The polyphosphate operon of Escherichia coli was overexpressed on a high copy plasmid under control of its native promoter. This operon contains the genes coding far polyphosphate kinase (PPK) and polyphosphatase (PPX), the enzymes responsible for polyphosphate synthesis and degradation, respectively The plasmid was engineered for improved stability by addition of the parB locus. The polyphosphate content of cells containing this plasmid was significantly higher than that of wildtype E. coli. Cells were subjected to phosphate limitation following a period of exponential growth, and then shifted back to high-phosphate conditions. Polyphosphate reserves were utilized during phosphate starvation and partially replenished after the shift PPK activity increased during phosphate starvation and dropped after the shift while PPX activity was highest when phosphate was in surplus. A ppk::lacZ reporter gene construct was used to investigate transcription from the ppk promoter. Although expression doubled upon phosphate starvation, it is unlikely that the Pho regulon is involved since both phoB and phoU mutant host strains gave similar responses.