Gene Inactivation Experiments Research Articles

Organisation of the Biosynthetic Gene Cluster and Tailoring Enzymes in the Biosynthesis of the Tetracyclic Quinone Glycoside Antibiotic Polyketomycin

Polyketomycin is a tetracyclic quinone glycoside produced by Streptomyces diastatochromogenes Tü6028. It shows cytotoxic and antibiotic activity, in particular against Gram-positive multi-drug-resistant strains (for example, MRSA). The polyketomycin biosynthetic gene cluster has been sequenced and characterised. Its identity was proven by inactivation of a alpha-ketoacyl synthase gene (pokP1) of the "minimal polyketide synthase II" system. In order to obtain valuable information about tailoring steps, we performed further gene-inactivation experiments. The generation of mutants with deletions in oxygenase genes (pokO1, pokO2, both in parallel and pokO4) and methyltransferase genes (pokMT1, pokMT2 and pokMT3) resulted in new polyketomycin derivatives, and provided information about the organisation of the biosynthetic pathway.

Efflux Unbalance in Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

Retrospective analysis of 189 nonredundant strains of Pseudomonas aeruginosa sequentially recovered from the sputum samples of 46 cystic fibrosis (CF) patients over a 10-year period (1998 to 2007) revealed that 53 out of 189 (28%) samples were hypersusceptible to the beta-lactam antibiotic ticarcillin (MIC < or = 4 microg/ml) (phenotype dubbed Tic(hs)). As evidenced by trans-complementation and gene inactivation experiments, the mutational upregulation of the efflux system MexXY was responsible for various degrees of resistance to aminoglycosides in a selection of 11 genotypically distinct strains (gentamicin MICs from 2 to 64 microg/ml). By demonstrating for the first time that the MexXY pump may evolve in CF strains, we found that a mutation leading to an F1018L change in the resistance-nodulation-cell division (RND) transporter MexY was able to increase pump-promoted resistance to aminoglycosides, cefepime, and fluoroquinolones twofold. The inactivation of the mexB gene (which codes for the RND transporter MexB) in the 11 selected strains showed that the Tic(hs) phenotype was due to a mutational or functional loss of function of MexAB-OprM, the multidrug efflux system known to contribute to the natural resistance of P. aeruginosa to beta-lactams (e.g., ticarcillin and aztreonam), fluoroquinolones, tetracycline, and novobiocin. Two of the selected strains synthesized abnormally low amounts of the MexB protein, and 3 of 11 strains expressed truncated MexB (n = 2) or MexA (n = 1) polypeptide as a result of mutations in the corresponding genes, while 7 of 11 strains produced wild-type though nonfunctional MexAB-OprM pumps at levels similar to or even higher than that of reference strain PAO1. Overall, our data indicate that while MexXY is necessary for P. aeruginosa to adapt to the hostile environment of the CF lung, the MexAB-OprM pump is dispensable and tends to be lost or inactivated in subpopulations of P. aeruginosa.

Characterization of the Tautomycetin Biosynthetic Gene Cluster from Streptomyces griseochromogenes Provides New Insight into Dialkylmaleic Anhydride Biosynthesis

Tautomycetin (TTN) is a highly potent and specific protein phosphatase inhibitor isolated from Streptomyces griseochromogenes. The biological activity of TTN makes it an important lead for drug discovery, whereas its rare dialkylmaleic anhydride moiety and structural similarity to tautomycin (TTM), another potent phosphatase inhibitor with tremendous medicinal potential, draws attention to novel biosynthetic chemistries responsible for its production. To elucidate the biosynthetic machinery associated with TTN production, the ttn biosynthetic gene cluster from S. griseochromogenes was isolated and characterized, and its involvement in TTN biosynthesis confirmed by gene inactivation and complementation experiments. The ttn cluster was localized to a 79 kb DNA region, consisting of 19 open reading frames that encode two modular type I polyketide synthases (TtnAB), one type II thioesterase (TtnH), eight proteins for dialkylmaleic anhydride biosynthesis (TtnKLMNOPRS), four tailoring enzymes (TtnCDFI), two regulatory proteins (TtnGQ), and one resistance protein (TtnJ). A model for TTN biosynthesis is proposed on the basis of functional assignments from sequence analysis, which agrees well with previous feeding experiments, has been supported by in vivo gene inactivation experiments, and is supported by analogy to the recently reported ttm cluster. These findings set the stage to fully investigate TTN biosynthesis and to biosynthetically engineer new TTN analogues.

