Isolation and characterization of a new plasmid from a Flavobacterium sp. which carries the genes for degradation of 2,4-dichlorophenoxyacetate

Abstract

A Flavobacterium sp. (strain 50001), capable of degrading 2,4-dichlorophenoxyacetate (2,4-D), 2-methyl-4-chlorophenoxyacetate, and 2-chlorobenzoate and imparting resistance to mercury, harbored a degradative plasmid, pRC10. Cured strains of the Flavobacterium sp. lost the plasmid as well as the ability to degrade these chlorinated compounds. Comparison of this plasmid with the well-characterized 2,4-D-degradative plasmid pJP4 from Alcaligenes eutrophus showed regions of homology between the two plasmids. Restriction fragments of plasmid pRC10 which shared homology with the regions conferring 2,4-D-degradative genes (tfd) of plasmid pJP4 were cloned into a broad-host-range plasmid and studied in Pseudomonas putida. From the results obtained, the cloned DNA fragment expressed the genes for 2,4-D monooxygenase (tfdA) and 2,4-dichlorophenol hydroxylase (tfdB). In spite of the similarity in function, the size (45 kilobases) and restriction pattern of plasmid pRC10 were considerably different from those of pJP4 (80 kilobases). This may be due to the difference in the microbial background during evolution of the two plasmids.Many of the microorganisms capable of metabolizing synthetic organic compounds harbor large degradative plasmids (15,22,36). The degradative plasmids which are involved in the catabolism of toluene and naphthalene are the best-characterized plasmids among this group (11,16,18,26,27,35). Recently, plasmids involved in the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) have also been under considerable investigation (1,(8)(9)(10)29). These plasmids range from 80 to 120 kilobase pairs (kb) in size and have been predominantly isolated from members of the genus Alcaligenes, obtained from soil or aquatic environments (1,8). One of these plasmids, pJP4 from Alcaligenes eutrophus, has been investigated more extensively (9,10,29).Several other microorganisms belonging to a variety of different bacterial genera, such as Acinetobacter, Arthrobacter, Corynebacterium, Flavobacterium, and Pseudomonas, have also been shown to degrade 2,4-D and related phenoxyacetates (2,4,8,12,14,28,(30)(31)(32)34 MATERIALS AND METHODSBacterial strains and plasmids. The bacteria and plasmids used in this study are listed in Table 1.Media and growth conditions. The Flavobacterium sp. and Alcaligenes eutrophus strains were grown at 28°C aerobically in minimal medium (MM) (5) supplemented with 2,4-D or other compounds as the sole source of carbon. Escherichia coli strains were grown in LB medium (5). The medium used for growing Pseudomonas putida has been described elsewhere (34). A nalidixic acid-resistant mutant of P. putida was obtained by selecting resistant colonies on heavily inoculated LB agar plates containing nalidixic acid (50 ,ug/ml) (Sigma Chemical Co.). The autoclaved medium was supplemented with antibiotics and metal salts by using sterile stock solutions as needed.Detection and isolation of plasmid DNA. An estimated 108 exponentially growing cells were suspended in E buffer (21) containing 25% sucrose, lysozy...

Characterization of a new 2,4-dichlorophenoxyacetic acid degrading plasmid pEST4011: physical map and localization of catabolic genes

“…This supports other evidence that 2,4-D degradative plasmids demonstrate appreciable homology between the genes encoding enzymes that catalyse the same steps in breakdown of 2,4-D and related compounds (Chaudhry & Huang, 1988). By using DNA-DNA hybridization, DNA cloning techniques and enzyme assays we established the location of at least three catabolic genes encoding the first three steps in the 2,4-D degradation pathway: 2,4-D monooxygenase ( @ A ) , 2,4-D dichlorophenol hydroxylase (tfdB) and chlorocatechol 1,2-dioxygenase (tfdC) on the 10.5 kbp BamHI-B fragment of pEST4011.…”

“…A second plasmid, pRC 10, present in a 2,4-D-degrading Flavobacteriurn sp. isolated in the USA, has also been characterized in detail (Chaudhry & Huang, 1988). Little information is, however, available on the nature and physical structure of other 2,4-D degradative plasmids.…”

“…Despite differences in the size and restriction pattern of 2,4-D plasmids there is considerable homology between the 2,4-D degradative genes (Don & Pemberton, 1981;Amy et al, 1985;Chaudhry & Huang, 1988;Ditzelmiiller et al, 1989). Comparison of other plasmids which determine the degradation of chlorinated compounds with pJP4 would be of great value in understanding the evolutionary process by which enzymes with novel specificities are evolved by bacteria and how they have spread amongst bacteria.…”

“…Plasmids involved in the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) have been under considerable investigation (Pemberton & Fisher, 1977;Pierce et al, 1981;Amy et al, 1985;Chaudhry & Huang, 1988). The 2,4-D degradative plasmid pJP4 from Alcaligenes eutrophus JMP134 (isolated in Australia) has become a model for the investigation of 2,4-D degradation (Don & Pemberton, 1985;Ghosal & You, 1988Harker et al, 1989;Perkins et al, 1990).…”

Degradation of 2,4-D herbicide by microorganisms isolated from Brazilian contaminated soil

“…Acinetobacter sp, Stenothrophomonas maltophilia and Flavobacterium sp had been previously described as 2,4-D herbicide degrading microorganisms in other studies (4,5,8,14,20), revealing a great importance in further studies aiming the bioremediation processes. The results are also noteworthy, considering that Serratia marcescens and Penicillium sp were isolated from tropical soil, and had never been reported as able to degrade 2,4-D before, a herbicide extensively used in Brazil.…”