Shu-Ming Li*, Emmanuel Wemakor and Lutz Heide
Pharmazeutisches Institut, Pharmazeutische Biologie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, D-72076 Tübingen, Germany
Abstract
Degenerate oligonucleotide primers were designed for the PCR amplification of 1-deoxy-D-xylulose 5-phosphate (DXP) synthase genes from genomic DNA of different Streptomyces strains. Prominent PCR fragments of the expected size (650 bp) were detected with all of the strains tested, and an additional band at 540 bp was observed with genomic DNA of Streptomyces spheroides. All of the PCR products showed high homology to DXP synthase (dxs) genes in the database. In total, 15 different dxs fragments were cloned from the five Streptomyces strains S. spheroides NCIMB 11891, S. hygroscopicus NRRL 3418, S. rishiriensis DSM 40489, S. coelicolor A3(2) and S. lividans TK 24. 11 of the 13 sequences could be placed into two different groups, distinguished by highly conserved motifs. Each examined organism contained at least one gene of each of the two groups. Three and five dxs isogenes, respectively, could be amplified from S. hygroscopicus NRRL 3418 and S. spheroides NCIMB 11891, which produce antibiotics with isoprenoid moieties. This is the first report of the existence of several putative dxs isogenes within single organisms.
Introduction
Isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) are the common precursors of isoprenoids found in all organisms, such as steroid hormones in mammals, carotenoids and other isoprenoids in plants, ubiquinones and/or menaquinones in bacteria, and membrane lipids such as sterols and hopanoids (Sacchettini and Poulter, 1997). It was previously believed that IPP is always synthesized by condensation of three molecules of acetyl coenzyme A via the mevalonate pathway. However, it has recently been found that in plants and bacteria IPP can also be synthesized via a mevalonate-independent pathway (Rohmer et al., 1993; Rohmer et al., 1996; Lichtenthaler, 1999; Rohmer, 1999). The actinomycetes, a group of Gram-positive bacteria, which produce a large variety of antibiotics, can use the mevalonate pathway, the mevalonate-independent pathway or both pathways for the biosynthesis of their terpenoids (Shin-ya et al., 1990; Seto et al., 1996; Seto et al., 1998).
Up to now, five genes have been identified which encode different enzymes of the mevalonate-independent pathway (Lüttgen et al., 2000 and references cited herein). The initial step of this pathway is the formation of 1-deoxy-D-xylulose 5-phosphate (DXP) by condensation of pyruvate and glyceraldehyde 3-phosphate, catalyzed by DXP synthase. The gene encoding DXP synthase (dxs) has been cloned from different bacteria and plants, e.g. from Escherichia coli (Sprenger et al., 1997; Lois et al., 1998), Mentha x piperita (Lange et al., 1998), Capsicum annuum L (Bouvier et al., 1998), Synechococcus leopoliensis (Miller et al., 1999), Rhodobacter capsulatus (Hahn et al., 2001), as well as from the Streptomyces sp. strain CL190 (Kuzuyama et al., 2000) which produces the hemiterpenoid secondary metabolite naphterpin.
The coumarin antibiotics novobiocin and clorobiocin (=chlorobiocin) are produced by Streptomyces spheroides NCIMB 11891 and S. hygroscopicus NRRL 3418 (Berger and Batcho, 1978), respectively, and contain a dimethylallyl moiety. We have shown that this dimethylallyl moiety is derived from the mevalonate-independent pathway (Li et al., 1998). Recently, we have cloned the biosynthetic gene cluster of novobiocin (Steffensky et al., 2000). The cluster contains a total of 23 open reading frames and is flanked on one side by the novobiocin resistance gene gyrBR, on the other side by an ABC transporter. However, neither the cluster nor the adjacent DNA regions contained genes involved in IPP formation. IPP for novobiocin biosynthesis may therefore be supplied by enzymes of the primary metabolism, or alternatively, specific genes for the biosynthesis of the isoprenoid moiety of novobiocin may exist, but be situated at a different locus of the genome. Screening of a genomic DNA library with one of the known genes of the mevalonate-independent pathway, e.g. dxs, as a probe may allow an investigation of the latter hypothesis.
