The structure and function of transposable elements that code for catabolic pathways involved in the biodegradation of organic compounds are reviewed. Seven of these catabolic transposons have structural features that place them in the Class I (composite) or Class II (Tn3-family) bacterial elements. One is a conjugative transposon. Another three have been found to have properties of transposable elements but have not been characterized sufficiently to assign to a known class. Structural features of the toluene (Tn4651/Tn4653) and naphthalene (Tn4655) elements that illustrate the enormous potential for acquisition, deletion and rearrangement of DNA within catabolic transposons are discussed. The recently characterized chlorobenzoate (Tn5271) and chlorobenzene (Tn5280) catabolic transposons encode different aromatic ring dioxygenases, however they both illustrate the constraints that must be overcome when recipients of catabolic transposons assemble and regulate complete metabolic pathways for environmental pollutants. The structures of the chlorobenzoate catabolic transposon Tn5271 and the related haloacetate dehalogenase catabolic element of plasmid pUO1 are compared and a hypothesis for their formation is discussed. The structures and activities of catabolic transposons of unknown class coding for the catabolism of halogenated alkanoic acids (DEH) and chlorobiphenyl (Tn4371) are also reviewed.

A key intermediate for biodegradation of various distinct aromatic growth substrates in Comamonas testosteroni is protocatechuate (Pca), which is metabolized by the 4,5-extradiol (meta) ring fission pathway. A locus harbouring genes from C. testosteroni BR6020 was cloned, dubbed pmd, which encodes the enzymes that degrade Pca. The identity of pmdAB, encoding respectively the α-and β-subunit of the Pca ring-cleavage enzyme, was confirmed by N-terminal sequencing and molecular mass determination of both subunits from the separated enzyme. Disruption of pmdA resulted in a strain unable to grow on Pca and a variety of aromatic substrates funnelled through this compound (m-and p-hydroxybenzoate, p-sulfobenzoate, phthalate, isophthalate, terephthalate, vanillate, isovanillate and veratrate). Growth on benzoate and o-aminobenzoate (anthranilate) was not affected in this strain, indicating that these substrates are metabolized via a different lower pathway. Tentative functions for the products of other pmd genes were assigned based on sequence identity and/or similarity to proteins from other proteobacteria involved in uptake or metabolism of aromatic compounds. This study provides evidence for a single lower pathway in C. testosteroni for metabolism of Pca, which is generated by different upper pathways acting on a variety of aromatic substrates.

Chlorobenzoate (3Cba)-degrading bacteria were isolated from the waters and sediments of flowthrough mesocosms dosed with various concentrations of 3Cba and inoculated with a 3Cba-degrading Alcaligenes sp., strain BR60. Bacteria capable of 3Cba degradation which were distinct from BR60 were isolated. They carried pBRC60, a plasmid introduced with Alcaligenes sp. strain BR60 that carries a transposable element (Tn5271) encoding 3Cba degradation. The isolates expressed these genes in different ways. The majority of pBRC60 recipients were motile, yellow-pigmented, gram-negative rods related to the group III pseudomonads and to BR60 by substrate utilization pattern. They were capable of complete 3Cba degradation at both millimolar and micromolar concentrations. Two isolates, Pseudomonas fluorescens PR24B(pBRC60) and Pseudomonas sp. strain PR120(pBRC60), are more distantly related to BR60 and both produced chlorocatechol when exposed to 3Cba at millimolar concentrations in the presence of yeast extract. These species showed poor growth in liquid 3Cba minimal medium but could degrade 3Cba in continuous cultures dosed with micromolar levels of the chemical. Laboratory matings confirm that pBRC60 can transfer from BR60 to species in both the beta and gamma subgroups of the proteobacteria and that 3Cba gene expression is variable between species. Selection pressures acting on pBRC60 recipients are discussed.

