A multifunctional enzyme is involved in bacterial ether lipid biosynthesis.

Fatty acid-derived ether lipids are present not only in most vertebrates but also in some bacteria. Here we describe what is to our knowledge the first gene cluster involved in the biosynthesis of such lipids in myxobacteria that encodes the multifunctional enzyme ElbD, which shows similarity to polyketide synthases. Initial characterization of elbD mutants in Myxococcus xanthus and Stigmatella aurantiaca showed the importance of these ether lipids for fruiting body formation and sporulation.

Characterization of four type IV pilin homologues in Stigmatella aurantiaca DSM17044 by heterologous expression in Myxococcus xanthus.

As prokaryotic models for multicellular development, Stigmatella aurantiaca and Myxococcus xanthus share many similarities in terms of social behaviors, such as gliding motility. Our current understanding of myxobacterial grouped-cell motilities comes mainly from the research on M. xanthus, which shows that filamentous type IV pili (TFP), composed of type IV pilin (also called PilA protein) subunits, are the key apparatus for social motility (S-motility). However, little is known about the pilin protein in S. aurantiaca. We cloned and sequenced four genes (pilA(Sa1~4)) from S. aurantiaca DSM17044 that are homologous to pilA(Mx) (pilA gene in M. xanthus DK1622). The homology and similarities among pilA(Sa) proteins and other myxobacterial homologues were systematically analyzed. To determine their potential biological functions, the four pilA(Sa) genes were expressed in M. xanthus DK10410 (ΔpilA(Mx)), which did not restore S-motility on soft agar or EPS production to host cells. After further analysis of the motile behaviors in a methylcellulose solution, the M. xanthus strains were categorized into three types. YL6101, carrying pilA(Sa1), and YL6104, carrying pilA(Sa4), produced stable but unretractable surface pili; YL6102, carrying pilA(Sa2), produced stable surface pili and exhibited reduced TFP-dependent motility in methylcellulose; YL6103, carrying pilA(Sa3), produced unstable surface pili. Based on these findings, we propose that pilA(Sa2) might be responsible for the type IV pilin production involved in group motility in S. aurantiaca DSM17044. After examining the developmental processes, it was suggested that the expression of PilA(Sa4) protein might have positive effects on the fruiting body formation of M. xanthus DK10410 cells. Moreover, the formation of fruiting body in M. xanthus cells with stable exogenous TFPSa were compensated by mixing them with S. aurantiaca DSM17044 cells. Our results shed some light on the features and functions of type IV pilin homologues in S. aurantiaca.

Evolutionary divergence of sedoheptulose 7-phosphate cyclases leads to several distinct cyclic products.

Sedoheptulose 7-phosphate cyclases are enzymes that utilize the pentose phosphate pathway intermediate, sedoheptulose 7-phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectant validamycin, and the natural sunscreens mycosporine-like amino acids. These proteins are phylogenetically related to the dehydroquinate (DHQ) synthases from the shikimate pathway and are part of the more recently recognized superfamily of sugar phosphate cyclases, which includes DHQ synthases, aminoDHQ synthases, and 2-deoxy-scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3-1. These enzymes catalyze the conversion of sedoheptulose 7-phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity, conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-like sugar phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic molecules.

Mutagenesis and biochemical studies on AuaA confirmed the importance of the two conserved aspartate-rich motifs and suggested difference in the amino acids for substrate binding in membrane-bound prenyltransferases.

AuaA is a membrane-bound farnesyltransferase from the myxobacterium Stigmatella aurantiaca involved in the biosynthesis of aurachins. Like other known membrane-bound aromatic prenyltransferases, AuaA contains two conserved aspartate-rich motifs. Several amino acids in the first motif NXxxDxxxD were proposed to be responsible for prenyl diphosphate binding via metal ions like Mg(2+). Site-directed mutagenesis experiments demonstrated in this study that asparagine, but not the arginine residue in NRxxDxxxD, is important for the enzyme activity of AuaA, differing from the importance of NQ or ND residues in the NQxxDxxxD or NDxxDxxxD motifs observed in some membrane-bound prenyltransferases. The second motif of known membrane-bound prenyltransferases was proposed to be involved in the binding of their aromatic substrates. KDIxDxEGD, also found in AuaA, had been previously speculated to be characteristic for binding of flavonoids or homogenisate. Site-directed mutagenesis experiments with AuaA showed that KDIxDxEGD was critical for the enzyme activity. However, this motif is very likely not specific for flavonoid or homogenisate prenyltransferases, because none of the tested flavonoids was accepted by AuaA or its mutant R53A in the presence of farnesyl, geranyl or dimethylallyl diphosphate.

