Variation in the ability of Pseudomonas sp. strain B13 cultures to utilize meta-chlorobenzoate is associated with tandem amplification and deamplification of DNA

Abstract

strain B13 (6, 7) and Pseudomonas putida AC858 (2, 4) which degrade Ben are, however, also capable of degrading meta-chlorobenzoate (3CB). In P. putida AC858, the 3CB+ determinants are located on a 117-kilobase (kb) plasmid, pAC25 (2). A 4.3-kb DNA region on this plasmid encodes three key chlorocatechol pathway enzymes (pyrocatechase II, cycloisomerase II, and hydrolase II) for 3CB utilization (10). On the other hand, the genetic and molecular functions involved in the utilization of Ben and 3CB by the B13 strain are still unclear. The 3CB+ determinants of B13 have been shown to be transferrable by conjugation to P. putida strains (16,20,21), but it is still unclear whether these determinants are carried on a plasmid in B13. DNA hybridization studies have revealed a high degree of homology between the 3CB+ DNA in B13 and AC858 (2). Furthermore, comparison of the nucleotide sequence of the 4.3-kb 3CB+ DNA from pAC25 with the amino acid sequence of the B13 enzymes responsible for chlorocatechol utilization suggests that the 3CB+ determinants in these two different strains are identical (10). Recently, the 3CB+ DNA from B13 has been cloned and expressed in plasmid vectors (27); this clone from B13 is, however, structurally different, especially with respect to the restriction endonuclease sites, from the 3CB+ DNA cloned from AC858.The elucidation of different functions involved in the elaboration of a pathway requires the use of mutants, each of which is defective in one or more of these functions. phenotype of B13 either is not uniformly expressed or is not stably inherited in every single-colony isolation. The question is whether the phenotypic variation is a consequence of genetic alterations, and if so, the variant phenotype would be expected to be inherited and maintained in all single-colony isolates of the variant. In an effort to resolve these questions, I studied the lineages of B13 colonies, and this report describes my observations on the variation in the ability of B13 colonies to utilize Ben and 3CB as sole carbon sources. The variation in phenotypic characteristics was also documented in biochemical studies, and variation in 3CB utilization was associated with tandem amplification or deamplification of the 4.3-kb 3CB+ DNA.

Intraclonal Polymorphism in Bacteria

“…In Pseudomonas sp. (strain B 13) the ability to utilize meta-chlorobenzoate is associated with the tandem amplification and deamplification of a 4.3-kbp DNA region which encodes three key enzymes in the chlorocatechol pathway (Rangnekar, 1988). Studies on switching between mucoid and nonmucoid strains of P. atlantica have shown that it occurs by precise insertion and deletion of a 1.2-kbp IS element into the eps gene (Bartlett et al, 1988).…”

Conjugative plasmid coding for the metabolism of 2-chlorobenzoic acid byPseudomonas aeruginosa

“…A high degree of variation in Pseudomonas sp. B13 carrying a plasmid coding for degradation of 3-chlorobenzoic acid and benzoic acid has also been reported (Rangnekar 1988). Results presented in Table 2 show that the putative plasmid is conjugative.…”

Chromosomal Integration of tcb Chlorocatechol Degradation Pathway Genes as a Means of Expanding the Growth Substrate Range of Bacteria To Include Haloaromatics

“…In 1988, the amplification of a 4.3-kb BglII fragment containing the clc genes in Pseudomonas sp. strain B13 clones expressing a 3-CBA ϩ phenotype was reported (27). Recently, the molecular basis for this phenomenon was discovered: the clc genes were found to reside on a 105-kb genetic element capable of site-specific chromosomal integration (28,29).…”