1887

Abstract

A 0·5 kb fragment of 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 β-synthase sequences. Probing an genomic library with the PCR product located a hybridizing colony from which pJV207 was isolated. Sequencing and analysis of the DNA insert in pJV207 detected two ORFs. The deduced amino acid sequence of ORF1 matched both cysteine synthase and cystathionine β-synthase sequences in GenBank, but its size favoured assignment as a cystathionine β-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 , but in the 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 gene, the product of which participates in cysteine synthesis by transsulfuration. Enzyme assays of mycelial extracts confirmed the formation of cysteine by thiolation of acetylserine, providing the first unambiguous detection of this activity in a streptomycete. Enzyme assays also detected cystathionine γ-synthase, cystathionine β-lyase and cystathionine γ-lyase activity in the extracts and showed that the substrate for cystathionine γ-synthase was succinyl-homoserine. Based on assay results, the mutation in VS263 does not inactivate the cysteine synthase gene but impairs expression in cultures grown in minimal medium.

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2002-07-01
2019-10-23
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