Discovery of the rhizopodin biosynthetic gene cluster in Stigmatella aurantiaca Sg a15 by genome mining.

The field of bacterial natural product research is currently undergoing a paradigm change concerning the discovery of natural products. Previously most efforts were based on isolation of the most abundant compound in an extract, or on tracking bioactivity. However, traditional activity-guided approaches are limited by the available test panels and frequently lead to the rediscovery of already known compounds. The constantly increasing availability of bacterial genome sequences provides the potential for the discovery of a huge number of new natural compounds by in silico identification of biosynthetic gene clusters. Examination of the information on the biosynthetic machinery can further prevent rediscovery of known compounds, and can help identify so far unknown biosynthetic pathways of known compounds. By in silico screening of the genome of the myxobacterium Stigmatella aurantiaca Sg a15, a trans-AT polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) gene cluster was identified that could not be correlated to any secondary metabolite known to be produced by this strain. Targeted gene inactivation and analysis of extracts from the resulting mutants by high performance liquid chromatography coupled to high resolution mass spectrometry (HPLC-HRMS), in combination with the use of statistical tools resulted in the identification of a compound that was absent in the mutants extracts. By matching with our in-house database of myxobacterial secondary metabolites, this compound was identified as rhizopodin. A detailed analysis of the rhizopodin biosynthetic machinery is presented in this manuscript.

Completing the puzzle of aurachin biosynthesis in Stigmatella aurantiaca Sg a15.

The aurachins are a family of secondary metabolites, with the main members aurachin A, B, C, and D, produced by the myxobacterium Stigmatella aurantiaca Sg a15. These isoprenoid quinoline alkaloids are classified as A-type or C-type aurachins according to the position of the farnesyl residue either at C4 or C3 of the quinoline core, respectively. Previous feeding studies revealed that the C-type aurachins are converted to A-type aurachins by late stage tailoring reactions. While the core gene cluster coding for the functionalities required for the biosynthesis of the basic structure aurachin D is known, neither of the genes encoding for the successively acting tailoring enzymes was known up to date, which was assumed to be due to a split cluster organisation. Here we describe the identification of a total of five genes, located upstream of the aurachin core cluster and at additional two loci elsewhere in the genome, encoding for the aforementioned functionalities. The generation and evaluation of respective inactivation mutants of S. aurantiaca Sg a15 allowed for the first time to propose an exhaustive model for aurachin biosynthesis. One of the deduced biosynthetic transformations corresponds to a pinacol rearrangement, an unprecedented tailoring reaction in secondary metabolite biosynthesis.

Unprecedented anthranilate priming involving two enzymes of the acyl adenylating superfamily in aurachin biosynthesis.

Biosynthesis of many polyketide-derived secondary metabolites is initiated by incorporating starter units other than acetate. Thus, understanding their priming mechanism is of importance for metabolic engineering. Insight into the loading process of anthranilate into the biosynthetic pathway for the quinoline alkaloids aurachins has been provided by the sequencing of a partial biosynthetic gene cluster in the myxobacterium Stigmatella aurantiaca. The cluster encodes a predicted aryl:CoA ligase AuaE that was hypothesized to activate and transfer anthranilate to the acyl carrier protein AuaB. However, gene inactivation and in vitro experiments described here contradicted this model. Aided by the genome sequence of S. aurantiaca, we identified an additional aryl:CoA ligase homologue, AuaEII, encoded in a different gene operon, which is additionally required for anthranilate priming. We report the characterization of both enzymes and the elucidation of a novel non-acetate priming strategy in thio-templated biosynthetic machineries.

AuaA, a membrane-bound farnesyltransferase from Stigmatella aurantiaca, catalyzes the prenylation of 2-methyl-4-hydroxyquinoline in the biosynthesis of aurachins.