Characterization of the Tautomycin Biosynthetic Gene Cluster from Streptomyces spiroverticillatus Unveiling New Insights into Dialkylmaleic Anhydride and Polyketide Biosynthesis

Tautomycin (TTM) is a highly potent and specific protein phosphatase inhibitor isolated from Streptomyces spiroverticillatus. The biological activity of TTM makes it an important lead for drug discovery, whereas its spiroketal-containing polyketide chain and rare dialkylmaleic anhydride moiety draw attention to novel biosynthetic chemistries responsible for its production. To elucidate the biosynthetic machinery associated with these novel molecular features, the ttm biosynthetic gene cluster from S. spiroverticillatus was isolated and characterized, and its involvement in TTM biosynthesis was confirmed by gene inactivation and complementation experiments. The ttm cluster was localized to a 86-kb DNA region, consisting of 20 open reading frames that encode three modular type I polyketide synthases (TtmHIJ), one type II thioesterase (TtmT), five proteins for methoxymalonyl-S-acyl carrier protein biosynthesis (Ttm-ABCDE), eight proteins for dialkylmaleic anhydride biosynthesis and regulation (TtmKLMNOPRS), as well as two additional regulatory proteins (TtmF and TtmQ) and one tailoring enzyme (TtmG). A model for TTM biosynthesis is proposed based on functional assignments from sequence analysis, which agrees well with previous feeding experiments, and has been further supported by in vivo gene inactivation experiments. These findings set the stage to fully investigate TTM biosynthesis and to biosynthetically engineer new TTM analogs.

Protein kinase C-delta mediates von Willebrand factor secretion from endothelial cells in response to vascular endothelial growth factor (VEGF) but not histamine.

Vascular endothelial growth factor (VEGF) and histamine induce von Willebrand factor (VWF) release from vascular endothelial cells. Protein kinase C (PKC) is involved in the control of exocytosis in many secretory cell types. We investigated the role of PKC and the interactions between PKC and Ca2+ signaling in both VEGF-induced and histamine-induced VWF secretion from human umbilical vein endothelial cells (HUVECs). Several PKC inhibitors (staurosporine, Ro31-8220, myristoylated PKC peptide inhibitor and Go6983) block VEGF-induced but not histamine-induced VWF secretion. PKC-alpha and novel PKCs (PKC-delta, PKC-epsilon, and PKC-eta), but not PKC-beta, are expressed in HUVECs. Both VEGF and histamine activate PKC-delta. However, gene inactivation experiments using small interfering RNA indicate that PKC-delta (but not PKC-alpha) is involved in the regulation of VEGF-induced but not histamine-induced secretion. Both VEGF and histamine induce a rise in cytosolic free Ca2+ ([Ca2+]c), but the response to VEGF is weaker and even absent in a significant subset of cells. Furthermore, VEGF-induced secretion is largely preserved when the rise in [Ca2+]c is prevented by BAPTA-AM. Our study identifies striking agonist specificities in signal-secretion coupling. Histamine-induced secretion is dependent on [Ca2+]c but not PKC, whereas VEGF-induced secretion is largely dependent on PKC-delta and significantly less on [Ca2+]c. Our data firmly establish the key role of PKC-delta in VEGF-induced VWF release, but suggest that a third, VEGF-specific, signaling intermediate is required as a PKC-delta coactivator.

