PMID:12563033

Citation	Gust, B, Challis, GL, Fowler, K, Kieser, T and Chater, KF (2003) PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc. Natl. Acad. Sci. U.S.A. 100:1541-6
Abstract	Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and lambda-Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriT(RK2) for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT(RK2) to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.
Links	PubMed PMC149868 Online version:10.1073/pnas.0337542100
Keywords	Amino Acid Sequence; Base Sequence; DNA Primers; Gas Chromatography-Mass Spectrometry; Genes, Bacterial; Molecular Sequence Data; Naphthols/metabolism; Odors; Polymerase Chain Reaction; Sequence Homology, Amino Acid; Soil; Streptomyces/genetics; Streptomyces/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
STRCO:CYC2		GO:0044550: secondary metabolite biosynthetic process	ECO:0006091: functional complementation evidence used in manual assertion		P		In this paper, the researchers used PCR targeting, gene replacement cassette, and transposon mutagenesis to determine whether genes cyc1 and cyc2 were both required for production of geosmin, a substance that gives soil its distinctive scent. GC-MS was used to detect geosmin production by each mutant. In Table 3, it is shown that when cyc2 is deleted, (strains J3001, J3002) there is no production of geosmin. When cyc1 is mutated using a transposon, geosmin is still produced. This indicates that cyc2, not cyc1, is required for the production of geosmin. To confirm this, the researchers reintroduced the cyc2 gene (complementation) via a vector into the strain, and geosmin production was restored.	complete CACAO 13559

References

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