Polyphosphate kinase (PPK) catalyzes the formation of polyphosphate (polyP). The PPK-encoding gene (ppk) has been cloned from Klebsiella aerogenes ATCC9621. The gene possessed an open reading frame of 2055 bp capable of encoding a putative polypeptide with a deduced M(r) of 80,157. This polypeptide showed 93% similarity to the Escherichia coli PPK. The nucleotide sequence of the promoter region of K. aerogenes ppk differed from that of the previously sequenced E. coli ppk. A putative pho box sequence was found in the promoter region of K. aerogenes ppk. The expression of lacZ from the ppk promoter was increased in E. coli MV1184 under conditions of phosphate (Pi) limitation, but not in E. coli ANCS3 (phoB-), indicating that the ppk promoter is regulated by the phoB product. Increased levels of specific PPK activity were shown by expressing the cloned ppk at high levels, resulting in increased accumulation of polyP in E. coli.