It has been reported that DXP is not only involved in the biosynthesis of IPP for the formation of isoprenoid primary and secondary metabolites, but also in the biosynthesis of thiamin (vitamin B1) (Thérisod et al., 1981; Begley 1996) and pyridoxol phosphate (vitamin B6) (Cane et al., 1999; Laber et al., 1999; Tazoe et al., 2000) in bacteria. Therefore, several DXP synthase genes may exist for different biosynthetic pathways in a single Streptomyces strain. For studies of the function of these enzymes, it would be useful to clone the encoding dxs genes.
We now report a convenient method for the PCR amplification of dxs fragments from different Streptomyces strains, and the identification of several putative dxs isogenes from each of the examined strains.
Results
Design of Oligonucleotide Primers and PCR Amplification of 1-Deoxy-D-Xylulose 5-Phosphate Synthase Genes
Two conserved regions of dxs from 9 different bacteria (Figure 1) were selected to design the PCR primers under consideration of the codon usage for Streptomyces (Wright and Bibb, 1992). The forward primer (5´-GGS ATG GCS TGG GAR GCS CTS AAC; R = G or A; S = G or C) was derived from the amino acid sequence Gly Met Ala Trp Glu Ala Leu Asn. This motif is part of the putative thiamin diphosphate binding site of DXP synthases (Sprenger et al., 1997; Lois et al., 1998). The amino acid sequence Asp Val Gly(Ala) Ile Ala Glu Gln His (Figure 1) served for construction of the reverse primer (5´- GTG CTG YTC SGC GAT SSC SAC GTC; R = G or A; S = G or C; Y = C or T). The expected size of the PCR products obtained with these two primers is between 640 and 660 bp.
Genomic DNA from five Streptomyces strains was used for PCR amplification: S. spheroides NCIMB 11891 and S. hygroscopicus NRRL 3418 are the producers of the aminocoumarin antibiotics novobiocin and clorobiocin which contain a dimethylallyl moiety; S. rishiriensis DSM 40489 is the producer of the aminocoumarin antibiotic coumermycin A1 which does not contain a terpenoid moiety (Berger and Batcho 1978); and S. coelicolor A3(2) and S. lividans TK 24 are the two best-examined Streptomyces strains, which are genetically very similar and which do not produce isoprenoid secondary metabolites (Kieser et al., 2000; p. 426). The genome of S. coelicolor A3(2) has been almost completely sequenced (Streptomyces coelicolor genome project: http://www.sanger.ac.uk/Projects/S.coelicolor).
Prominent bands at about 650 bp were observed on agarose gels after PCR amplification with genomic DNA from all of the tested strains. With genomic DNA from S. spheroides, an additional band at about 540 bp was observed. The formation of these PCR products was dependent on the presence of genomic DNA and of both primers (data not shown).
Identification and Characterization of the Cloned dxs Fragments
The PCR products were cloned into the SmaI site of pBluescript SK(-) and the inserts were sequenced on both strands with standard M13 primers. All of 28 clones sequenced revealed very high similarity to the 1-deoxy-D-xylulose 5-phosphate synthases in the database. In total, the 28 clones represented 13 different genes: four from the novobiocin producer S. spheroides, three from the clorobiocin producer S. hygroscopicus, and two each from S. coelicolor, S. lividans and S. rishiriensis. As shown in Figures 2 and 3, the deduced amino acid sequences of these genes showed very high homology with each other (approximately 70 % identity at the amino acid level within 11 of the 13 genes). Especially, the first 50 amino acids of the examined sequences are very strongly conserved (>94 % identity within 11 of the 13 genes). The 540 bp fragment dxsD-Ss from S. spheroides showed a deletion of 38 amino acids from position 48 to 86, and a smaller deletion from position 148-151.
