Manipulation of independent synthesis and degradation of polyphosphate in Escherichia coli for investigation of phosphate secretion from the cell.
The genes involved in polyphosphate metabolism in Escherichia coli were cloned behind different inducible promoters on separate plasmids. The gene coding for polyphosphate kinase (PPK), the enzyme responsible for polyphosphate synthesis, was placed behind the Ptac promoter. Polyphosphatase, a polyphosphate depolymerase, was similarly expressed by using the arabinose-inducible PBAD promoter. The ability of cells containing these constructs to produce active enzymes only when induced was confirmed by polyphosphate extraction, enzyme assays, and RNA analysis. The inducer concentrations giving optimal expression of each enzyme were determined. Experiments were performed in which ppk was induced early in growth, overproducing PPK and allowing large amounts of polyphosphate to accumulate (80 mumol in phosphate monomer units per g of dry cell weight). The ppx gene was subsequently induced, and polyphosphate was degraded to inorganic phosphate. Approximately half of this polyphosphate was depleted in 210 min. The phosphate released from polyphosphate allowed the growth of phosphate-starved cells and was secreted into the medium, leading to a down-regulation of the phosphate-starvation response. In addition, the steady-state polyphosphate level was precisely controlled by manipulating the degree of ppx induction. The polyphosphate content varied from 98 to 12 mumol in phosphate monomer units per g of dry cell weight as the arabinose concentration was increased from 0 to 0.02% by weight.
Van Dien
S J
SJ
Department of Chemical Engineering, University of California, Berkeley 94720-1462, USA.
Keyhani
S
S
Yang
C
C
Keasling
J D
JD
D017766:Acid Anhydride Hydrolases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D000469:Alkaline Phosphatase / Q000096:biosynthesis; D001089:Arabinose / Q000378:metabolism; D003001:Cloning, Molecular; D015536:Down-Regulation; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D010710:Phosphates / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011122:Polyphosphates / Q000032:analysis / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis
D010710:Phosphates; D011122:Polyphosphates; D012329:RNA, Bacterial; D012333:RNA, Messenger; D001089:Arabinose; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase; D000469:Alkaline Phosphatase; D017766:Acid Anhydride Hydrolases; C022563:endopolyphosphatase