Construction of chlorobenzene-utilizing recombinants by progenitive manifestation of a rare event.
Separate continuous cultures of Pseudomonas putida R5-3, grown on toluene, and Pseudomonas alcaligenes C-O, grown on benzoate, were concentrated and continuously amalgamated on a ceramic bead column, which was subjected to a continuous stream of chlorobenzene vapors. A recombinant strain, P. putida CB1-9, was isolated in less than 1 month. P. alcaligenes C-0 grew on benzoate and 3-chlorobenzoate but not on toluene, P. putida R5-3 grew on benzoate and toluene but not on 3-chlorobenzoate, and neither strain grew on chlorobenzene or 1,4-dichlorobenzene; however, the recombinant P. putida CB1-9 grew on all of these substrates. Chlorobenzene-utilizing strains were not found in continuous cultures run at the lowest growth rate (0.05/h) or in the absence of the donor strain, P. alcaligenes C-0. Chloride was released in stoichiometric amounts when P. putida CB1-9 was grown on either chlorobenzene or 1,4-dichlorobenzene. The recombinant strain was related to P. putida R5-3, phenotypically and genetically. Restriction enzyme digests of the single 57-kilobase (kb) plasmid in R5-3 and of the single 33-kb plasmid in CB1-9 were similar, but also indicated rearrangement of plasmid DNA. Coincidental or causal to the loss of the 24-kb fragment was the observation that the recombinant--unlike its parent, R5-3--did not grow on xylenes or methylbenzoates. Although both ortho-pyrocatechase (OP) and meta-pyrocatechase (MP) were found in CB1-9 and R5-3, MP activity was 20- to 50-fold higher in R5-3 cells grown on 4-methylbenzoate than in the same cells grown on benzene.(ABSTRACT TRUNCATED AT 250 WORDS)
Kröckel
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Department of Soil and Environmental Sciences, University of California, Riverside 92521.
Focht
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D001565:Benzoates / Q000378:metabolism; D002722:Chlorobenzenes / Q000378:metabolism*; D002723:Chlorobenzoates / Q000378:metabolism; D003227:Conjugation, Genetic; D003470:Culture Media; D004274:DNA, Recombinant; D005838:Genotype; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D014050:Toluene / Q000378:metabolism
D001565:Benzoates; D002722:Chlorobenzenes; D002723:Chlorobenzoates; D003470:Culture Media; D004274:DNA, Recombinant; C036427:3-chlorobenzoic acid; D014050:Toluene