Genetically altered levels of inorganic polyphosphate in Escherichia coli

Abstract

The ppk gene encoding polyphosphate kinase (PPK), the enzyme in Escherichia coli that makes long chains of polyphosphate (polyP) reversibly from ATP, was disrupted by insertion of a kanamycin resistance gene. Expression of the exopolyphosphatase gene (ppx) immediately downstream of ppk in the operon was likewise disrupted. Cells were also transformed with a high-copy-number plasmid bearing ppk. Genetically altered polyP levels were estimated in cell extracts by the PPK conversion of ADP to ATP. PolyP levels (microgram/10(11) cells) near 2.0 were reduced in the ppk(-)-ppx- mutants to 0.16 and increased more than 100-fold (e.g. 220) in cells transformed with multiple copies of ppk. Mutant cells, lacking the long polyP chains, showed a growth lag following dilution of a stationary-phase culture. PolyP-deficient cells exhibit a striking phenotype in their failure to survive in stationary phase and loss of resistance to heat (55 degrees C) and to oxidants (42 mM H2O2). High polyP levels are also associated with reduced survival.