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• W2046292599 abstract "Short chain carboxylic acids are well known as the precursors of fatty acid and polyketide biosynthesis. Iso-fatty acids, which are important for the control of membrane fluidity, are formed from branched chain starter units (isovaleryl-CoA and isobutyryl-CoA), which in turn are derived from the degradation of leucine and valine, respectively. Branched chain carboxylic acids are also employed as starter molecules for the biosynthesis of secondary metabolites, e.g. the therapeutically important anthelmintic agent avermectin or the electron transport inhibitor myxothiazol. During our studies on myxothiazol biosynthesis in the myxobacterium, Stigmatella aurantiaca, we detected a novel biosynthetic route to isovaleric acid. After cloning and inactivation of the branched chain keto acid dehydrogenase complex, which is responsible for the degradation of branched chain amino acids, the strain is still able to produce iso-fatty acids and myxothiazol. Incorporation studies employing deuterated leucine show that it can only serve as precursor in the wild type strain but not in theesg mutant. Feeding experiments using13C-labeled precursors show that isovalerate is efficiently made from acetate, giving rise to a labeling pattern in myxothiazol that provides evidence for a novel branch of the mevalonate pathway involving the intermediate 3,3-dimethylacryloyl-CoA. 3,3-Dimethylacrylic acid was synthesized in deuterated form and fed to the esg mutant, resulting in strong incorporation into myxothiazol and iso-fatty acids. Similar experiments employingMyxococcus xanthus revealed that the discovered biosynthetic route described is present in other myxobacteria as well. Short chain carboxylic acids are well known as the precursors of fatty acid and polyketide biosynthesis. Iso-fatty acids, which are important for the control of membrane fluidity, are formed from branched chain starter units (isovaleryl-CoA and isobutyryl-CoA), which in turn are derived from the degradation of leucine and valine, respectively. Branched chain carboxylic acids are also employed as starter molecules for the biosynthesis of secondary metabolites, e.g. the therapeutically important anthelmintic agent avermectin or the electron transport inhibitor myxothiazol. During our studies on myxothiazol biosynthesis in the myxobacterium, Stigmatella aurantiaca, we detected a novel biosynthetic route to isovaleric acid. After cloning and inactivation of the branched chain keto acid dehydrogenase complex, which is responsible for the degradation of branched chain amino acids, the strain is still able to produce iso-fatty acids and myxothiazol. Incorporation studies employing deuterated leucine show that it can only serve as precursor in the wild type strain but not in theesg mutant. Feeding experiments using13C-labeled precursors show that isovalerate is efficiently made from acetate, giving rise to a labeling pattern in myxothiazol that provides evidence for a novel branch of the mevalonate pathway involving the intermediate 3,3-dimethylacryloyl-CoA. 3,3-Dimethylacrylic acid was synthesized in deuterated form and fed to the esg mutant, resulting in strong incorporation into myxothiazol and iso-fatty acids. Similar experiments employingMyxococcus xanthus revealed that the discovered biosynthetic route described is present in other myxobacteria as well. fatty acid 3,3-Dimethyl-d6-acrylic acid isovaleric acid isobutyric acid 2-methyl butyric acid high pressure liquid chromatography Fatty acids (FA)1 are important building blocks of cell membranes. The various groups of prokaryotes differ remarkably in the structure and synthesis of fatty acid-derived lipids, which serve as reliable systematic marker molecules in chemotaxonomy (1Fuchs G. Lengeler J. Drews G. Schlegel H. Biology of the Prokaryotes. Blackwell Science, Stuttgart, Germany1999: 110-157Google Scholar). Unsaturated and branched chain (or iso-) FA increase the fluidity of the membrane and fulfill the function of thermal adaptation. The higher the content of these FA, the lower is the solid-to-liquid phase transition temperature of lipids. The biosynthesis of FA has been elucidated in detail, revealing that the difference between the biosynthetic pathways to FA and iso-FA lies only in the respective acylated primer acyl carrier proteins and in the condensing enzymes, which prefer modified primer acyl chains for elongation instead of the “normal” acetyl primer (1Fuchs G. Lengeler J. Drews G. Schlegel H. Biology of the Prokaryotes. Blackwell Science, Stuttgart, Germany1999: 110-157Google Scholar, 2Michal G. Biochemical Pathways, 1. Spektrum Akad. Verlag, Heidelberg, Germany1999Google Scholar). The modified primers used for iso-FA biosynthesis are the coenzyme A esters of isovaleric