Phylogenetic analysis (Figure 3) of the PCR products revealed that, except dxsD-Ss and dxsC-Sh, the genes identified in this study can be placed into two groups. The distinction between these two groups is also obvious from the sequence alignment in Figure 2, most notably by the highly conserved motifs in position 152-161 (KIHPDTGLPI in group A and V(A)MDPLTCA(E)PL in group B).
At present, 98 % of the genome of S. coelicolor A3(2) have been sequenced, and the database contains two genes from this organism with homologies to DXP synthase. Indeed, our PCR method led to the amplification of fragments of both these genes: dxsA-Sc (656 bp) was identical (except for one base, and minor differences caused by wobbles in the backward primer) to an ORF sequenced on two overlapping cosmids in the database, SC1C3.01 and SC7B7.10 (AL023702 and AL009199, respectively), and dxsB-Sc (659 bp) was completely identical to the ORF SC6A5.17 (bp 17123-17781 of AL049485). dxsA-Sc and dxsB-Sc showed 68 % identity with each other on the amino acid level. Two corresponding genes were amplified from S. lividans TK 24, which is genetically very similar to S. coelicolor.
Since the dxs isogenes show very high homology, it may be expected that homologous recombination events can occur between these sequences in the bacterial genomes. Indeed, two of the 28 clones obtained in this study represented sequences which were likely to have originated from such recombination events: the fragment dxsAxC-Ss from S. spheroides NCIMB 11891 was completely identical to dxsA-Ss in the region from bp 1-449, and completely identical to dxsC-Ss in the region from bp 450-660, except for differences caused by wobbles in the primer sequence. Likewise the fragment dxsBxA-Sc from S. coelicolor A3(2) was identical (except for 1 bp) to dxsB-Sc in the region from bp 1-367, and to dxsA-Sc from bp 368-660. Since these two sequences apparently did not represent independent genes, they were not included in Figures 2 and 3. They have been deposited in the GenBank database under accession number AF283711 (dxsBxA-Sc) and AF283721 (dxsAxC-Ss).
Discussion
With two degenerate primers derived from conserved regions of dxs genes from different bacteria, we have amplified a total of 13 putative dxs fragments using genomic DNA from five different Streptomyces strains.
The described method proved to be specific to putative dxs sequences. All of the 28 amplified PCR products showed very high homology to 1-deoxy-D-xylulose 5-phosphate synthase genes. Two to four different dxs fragments were cloned from each of the tested strains. The method is suitable for the amplification of probes for hybridization; the size of the PCR products (about 650 bp) is convenient to be subcloned and to be used as a probe for Southern hybridization.
The alignment (Figure 2) clearly showed that 11 of the 13 amplified genes could be placed into two groups (group A and group B), distinguished by motifs which are highly conserved within each group. Noteworthy, each of the examined Streptomyces strains contained at least one gene of group A and one gene of group B. The very high identity between these 11 genes and the functionally identified DXP synthase from Streptomyces sp. CL190 (Kuzuyama et al., 2000) (>70 % on the amino acid level) makes it likely that all these genes encode DXP synthases. Of the two S. coelicolor dxs genes contained in the database, dxsA-Sc (= SC1C3.01/SC7B7.10) belongs to group A, and dxsB-Sc (= SC6A5.17) to group B. dxsB-Sc is situated in the S. coelicolor genome within an hopanoid/isoprenoid biosynthetic gene cluster, in the vicinity of a polyprenyl synthase, a squalen (or phytoene) synthase, a squalen (or phytoene) dehydrogenase and a squalene cyclase (Poralla et al., 2000). This suggests that dxsB-Sc may be involved in IPP biosynthesis, and the same may be true for the other group B genes identified in our study. The genes of group A may represent biosynthetic genes engaged in thiamin and/or pyridoxol biosynthesis in Streptomyces. dxs genes from other organisms (e.g. Mycobacterium tuberculosis, E. coli, Synechococcus leopoliensis and others) could not be placed into one of the above groups and will probably deal with both biosynthetic pathways.