In Comamonas testosteroni BR60 (formerly Alcaligenes sp. strain BR60), catabolism of the pollutant 3-chlorobenzoate (3CBA) is initiated by enzymes encoded by cbaABC, an operon found on composite transposon Tn5271 of plasmid pBRC60. The cbaABC gene product CbaABC converts 3CBA to protocatechuate (PCA) and 5-Cl-PCA, which are then metabolized by the chromosomal PCA meta (extradiol) ring fission pathway. In this study, cbaA was found to possess a 70 type promoter. O 2 uptake experiments with whole cells and expression studies with cbaA-lacZ constructs showed that cbaABC was induced by 3CBA. Benzoate, which is not a substrate of the 3CBA pathway, was a gratuitous inducer, and CbaR, a MarR family repressor coded for by a divergently transcribed gene upstream of cbaABC, could modulate induction mediated by benzoate. Purified CbaR bound specifically to two regions of the cbaA promoter (P cbaA ); site I, a high-affinity site, is between the transcriptional start point (position ؉1) and the start codon of cbaA, while site II, a lower-affinity site, overlaps position ؉1. 3CBA at concentrations as low as 40 M interfered with binding to P cbaA . PCA also interfered with binding, while benzoate only weakly disrupted binding. Unexpectedly, benzoate with a hydroxyl or carboxyl at position 3 improved CbaR binding. Data are also presented that suggest that an unidentified regulator is encoded on the chromosome that induces cbaABC in response to benzoate and 3CBA.The chlorinated benzoic acids (CBA) are a common class of pollutants that occur in the environment as a result of intentional introduction (e.g., in the form of herbicides) or incomplete bacterial metabolism of some accidentally released chemicals (e.g., polychlorinated biphenyls) (46). Bacteria possess a remarkable assortment of metabolic pathways for biodegradation of CBA, and the innate ability of bacteria to degrade CBA has been exploited for bioremediation of contaminated sites (47). Several aerobic degradation pathways have been characterized at the biochemical and genetic levels. The most intensively studied pathway is encoded by the clc genes of Pseudomonas putida that specify intradiol ring fission of 3-chlorocatechol, a metabolite generated by nonspecific activity of benzoate or toluate dioxygenases with 3-chlorobenzoate (3CBA) (19). In contrast, the cba-encoded pathway involves a dioxygenase and a dehydrogenase that convert 3CBA, 4CBA, or 3,4-dichlorobenzoate to the vicinal diol intermediates protocatechuate (PCA) and 5-Cl-PCA (Fig. 1A) (40, 41). Other CBA degradation operons include the cbd-encoded pathway of Burkholderia cepacia (22) and the ohb-encoded pathway of Pseudomonas aeruginosa (60), both of which specify dioxygenase-mediated conversion of 2CBA to catechol; the fcb pathway of Arthrobacter globiformis for conversion of 4CBA to 4-hydroxybenzoate by a coenzyme A ligase and a hydrolase (61); and an Alcaligenes sp. pathway that converts 3CBA to 3-hydroxybenzoate (31, 32). Proven or putative regulatory factors for these pathways are encoded by genes close...

A chlorobenzoate-catabolic transposon (TnS271) was introduced on a conjugative plasmid (pBRC60) in the natural host, Alcaligenes sp. strain BR60, into lake water and sediment flowthrough microcosms. Experimental microcosms were exposed to micromolar levels of 3-chlorobenzoate, 4-chloroaniline, 2,4-dichlorophenoxyacetate, or 3-chlorobiphenyl. The populations of the host, BR60, and organisms carrying TnS271 were monitored over a 100-day period by use of selective plate counts and the most-probable-number-DNA hybridization method. Populations of TnS271-carrying bacteria were significantly higher in microcosms dosed with 3-chlorobenzoate, 4-chloroaniline, and 3-chlorobiphenyl than in the control microcosms, indicating that each of these chemicals exerts a selective force on this particular genotype in natural systems. The rates of 3-chlorobenzoate uptake and respiration correlated with Tn5271-carrying populations, as did the rates of 4-chloroaniline uptake and respiration. Plasmid transfer in the 3-chlorobenzoate-and 3-chlorobiphenyl-dosed microcosms resulted in the selection of three phenotypic clusters of chlorobenzoate degraders, only one of which was closely related to the original pBRC60 (Tn5271) donor, Alcaligenes sp. strain BR60. Bacteria dominating 4-chloroaniline-dosed microcosms carried IS1071, the class II insertion sequence that brackets TnS271, on a plasmid unrelated to pBRC60. The importance of plasmid transfer and transposition during chemical adaptation is discussed.