acid (IVA, resulting in iso-odd FA with an uneven number of carbon atoms), isobutyric acid (IBA, resulting in iso-even FA with an even number of carbon atoms), or 2-methyl butyric acid (2MBA, resulting in ante-iso FA, in which the methyl branch is located on the third carbon atom counted from the ω-end of the chain), which can be generated via the degradation pathway from branched chain amino acids or from intermediates of the biosyntheses of these essential amino acids (Fig. 1). The branched chain amino acids leucine, valine, and isoleucine are transaminated by a separate enzyme and then oxidatively decarboxylated by the Bkd (branched chain keto aciddehydrogenase) complex, resulting in IVA-CoA, IBA-CoA, and 2MBA-CoA, respectively (3Voet D. Voet J. Biochemistry. 2nd Ed. John Wiley & Sons, Inc., New York1995Google Scholar). These precursors are not only employed in iso-FA biosynthesis but can as well serve as starter molecules for the formation of polyketides. These represent a group of immensely diverse natural products with biological activity, many of which are widely used in the clinic. Examples of polyketides with a branched chain carboxylic acid starter moiety are the anthelmintic agent avermectin produced by Streptomyces avermitilis (4Ikeda H. Omura S. Chem. Rev. 1997; 7: 2591-2609Crossref Scopus (140) Google Scholar), the clinically used virginiamycin group of antibiotics, and the electron transport inhibitors myxothiazol (5Trowitzsch-Kienast W. Wray V. Gerth K. Reichenbach H. Ho¨fle G. Liebigs Ann. Chem. 1986; 10: 93-98Crossref Scopus (26) Google Scholar, 6Silakowski B. Schairer H.U. Ehret H. Kunze B. Weinig S. Nordsiek G. Brandt P. Blo¨cker H. Ho¨fle G. Beyer S. Mu¨ller R. J. Biol. Chem. 1999; 274: 37391-37399Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) (compare Fig. 4) and myxalamid (7Jansen R. Reifenstahl G. Gerth K. Reichenbach H. Ho¨fle G. Liebigs Ann. Chem. 1983; 7: 1081-1095Crossref Scopus (53) Google Scholar, 8Silakowski B. Nordsiek G. Kunze B. Blo¨cker H. Mu¨ller R. Chem. Biol. 2001; 8: 59-69Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) from the myxobacterium Stigmatella aurantiaca. During our studies on the biosynthesis of the latter secondary metabolites in myxobacteria, we became interested in the Bkd locus of S. aurantiaca DW4/3-1, because it seems to regulate the production of starter molecules for primary metabolism (iso-FA) and secondary metabolism. In addition, myxobacteria show a so-called social behavior that culminates in the formation of multicellular fruiting bodies under starvation conditions (9Dworkin M. Microbiol. Rev. 1996; 60: 70-102Crossref PubMed Google Scholar). The model organism Myxococcus xanthus has been studied extensively to reveal the mechanisms underlying this process. It has been shown in M. xanthusthat a knockout of the bkd gene leads to a development-defective phenotype (10Downard J. Ramaswamy S.V. Kil K.S. J. Bacteriol. 1993; 175: 7762-7770Crossref PubMed Google Scholar, 11Downard J. Toal D. Mol. Microbiol. 1995; 16: 171-175Crossref PubMed Scopus (74) Google Scholar). The signal interrupted is called the E signal, the nature and function of which is not well understood. Because the mutant was detected in a screen for developmentally defective strains, the gene affected was calledesg (for Esignal gene) instead of bkd. Recently, Shimkets and co-workers (12Kearns D.B. Campbell B.D. Shimkets L.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11505-13994Crossref PubMed Scopus (37) Google Scholar, 13Kearns D.B. Venot A. Bonner P.J. Stevens B. Boons G.-J. Shimkets L.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13990-13994Crossref PubMed Scopus (37) Google Scholar) have shed light on the nature of the signal itself and proposed that it might be a lipid.Figure 413C NMR signals of myxothiazol from S. aurantiaca.A, natural abundance signals. B, after feeding of [2-13C]acetate to strain DW4/3-1. C, after feeding of [2-13C]acetate to strain EBS7. D, after feeding of [1,2-13C2]acetate to strain DW4/3-1. E, after feeding of [1,2-13C2]acetate to strain EBS7.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Assuming that the esg gene of S. aurantiacaDW4/3-1 might have similar functions during the development of the bacterium, we suspected the encoding gene to be a good candidate to study to reveal the connections not only between primary and secondary metabolism but also to development in myxobacteria. Additionally, FA analyses of esg mutants of M. xanthus published by the Shimkets and the Downard groups (11Downard J. Toal D. Mol. Microbiol. 1995; 16: 171-175Crossref PubMed Scopus (74) Google Scholar, 13Kearns D.B. Venot A. Bonner P.J. Stevens B. Boons G.-J. Shimkets L.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13990-13994Crossref PubMed Scopus (37) Google Scholar, 21Toal D.R. Clifton S.W. Roe B.A. Downard J. Mol. Microbiol. 