marine Actinomycete Verrucosispora Induce Upregulation of p53 and the Cyclin Kinase Inhibitor p21

Netropsin (1) and distamycin (2) are naturally occurring gpeptides with antiviral and antibacterial activity (Figure 1). Netropsin, formerly named congocidine, was isolated from S. netropsis in 1951, while distamycin was isolated from S. distallicus in 1964. Both compounds bind in the minor groove of DNA, and they were arguably the first compounds for which AT-selective DNA binding was demonstrated. The design of synthetic derivatives capable of addressing a specific sequence of DNAwould allow the selective inhibition of gene expression and thus result in compounds that act as antitumor agents. As a consequence, netropsin and distamycin were used as the basic structures for the synthesis of numerous analogues to enable the investigation and modulation of their minor-groove binding. This was also achieved by using combinatorial approaches. A particular challenge was to generate selective binders for GC base pairs. One approach was based on the assumption that the introduction of a hydrogen-bond acceptor in the pyrrole

Nataxazole, a New Benzoxazole Derivative with Antitumor Activity Produced by Streptomyces sp. Tü 6176 †

The new benzoxazole derivative nataxazole was isolated from Streptomyces sp. (strain Tü 6176). Nataxazole is related in structure to the potent antitumor compounds UK-1 and AJI9561 and showed similar strong growth inhibitory activity against various human tumor cell lines.