Genetic analysis of the biosynthesis of non-ribosomal peptide- and polyketide-like antibiotics, iron uptake and biofilm formation by Bacillus subtilis A1/3

The Bacillus subtilis strain A1/3 shows exceptionally diverse antibiotic capacities compared to other B. subtilis strains. To analyze this phenomenon, mutants for the putative pantotheinyltransferase gene ( pptS), and for several genes involved in non-ribosomal peptide synthesis and polyketide synthesis were constructed and characterized, using bioassays with blood cells, bacterial and fungal cells, and mass spectrometry. Among at least nine distinct bioactive compounds, five antibiotics and one siderophore activity were identified. The anti-fungal and hemolytic activities of strain A1/3 could be eliminated by mutation of the fen and srf genes essential for the synthesis of fengycins and surfactins. Both pptS - and dhb -type mutants were defective in iron uptake, indicating an inability to produce a 2,3-dihydroxybenzoate-type iron siderophore. Transposon mutants in the malonyl CoA transacylase gene resulted in the loss of hemolytic and anti-fungal activities due to the inhibition of bacillomycin L synthesis, and this led to the discovery of bmyLD-LA-LB* genes. In mutants bearing disruption mutations in polyketide ( pksM - and/or pksR -like) genes, the biosynthesis of bacillaene and difficidins, respectively, was inactivated and was accompanied by the loss of discrete antibacterial activities. The formation of biofilms (pellicles) was shown to require the production of surfactins, but no other lipopeptides, indicating that surfactins serve specific developmental functions.Communicated by A. Kondorosi     

Galiellalactone and its biogenetic precursors as chemotaxonomic markers of the Sarcosomataceae (Ascomycota)

(-)-Galiellalactone is a hexaketide metabolite with interesting pharmacological activities which was detected in four strains of Galiella rufa (Sarcosomataceae, Ascomycota) and in two unidentified fungi shown by their 18S rDNA sequences also to belong to the Sarcosomataceae. These were a wood-inhabiting apothecial species from Chile and an endophytic isolate from Cistus salviifolius (Sardinia). Other members of the family (Urnula helvelloides, one Strumella coryneoidea isolate) produced no galiellalactone but merely hexaketides structurally related to galiellalactone precursors, whereas a third group of species (Sarcosoma latahensis, Strumella griseola, one S. coryneoidea isolate) lacked hexaketide production altogether. Despite thorough screening programmes, galiellalactone and its precursors have not yet been found in any fungus outside the Sarcosomataceae and may thus be a chemotaxonomic marker of the family, supporting its current phylogenetic definition. Two pentaketide derivatives of the 6-pentyl-alpha-pyrone type were found in all G. rufa strains as well as in A111-95 and the hexaketide-producing S. coryneoidea isolate.     

Natural products with calmodulin inhibitor properties

This review summarizes properties of various naturally occurring compounds with reported calmodulin (CaM)-inhibitory properties which include about 159 natural products belonging to different structural classes. Most inhibitors are alkaloid and peptide type of compounds and have been isolated from a wide variety of natural sources, including many plant species. Among the most potent natural anti-CaM substances, however, are several animal venoms and the antibiotic polymixin. The largest number of compounds described were discovered by means of enzymatic functional assays.     

Phomoxins B and C: polyketides from an endophytic fungus of the genus Eupenicillium

Chemical investigations of the culture broth from an endophytic fungus Eupenicillium sp. have afforded two natural products phomoxins B (1) and C (2) as well as the previously reported fungal metabolite eupenoxide (3). Compounds 1 and 2 both contain a cyclic carbonate moiety that is rare among natural products. This paper reports the full spectroscopic characterisation of phomoxins B (1) and C (2) by NMR, UV, IR and MS data. All compounds were inactive against a panel of nosocomial microbes.     

A sensitive and robust method for quantification of intracellular short-chain coenzyme A esters

A procedure for the analysis of short-chain intracellular coenzyme A (CoA) esters and adenine nucleotide pools in microbial cells is described. The simultaneous isolation of bacterial cells from media, quenching of their metabolism, and extraction of metabolites was accomplished by centrifugation of cells through a layer of silicone oil into a denser solution of trichloroacetic acid. The acid was neutralized by extraction into Freon containing tri-n-octylamine to provide a salt-free solution of cell metabolites. After high-performance liquid chromatography separation, CoA, CoA esters, and adenine-containing nucleotides were derivatized by postcolumn reaction with bromoacetaldehyde to form the fluorescent 1,N6-ethenoadenine adducts which were analyzed by a fluorescence detector at picomolar levels.     