1995; 16: 177-189Crossref PubMed Scopus (60) Google Scholar) showed that the mutant strain was still able to produce iso-FA, which left both groups with the question how this might be accomplished. In general,M. xanthus contains an immense diversity of FA, which include saturated, unsaturated, branched chain, and hydroxylated species (14Ware J. Dworkin M. J. Bacteriol. 1973; 115: 253-261Crossref PubMed Google Scholar). We here describe the cloning and inactivation of the esggene in S. aurantiaca DW4/3-1 and provide evidence for an unexpected biosynthetic pathway complementing Bkd disruption in myxobacteria. This pathway was characterized in feeding experiments with labeled precursors and intermediates. It must be taken into consideration when analyzing the nature of the developmental E signal in M. xanthus and S. aurantiaca. Southern analysis of genomic DNA was performed using the standard protocol for homologous probes of the DIG High Prime DNA labeling and detection starter kit II (Roche Molecular Biochemicals). PCR was carried out using Taq polymerase (Invitrogen) according to the manufacturer’s protocol. 5% Me2SO was added to the mixture. The conditions for amplification with the Eppendorf Mastercycler gradient were as follows: denaturation 30 s at 95 °C, annealing 30 s at 55 °C, and extension 45 s at 72 °C for 30 cycles and a final extension at 72 °C for 10 min. Sequencing was performed using the Big Dye RR terminator cycle sequencing kit (PerkinElmer Biosystems), and the gels were run on ABI-377 sequencers. All other DNA manipulations were performed according to standard protocols (15Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Amino acid and DNA alignments were done using the programs of the Lasergene software package (DNASTAR Inc.) and Clustal W (16Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar). A set of three oligonucleotide combinations designed to bind to the M. xanthus esg sequence enabled the PCR amplification of a esg gene fragment from genomic DNA of S. aurantiaca DW4/3-1 (primers used were esgup, 5′-ggcatccgcgggtccatct-3′, and esgdown, 5′-gcggggccagcagcgagtag-3′). The amplified fragment (592 bp) was cloned into pCR2.1/TOPO (Invitrogen) (resulting in pEBS5). Subsequently primers esgup and esgd2 (5′-aggccttgatgagctcccagtcg-3′) were used to identify a cosmid harboring the complete gene (EBS15) from a pooled library of S. aurantiaca DW4/3-1 as described (6Silakowski B. Schairer H.U. Ehret H. Kunze B. Weinig S. Nordsiek G. Brandt P. Blo¨cker H. Ho¨fle G. Beyer S. Mu¨ller R. J. Biol. Chem. 1999; 274: 37391-37399Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). A 3-kilobase pair BamHI/XhoI fragment from EBS17 was shown to hybridize with the probe and next cloned into pSK(−) (Stratagene), resulting in pEBS22. The sequence of the insert was obtained via primer walking. Based on the sequence, a 476-bp fragment of the β-subunit of the esg complex was amplified (using oligonucleotides esgup and esgd2) and cloned into a vector harboring a kanamycin resistance gene (pCR2.1/TOPO). The resulting plasmid pEBS7 was electroporated into S. aurantiaca DW4/3-1 (6Silakowski B. Schairer H.U. Ehret H. Kunze B. Weinig S. Nordsiek G. Brandt P. Blo¨cker H. Ho¨fle G. Beyer S. Mu¨ller R. J. Biol. Chem. 1999; 274: 37391-37399Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 17Stamm I. Leclerque A. Plaga W. Arch. Microbiol. 1999; 172: 175-181Crossref PubMed Scopus (12) Google Scholar). The correctness of the integration of the plasmid into the chromosome of kanamycin resistant colonies (S. aurantiaca EBS7) was verified using Southern hybridization (data not shown). Fatty acid methyl esters were obtained from 40 mg of cells scraped from Petri dishes by saponification, methylation, and extraction using minor modifications of the methods of Miller (18Miller L.T. J. Clin. Microbiol. 1982; 16: 584-586Crossref PubMed Google Scholar) and Kuykendall et al. (19Kuykendall L.D. Roy M.A. O'Neill J.J. Devine T.E. Int. J. Syst. Bact. 1988; 38: 358-361Crossref Scopus (861) Google Scholar). The fatty acid methyl ester mixtures were separated using a Sherlock microbial identification system (Microbial ID, Newark, DE), which consisted of a Hewlett-Packard model 5980 gas chromatograph fitted with a 5% phenyl-methyl silicone capillary column (0.2 mm × 25 m), a flame ionization detector, a Hewlett-Packard model 7673A automatic sampler, and a Hewlett-Packard model KAYAK XA computer (Hewlett-Packard Co., Palo Alto, CA). The peaks were automatically integrated, and the fatty acid names and percentages were calculated by the microbial identification system standard software (Microbial ID). The gas chromatographic parameters were as follows: carrier gas, ultra-high purity hydrogen; column head pressure, 60 kPa; injection