Polyphosphate metabolism in the archaeon Sulfolobus acidocaldarius

摘要

In response to nutrient limitation and during stationary phase, bacteria dynamically accumulate reserve polymers as glycogen and polyphosphates (polyP). PolyP is synthesized by polyphosphate kinase (PPK), having a role in the survival during these stressful conditions. At present no quantitative study of polyP has been done nor its relationship with environmental changes has been established in the domain Archaea. Our purpose is to study, in the thermoacidophilic archaeon Sulfolobus acidocaldarius, the components of the polyP metabolism and their connection with survival under nutrient limitation. PPK activity was assayed in membrane fractions of cells grown in normal and phosphate starved conditions. Taking advantage of the reported purification in S. acidocaldarius of a glycogen-bound protein of 60 kDa with PPK as well as glycosyl-transferase activities (R. Skorko, J. Osipiuk and K. Stetter. J. Bacteriol. 171:5162-5164,1989), we purified the glycogen-protein complex, identified the 60 kDa protein (P60) and cloned its gene by using reverse genetics. Phosphorylation of P60 was studied in vivo in normal and phosphate-starved conditions. Recombinant P60 (rP60) was overexpressed in E. coli and after purification it was assayed for both PPK and glycosyl-transferase activities. Although PPK activity was higher during the exponential phase of growth of S. acidocaldarius, PolyP accumulated mainly during the stationary phase. PPK activity was higher in cultures starved for phosphate. Under these conditions P60 was phosphorylated in vivo. Unexpectedly, the deduced amino acid sequence of the p60 gene showed a high percentage of identity with bacterial and archaeal glycogen synthases and no similarity with the 18 PPK bacterial sequences reported so far. In agreement with these results, the recombinant protein did not show any PPK activity but only a glycosyl-transferase activity, according to with its homology to glycogen synthases. Our results strongly suggest that P60 is a glycogen synthase which is not responsible for the PPK activity present in crude extracts ofS. acidocaldarius. Further research will be required to elucidate the metabolic pathway of synthesis of polyP in Archaea specially in view of the absence of a PPK gene in the finished sequences of Archaeal genomes.