Cloning and characterization of polyphosphate kinase and exopolyphosphatase genes from Pseudomonas aeruginosa 8830

Pseudomonas aeruginosa accumulates polyphosphates in response to nutrient limitations. To elucidate the function of polyphosphate in this microorganism, we have investigated polyphosphate metabolism by isolating from P. aeruginosa 8830 the genes encoding polyphosphate kinase (PPK) and exopolyphosphatase (PPX), which are involved in polyphosphate synthesis and degradation, respectively. The 690- and 506-amino-acid polypeptides encoded by the two genes have been expressed in Escherichia coli and purified, and their activities have been tested in vitro. Gene replacement was used to construct a PPK-negative strain of P. aeruginosa 8830, Low residual PPK activity in the ppk mutant suggests a possible alternative pathway of polyphosphate synthesis in this microorganism, Primer extension analysis indicated that ppk is transcribed from a sigma(E)-dependent promoter, which could be responsive to environmental stresses. However, no coregulation between ppk and ppx promoters has been demonstrated in response to osmotic shock or oxidative stress.