volume, 2 μl; column split ratio, 100:1; septum purge, 5 ml/min; column temperature, 170–270 °C at 5 °C/min; injection port temperature, 250 °C; and detector temperature, 300 °C. FA analysis using gas chromatography-mass spectrometry was done on a Finnigan MAT GCQ gas chromatograph (San Jose, CA) connected to an ion trap mass spectrometer running in the EI mode (70 eV) under conditions similar to those described above. Myxothiazol was isolated fromS. aurantiaca DW4/3-1 and its decendants by repeated methanol extraction of cells and adsorber resin (see below) followed by evaporation of the solvent. The resulting aqueous phase was extracted with ethyl acetate, and the organic solvent was evaporated to dryness. The combined residues were dissolved in a small amount of methanol and extracted repeatedly with n-heptane. The resulting methanolic solution was concentrated under reduced pressure and subjected to preparative reverse phase HPLC on a Nucleodur 100–10 C18 EC column (259 mm length/21 mm Ø (Macherey & Nagel, Du¨ren, Germany); the solvent was methanol/water (8/2) with a flow rate of 12 ml/min; detection was at 234 nm) resulting in 2–6 mg/liter of myxothiazol. The 13C NMR spectra were recorded on a Bruker AMX400 at 100.6 MHz in CD3OD with the solvent as internal standard. Feeding experiments using [13C]acetate were performed withS. aurantiaca in one or two 400-ml cultures of probione medium (20Silakowski B. Kunze B. Mu¨ller R. Gene (Amst.). 2001; 275: 233-240Crossref PubMed Scopus (65) Google Scholar) with 1% of the adsorber resin XAD-16 (Rohm & Haas, Germany) in 1000-ml Erlenmeyer flasks. Mutant strains were grown in the same medium after the addition of kanamycin to a final concentration of 50 μg/ml. After inoculation with 1 × 107 cells/ml from a preculture in tryptone starch medium (10 g of tryptone, 2 g of MgSO4·7H2O, 4 g of soluble starch, 11.9 g of HEPES Pufferan buffer/sliter; pH adjusted to 7.2 with KOH), the cells were grown at 30 °C for 93 h before harvest. After 20, 29, 44, and 53 h, 50 mg of labeled acetate were added to each flask. Feeding of IVA, 2MBA, and IBA was done in 50 ml of tryptone medium (6Silakowski B. Schairer H.U. Ehret H. Kunze B. Weinig S. Nordsiek G. Brandt P. Blo¨cker H. Ho¨fle G. Beyer S. Mu¨ller R. J. Biol. Chem. 1999; 274: 37391-37399Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) after inoculation with 1 × 107 cells/ml for S. aurantiaca and as described by Downard and co-workers (21Toal D.R. Clifton S.W. Roe B.A. Downard J. Mol. Microbiol. 1995; 16: 177-189Crossref PubMed Scopus (60) Google Scholar) forM. xanthus. M. xanthus DK1622 (22Kaiser A. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 5952-5956Crossref PubMed Scopus (390) Google Scholar) and the transposon esg mutant M. xanthus Ω258 (21Toal D.R. Clifton S.W. Roe B.A. Downard J. Mol. Microbiol. 1995; 16: 177-189Crossref PubMed Scopus (60) Google Scholar) were used in this study. The branched chain acids were added individually in concentrations of 1 mm and as a mixture of all three of them (each 1 mm). The cells were counted and harvested after 48 h. Growth was almost identical except for the cells grown in the mixture, which did not grow at all. FA analysis was additionally performed on the wild type and the mutant cells of S. aurantiaca DW4/3-1 to analyze the FA composition in correlation with the growth phase. The cells were harvested after 0, 14.5, 24 and 38.5 h. Feeding of 3,3-dimethylacrylic acid (DMAA) and leucine to S. aurantiaca and M. xanthus strains was performed in tryptone or CTT medium (23Hodgkin J. Kaiser D. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 2932-2942Crossref Scopus (249) Google Scholar) after the addition of 1 mm of [D6]DMAA ordl-[D10]leucine (Campro Scientific, Berlin, Germany) and inoculation of 50 ml cultures with 1 × 107 cells/ml. The cultures were harvested after 70 h (S. aurantiaca) and 52 h (M. xanthus) for fatty acid analysis. For the incorporation of [D6]DMAA or [D10]dl-leucine into myxothiazol, S. aurantiaca was cultivated in 50 ml of probione medium with XAD, and 1 mg of the precursor was added at 18, 48, 66, and 72 h. The cells were harvested after 92 h, and the incorporation of the precursors into myxothiazol was determined after methanol extraction of cells and XAD by HPLC-MS (Series 1100 HPLC, Hewlett-Packard, Palo Alto, CA; SCIEX API 2000 ESI-MS, PerkinElmer). To a solution of diisopropylamine (1.5 ml, 10.8 mmol) in 20 ml of anhydrous tetrahydrofurane was added 2.5 mbutyllithium solution (4.3 ml,10.8 mmol) in hexane at −15 °C under an argon atmosphere, and the mixture was stirred for 15 min at room temperature. It was then cooled to −78 °C, and 2.16 ml (10.8 mmol) of triethylphosphonoacetate was added slowly. The reaction mixture was allowed to warm to room temperature for 30 min and then cooled again