More than two dxs genes were found in S. hygroscopicus NRRL 3418 (3 genes) and S. spheroides NCIMB 11891 (4 genes). These organisms are the producers of clorobiocin and novobiocin, which contain an isoprenoid moiety formed via DXP. Therefore the identified, additional dxs isogenes may be involved in the biosynthesis of novobiocin and clorobiocin. However, some of the identified genes may also be non-functional, at least under the present culture conditions. Streptomycetes have a comparatively large genome (approximately 8000 kb) and quite commonly they contain gene clusters which are not functionally expressed (Hopwood, 1999 and references cited herein).
Streptomyces sp. CL190 (syn.: S. aeriouvifer) produces, besides the ubiquitous menaquinones, the hemiterpenoid secondary metabolite naphterpin (Seto et al., 1996). Kuzuyama et al., (Kuzuyama et al., 2000) have amplified, cloned and expressed a single dxs gene from this organism. This gene clearly belongs to group B (Figure 2). It may be interesting to examine whether additional dxs genes can be identified from this organism, using our PCR method.
The present study describes, for the first time, the existence of several putative dxs isogenes in a single organism, and we developed a convenient method to amplify these genes from different Streptomyces strains. Given the importance of DXP synthase for the newly discovered isoprenoid biosynthetic pathway (which may prove an important target for new antibiotics and herbicides [Rohmer, 1998]), and for thiamin and pyridoxol biosynthesis, this PCR method may be a valuable tool for the future investigation of these pathways, enabling the cloning of several dxs isogenes from Streptomyces strains.
Experimental Procedures
Bacterial Strains, Plasmids and Culture Conditions
Plasmid vector pBluescript SK(-) was purchased from Stratagene (Heidelberg, Germany) and used for cloning of the PCR fragments. Escherichia coli XL1 Blue MRF' (Stratagene, Heidelberg, Germany) was used for the preparation of recombinant plasmids and grown in liquid Luria-Bertani (LB) medium or on solid LB medium (1.5 % agar) at 37°C (Sambrook et al., 1989). Carbenicillin (50 µg/ml) was used for selection of recombinant strains.
S. hygroscopicus NRRL 3418 was from the Agricultural Research Service Culture Collection (Peoria, Illinois, USA). S. rishiriensis DSM 40489 was purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). S. spheroides NCIMB 11891, S. lividans TK 24 and S. coelicolor A3(2) were obtained from E. Cundliffe (Leicester, UK) and D.A. Hopwood (Norwich, UK), respectively.
For preparation of genomic DNA, the Streptomyces strains were grown at 28°C and 175 rpm for 2-4 days in baffled shake flasks in YMG medium containing 1.0 % malt extract, 0.4 % yeast extract, 0.4 % glucose, and 1.0 mM CaCl2 (pH 7.3) (Steffensky et al., 2000).
DNA Isolation, Manipulation and Cloning
The genomic DNA of Streptomyces strains was isolated according to the method described by Kieser et al., (Kieser et al., 2000).
Isolation of PCR fragments from agarose gels for cloning was carried out with the Qiagen QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany).
Isolation of plasmids for sequencing was carried out with ion exchange columns (Nucleobond AX Kits, Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol.
Polymerase Chain Reaction (PCR) and Cloning of PCR Products
PCR was performed on a GeneAmp PCR System 9700 (Perkin-Elmer, Weiterstadt, Germany). The PCR reaction mixture (100 µl total volume) contained 0.5 µM of each primer (synthesized by Applied Biosystems, Weiterstadt, Germany), 500 ng genomic DNA of Streptomyces, 0.2 mM of each deoxynucleoside triphosphate, 1.5 mM MgCl2, 5 % DMSO and 2.5 units of Taq DNA polymerase (Promega, Madison, USA). After denaturation at 96°C for 5 min, 25 cycles were carried out with 1.5 min at 95°C, 1.5 min at 65°C and 2.0 min at 72°C, and the final extension was performed at 72°C for 10 min. The PCR products were isolated from agarose gel and cloned blunt-end into the SmaI site of pBluescript SK(-) using the SureCloning Kit (Pharmacia, Freiburg, Germany).
