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

Pseudomonas aeruginosa accumulates polyphosphates in response to nutrient l imitations. To elucidate the function of polyphosphate in this microorganis m, we have investigated polyphosphate metabolism by isolating from P. aerug inosa 8830 the genes encoding polyphosphate kinase (PPK) and exopolyphospha tase (PPX), which are involved in polyphosphate synthesis and degradation, respectively. The 690- and 506-amino-acid polypeptides encoded by the two g enes have been expressed in Escherichia coli and purified, and their activi ties have been tested in vitro. Gene replacement was used to construct a PP K-negative strain of P. aeruginosa 8830, Low residual PPK activity in the p pk mutant suggests a possible alternative pathway of polyphosphate synthesi s in this microorganism, Primer extension analysis indicated that ppk is tr anscribed from a sigma(E)-dependent promoter, which could be responsive to environmental stresses. However, no coregulation between ppk and ppx promot ers has been demonstrated in response to osmotic shock or oxidative stress.