Biosynthesis and Structures of Cyclomarins and Cyclomarazines, Prenylated Cyclic Peptides of Marine Actinobacterial Origin

Two new diketopiperazine dipeptides, cyclomarazines A and B, were isolated and characterized along with the new cyclic heptapeptide cyclomarin D from the marine bacterium Salinispora arenicola CNS-205. These structurally related cyclic peptides each contain modified amino acid residues, including derivatives of N-(1,1-dimethylallyl)-tryptophan and delta-hydroxyleucine, which are common in the di- and heptapeptide series. Stable isotope incorporation studies in Streptomyces sp. CNB-982, which was first reported to produce the cyclomarin anti-inflammatory agents, illuminated the biosynthetic building blocks associated with the major metabolite cyclomarin A, signifying that this marine microbial peptide is nonribosomally derived largely from nonproteinogenic amino acid residues. DNA sequence analysis of the 5.8 Mb S. arenicola circular genome and PCR-targeted gene inactivation experiments identified the 47 kb cyclomarin/cyclomarazine biosynthetic gene cluster (cym) harboring 23 open reading frames. The cym locus is dominated by the 23 358 bp cymA, which encodes a 7-module nonribosomal peptide synthetase (NRPS) responsible for assembly of the full-length cyclomarin heptapeptides as well as the truncated cyclomarazine dipeptides. The unprecedented biosynthetic feature of the megasynthetase CymA to synthesize differently sized peptides in vivo may be triggered by the level of beta oxidation of the priming tryptophan residue, which is oxidized in the cyclomarin series and unoxidized in the cyclomarazines. Biosynthesis of the N-(1,1-dimethyl-2,3-epoxypropyl)-beta-hydroxytryptophan residue of cyclomarin A was further illuminated through gene inactivation experiments, which suggest that the tryptophan residue is reverse prenylated by CymD prior to release of the cyclic peptide from the CymA megasynthetase, whereas the cytochrome P450 CymV installs the epoxide group on the isoprene of cyclomarin C post-NRPS assembly. Last, the novel amino acid residue 2-amino-3,5-dimethylhex-4-enoic acid in the cyclomarin series was shown by bioinformatics and stable isotope experiments to derive from a new pathway involving condensation of isobutyraldehyde and pyruvate followed by S-adenosylmethionine methylation. Assembly of this unsaturated, branched amino acid is unexpectedly related to the degradation of the environmental pollutant 3-(3-hydroxyphenyl)propionic acid.

Identification of Fredericamycin E from Streptomyces griseus: Insights into Fredericamycin A Biosynthesis Highlighting Carbaspirocycle Formation

Fredericamycin (FDM) A ( 1), a pentadecaketide featuring two sets of peri-hydroxy tricyclic aromatic moieties connected through a unique asymmetric carbaspiro center, exhibits potent cytotoxicity and represents a novel anticancer drug lead. We have localized previously the fdm gene cluster to a 33 kb DNA segment of Streptomyces griseus ATCC49344, the involvement of which in the biosynthesis of 1 was confirmed by gene inactivation, complementation, and heterologous expression experiments. We now report the isolation and characterization of FDM E ( 5), a heretofore undetected intermediate for 1 biosynthesis from S. griseus, shedding new insight into the mechanism of carbaspirocycle formation. The structure of 5 was elucidated through the combination of spectroscopic methods and isotope-labeling experiments. The core spiro[4.5]decane scaffold of 5 is characterized by a unique cyclohexa-1,2,4-triketone moiety. Transformation of the spiro[4.5]decane 5 into the spiro[4.4]nonane 1 can be rationalized by a biosynthetic benzilic acid-like rearrangement. This unusual rearrangement can be mimicked in vitro by proceeding under aerobic conditions in the absence of enzyme. FDM E displays cytotoxic activity on par with 1 against a selected set of cancer cells, a finding that further supports the unique molecular topology, resulting from the unprecedented carbaspirocycle as exemplified by 1 and 5, as a novel pharmacophore for this family of anticancer agents.