DNA Sequencing and Computer-Assisted Sequence Analysis
DNA sequencing was performed by the dideoxynucleotide chain termination method using M13 primers on a LI-COR automatic sequencer (MWG-Biotech, Ebersberg, Germany).
The DNASIS software package (version 2.1, 1995; Hitachi Software Engineering, San Bruno, CA, USA) was used for sequence analysis. Amino acid sequence homology searches were carried out in the GenBank database using the BLAST programme (release 2.0).
Acknowledgments
This work was financially supported by the Deutsche Forschungsgemeinschaft (to L. H. and S.-M. L.)
References
Begley, T. P. 1996. The biosynthesis and degradation of thiamin (vitamin B1). Nat. Prod. Rep. 13:177-185.
Berger, J., and Batcho, A. D. 1978. Coumarin-glycoside antibiotics. J. Chromatogr. Libr. 15:101-158.
Bouvier, F., d'Harlingue, A., Suire, C., Backhaus, R. A., and Camara, B. 1998. Dedicated roles of plastid transketolases during the early onset of isoprenoid biogenesis in pepper fruits. Plant Physiol. 117:1423-1431.
Cane, D. E., Du, S., Robinson, J. K., Hsiung, Y., and Spenser, I. D. 1999. Biosynthesis of vitamin B6: enzymatic conversion of 1-deoxy-D-xylulose-5-phosphate to pyridoxol phosphate. J. Am. Chem. Soc. 121:7722-7723.
Hahn, F. M., Eubamks, L. M., Testa, C. A., Blagg, B. S. J., Baker, J. A., and Poulter, C. D. 2001. 1-Deoxy-D-xylulose 5-phosphate synthase, the gene product of open reading frame (ORF) 2816 and 2895 in Rhodobacter capsulatus. J. Bacteriol. 183:1-11.
Hopwood, D.A. 1999. Forty years of genetics with Streptomyces: from in vivo through in vitro to in silico. Microbiology 145:2183-2202.
Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F., and Hopwood, D. A 2000. Practical Streptomyces genetics. The John Innes Foundation, Norwich, England.
Kuzuyama, T., Takagi, M., Takahashi, S., and Seto, H. 2000. Cloning and characterization of 1-deoxy-D-xylulose 5-phosphate synthase from Streptomyces sp. strain CL190, which uses both the mevalonate and nonmevalonate pathways for isopentenyl diphosphate biosynthesis. J. Bacteriol. 182:891-897.
Laber, B., Maurer, W., Scharf, S., Stepusin, K., and Schmidt, F. S. 1999. Vitamin B6 biosynthesis: formation of pyridoxine 5´-phosphate from 4-(phosphohydroxy)-L-threonine and 1-deoxy-D-xylulose-5-phosphate by PdxA and PdxJ protein. FEBS Lett. 449:45-48.
Lange, B. M., Wildung, M. R., McCaskill, D., and Croteau. R. 1998. A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway. Proc. Natl. Acad. Sci. USA 95:2100-2104.
Li, S.-M., Hennig, S., and Heide, L. 1998. Biosynthesis of the dimethylallyl moiety of novobiocin via a non-mevalonate pathway. Tetrahedron Lett. 39:2717-2720.
Lichtenthaler, H. K. 1999. The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50:47-65.
Lois, L. M., Campos, N., Rosa-Putra, S., Danielsen, K., Rohmer, and M., Boronat, A. 1998. Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamine, and pyridoxol biosynthesis. Proc. Natl. Acad. Sci. USA 95:2105-2110.
