A New Method for Bacterial Strain Construction in Biodegradation of Xenobiotic Compounds
Separate continuous cultures of Pseudomonas putida R5-3 (benzene+, toluene+, xylene+, benzoate+), grown on toluene, and Pseudomonas alcaligenes C-O (benzoate+, 3-chlorobenzoate+), 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 of P. putida CB1-9, able to grow on chlorobenzene, 1, 4-dichlorobenzene (1,4-DCB), and 3-chlorobenzoate, was isolated in less than one month. Chloride was released in stoichiometric amounts when P. putida CB1-9 was grown on either chlorobenzene or 1,4-DCB. Chlorobenzeneutilizing strains were not found in continuous cultures run at the lowest dilution rate (0.05 hr−1) or in the absence of the donor strain, P. alcaligenes C-0. The recombinant strain was related to P. putida R5-3, phenotypically and genetically. The recombinant, however, lost its ability to grow on p- and m-xylenes and the corresponding methylbenzoates. P. putida R5-3 carries a nonconjugative XYL-like plasmid with induction patterns similar to the TOL plasmid pWWO. P. putida CB1-9 carries a 33-kb plasmid which is derived from the 57-kb plasmid of P. putida R5-3, probably by deletion of genes coding for key enzymes in xylene and toluate catabolism.