Chromosomal integration of the Vitreoscilla hemoglobin gene in Burkholderia and Pseudomonas for the purpose of producing stable engineered strains with enhanced bioremediating ability

Using the pUT-miniTn5 vector system developed by the laboratory of K.N. Tim mis, the Vitreoscilia hemoglobin gene (vgb) was integrated into the chromos omes of Pseudomonas aeruginosa and Burkholderia cepacia; Vitreoscilla hemog lobin (VHb) was expressed at 8.8 and 0.8 nmol/g wet weight of cells in the respective engineered strains. The vgb-bearing P. aeruginosa outgrew wild-t ype P. aeruginosa and degraded benzoic acid faster than the latter strain a t both normal and low aeration. In contrast, the vgb-bearing B. cepacia str ain had a growth advantage over the wild type strain at ca. 90 ppm, but not at ca. 120 ppm 2,4-dinitrotoluene (DNT); no difference in DNT degradation was seen between the two strains at either normal or low aeration. The resu lts demonstrate the practicality of enhancing bioremediation with vgb stabl y integrated into the chromosome, but also suggest that a minimal level of VHb expression is required for its beneficial effects to be seen.