• Microbiology and Biotechnology Letters
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• v.18 no.5
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• pp.454-459
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• 1990

For the genetic multipurpose of antagonistic abilities of Pseudomom etutzeri YPL-1 aganist Fusarium solani causing root rot of many important crops by genetic engineering, optimal conditions for transformation of P-stutzeri YPL-1 by pKT230 were investigated. Maxium frequency of the transformation was achieved when cells were harvested at early exponential growth phase. The highest transformation efficiency was obtained when the competent cells were exposed to chilled transformation buffer containing 20 mM RbCI, 100 mM $CaCl_2$ and added l${\mu}g$/ml of plasmid DNA. The pH optimum for transformation was 6.5. When the bacterial cells that were incubated during 60 minutes for the competence were brought in contact with plasmid DNA, the transformations were obtained in the best frequency. It was formed that transformation frequency was 2 ~$6 \times 10^{-6}$ under the optimal conditions.

• Proceedings of the Korean Society of Soil and Groundwater Environment Conference
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• 2000.11a
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• pp.207-208
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• 2000

Since trichloroethylene (TCE), dichloroethylene (DCE), and vinyl chloride (VC) arise from anaerobic degradation of tetrachloroethylene (PCE) and TCE, there is interest in creating aerobic remediation systems that avoid the highly toxic VC and cis-DCE which predonominate in anaerobic degradation. However, it seemed TCE could not be degraded aerobically without an inducing compound (which also competitively inhibits TCE degradation). It has been shown that TCE induces expression of both the toluene dioxygenase of p. putida F1 as well as toluene-p-monooxygenase of P.mendocina KRI. We investigated here the ability of PCE, TCE, and chlorinated phenols to induce toluene-o-xylene monooxygenase (ToMO) from P.stutzeri OX1. ToMO has a relaxed regio-specificity since it hydroxylates toluene in the ortho, meta, and para positions; it also has a broad substrate range as it oxidizes o-xylene, m-xylene, p-xylene, toluene, benzene, ethylbenzene, styrene, and naphthalene; chlorinated compounds including TCE, 1, 1-DCE, cis-DCE, trans-DCE, VC, and chloroform : as well as mixtures of chlorinated aliphatics (Pseudomonas 1999 Maui Meeting). ToMO is a multicomponent enzyme with greatest similarity to the aromatic monooxygenases of Burkholderia pickettii PKO1 and P.mendocina KR1. Using P.sturzeri OX1, it was found that PCE induces P.mendocina KR1 Using P.situtzeri OX1, it was found that PCE induces ToMO activity measured as naphthalene oxygenase activity 2.5-fold, TCE induces 2.3-fold, and toluene induces 3.0 fold. With the mutant P.stutzeri M1 which does not express ToMO, it was also found there was no naphthalene oxygenate activity induced by PCE and TCE; hence, PCE and TCE induce the tow path. Using P.putida PaW340(pPP4062, pFP3028) which has the tow promoter fused to the reporter catechol-2, 3-dioxygenase and the regulator gene touR, it was determined that the tow promoter was induced 5.7-, 7.1-, and 5.2-fold for 2-, 3-, 4-chlorophenol, respectively (cf. 8.9-fold induction with o-cresol) : however, TCE and PCE did not directly induce the tou path. Gas chromatography and chloride ion analysis also showed that TCE induced ToMO expression in P.stutzeri OX1 and was degraded and mineralized. This is the first report of significant PCE induction of any enzyme as well as the first report of chlorinated compound induction of the tou operon. The results indicate TCE and chlorinated phenols can be degraded by P.stutzeri OX1 without a separate inducer of the tou pathway and without competitive inhibition.