The effect of polyphosphate kinase gene deletion on polyhydroxyalkanoate accumulation and carbon metabolism in Pseudomonas putida KT2440

Abstract

The primary enzyme involved in polyphosphate (polyP) synthesis, polyP kinase (ppk), has been deleted in Pseudomonas putidaKT2440. This has resulted in a threefold to sixfold reduction in polyhydroxyalkanoate (PHA) accumulation compared with the wild type under conditions of nitrogen limitation, with either temperature or oxidative (H2O2) stress, when grown on glucose. The accumulation of PHA by ppk mutant was the same as the wild type under nitrogen-limiting growth conditions. There was no difference in polyP levels between wild-type and ppk strains under all growth conditions tested. In the ppk mutant proteome, polyP kinase (PPK) was undetectable, but up-regulation of the polyp-associated proteins polyP adenosine triphosphate (ATP)/nicotinamide adenine dinucleotide (NAD) kinase (PpnK), a putative polyP adenosine monophosphate (AMP) phosphotransferase (PP_1752), and exopolyphosphatase was observed. ppk strain exhibited significantly retarded growth with glycerol as carbon and energy source (42h of lag period compared with 24h in wild-type strain) but similar growth to the wild-type strain with glucose. Analysis of gene transcription revealed downregulation of glycerol kinase and the glycerol facilitator respectively. Glycerol kinase protein expression was also downregulated in the ppk mutant. The deletion of ppk did not affect motility but reduced biofilm formation. Thus, the knockout of the ppk gene has resulted in a number of phenotypic changes to the mutant without affecting polyP accumulation.