to −78 °C. Acetone-d6 (1.6 ml, 21.6 mmol) of was then added, and the mixture was stirred at room temperature for 1.5 h. The reaction was quenched with water at −78 °C, and the mixture allowed to warm to room temperature. The product was extracted with three portions of 10 ml of ethyl ether, and the combined organic layer was washed with brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (eluent: hexane/ethyl ether = 9:1, v/v). Concentration of the appropriate fractions afforded ethyl 3,3-dimethyl-d6-acrylate (1.2 g, 8.96 mmol, 83%) as a yellowish liquid; Rf 0.46 (hexane/ethyl ether, 5/1 v/v). Electrospray MS m/z 135.1 [M + H]+. Electrospray-high resolution mass spectroscopy: m/z 135.1281 [M + H]+, Calculated for C7H7D6O2: 135.1292.1H NMR (300 MHz, CDCl3): δH 1.26 (3H, t, J = 7.3 Hz), 4.12 (2H, dd,J = 7.3, 14.0 Hz), 5.65 (1H, s). To a solution of ethyl-3,3-dimethyl-3,3-d6-acrylate (1.1 g, 8.2 mmol) in 3 ml of methanol and 4.8 ml of tetrahydrofurane was added 8 ml of 2 m LiOH solution. The mixture was stirred for 6 h at room temperature. The organic solvent was removed in vacuo, and 1 nHCl solution was added to adjust the pH to 2–3. The product was extracted with five portions of 15 ml of ethyl acetate, and the combined organic layer was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (eluent: hexane/ethyl acetate = 2/1 v/v) to give 3,3-dimethyl-d6-acrylic acid (677 mg, 6.38 mmol, 78%) as colorless crystal; Rf 0.2 (hexane/ethyl acetate, 2/1, v/v). Electrospray MS m/z 107.1 [M + H]+. Electrospray-high resolution mass spectroscopy: m/z 107.0978 [M + H]+, Calculated for C5H3D6O2: 107.0979.1H NMR (300 MHz, CDCl3): δH 5.81 (1H, s). The nucleotide sequence reported here is available in the EMBL Nucleotide Sequence Data base under the accession number AJ439341. We became interested in the branched chain keto acid dehydrogenase gene locus from S. aurantiacaDW4/3-1, because of the possibility of using it for mutasynthesis to generate novel myxothiazol derivatives. We speculated that inactivation of the encoding genes would result in an inability of the bacteria to degrade leucine, which would in turn lead to a lack of precursors (IVA-CoA) for secondary metabolite production and therefore shut down myxothiazol biosynthesis. Feeding of alternative starter units to theesg mutant was speculated to result in the formation of novel myxothiazols. Cloning of the esg locus from S. aurantiacaDW4/3-1 was performed based on the known esg sequence fromM. xanthus (21Toal D.R. Clifton S.W. Roe B.A. Downard J. Mol. Microbiol. 1995; 16: 177-189Crossref PubMed Scopus (60) Google Scholar). A 3-kilobase pair fragment was sequenced that encodes the α- and the β-subunit of the Bkd complex. The α-subunit comprises 885 bp and starts with ATG, which is preceded by a putative ribosome-binding site (GAGGAG). A 2-bp intergenic region can be found between the α- and the β-subunit. The latter is encoded by a gene 1056 bp in size, which starts with an ATG preceded by the putative RBS GAGAAG. The DNA sequence is highly homologous to the known sequence from the M. xanthus esg locus (the DNA levels were 82.4% identity for the α-subunit and 83.3% for the β-subunit; the protein levels were 82.7% for the α-subunit and 82.3% for the β-subunit). After homologous recombination of the plasmid pEBS7 into the chromosome of S. aurantiaca DW4/3-1, the esg locus is inactivated, because the mutants harbor two truncated versions of the target gene (one copy with a deletion of the 5′-end and one copy with a deletion at the 3′-end of the gene). The resulting esgmutant S. aurantiaca EBS7 was further analyzed in comparison with the wild type. Secondary metabolite production was analyzed as described previously (6Silakowski B. Schairer H.U. Ehret H. Kunze B. Weinig S. Nordsiek G. Brandt P. Blo¨cker H. Ho¨fle G. Beyer S. Mu¨ller R. J. Biol. Chem. 1999; 274: 37391-37399Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Myxothiazol is still produced in the esg mutant. Nevertheless, an approximately 3-fold decrease in productivity was observed (as judged by the comparison of the peak areas representing myxothiazol in HPLC analysis). The analysis of the fatty acid composition of both S. aurantiaca strains revealed some striking differences compared with the fatty acid analysis of M. xanthus and the corresponding esg mutant Ω258. In general, production of leucine- and valine-derived fatty acids was decreased in the esg mutants, whereas the production of saturated and unsaturated fatty acids was increased (Fig. 2). Feeding of the short chain carboxylic acids IVA, IBA, and 2MBA to the S. aurantiaca DW4/3-1 esg mutant was performed and resulted in an almost complete restoration of wild type fatty acid levels in the IVA feeding experiment. The addition of IBA did not result in complete restoration, whereas 2MBA feeding