We describe a rapid procedure for extracting purified chromosomal DNA from Pseudomonas putida, and some minor modifications needed for use in other organisms, including Gram‐positive strains. The technique is rapid and generally produces between 1 and 5 μg of DNA from 1 ml of liquid culture. The DNA is highly purified and can be readily cut with small quantities of restriction endo‐nucleases, cloned into plasmid vectors and used as a substrate for hybridization with labelled DNA probes. Genetical analysis and cloning of genomic DNA from environmentally important organisms like Ps. putida has become increasingly important. Bacterial chromosomal DNA is usually prepared from large volumes of liquid culture, and involves time‐consuming steps necessary to purify the DNA sufficiently for use in cloning experiments (e. g. Marmur 1961). We have developed a method for preparing small quantities of genomic DNA, which involves whole cell lysis and purification by precipitation and centrifugation. Loss of DNA occurs by shearing, but the yield of DNA is sufficient, and the quality is high. The method has been used to prepare DNA from a variety of organisms and is particularly applicable where the preparation and analysis of DNA from a large number of isolates is required.
Favourable mutations involving the two dehalogenases (DehI and DehII) of Pseudomonas putida PP3 and derivative strains containing the cloned gene for DehI (dehI) occurred in response to specific environmental conditions, namely: starvation conditions; the presence of dehalogenase substrates (halogenated alkanoic acids--HAAs) which were toxic to P. putida; and/or the presence of a potential growth substrate. Fluctuation tests showed that these mutations were environmentally directed by the presence of HAAs. The mutations were associated with complex DNA rearrangements involving the movement of dehI located on a transposon DEH. Some mutations resulted in switching off the expression of either one or both of the dehalogenases, events which were effective in protecting P. putida from toxic compounds in its growth environment. Other mutations partially restored P. putida's dehalogenating capability under conditions where toxic substrates were absent. Restoration of the capability to untilize HAAs was favoured when normal growth substrates were present in the environment.
A rapid and inexpensive method for the measurement of copy number of small plasmids, ranging from 8.7 to 13 kb, in under 1 ml of liquid culture is described. The method involves whole cell lysis, electrophoretic separation of plasmid and chromosomal DNA followed by relative densitometric measurement of each, to give an estimation of the plasmid copies per chromosome. Results can be obtained in under 8 h and the method proved to be reproducible, fast and ideal for processing large numbers of samples from batch or continuous culture.
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