Introduction of a plasmid-encoded phoA gene for constitutive overproduction of alkaline phosphatase in three subsurface Pseudomonas isolates

Abstract

Three bacterial isolates, Pseudomonas fluorescens F1, Pseudomonas rhodesiae R1 and Pseudomonas veronii V1 were genetically modified by introduction of a plasmid, pJH123, with a phoA hybrid gene that directed constitutive overproduction of the enzyme alkaline phosphatase. The presence of the plasmid in the bacterial hosts elevated extracytoplasmic alkaline phosphatase production from 100- to 820-fold. The growth and survival of the plasmid-bearing hosts in sterilized soil slurries was comparable to parental control strains. In the absence of antibiotic selection, pJH123 was maintained in two of the three hosts ( P. fluorescens F1 and P. veronii V1) during incubation in minimal medium. The effects of the genetically enhanced pseudomonads on the liberation of inorganic phosphate (PO 4 3−) were determined in sterilized soil slurries following the addition of an organophosphorus compound, glycerol-3-phosphate. A significant accumulation of PO 4 3− was measured in soil slurries amended with 10 mM glycerol-3-phosphate and any of the three phosphatase-enhanced pseudomonad isolates. In contrast, soil slurries containing unmodified parental strains did not exhibit significant PO 4 3− accumulation. Two of the three enhanced phosphate-liberating strains released sufficient PO 4 3− that cell-free supernatants from sterilized soil slurry incubations removed significant amounts of uranium (as much as 69%) from solution.