Detection, isolation, and stability of megaplasmid-encoded chloroaromatic herbicide-degrading genes within...

Dicamba is used as a model system for microbial degradation of chloroaromatic benzoic acids. The detection, isolation, and stability of a megaplasmid within a Pseudomonas sp. is described as the first step in optimizing the growth of this microorganism and other microorganisms similar to it. A large plasmid, pDK1, consisting of approximately 250 kb, was purified from dicamba-degrading Pseudomonas sp. PXM. This plasmid was purified by the method of Allen (personal communication, 1994), which is a modified version of several that have been attempted for the isolation of large plasmids (Lee and Rasheed, 1990). The restriction analysis of this plasmid (pDK1) from PXM. revealed many distinctive bands on agarose gel electrophoresis. Based on the preliminary restriction enzyme analysis, the estimated size of this plasmid is 250 kb, which could make it one of the largest procaryotic plasmids encoding for chloroaromatic degrading enzymes. Allen's methodology results in very high purity and reproducibility compared to the other methods used in this study. As described in this work, the method of Kado and Liu (1981) is easier to perform and results in a more reproducible plasmid preparation than the method of Casse et al. (1979). Casse's protocol requires the use of a highly alkaline SDS solution (pH 12.45) in order to eliminate the chromosomal DNA. However, only incomplete removal of the chromosomal DNA results. Compared to the Casse et al. protocol, the Kado and Liu protocol requires the use of a highly alkaline solution (pH 12.6) and a high temperature (55-65 degrees C) to eliminate the chromosomal DNA. This results in a nearly complete removal of the chromosomal DNA. The high temperature treatment also quickly eliminates the RNA. Another advantage of the protocol of Kado and Liu over the protocol of Casse et al. is that the former uses phenol-chloroform extraction while the latter uses only phenol extraction. The phenol-chloroform extraction step denatures the DNA along with the proteins. In addition to this, the phenol-chloroform mixture minimizes the formation of a brown oxidation pigment that usually occurs with phenol extraction alone. Finally, the time needed to complete the Kado and Liu protocol is much shorter (2 hr) than the time needed to complete the Casse protocol (8 hr). As described previously, a highly purified plasmid preparation with minimal chromosomal DNA was prepared by following the suggestions of L. Allen.(ABSTRACT TRUNCATED AT 400 WORDS)