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Biosynthesis of sulfur-containing amino acids in Streptomyces venezuelae ISP5230: roles for cystathionine ß-synthase and transsulfuration^b

Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1¹

Author for correspondence: L. C. Vining. Tel: +1 902 494 2040. Fax: +1 902 494 3736. e-mail: Leo.Vining{at}Dal.Ca

A 0·5 kb fragment of Streptomyces venezuelae ISP5230 genomic DNA was amplified by PCR using primers based on consensus sequences of cysteine synthase isozyme A from bacteria. The deduced amino acid sequence of the PCR product resembled not only cysteine synthase sequences from prokaryotes and eukaryotes but also eukaryotic cystathionine ß-synthase sequences. Probing an Str. venezuelae genomic library with the PCR product located a hybridizing colony from which pJV207 was isolated. Sequencing and analysis of the Str. venezuelae DNA insert in pJV207 detected two ORFs. The deduced amino acid sequence of ORF1 matched both cysteine synthase and cystathionine ß-synthase sequences in GenBank, but its size favoured assignment as a cystathionine ß-synthase. ORF2 in the pJV207 insert was unrelated in function to ORF1; in its sequence the deduced product resembled acetyl-CoA transferases, but disruption of the ORF did not cause a detectable phenotypic change. Disruption of ORF1 failed to elicit cysteine auxotrophy in wild-type Str. venezuelae, but in the cys-28 auxotroph VS263 it prevented restoration of prototrophy with homocysteine or methionine supplements. The change in phenotype implicated loss of the transsulfuration activity that in the wild-type converts these supplements to cysteine. This study concludes that disruption of ORF1 inactivates a cbs gene, the product of which participates in cysteine synthesis by transsulfuration. Enzyme assays of Str. venezuelae mycelial extracts confirmed the formation of cysteine by thiolation of O-acetylserine, providing the first unambiguous detection of this activity in a streptomycete. Enzyme assays also detected cystathionine -synthase, cystathionine ß-lyase and cystathionine -lyase activity in the extracts and showed that the substrate for cystathionine -synthase was O-succinyl-homoserine. Based on assay results, the cys-28 mutation in Str. venezuelae VS263 does not inactivate the cysteine synthase gene but impairs expression in cultures grown in minimal medium.

Keywords: cysteine auxotroph, sulfur metabolism, gene disruption, streptomycetes

Abbreviations: CBL, cystathionine ß-lyase; CBS, cystathionine ß-synthase; CGL, cystathionine -lyase; CGS, cystathionine -synthase; CS, cysteine synthase; OAH, O-acetylhomoserine; OSH, O-succinylhomoserine

^b The GenBank accession number for the sequence reported in this paper is AF319543.

^a Present address: Department of Microbiology, University of Minnesota, 1030 Mayo Building, 420 Delaware Street SE, Minneapolis, MN 55455, USA.

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