Involvement of BmoR and BmoG in n-alkane metabolism in ‘Pseudomonas butanovora’

'Pseudomonas butanovora' uses an alcohol-inducible alkane monooxygenase (BMO) to grow on C(2)-C(9) n-alkanes. Five ORFs were identified flanking the BMO structural genes. Two of the ORFs, bmoR, encoding a putative sigma(54)-transcriptional regulator BmoR, and bmoG, encoding a putative GroEL chaperonin BmoG, were analysed by gene-inactivation experiments. The BmoR-deficient mutant grew at slower growth rates than the wild-type on C(2)-C(5) n-alkanes and showed little to no growth on C(6)-C(8) n-alkanes within 7 days. A BmoR-deficient mutant was constructed in the 'P. butanovora' bmoX : : lacZ reporter strain and used to test whether bmoR was involved in bmoX induction after growth on C(2)-C(8) carbon sources. In acetate- or lactate-grown cells, C(2)-C(8) n-alcohols failed to induce beta-galactosidase activity. In contrast, in propionate-, butyrate- or pentanoate-grown cells, n-butanol induced approximately 45 % of the beta-galactosidase activity observed in the control bmoX : : lacZ strain. In propionate-grown cells, C(2)-C(5) n-alcohols induced beta-galactosidase activity, whereas C(7) and C(8) n-alcohols did not. BmoR may act as a sigma(54)-transcriptional regulator of bmo that is controlled by the n-alcohol produced in the alkane oxidation. During growth on short-chain-length fatty acids, however, another BMO regulatory system seems to be activated to promote transcription of bmo by short-chain-length alcohols (i.e. </=C(6)). The bmoG-deficient mutant did not grow on C(2)-C(8) n-alkanes; however, it was capable of transcribing bmoX and bmoC of the BMO operon. BmoG may act as a chaperonin to assemble competent BMO.

Multi-oxygenase Complexes of the Gilvocarcin and Jadomycin Biosyntheses

A series of gene inactivation and gene recombination experiments involving oxygenase encoding genes of the gilvocarcin and jadomycin biosynthetic pathways showed that the enigmatic oxidative rearra...

Sequencing and Analysis of the Biosynthetic Gene Cluster of the Lipopeptide Antibiotic Friulimicin in Actinoplanes friuliensis

Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin, which is a cyclic peptide with one exocyclic amino acid linked to a branched-chain fatty acid acyl residue. The structural relationship to daptomycin and the excellent antibacterial performance of friulimicin make the antibiotic an attractive drug candidate. The complete friulimicin biosynthetic gene cluster of 24 open reading frames from A. friuliensis was sequenced and analyzed. In addition to genes for regulation, self-resistance, and transport, the cluster contains genes encoding peptide synthetases, proteins involved in the synthesis and linkage of the fatty acid component of the antibiotic, and proteins involved in the synthesis of the nonproteinogenic amino acids pipecolinic acid, methylaspartic acid, and 2,3-diaminobutyric acid. By using heterologous gene expression in Escherichia coli, we provide biochemical evidence for the stereoselective synthesis of L-pipecolinic acid by the deduced protein of the lysine cyclodeaminase gene pip. Furthermore, we show the involvement of the dabA and dabB genes in the biosynthesis of 2,3-diaminobutyric acid by gene inactivation and subsequent feeding experiments.

Thiosulphate oxidation in the phototrophic sulphur bacterium Allochromatium vinosum

