| One strategy to detoxify soil and water contaminated with stable toxic compounds composed of aromatic rings is bioremediation. Microorganisms such as Burkholderia cepacia, Burkholderia sp. strain DNT, Xanthomonas maltophilia and Pseudomonas aeruginosa have the ability to degrade aromatic ring structures. B. sp. strain DNT can degrade the toxic compound 2,4-dinitrotoluene (DNT) and the soil bacterium Pseudomonas aeruginosa can degrade benzoic acid. In many cases, however, these toxic compounds exist in places with low oxygen levels and limited nutrients, both limitations to their bioremediation. These microorganisms all are obligately aerobic, so that their metabolism is decreased under oxygen limited conditions. The hemoglobin (VHb) from the bacterium Vitreoscilla has been shown to enhance growth, productivity as well as bioremediation when transformed into heterologous bacteria especially under oxygen limited conditions. In this study wild type and site-directed mutant VHb's were cloned into B. sp. strain DNT, and P. aeruginosa using the transposon vector pUTminiTn5 for chromosomal DNA integration to produce strains BsYS, BsYS5, BsYS6, PaYS5, PaYS6 and PaYS8. The VHb's expressed by these mutant vgb's had Kd's approximately ½ and twice the Kd of the wild type in order to test the effect of varying the oxygen affinity of VHb on its effectiveness in bioremediation.; Recombinant strain BsYS showed two-fold improved growth in cell mass compared with wild type strain BsWT under hypoxic conditions and degraded 28% more DNT than BsWT under these conditions. Both VHb mutant strains BsYS5 and BsYS6 outgrew BcJC under hypoxic conditions and increased degradation of DNT relative to BcJC and BsWT.; Similarly, recombinant P. aeruginosa strains PaJC, PaYS5, PaYS6 and PaYS8 showed 23% increased growth after 120 hours compared to PaWT under hypoxic conditions. The mutant VHb strains showed 12% increased growth compared with wild type VHb strain PaJC under hypoxic conditions. In the case of benzoic acid degradation, the mutant VHb strains improved benzoic acid degradation by 18% compared with PaJC and by 26% compared with PaWT. The finding that mutant VHb's with oxygen affinities both higher and lower than that of the wild type may indicate that VHb has multiple cellular effects, e.g., oxygen delivery to the cytochrome bo terminal oxidase increased in some cases and oxygen delivery to the aromatic oxygenases increased in others.; Since only shuttle vectors or broad host range vectors have been used for cloning in species like P. aeruginosa, survival in P. aeruginosa of pUC-based plasmid pUC8:16 (containing vgb and ColE1 ori) was investigated using direct transformation into P. aeruginosa. Survival and replication of pUC8:16 in P. aeruginosa was confirmed by PCR, Southern hybridization, and CO difference spectra. |