had no effect (Fig. 2). The addition of all three acids abolished growth, whereas the addition of a single compound had no effect on the cell count after 48 h. A time course study revealed that the fatty acid composition does not differ significantly over time (the cells were harvested after 15, 24, 39, and 48 h prior to the analysis). Furthermore, feeding of IVA, IBA, or 2MBA to the wild type had no effect on the fatty acid composition (data not shown). Analysis of fatty acid composition in M. xanthus DK1622 and strain Ω258 was performed as well, revealing as expected an enrichment of iso-even FA, iso-odd FA, and ante-iso FA after the addition of IBA, IVA, and 2MBA, respectively (data not shown; compare Refs. 13Kearns D.B. Venot A. Bonner P.J. Stevens B. Boons G.-J. Shimkets L.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13990-13994Crossref PubMed Scopus (37) Google Scholar and 21Toal D.R. Clifton S.W. Roe B.A. Downard J. Mol. Microbiol. 1995; 16: 177-189Crossref PubMed Scopus (60) Google Scholar). An approximate restoration of wild type levels of fatty acids could only be seen in IVA and DMAA fed mutants. Thus, DMAA could replace the effect of IVA in M. xanthus and in S. aurantiaca mutants. Feeding of IVA had been reported to increase the amount of iso-odd FA (mostly FA iso 15:0) in the M. xanthus esg mutant 2-fold, whereas FA 16:1 ω5c was dramatically reduced under these conditions (13Kearns D.B. Venot A. Bonner P.J. Stevens B. Boons G.-J. Shimkets L.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13990-13994Crossref PubMed Scopus (37) Google Scholar). We only observe an increase of iso-odd FA by a factor of 1.5 (Ω258; data not shown), which compares very well to the data reported by the Downard group (21Toal D.R. Clifton S.W. Roe B.A. Downard J. Mol. Microbiol. 1995; 16: 177-189Crossref PubMed Scopus (60) Google Scholar). In S. aurantiaca, only restoration but no increase above wild type levels of iso-odd FA can be detected in the esg mutant (Fig. 2). A significant decrease of FA 16:1 ω5c below the wild type level was not detected, neither inM. xanthus nor in S. aurantiaca esg mutants after IVA feeding. Because inactivation of theesg gene did not result in loss of production of myxothiazol and iso-fatty acids, we suspected that the strain carries an isogene ofesg. Comparable results regarding iso-FA production inM. xanthus esg mutants have led to similar speculations (10Downard J. Ramaswamy S.V. Kil K.S. J. Bacteriol. 1993; 175: 7762-7770Crossref PubMed Google Scholar, 13Kearns D.B. Venot A. Bonner P.J. Stevens B. Boons G.-J. Shimkets L.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13990-13994Crossref PubMed Scopus (37) Google Scholar). Based on conserved sequences within known bkdgenes, degenerate primers were constructed to amplify genomic sequences encoding further bkd genes. In addition, Southern blot analyses under low stringency conditions were performed using theesg gene as a probe (data not shown). No indication of the presence of a second esg gene was found. Because no iso-gene could be detected, we wanted to analyze biochemically whether leucine could still be incorporated into myxothiazol.dl-[D10]leucine was fed to the wild type and to the mutant strain of S. aurantiaca DW4/3-1. Subsequently, myxothiazol was isolated and analyzed by mass spectrometry. The data generated clearly show that leucine is incorporated into myxothiazol in the wild type (∼50%; Fig. 3), whereas no incorporation could be detected in the esg mutant. This shows that the mutant strain is not able to use leucine as precursor for myxothiazol biosynthesis and indicates that a previously unidentified pathway must be responsible for the formation of activated IVA as a starter molecule for myxothiazol biosynthesis. To shed light onto this pathway [1-13C]acetate, [2-13C]acetate, and [1,2-13C2]acetate were fed to the mutant strain and to the wild type. Incorporation of the labeled acetate into myxothiazol was analyzed after purification of the compound by13C NMR spectroscopy. Almost no incorporation of acetate into the starting unit in the wild type was observed (TableI), whereas in the esgmutant, the incorporation was almost half of the incorporation of other malonyl-CoA derived carbon atoms (Fig. 4and Table I). The labeling pattern corresponds to intact incorporation of acetate units from C1 to C2 and from C3 to C4 of the IVA starter molecule (identical to C10-C11 and C12-C13 (or C12-C14; C13 and C14 cannot be distinguished via NMR) in myxothiazol; see Fig. 4). The methylbranch carbon (C14 or C13) is derived from the C2 of acetate.Table IRelative 13C-enrichments in comparison with natural abundance in myxothiazol after feeding of [1-13C], [2-13C], and [1,2-13C2]acetate to S. aurantiaca DW4/3–1 and EBS7 and coupling constants obtained after feeding [1,2-13C2]acetateC atomδc[1-13C]acetate[2-13C]acetate[1,2-13C2]acetateDW4/3–1EBS7DW4/3–1EBS7DW4/3–1EBS7Jcc1-aAll spectra were recorded at 100.6 MHz in CD3OD.ppmHz8133.71021—1-b—, no enrichment detected.