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.

Aerobic uranium (VI) bioprecipitation by metal‐resistant bacteria isolated from radionuclide‐ and metal‐contaminated subsurface soils

“…The activity of a NSAP constitutively expressed by strain N14 was directly implicated in the precipitation of greater than 90% of U(VI) from solution as a uranyl phosphate precipitate (Macaskie et al ., 1994). A number of studies have subsequently demonstrated comparable U‐phosphate precipitation with diverse genera, including Deinococcus , Escherichia and Pseudomonas (Basnakova et al ., 1998; Powers et al ., 2002; Appukuttan et al ., 2006). However, such activity only occurred following the introduction of recombinant acid [ phoN (Basnakova et al ., 1998; Appukuttan et al ., 2006)] or alkaline [ phoA (Powers et al ., 2002)] phosphatase genes.…”

Biomineralization of Uranium by PhoY Phosphatase Activity Aids Cell Survival in Caulobacter crescentus

“…BSAR-1, Arthrobacter, Rahnella, and Bacillus has been found to facilitate U(VI) precipitation through the formation of uranium phosphate complexes (17)(18)(19). Phosphatases from these organisms have been cloned and expressed in Escherichia coli and other organisms, and the resulting engineered strains have been reported to efficiently precipitate uranium (20)(21)(22). Among all these systems, however, little attention has been paid to the cellular benefits of the native or heterologously expressed phosphatases toward U(VI) resistance.…”

Biomineralization of U(VI) phosphate promoted by microbially-mediated phytate hydrolysis in contaminated soils

“…Previous studies have proposed that microbial phosphatases are overexpressed in contaminated environments as detoxification mechanism resulting from exposure to heavy metals (Macaskie et al, 1994;Montgomery et al, 1995;Powers et al, 2002;Sobecky et al, 1996;Yong and Macaskie, 1995). The disparity in the lag phase of inorganic phosphate production between pH 5.5 phytate-amended reactors with and without glycerol was not only eliminated, but unexpectedly reversed in the presence of 200 µM U(VI) ( Figure 2).…”