Lüttgen, H., Rohdich, F., Herz, F., Wungsintaweekul, J., Hecht, S, Schuhr, C. A., Fellermeier, M., Sagner, S., Zenk, M. H., Bacher, A., and Eisenreich, W. 2000. Biosynthesis of terpenoids: YchB protein of Escherichia coli phosphorylates the 2-hydroxy group of 4-diphosphocytidyl-2C-methyl-D-erythritol. Proc. Natl. Acad. Sci. USA 97:1062-1067.
Miller, B., Heuser, T., and Zimmer W. 1999. A Synechococcus leopoliensis SAUG 1402-1 operon harboring the 1-deoxyxylulose 5-phosphate synthase gene and two additional open reading frames is functionally involved in the dimethylallyl diphosphate synthesis. FEBS Lett. 460:485-490.
Poralla, B., Muth, G. and Härtner, T. 2000. Hopanoids are formed during transition from substrate to aerial hyphae in Streptomyces coelicolor A3(2). FEMS Microbiol. Lett. 189:93-95.
Rohmer, M. 1998. Isoprenoid biosynthesis via the mevalonate-independent route, a novel target for antibacterial drugs? Drug Res. 50:136-154.
Rohmer, M. 1999. A mevalonate-independent route to isopentenyl diphosphate. In: Cane DE (ed) Comprehensive natural products chemistry. Isoprenoids including carotenoids and steroids. Vol 2, Elsevier: Oxford, U.K, pp 45-67.
Rohmer, M., Knani, M., Simonin, P., Sutter, B., and Sahm, H. 1993. Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. Biochem. J. 295:517524.
Rohmer, M., Seemann, M., Horbach, S., Bringer-Meyer, S., and Sahm, H. 1996. Glyceraldehyde 3-phosphate and pyruvate as precursors of isoprenic units in an alternative non-mevalonate pathway for terpenoid biosynthesis. J. Am. Chem. Soc. 118:2564-2566.
Sacchettini, J. C., and Poulter, C. D. 1997. Creating isoprenoid diversity. Science 277:1788-1789.
Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989 Molecular cloning: a laboratory manual 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Seto, H., Watanabe, H., and Furihata, K. 1996. Simultaneous operation of the mevalonate and non-mevalonate pathways in the biosynthesis of isopentenyl diphosphate in Streptomyces aeriouvifer. Tetrahedron Lett. 37:7979-7982.
Seto, H., Orihara, N., and Furihata, K. 1998. Studies on the biosynthesis of terpenoids produced by actinomycetes. Part 4. Formation of BE-40644 by the mevalonate and nonmevalonate pathways. Tetrahedron Lett. 39:9497-9500.
Shin-ya, K., Furihata, K., Hayakawa, Y., and Seto, H. 1990. Biosynthetic studies of naphterpin, a terpenoid metabolite of Streptomyces. Tetrahedron Lett. 31:6025-6026.
Sprenger, G. A., Schörken, U., Wiegert, T., Grolle, S., de Graaf, A. A., Taylor, S. V., Begley, T. P., Bringer-Meyer, S., and Sahm, H. 1997. Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol. Proc. Natl. Acad. Sci. USA 94:12857-12862.
Steffensky, M., Mühlenweg, A., Wang, Z.-X., Li, S.-M., and Heide, L. 2000. Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides NCIB 11891. Antimicrob. Agents Chemother. 44:1214-1222.
Tazoe, M., Ichikawa, K., Hoshino, T. 2000. Biosynthesis of vitamin B6 in Rhizobium. J. Biol. Chem. 275:11300-11305.
Thérisod, M., Fischer, J. C., and Estramareix, B. 1981. The origin of the carbon chain in the thiazole moiety of thiamine in Escherichia coli: incorporation of deuterated 1-deoxy-D-threo-2-pentulose. Biochem. Biophys. Res. Commun. 98:374-379.
Wright, F., and Bibb, M. J. 1992. Codon usage in the G+C-rich Streptomyces genome. Gene 113:55-65.