Journal of Molecular Biology
Regular articleThe first gene in the biosynthesis of the polyketide antibiotic TA Of Myxococcus xanthus codes for a unique PKS module coupled to a peptide synthetase1
Introduction
Polyketides constitute a large and highly diverse group of secondary metabolites, synthesized by bacteria, fungi and plants, with a broad range of biological activities and medical applications (Katz & Donadio, 1993). They include anticancer agents (Daunorubicin), antibiotics (tetracyclines, erythromycin, etc.), immunosuppressants (macrolide FK506), and compounds with mycotoxic activity (aflatoxins, ochratoxins, ergochromes, patulin, etc.). Polyketides are synthesized by repetitive condensations of acetate or propionate monomers in a similar way to that of fatty acid biosynthesis (Hopwood & Sherman, 1990). Structural diversity of polyketides is achieved through different thioester primers, varying chain extension units used by the polyketide synthases (PKSs), and variations in the stereochemistry and the degree of reduction of the intermediates. Diversity is also achieved by subsequent processing, such as alkylations, oxidations, O-methylations, glycosylations and cyclizations. Genetic studies (Donadio et al., 1991) indicated that gene organization of functional units and motif patterns of various PKSs are similar Donadio and Katz 1992, Malpartida et al 1987. This similarity was used to obtain new PKS systems in Gram negative (Schupp et al., 1995) and Gram positive bacteria Kakinuma et al 1991, Malpartida et al 1987, Sherman et al 1989.
PKS systems are classified into two types: type I PKSs are large, multifunctional enzymes, containing a separate site for each condensation or modification step. These represent “modular PKSs” in which the functional domains encoded by the DNA sequence are usually ordered parallel with the sequence of reactions carried out on the growing polyketide chain. Type II PKSs are systems made up of individual enzymes, in which each catalytic site is used repeatedly during the biosynthetic process.
Genetic studies on prokaryotic PKSs have focused on Gram positive microorganisms, particularly on actinomycetes. Myxobacteria are Gram negative bacteria that produce a large number of secondary metabolites, including polyketides (Reichenbach & Hofle, 1993). They are soil microorganisms feeding on proteins and peptides and undergoing a complex life cycle, which includes cell to cell interactions, signaling, myxosporulation and fruiting body formation (Shimkets, 1990). Myxococcus xanthus produces TA (Figure 1), an antibacterial antibiotic which has been studied extensively in our laboratory (Rosenberg et al., 1973). The only other myxococcal PKS to be studied genetically is that of Soraphen A, an antifungal macrolide antibiotic produced by Sorangium cellulosum. The genes participating in the biosynthesis of Soraphen A were assigned to a DNA fragment of approximately 32 kb which contains sequences displaying homology to PKS genes from actinomycetes (Schupp et al., 1995).
The antibiotic TA was crystalized and its chemical properties were determined Rosenberg et al 1982, Trowitzsch et al 1982. It is a macrocyclic polyketide synthesized through the incorporation of acetate, methionine, and glycine Fytlovitch et al 1982, Trowitzsch et al 1982. TA inhibits cell wall synthesis by interfering with the polymerization of the lipid-disaccharide-pentapeptide (Zafriri et al., 1981) and its ability to adhere avidly to tissues and inorganic surfaces (Rosenberg et al., 1984) makes it potentially useful in a wide range of clinical applications, such as treating gingivitis (Manor et al., 1989). A DNA fragment of at least 36 kb, encoding genes involved in TA production, identified through transposon mutagenesis (Varon et al., 1992) as well as in vivo lacZ(Kroos & Kaiser, 1984) fusions, suggested that the genes in the cluster are co-regulated Tolchinsky et al 1992, Varon and Rosenberg 1996, Varon et al 1997. Here, a 7.2kb DNA fragment, derived from a region essential for TA biosynthesis, was cloned and sequenced. Analysis of this sequence identified part of a single, large open reading frame (ORF) which contains two regions of similarity to other proteins. One region (at the 3′ end) displays homology to large multifunctional type I modular PKSs, demonstrating that TA PKSs are organized in a type I modular gene organization. However, this polypeptide is unique, as the other region (at the 5′ end) codes for a peptide synthase amino acid activating domain. The unusual presence of these two regions on one polypeptide is explained by the chemical structure of TA, which contains an amino acid within its polyketide carbon chain.
Section snippets
Analysis of the TA gene cluster by chromosomal restriction map
Chromosomal DNA of several transposition mutants (ER-2514, ER-1037, ER-1030, ER-1311, ER-7513, ER-3708, ER-4639 and ER-6199; Varon et al., 1992) was extracted, digested with restriction enzymes that cut within the transposon, and analyzed by Southern hybridization with six different probes (originating from Tn V and Tn5 lac; see Materials and Methods). We used probes designed to hybridize either to the entire transposon, or to its 5′ or 3′ ends. A chromosomal restriction map of the whole gene
Discussion
The biosynthesis of the polyketide antibiotic TA is a multi-step process, in which a glycine-containing molecule is used as a unique starter unit, and elongated by condensation of 11 acetate molecules by PKSs. The completion of the polyketide chain is achieved via a number of post-modification steps, such as a specific hydroxylation (C20), two methylation reactions (C30 and C31) and O-methylation (C35). The results obtained here demonstrate, for the first time, that the TA biosynthetic genes
Bacterial strains and plasmids
The M. xanthus strains used here were the wild-type strain ER-15, the Tn5 lac mutants ER-2514, ER-1037, ER-1030, ER-7512 and the Tn V mutants ER-4639, ER-1311 and ER-6199. All the mutants are blocked in TA production and have been described (Varon et al., 1992). Escherichia coli TG1 (Bethesda Research Laboratories), DH10ß (F−mcrA (mrr, hsdRMS, mcrBC) f80d lacZ,M15, lacX74, endA1, recA1, deoR (ara-leu) 7697 araD139, galK, nupG, rpsL), and XL1-Blue MR (Stratagene, La Jolla, CA) were used for
Acknowledgements
We thank Dr D. H. Sherman for providing the gra I DNA fragments and Dr P. F. Leadlay for providing the ery AI, ery AII and DEBS II DNA fragments.
This work was supported in part by the Pasha Gol Chair for Applied Microbiology (E.R.), the Morris and Manja Leigh Chair for Biophysics and Biotechnology (E.Z.R.), a British Council Clore Foundation Scholarship awarded to Y.P., and Cancer Research Campaign (no. SP1937/0301) and the Wellcome Trust (no. 038060/Z/93/Z) grants to E.O.
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