Two different pathways for thiosulphate oxidation are present in the purple sulphur bacterium Allochromatium vinosum: oxidation to tetrathionate and complete oxidation to sulphate with obligatory formation of sulphur globules as intermediates. The tetrathionate:sulphate ratio is strongly pH-dependent with tetrathionate formation being preferred under acidic conditions. Thiosulphate dehydrogenase, a constitutively expressed monomeric 30 kDa c-type cytochrome with a pH optimum at pH 4.2 catalyses tetrathionate formation. A periplasmic thiosulphate-oxidizing multienzyme complex (Sox) has been described to be responsible for formation of sulphate from thiosulphate in chemotrophic and phototrophic sulphur oxidizers that do not form sulphur deposits. In the sulphur-storing A. vinosum we identified five sox genes in two independent loci (soxBXA and soxYZ). For SoxA a thiosulphate-dependent induction of expression, above a low constitutive level, was observed. Three sox-encoded proteins were purified: the heterodimeric c-type cytochrome SoxXA, the monomeric SoxB and the heterodimeric SoxYZ. Gene inactivation and complementation experiments proved these proteins to be indispensable for thiosulphate oxidation to sulphate. The intermediary formation of sulphur globules in A. vinosum appears to be related to the lack of soxCD genes, the products of which are proposed to oxidize SoxY-bound sulphane sulphur. In their absence the latter is instead transferred to growing sulphur globules.

The Biosynthetic Gene Clusters of Aminocoumarin Antibiotics

Plants and microorganisms are the most important sources of secondary metabolites in nature. For research in the functional genomics of secondary metabolism, and for the biotechnological application of such research by genetic engineering and combinatorial biosynthesis, most microorganisms offer a unique advantage to the researcher: the biosynthetic genes for a specific secondary metabolite are not scattered over the genome, but rather are clustered in a well-defined, contiguous region - the biosynthetic gene cluster of that metabolite. This is exemplified in this review for the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A (1), which are potent inhibitors of DNA gyrase. Cloning, sequencing and analysis of the biosynthetic gene clusters of these three antibiotics revealed that the structural differences and similarities of the compounds are perfectly reflected by the genetic organisation of the biosynthetic gene clusters. The function of most biosynthetic genes could be identified by gene inactivation experiments as well as by heterologous expression and biochemical investigation. The prenylated benzoic acid moiety of novobiocin and clorobiocin, involved in the interaction with gyrase, is structurally similar to metabolites found in plants. However, detailed investigations of the biosynthesis revealed that the biosynthetic pathway and the enzymes involved are totally different from those identified in plants.

Utilization of the Methoxymalonyl-Acyl Carrier Protein Biosynthesis Locus for Cloning of the Tautomycin Biosynthetic Gene Cluster from Streptomyces spiroverticillatus

Tautomycin (TTM), a potent protein phosphatase inhibitor, consists of a polyketide chain containing a spiroketal moiety and an acyl chain bearing a dialkylmaleic anhydride structure. PCR using degenerate primers was used to clone genes from Streptomyces spiroverticillatus for formation of the methoxymalonyl-acyl carrier protein. This locus was found to contain five genes (ttmC, ttmA, ttmD, ttmB, and ttmE), one of which was used as a probe to clone the 110-kb TTM biosynthetic gene cluster. The involvement of the ttmA gene in TTM biosynthesis was confirmed by gene inactivation and mutation complementation experiments.

Phenolic lipid synthesis by type III polyketide synthases is essential for cyst formation in Azotobacter vinelandii.

Cysts of Azotobacter vinelandii are resting cells that are surrounded by a protective coat, conferring resistance to various chemical and physical agents. The major chemical components of the cyst coat are alkylresorcinols, which are amphiphilic molecules possessing an aromatic ring with a long aliphatic carbon chain. Although alkylresorcinols are widely distributed in bacteria, fungi, plants, and animals, no enzyme systems for their biosynthesis are known. We report here an ars operon in A. vinelandii that is responsible for the biosynthesis of the alkylresorcinols in the cysts. The ars operon consisted of four genes, two of which encoded a type III polyketide synthase, ArsB and ArsC. In vitro experiments revealed that ArsB and ArsC, sharing 71% amino acid sequence identity, were an alkylresorcinol synthase and an alkylpyrone synthase, respectively, indicating that ArsB and ArsC are not isozymes but enzymatically distinct polyketide synthases. In addition, ArsB and ArsC accepted several acyl-CoAs with various lengths of the side chain as a starter substrate and gave corresponding alkylresorcinols and alkylpyrones, respectively, which suggests that the mode of the ring folding is uninfluenced by the structure of the starter substrates. The importance of the alkylresorcinols for encystment was confirmed by gene inactivation experiments; the lack of alkylresorcinols synthesis caused by ars mutations resulted in the formation of severely impaired cysts, as observed by electron microscopy.

