PRODUCTION AND RELEASE OF POLYPHOSPHATE BY A GENETICALLY-ENGINEERED STRAIN OF ESCHERICHIA-COLI

A recombinant strain of Escherichia coli MV1184, which contains plasmi d-borne genes encoding the phosphate-specific transport (Pst) system a nd polyphosphate (polyP) kinase, accumulated high levels of P-i, and r eleased polyP into the medium. PolyP could be separated from the cultu re supernatant by DEAE-Toyopearl 650M chromatography and identified by high-resolution P-31 nuclear magnetic resonance spectroscopy. Once E. coli recombinants accumulated high levels of polyP, they released pol yP concomitantly with P-i uptake. PolyP release did not accompany the decrease in the cell density, indicating that it is not simply a resul t of cell lysis. PolyP release ceased when P-i became depleted in the medium and resumed upon addition of P-i to the medium. When P-i uptake was inhibited by 0.1 mM carbonyl cyanide m-chlorophenylhydrazone (CCC P), no polyP release was observed. Furthermore, neither P-i uptake nor polyp release occurred when cells were incubated at 4 degrees C. Thes e findings suggest that the occurrence of polyP release is a possible mechanism that limits a further increase in the cellular polyP concent ration in E. coli recombinants. High-resolution P-31 nuclear magnetic resonance spectroscopy also detected a surface pool of polyp in intact cells of the E. coli recombinant. The polyp resonance increased when cells were treated with EDTA and broadened upon the addition of a shif t reagent, praseodymium. Although the mechanism of surface polyP accum ulation is unclear, surface polyP seems to serve as the source for pol yP release.