Biosynthesis of sulfur-containing amino acids in Streptomyces venezuelae ISP5230: roles for cystathionine β-synthase and transsulfuration b bThe GenBank accession number for the sequence reported in this paper is AF319543.

Abstract

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 β-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 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 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 Oacetylserine, 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 O-succinylhomoserine. 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.

Conversion of Methionine to Cysteine in Bacillus subtilis and Its Regulation

“…In contrast, cystathionine ␤-synthase activity was only observed in vitro with YrhA. Cystathionine ␤-synthases are usually 150 amino acids longer in their C-terminal region than OAS thiollyases (14,15,41). YrhA is more closely related to OAS thiollyases, as indicated by its size and its similarities.…”

“…Despite the fact that yrhA is constitutively expressed at a high level in the cysK mutant ( Table 2) (1), CysK still represents 95% of the OAS thiol-lyase activity. In contrast, the ⌬yrhA mutant grows poorly in the presence of methionine, as observed for an yrhA-like mutant of Lactococcus lactis and a cbs (cystathionine ␤-synthase probable gene) mutant of Streptomyces venezuelae (14,52). The drastic growth defect of the B. subtilis ⌬yrhA mutant in the presence of methionine is most probably due to the cystathionine ␤-synthase activity of YrhA rather than to its low OAS thiol-lyase activity in the presence of the major OAS thiol-lyase, CysK.…”

Cytochrome bd Oxidase, Oxidative Stress, and Dioxygen Tolerance of the Strictly Anaerobic Bacterium Moorella thermoacetica

“…Expression of cysK has been reported to be a general response to stress in bacteria and plants (5,57,71,73). In bacteria, cysteine is synthesized from O-acetylserine and H 2 S catalyzed by O-acetylserine sulfhydrolase (CysK) while O-acetylserine is produced from serine and acetyl-CoA catalyzed by serine acetyltransferase (CysE) (12,29). In the M. thermoacetica genome, a CysE homolog is encoded by gene 521 on contig 282.…”

Generation and study of the strains of streptomycetes-heterologous hosts for the production of moenomycin

“…To produce spores the streptomycetes were grown on oatmeal agar [14] or MYM [15] from 2 to 8 days (depending on the strain) at 28 °C, ( S. ghanaensis и S. thermospinosisporus – при 37 °C). To examine NoA 2 biosynthesis, moeno38-6 + Streptomyces strains were grown in liquid R5A [4] or TSB (BD, USA) and on solid agar soybean medium (g/l: soy flour – 20, NaCl – 5, agar - 16).…”