Genome-wide Transcriptional Profile of Escherichia coli in Response to High Levels of the Second Messenger 3′,5′-Cyclic Diguanylic Acid

Cyclic diguanylic acid (c-di-GMP; cGpGp) is a global second messenger controlling motility and adhesion in bacterial cells. Intracellular concentrations of c-di-GMP depend on two opposite activities: diguanylate cyclase, recently assigned to the widespread GGDEF domain, and c-di-GMP-specific phosphodiesterase, associated with proteins harboring the EAL domain. To date, little is known about the targets of c-di-GMP in the cell or if it affects transcriptional regulation of certain genes. In order to expand our knowledge of the effect of this molecule on the bacterial metabolism, here we report on the Escherichia coli transcriptional profile under high levels of c-di-GMP. We show that an important number of genes encoding cell surface and membrane-bound proteins are altered in their transcriptional activity. On the other hand, genes encoding several transcriptional factors, such as Fur, RcsA, SoxS, and ZraR, are up-regulated, and others, such as GadE, GadX, GcvA, and MetR, are down-regulated. Transcription of motility and cell division genes were altered, and consistent with this was the physiological analysis of cells overexpressing yddV, a diguanylate cyclase; these cells displayed an abnormal cell division process when high levels of c-di-GMP were present. We also show evidence that the diguanylate cyclase gene yddV is co-transcribed with dos, a heme base oxygen sensor with c-di-GMP-specific phosphodiesterase activity. A delta dos::kan mutation rendered the cells unable to divide properly, suggesting that dos and yddV may be part of a fine-tuning mechanism for regulating the intracellular levels of c-di-GMP.

Cloning, Sequencing, Analysis, and Heterologous Expression of the Fredericamycin Biosynthetic Gene Cluster from Streptomyces griseus

Fredericamycin (FDM) A, a pentadecaketide featuring two sets of peri-hydroxy tricyclic aromatic moieties connected through a unique chiral spiro carbon center, exhibits potent cytotoxicity and has been studied as a new type of anticancer drug lead because of its novel molecular architecture. The fdm gene cluster was localized to 33-kb DNA segment of Streptomyces griseus ATCC 49344, and its involvement in FDM A biosynthesis was proven by gene inactivation, complementation, and heterologous expression experiments. The fdm cluster consists of 28 open reading frames (ORFs), encoding a type II polyketide synthase (PKS) and tailoring enzymes as well as several regulatory and resistance proteins. The FDM PKS features a KSalpha subunit with heretofore unseen tandem cysteines at its active site, a KSbeta subunit that is distinct phylogenetically from KSbeta of hexa-, octa-, or decaketide PKSs, and a dedicated phosphopantetheinyl transferase. Further study of the FDM PKS could provide new insight into how a type II PKS controls chain length in aromatic polyketide biosynthesis. The availability of the fdm genes, in vivo characterization of the fdm cluster in S. griseus, and heterologous expression of the fdm cluster in Streptomyces albus set the stage to investigate FDM A biosynthesis and engineer the FDM biosynthetic machinery for the production of novel FDM A analogues.

Insights into the Biosynthesis of the Benzoquinone Ansamycins Geldanamycin and Herbimycin, Obtained by Gene Sequencing and Disruption