Aurachins are quinoline alkaloids isolated from the myxobacterium Stigmatella aurantiaca. They are substituted with an isoprenoid side chain and act as potent inhibitors in the electron transport chain. A biosynthetic gene cluster that contains at least five genes (auaA-auaE) has been identified for aurachin biosynthesis. In this study, auaA, the gene encoding a putative prenyltransferase of 326 amino acids, was cloned and overexpressed in Escherichia coli. Biochemical investigations showed that AuaA catalyzes the prenylation of 2-methyl-4-hydroxyquinoline in the presence of farnesyl diphosphate (FPP), thereby resulting in the formation of aurachin D. The hydroxyl group at position C4 of the quinoline ring is essential for an acceptance by AuaA; this was concluded by testing 18 quinoline derivatives or analogues with AuaA and FPP. (1) H NMR and HR-EI-MS analyses of six isolated enzyme products revealed the presence of a farnesyl moiety at position C3 of the quinoline ring. K(M) values of 43 and 270 µM were determined for FPP and 2-methyl-4-hydroxyquinoline, respectively. Like other known membrane-bound prenyltransferases, the reaction catalyzed by AuaA is dependent on the presence of metal ions such as Mg(2+) , Mn(2+) and Co(2+) , although no typical (N/D)DXXD binding motif was found in the sequence.

Myxochromide B3, a new member of the myxochromide family of secondary metabolites.

Myxochromides are cyclic depsipeptides with an unsaturated polyketide side chain, which have been reported from different myxobacterial species, e.g., Myxococcus xanthus and Stigmatella aurantiaca. To date, myxochromides are subdivided into the groups A and S, according to their peptidic core structure. The peptide moiety of the new myxochromide B3 (1), which was isolated from a myxobacterial strain of the genus Myxococcus, differs from that of myxochromides A and S. Compound 1 thus is the first representative of a new group of myxochromides. For myxochromide A3 (2) the complete and assigned spectroscopic data are described. For the structure elucidation one- and two-dimensional NMR spectroscopy as well as mass spectrometry have been applied. Configurational analysis has been accomplished by chiral GC-MS and HPLC.

Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition.

Horizontal gene transfer by transposition has been widely used for transgenesis in prokaryotes. However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range. We examine the possibility that transposons can be used to deliver large transgenes to heterologous hosts. This possibility is particularly relevant to the expression of large secondary metabolite gene clusters in various heterologous hosts. Recently, we showed that the engineering of large gene clusters like type I polyketide/nonribosomal peptide pathways for heterologous expression is no longer a bottleneck. Here, we apply recombineering to engineer either the epothilone (epo) or myxochromide S (mchS) gene cluster for transpositional delivery and expression in heterologous hosts. The 58-kb epo gene cluster was fully reconstituted from two clones by stitching. Then, the epo promoter was exchanged for a promoter active in the heterologous host, followed by engineering into the MycoMar transposon. A similar process was applied to the mchS gene cluster. The engineered gene clusters were transferred and expressed in the heterologous hosts Myxococcus xanthus and Pseudomonas putida. We achieved the largest transposition yet reported for any system and suggest that delivery by transposon will become the method of choice for delivery of large transgenes, particularly not only for metabolic engineering but also for general transgenesis in prokaryotes and eukaryotes.

DKxanthene biosynthesis--understanding the basis for diversity-oriented synthesis in myxobacterial secondary metabolism.

The DKxanthenes are a family of yellow pigments which play a critical role in myxobacterial development. Thirteen unique structures from Myxococcus xanthus DK1622 differ in the length of their characteristic polyene functionality, as well as the extent of methyl branching. We aimed to understand the mechanistic basis for this "molecular promiscuity" by analyzing the gene cluster in DK1622, and comparing it to the DKxanthene biosynthetic locus in a second myxobacterium, Stigmatella aurantiaca DW4/3-1, which produces a more limited range of compounds. While the core biosynthetic machinery is highly conserved, M. xanthus contains a putative asparagine hydroxylase function which is not present in S. aurantiaca. This observation accounts, in part, for the significantly larger metabolite family in M. xanthus. Detailed analysis of the encoded hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) assembly line provides direct evidence for the mechanism underlying the variable polyene length and the observed pattern of methyl functionalities.

Integration into the phage attachment site, attB, impairs multicellular differentiation in Stigmatella aurantiaca.