—137729133.3——18141577210128.1—13———47111143.1———7—4711232.3—10———43513/1422.7———6—41-cEnrichment of intact acetate was determined from the overall enrichment of C-13/C-14 in analogy to C-12.3517132.9——1917168481886.21625——208482194.4——21171196922172.13025——15969No 13C enrichment was observed for other carbon atoms.1-a All spectra were recorded at 100.6 MHz in CD3OD.1-b —, no enrichment detected.1-c Enrichment of intact acetate was determined from the overall enrichment of C-13/C-14 in analogy to C-12. Open table in a new tab No 13C enrichment was observed for other carbon atoms. From the labeling pattern observed, a hypothetical pathway to IVA branching from the mevalonate biosynthetic route is postulated as shown in Fig.5. This pathway putatively involves 3,3-dimethylacrylyl-CoA as an intermediate. Therefore, the corresponding free acid was synthesized in deuterated form ([D6]DMAA) and fed to the culture broth of theesg mutants and wild type strains of S. aurantiaca and M. xanthus. Analysis of myxothiazol from S. aurantiaca using mass spectrometry revealed that [D6]DMAA was incorporated with an efficiency of 45% in the esg mutant but gave almost no incorporation in the wild type. [D6]DMAA was also incorporated into iso-FA, which could be detected by gas chromatography-mass spectrometry analysis via the expected mass increase of the fatty acid methyl esters by 6 units (TableII). Most significantly, DMAA was incorporated into iso-even and iso-odd-FA (∼75%) inM. xanthus and S. aurantiaca esg mutants. DMAA feeding almost restored wild type levels of iso-fatty acids (data not shown). In contrast to S. aurantiaca EBS7, there was still a low level of incorporation of dl-[D10]leucine into iso-FA in M. xanthus mutant Ω258, indicating that the mutant is leaky.Table IIIncorporation (in %) of [D6]DMAA (A) and [D10]leucine (B) into selected iso-even and iso-odd fatty acidsFatty acidDW4/3–1EBS7DK1622Ω258[D6]DMAA[D10]leucine[D6]DMAA[D10]leucine[D6]DMAA[D10]leucine[D6]DMAA[D10]leucineIso-15:006700013852Iso-15:0 3OH—2-a—, Iso-15:0 3OH was not found in S. aurantiaca.———09701Iso-17:007800011904Iso-17:0 3OH011770012645Iso-14:0 3OH01271009783Iso FA 15:0 3OH was not found in S. aurantiaca DW4/3–1.2-a —, Iso-15:0 3OH was not found in S. aurantiaca. Open table in a new tab Iso FA 15:0 3OH was not found in S. aurantiaca DW4/3–1. During the course of this study an unexpected biosynthetic pathway to IVA in S. aurantiaca DW4/3-1 and M. xanthuswas detected. The initial indication for a second route in the strain leading to the formation of IVA came from the experiment that showed that the esg mutant of S. aurantiaca DW4/3-1 is unable to create iso-FA and myxothiazol from leucine. The subsequent feeding experiments employing different forms of13C-labeled acetate provided evidence for a previously unknown biosynthetic route branching from HMG-CoA as depicted in Fig.5. Further evidence for the existence of this route comes from feeding experiments using DMAA, which was synthesized in labeled form and is incorporated into myxothiazol and iso-FA with high efficiency. One could argue that DMAA is unspecifically reduced to IVA in the bacterial cell and subsequently serves as precursor unit. This is rather unlikely, because no incorporation of DMAA into iso-FA and myxothiazol was found in wild type cells of either S. aurantiaca DW4/3-1 or M. xanthus. Unspecific reduction of DMAA should have occurred under these conditions as well. In addition, we fed DMAA to several other bacterial species known to produce iso-FA and analyzed the FA. In agreement with the data presented here, no incorporation into iso-FA was found, again arguing against unspecific reduction of DMAA. 2H. B. Bode, T. Mahmud, and R. Mu¨ller, unpublished data. DMAA and IVA need to be activated in the cell by an CoA ligase, which has already been speculated to be present in M. xanthus (10Downard J. Ramaswamy S.V. Kil K.S. J. Bacteriol. 1993; 175: 7762-7770Crossref PubMed Google Scholar, 13Kearns D.B. Venot A. Bonner P.J. Stevens B. Boons G.-J. Shimkets L.