Geldanamycin and the closely related herbimycins A, B, and C were the first benzoquinone ansamycins to be extensively studied for their antitumor properties as small-molecule inhibitors of the Hsp90 protein chaperone complex. These compounds are produced by two different Streptomyces hygroscopicus strains and have the same modular polyketide synthase (PKS)-derived carbon skeleton but different substitution patterns at C-11, C-15, and C-17. To set the stage for structural modification by genetic engineering, we previously identified the gene cluster responsible for geldanamycin biosynthesis. We have now cloned and sequenced a 115-kb segment of the herbimycin biosynthetic gene cluster from S. hygroscopicus AM 3672, including the genes for the PKS and most of the post-PKS tailoring enzymes. The similarities and differences between the gene clusters and biosynthetic pathways for these closely related ansamycins are interpreted with support from the results of gene inactivation experiments. In addition, the organization and functions of genes involved in the biosynthesis of the 3-amino-5-hydroxybenzoic acid (AHBA) starter unit and the post-PKS modifications of progeldanamycin were assessed by inactivating the subclusters of AHBA biosynthetic genes and two oxygenase genes (gdmM and gdmL) that were proposed to be involved in formation of the geldanamycin benzoquinoid system. A resulting novel geldanamycin analog, KOS-1806, was isolated and characterized.

Salicylate Biosynthesis: Overexpression, Purification, and Characterization of Irp9, a Bifunctional Salicylate Synthase fromYersinia enterocolitica

In some bacteria, salicylate is synthesized using the enzymes isochorismate synthase and isochorismate pyruvate lyase. In contrast, gene inactivation and complementation experiments with Yersinia enterocolitica suggest the synthesis of salicylate in the biosynthesis of the siderophore yersiniabactin involves a single protein, Irp9, which converts chorismate directly into salicylate. In the present study, Irp9 was for the first time heterologously expressed in Escherichia coli as a hexahistidine fusion protein, purified to near homogeneity, and characterized biochemically. The recombinant protein was found to be a dimer, each subunit of which has a molecular mass of 50 kDa. Enzyme assays, reverse-phase high-pressure liquid chromatography and 1H nuclear magnetic resonance (NMR) spectroscopic analyses confirmed that Irp9 is a salicylate synthase and converts chorismate to salicylate with a K(m) for chorismate of 4.2 microM and a k(cat) of 8 min(-1). The reaction was shown to proceed through the intermediate isochorismate, which was detected directly using 1H NMR spectroscopy.

Structure and Biosynthesis of Myxochromides S1–3 in Stigmatella aurantiaca: Evidence for an Iterative Bacterial Type I Polyketide Synthase and for Module Skipping in Nonribosomal Peptide Biosynthesis

The myxobacterium Stigmatella aurantiaca DW4/3-1 harbours an astonishing variety of secondary metabolic gene clusters, at least two of which were found by gene inactivation experiments to be connected to the biosynthesis of previously unknown metabolites. In this study, we elucidate the structures of myxochromides S1-3, novel cyclic pentapeptide natural products possessing unsaturated polyketide side chains, and identify the corresponding biosynthetic gene locus, made up of six nonribosomal peptide synthetase modules. By analyzing the deduced substrate specificities of the adenylation domains, it is shown that module 4 is most probably skipped during the biosynthetic process. The polyketide synthase MchA harbours only one module and is presumably responsible for the formation of the variable complete polyketide side chains. These data indicate that MchA is responsible for an unusual iterative polyketide chain assembly.

Genes Involved in Formation and Attachment of a Two-Carbon Chain as a Component of Eurekanate, a Branched-Chain Sugar Moiety of Avilamycin A

Eurekanate belongs to the important class of branched-chain carbohydrates present in a wide variety of natural sources. It is a component of avilamycin A, a potent inhibitor of bacterial protein synthesis targeting the 50S ribosomal subunit. The present work provides experimental proof for the function of two genes of the avilamycin biosynthetic gene cluster, aviB1 and aviO2, that are both involved in avilamycin structure modification. The functions of both genes were identified by gene inactivation experiments and nuclear magnetic resonance analyses of extracts produced by the mutants. We suggest that both AviO2 and AviB1 are involved in the biosynthesis of eurekanate within avilamycin biosynthesis. Moreover, two other genes (aviO1 and aviO3) have been inactivated, resulting in a breakdown of avilamycin production in the mutants ITO1 and ITO3, which clearly shows the essential role of both enzymes in avilamycin biosynthesis. The exact functions of both aviO1 and aviO3 remained unknown.