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Stringent Response in Streptomyces coelicolor

Stringent Response in Streptomyces coelicolor. Differentiation: formation of aerial mycelia, followed by sporulation Antibiotics also produced near time of differentiation. Streptomyces rel mutants. S. equisilimus RelA - properties of both E. coli ’s SpoT and RelA

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Stringent Response in Streptomyces coelicolor

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  1. Stringent Response in Streptomyces coelicolor • Differentiation: formation of aerial mycelia, followed by sporulation • Antibiotics also produced near time of differentiation

  2. Streptomycesrel mutants • S. equisilimus RelA - properties of both E. coli’s SpoT and RelA • S. coelicolor homolog (formerly ORF1) • discovered when deletion prevented antibiotic production • strong homology to E. coli proteins

  3. Possible Homology Situations Concentrated at N-terminus Concentrated at C-terminus Consistent throughout

  4. E. coli RelA and E. coli SpoT E. coli RelA and S. coelicolor Rel Identities = 278/734 (37%), Positives = 425/734 (57%), Gaps = 53/734 (7%) E. coli SpoT and S. coelicolor Rel Identities = 295/741 (39%), Positives = 429/741 (57%), Gaps = 49/741 (6%) Identities = 216/680 (31%), Positives = 354/680 (51%), Gaps = 39/680 (5%)

  5. Correlation between structure and function? • Not apparent from sequence homologies • Expressed in E. coli, has SpoT properties • In S. coelicolor mutants, has both RelA and SpoT properties (similar to S. equisilimus relA) • Deletion mutants may elucidate a structure/function connection, if one exists

  6. Deletion mutants spoT rel relA rel Full length Δ(1-92) Δ(1-229) Δ(ATP/GTP) Δ(490-847) Δ(1-358,490-847) Δ(1-229,423-847) Δ(1-266,454-847)

  7. Chromatography basics F1blue – F2blue = ΔFblue F1red – F2red = ΔFred ΔFblue ΔFred F1 : Impelling force hydrodynamic Polar and steric interactions between the solvent and solute determine the degree of separation. F2: Repelling forces Surface energy, Van der Waals forces, steric specificity

  8. ppGpp production Full length Δ(1-92) Δ(1-229) Δ(ATP/GTP) Δ(490-847) Δ(1-358,490-847) Δ(1-229,423-847) Poor expression Δ(1-266,454-847) Positive result indicates ppGpp was detected.

  9. ppGpp accumulation Full length Δ(1-92) Δ(1-229) Δ(ATP/GTP) Δ(490-847) Δ(1-358,490-847) n/a Δ(1-229,423-847) n/a Poor expression Δ(1-266,454-847) Positive result indicates ppGpp content increased after addition of serine hydroxamate, indicating dependence on ribosomes.

  10. possible ribosome binding modulatory region ATP/GTP binding motif catalytic domains NH2 COOH 1 93 267 397 453 847 possible ribosome binding modulatory region phosphohydrolase synthetase

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