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13990-13994Crossref PubMed Scopus (37) Google Scholar). Another example for a previous non-textbook biosynthetic pathway has been extensively studied recently and is responsible for the generation of isopentenyl pyrophosphate independently of mevalonate (24Rohdich F. Kis K. Bacher A. Eisenreich W. Curr. Opin. Chem. Biol. 2001; 5: 535-540Crossref PubMed Scopus (162) Google Scholar). The data presented here show again that standard biochemistry does not always apply in nature and provide further evidence for the assumption that novel biochemistry can still be detected by close analysis of not only secondary but also primary metabolism. The pathway proposed in Fig. 5 builds up myxothiazol and iso-FA from 3,3-dimethylacrylyl-CoA directly. Alternatively, IVA could be generated from 3-methyl-3-butenoyl-CoA after isomerization of the endo- to the exo-double bond. The latter compound is speculated to be derived from HMG-CoA by decarboxylation and dehydration (similar to isopentenylpyrophosphate from activated mevalonic acid). DMAA could as well be formed from HMG-CoA via 3-methyl-glutaconyl-CoA (after dehydration) and subsequent decarboxylation. Intriguingly, DMAA is incorporated into secondary metabolites and primary metabolites in form of not only iso-odd FA but also iso-even FA, which came as a surprise. We therefore speculate that DMAA can serve as precursor for IBA biosynthesis as well (e.g. via hydroxylation, dehydrogenation, and oxidative decarboxylation of IVA and subsequent activation of IBA). It will be interesting to see whether the pathway described is present in all myxobacteria or even in other organisms. We performed several feeding experiments employing DMAA with eubacteria and other myxobacterial species. 3H. B. Bode and R. Mu¨ller, unpublished data. Although these experiments did not result in any detectable incorporation into iso-FA, it seems likely that the pathway is present in at least other myxobacterial species and possibly other organisms, because incorporation of DMAA could be detected in two myxobacterial species already (S. aurantiaca DW4/3-1 and M. xanthus esg mutants). There was no detectable incorporation in wild type cells, which implies that the bypass via DMAA is only used as rescue pathway in esg mutants, which are currently not available for the other strains tested. This finding also indicates that the pathway is only expressed when the degradation of branched chain amino acids is impaired. Currently, the expected selective expression of the pathway genes in esg mutants is being analyzed using two-dimensional gel electrophoresis of proteins involved in the novel pathway based on the M. xanthus genome sequence (available at Cereon Genomics for academic use). Shimkets and co-workers (13Kearns D.B. Venot A. Bonner P.J. Stevens B. Boons G.-J. Shimkets L.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13990-13994Crossref PubMed Scopus (37) Google Scholar) describe in the esg mutant ofM. xanthus selective enrichment of the FA 16:1 ω5c, the production of which they correlate to the E signal. Using an excitation assay for FA content, chemically synthesized 1,2-O-Bis(11-(Z)-hexadecenoyl)-sn-glycero-3-phosphoethanolamine (16:1 ω5c/16:1 ω5c) was shown to elicit chemotactic activity ofM. xanthus cells in much lower concentrations than other phosphoethanolamines tested. Although the general outcome of our feeding experiments is in good agreement with the data from the Shimkets group, we did not see a comparable increase over wild type levels of 15:0 iso-FA in Ω258 fed with IVA, and saw no increase at all in S. aurantiaca EBS7. Neither did we see the strong reduction of 16:1 ω5c in both strains after IVA feeding as described for Ω258. Nevertheless, the general response of the strains seems to be consistent in that they up-regulate iso-odd FA if lower amounts of unsaturated FA (e.g. FA 16:1 ω5c) can be generated. Our data would explain why the reversal period reported for the extract from Ω258 fed with IVA differs only very little from the wild type (13Kearns D.B. Venot A. Bonner P.J. Stevens B. Boons G.-J. Shimkets L.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13990-13994Crossref PubMed Scopus (37) Google Scholar). Kearns et al. (13Kearns D.B. Venot A. Bonner P.J. Stevens B. Boons G.-J. Shimkets L.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13990-13994Crossref PubMed Scopus (37) Google Scholar) suggested that the question of what role the lipid plays in development can be best addressed by devising a method to limit the concentration of phosphoethanolamine containing 16:1 ω5c FA during development to examine the morphological and behavioral consequences, and they pointed out that this is difficult to achieve. Alternatively, one could prove the involvement of FA 16:1 ω5c by further selectively enriching the phosphoethanolamines in this FA. If genes involved in the bypass pathway to IVA described in this paper can be isolated and inactivated in addition to the esg gene, one should expect this to result in mutants unable to generate iso-FA and thus to increase the relative amount of 16:1 ω5c FA. We gratefully acknowledge the critical evaluation of this manuscript by Heinz G. Floss and Thomas Hartmann. We thank Dale Kaiser for M. xanthus DK1622 and John Downard for providing mutant Ω258." @default.
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