The ergot alkaloid gene cluster in Claviceps purpurea: Extension of the cluster sequence and intra species evolution
Graphical abstract
The extension of the ergot alkaloid cluster and the comparison of its structure in two chemical races of Claviceps purpurea are reported.
Introduction
The ergot disease of grasses and cereals caused by the ergot fungus Claviceps purpurea is characterized by the formation of overwintering structures, so-called sclerotia, containing variable amounts of mycotoxins, the ergopeptines. This is a class of cyclol-structured alkaloid peptides containing D-lysergic acid. Since some of these substances have considerable pharmacological importance, physiology, biochemistry and genetics of their formation have been studied in detail (Stadler and Giger, 1984, Tudzynski et al., 2001). Tsai et al. (1995) identified the gene (dmaW) for the first specific enzyme of the pathway, dimethylallyltryptophan synthase (see Fig. 1), in C. fusiformis, and Tudzynski et al. (1999) described a putative ergot alkaloid gene cluster in the C. purpurea strain P1; the putative cluster included a homologue of dmaW, cpd1, and a trimodular NRPS gene, cpps1, encoding lysergylpeptidyl synthetase 1 (LPS1) involved in the attachment of the tripeptide moiety to dehydrolysergic acid (Riederer et al., 1996). Recently, a second NRPS gene in the cluster region, cpps2, was shown to encode LPS2, the D-lysergic acid activating NRPS module (Correia et al., 2003; see Fig. 1). The function of the other genes identified in the neighborhood of cpd1, cpps1 and cpps2 – among them several oxidase/oxygenase genes – remains to be elucidated. Here, we show that the putative cluster region extends on both sides of the originally described sequence, covering at least 68.5 kb. It contains two additional NRPS genes, cpps3 and cpps4, the functions of which remain to be determined. A second major aspect of the present paper is the comparison of the putative ergot alkaloid cluster in two chemical races: strain P1 (major alkaloid: ergotamine) and ECC93 (ergocristine) to study variability/evolution of the cluster sequence within the species C. purpurea and to determine if the difference in the primary ergopeptine produced is mainly due to internal amino acid pools (as postulated by Riederer et al., 1996) or to differences in critical substrate specificity conferring amino acids of the NRPS modules.
Section snippets
Defining the ergot alkaloid gene cluster in strain P1
The core region of the ergot alkaloid gene cluster (Correia et al., 2003) stretches from a putative oxidase gene (cpox3) to the LPS1 encoding gene cpps1 over 40 kb. Chromosome walking using clones of an EMBL3 genomic library of strain P1 extended the available genomic sequence downstream of cpox3 (“left” side) over 15 kb (see Fig. 2). This region contains an additional NRPS gene, cpps3, and a gene probably involved in primary metabolism, cpimd (encoding an isopropylmalate-dehydratase). Cpps3
Strains and culture conditions
Claviceps purpurea strains P1 and ECC93, which produce mainly ergotamine and ergocristine, respectively, have been described previously (Tudzynski et al., 1999, Keller et al., 1988), as were the standard media and culture conditions (Tudzynski et al., 1999).
For northern analyses, mycelia were cultivated in Mantle A medium with low (0.5 g/l KH2PO4) and high (2.0 g/l KH2PO4) amounts of phosphate, respectively. After 5 d mycelia were harvested and RNA was prepared using the RNAgents® Total RNA
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft (Tu 50/13, SPP 1152: Evolution of metabolic diversity, to P.T.) and the United States Department of Agriculture (2001-35319-10930 and 58-6401-2-0025 to D.G.P. and C.L.S.). We thank N. Lorenz for discussion and for support in the northern analyses, and U. Keller (Berlin) for providing us with unpublished data.
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