Indole-3-Glycerol-Phosphate Synthase Is Recognized by a Cold-Inducible Group II Chaperonin in Thermococcus kodakarensis

Abstract

ABSTRACT Thermococcus kodakarensis optimally grows at 85°C and possesses two chaperonins, cold-inducible CpkA and heat-inducible CpkB. Gene disruptants DA1 (Δ cpkA ) and DB1 (Δ cpkB ) showed decreased cell growth at 60°C and 93°C, respectively. The DB2 mutant (Δ cpkA ∷ cpkB Δ cpkB ), whose cpkB gene was expressed under the control of the cpkA promoter, did not grow at 60°C, and the DB3 mutant [Δ cpkA (1–524)∷ cpkB (1–524) Δ cpkB ], whose CpkA amino acid residues 1 to 524 were replaced with corresponding CpkB residues that maintained the C-terminal region intact, grew at 60°C, implying that the CpkA C-terminal region plays a key role in cell growth at 60°C. To screen for specific CpkA target proteins, comparative pulldown studies with anti-Cpk were performed using cytoplasmic fractions from DA1 cells cultivated at 93°C and DB1 cells cultivated at 60°C. Among the proteins coprecipitated with anti-Cpk, TK0252, encoding indole-3-glycerol-phosphate synthase (TrpC), showed the highest Mascot score. Counter-pulldown experiments were also performed on DA1 and DB1 extracts using anti-TrpC. CpkA coimmunoprecipitated with anti-TrpC while CpkB did not. The results obtained indicate that TrpC is a specific target for CpkA. The effects of Cpks on denatured TrpC were then examined. The refolding of partially denatured TrpC was accelerated by the addition of CpkA but not by adding CpkB. DA1 cells grew optimally in minimal medium only in the presence of tryptophan but hardly grew in the absence of tryptophan at 60°C. It has been suggested that a lesion of functional TrpC is caused by cpkA disruption, resulting in tryptophan auxotrophy.