Stigmatella aurantiaca displays a complex developmental life cycle in response to starvation conditions that results in the formation of tree-like fruiting bodies capable of producing spores. The phage Mx8, first isolated from the close relative Myxococcus xanthus, is unable to infect S. aurantiaca cells and integrate into the genome. However, plasmids containing Mx8 fragments encoding the integrase and attP are able to integrate at the attB locus in the S. aurantiaca genome by site-specific recombination. After recombination between attP and attB, the S. aurantiaca cells were incapable of building normal fruiting bodies but formed clumps and fungus-like structures characteristic of intermediate stages of development displayed by the wild type. We identified two tRNA genes, trnD and trnV, encoding tRNA(Asp) and tRNA(Val), respectively, composing an operon at the attB locus of S. aurantiaca. Integration of attP-containing plasmids resulted in the incorporation of the t(Mx8) terminator sequence, in addition to a short sequence of Mx8 DNA downstream of trnD. The integrant was unable to process the trnD transcript at the normal 3' processing site and displayed a lower level of expression of the trnVD operon. In addition, several developmentally regulated proteins were no longer produced in mutants following insertion at the attB locus. We hypothesize that the integration of the t(Mx8) terminator sequence results in reduced levels of mature tRNA(Asp) and tRNA(Val) and that altered protein production during development is thereby responsible for the observed phenotype. The trnVD locus thus defines a new developmental checkpoint for Stigmatella aurantiaca.

Novel insights into siderophore formation in myxobacteria.

The myxochelins are catecholate-type siderophores produced by a number of myxobacterial strains, and their corresponding biosynthetic gene clusters have been identified in Stigmatella aurantiaca Sg a15, and Sorangium cellulosum So ce56; the latter being presented in this work. Biochemical and genetic studies described here further clarify myxochelin biosynthesis. In addition to the myxochelin A biosynthetic complex, the aminotransferase MxcL is required in order to form myxochelin B, starting from 2,3-dihydroxy benzoic acid and L-lysine. Additionally, the substrate specificity of the myxochelin A biosynthetic complex was analyzed in vitro; this led to the formation of novel myxochelin derivatives. Furthermore, MxcD was over-expressed and its function as an active isochorismic acid synthase in Escherichia coli was verified by complementation studies, as was activity in vitro. The organization of the myxochelin gene cluster of S. cellulosum So ce56 was compared to that of the Sg a15 gene cluster. The comparison revealed that although the organization of the biosynthetic genes is completely different, the biosynthesis is most probably extremely similar.

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.

A biosynthetic pathway to isovaleryl-CoA in myxobacteria: the involvement of the mevalonate pathway.

A biosynthetic shunt pathway branching from the mevalonate pathway and providing starter units for branched-chain fatty acid and secondary metabolite biosynthesis has been identified in strains of the myxobacterium Stigmatella aurantiaca. This pathway is upregulated when the branched-chain alpha-keto acid dehydrogenase gene (bkd) is inactivated, thus impairing the normal branched-chain amino acid degradation process. We previously proposed that, in this pathway, isovaleryl-CoA is derived from 3,3-dimethylacrylyl-CoA (DMA-CoA). Here we show that DMA-CoA is an isomerization product of 3-methylbut-3-enoyl-CoA (3MB-CoA). This compound is directly derived from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by a decarboxylation/ dehydration reaction resembling the conversion of mevalonate 5-diphosphate to isopentenyl diphosphate. Incubation of cell-free extracts of a bkd mutant with HMG-CoA gave product(s) with the molecular mass of 3MB-CoA or DMA-CoA. The shunt pathway most likely also operates reversibly and provides an alternative source for the monomers of isoprenoid biosynthesis in myxobacteria that utilize L-leucine as precursor.

Signaling in myxobacteria.

Myxobacteria use soluble and cell-contact signals during their starvation-induced formation of fruiting bodies. These signals coordinate developmental gene expression with the cell movements that build fruiting bodies. Early in development, the quorum-sensing A-signal in Myxococcus xanthus helps to assess starvation and induce the first stage of aggregation. Later, the morphogenetic C-signal helps to pattern cell movement and shape the fruiting body. C-signal is a 17-kDa cell surface protein that signals by contact between the ends of two cells. The number of C-signal molecules per cell rises 100-fold from the beginning of fruiting body development to the end, when spores are formed. Traveling waves, streams, and sporulation have increasing thresholds for C-signal activity, and this progression ensures that spores form inside fruiting bodies.