Variation in chlorobenzoate catabolism by Pseudomonas putida P111 as a consequence of genetic alterations

Abstract

Pseudomonas putida P111 is able to utilize a broad range of monochlorinated, dichlorinated, and trichlorinated benzoates. The involvement of two separate dioxygenases was noted from data on plasmid profiles and DNA hybridization. The benzoate dioxygenase, which converts 3-chlorobenzoate (3-CB), 4-CB, and benzoate to the corresponding catechols via reduction of a dihydrodiol, was shown to be chromosomally coded. The chlorobenzoate-1,2-dioxygenase that converts ortho-chlorobenzoates to the corresponding catechols without the need of a functional dioldehydrogenase was shown to be encoded on plasmid pPB111 (75 kb). Cured strains were unable to utilize ortho-chlorobenzoates for growth. DNA hybridization data indicated that catabolism of the corresponding chlorocatechols was coded on chromosomal genes. Maintenance of plasmid pPB111 was dependent on the presence of ortho-chlorobenzoates in the growth media. A unique variant of P111 (P111D), able to grow on 3,5-dichlorobenzoate (3,5-DCB), was obtained by continuous subculturing from media containing progressively lower and higher concentrations of 3-CB and 3,5-DCB, respectively. The low frequency of segregants able to grow on 2,5-DCB, 2,3-DCB, and 2,3, 5-trichlorobenzoate was evident by lag periods greater than 200 h. Continued subculture on 3,5-DCB resulted in the formation of new plasmid pPH111 (120 kb), which was homologous to pPB111. A probe from the clc operon, which encodes for the chlorocatechol pathway, hybridized to plasmid pPH111 and to the chromosome of the wild-type strain P111 but not to its plasmid pPB111 nor to the chromosome of strain P111A, which had lost the ability to utilize chlorobenzoates.(ABSTRACT TRUNCATED AT 250 WORDS)