Analysis of myxobacterial secondary metabolism goes molecular

During the last 20 years myxobacteria have made their way from highly exotic organisms to one of the major sources of microbial secondary metabolites besides actinomycetes and fungi. The pharmaceutical interest in these peculiar prokaryotes lies in their ability to produce a variety of structurally unique compounds and/or metabolites with rare biological activities. This review deals with the recent progress toward a better understanding of the biology, the genetics, the biochemistry and the regulation of secondary metabolite biosynthesis in myxobacteria. These research efforts paved the way to sophisticated in vitro studies and to the heterologous expression of complete biosynthetic pathways in conjunction with their targeted manipulation. The progress made is a prerequisite for using the vast resource of myxobacterial diversity regarding secondary metabolism more efficiently in the future.     

The aureolic acid family of antitumor compounds: structure, mode of action, biosynthesis, and novel derivatives

Members of the aureolic acid family are tricyclic polyketides with antitumor activity which are produced by different streptomycete species. These members are glycosylated compounds with two oligosaccharide chains of variable sugar length. They interact with the DNA minor groove in high-GC-content regions in a nonintercalative way and with a requirement for magnesium ions. Mithramycin and chromomycins are the most representative members of the family, mithramycin being used as a chemotherapeutic agent for the treatment of several cancer diseases. For chromomycin and durhamycin A, antiviral activity has also been reported. The biosynthesis gene clusters for mithramycin and chromomycin A₃ have been studied in detail by gene sequencing, insertional inactivation, and gene expression. Most of the biosynthetic intermediates in these pathways have been isolated and characterized. Some of these compounds showed an increase in antitumor activity in comparison with the parent compounds. A common step in the biosynthesis of all members of the family is the formation of the tetracyclic intermediate premithramycinone. Further biosynthetic steps (glycosylation, methylations, acylations) proceed through tetracyclic intermediates which are finally converted into tricyclic compounds by the action of a monooxygenase, a key event for the biological activity. Heterologous expression of biosynthetic genes from other aromatic polyketide pathways in the mithramycin producer (or some mutants) led to the isolation of novel hybrid compounds.Felipe Lombó and Nuria Menéndez have equally contribute to this work.     

Starter unit specificity directs genome mining of polyketide synthase pathways in fungi

Search of the protein database with the aflatoxin pathway polyketide synthase (PKS) revealed putative PKSs in the pathogenic fungi Coccidioides immitis and Coccidioides posadasii that could require partnerships with a pair of fatty acid synthase (FAS) subunits for the biosynthesis of fatty acid-polyketide hybrid metabolites. A starter unit:acyl-carrier protein transacylase (SAT) domain was discovered in the nonreducing PKS. This domain is thought to accept the fatty acid product from the FAS to initiate polyketide synthesis. We expressed the C. immitis SAT domain in Escherichia coli and showed that this domain, unlike that from the aflatoxin pathway PKS, transferred octanoyl-CoA four times faster than hexanoyl-CoA. The SAT domain also formed a covalent octanoyl intermediate and transferred this group to a free-standing ACP domain. Our results suggest that C. immitis/posadasii, both human fungal pathogens, contain a FAS/PKS cluster with functional similarity to the aflatoxin cluster found in Aspergillus species. Dissection of the PKS and determination of in vitro SAT domain specificity provides a tool to uncover the growing number of similar sequenced pathways in fungi, and to guide elucidation of the fatty acid-polyketide hybrid metabolites that they produce.     

Acetogenic anthraquinones: biosynthetic convergence and chemical evidence of enzymatic cooperation in nature

Phenylanthraquinones belong to the quite rare class of fully unsymmetric biaryls, consisting of two different molecular portions, an anthraquinone part, chrysophanol, and a phenyl part, 2,4-dihydroxy-6-methoxyacetophenone, linked together by phenol-oxidative coupling. The biosynthesis of these two moieties, from eight and four acetate units, respectively, bears some unique features: Chrysophanol is the first example of an acetogenic natural product that is, in an organism-specific manner, formed via more than one folding mode: In eukaryotes, like, e.g., in fungi, in higher plants, and in insects, it is formed via folding mode F, while in prokaryotes it originates through mode S. It has, more recently, even been found to be synthesized by a third pathway, which we have named mode S′. It is thus the first example of biosynthetic convergence in polyketide biosynthesis. The monocyclic “southern” portion of the molecule, which is much simpler (arising from only four acetate units and without decarboxylation), unexpectedly does not show the anticipated randomization of the C₂-labeling in the aromatic ring, but has clearly localized C₂ units, excluding any symmetric intermediate like, e.g., 2,4,6-trihydroxyacetophenone. This is confirmed by competitive feeding experiments with specifically ¹³C₂-labeled acetophenones, showing the O-methylation to be the decisive symmetry-preventing step, which hints at a close collaboration of the participating enzymes. The results make knipholone an instructive example of structure, function, and evolution of polyketide synthases and O-methyltransferases, and their cooperation.