Naturally occurring TOL plasmids in Pseudomonas strains carry either two homologous or two nonhomologous catechol 2,3-oxygenase genes

Abstract

Structural genes for catechol 2,3-oxygenase (C230) were cloned from the TOL plasmids pWW5, pWW14, pWW74, pWW84, and pWW88 isolated fro*n Pseudomonas strains of diverse geographical origins. Each pKT230-based C230+ recombinant plasmid carried a 2.05-kilobase XhoI insert which showed strong homology in Southern hybridizations with the xyLE gene from the archetype TOL plasmid pWWO. Fragments were mapped for restriction endonuclease sites and were classified into two closely related groups on the basis of restriction maps. C230 structural genes were located on cloned fragments by a combination of subcloning and site-specific mutagenesis. All five TOL plasmids examined yielded clones whose maps differed from that of xyLE of pWWO by only a single XbaI site, but in addition plasmids pWW5, pWW74, and pWW88 carried a second, homologous C230 gene with seven further restriction site differences. The remaining plasmids, pWW14 and pWW84, carried a second nonhomologous C230 gene related to the second C230 gene (C2301) of TOL plasmid pWW15 described previously (H. Keil, M. R. Lebens, and P. A. Williams, J. Bacteriol. 163:248&255, 1985). Thus, each naturally occurring TOL plasmid in this study appears to carry genes for two meta cleavage dioxygenases.The degradation of toluene and some substituted toluenes via the meta pathway by Pseudomonas spp. is often plasmid mediated (7,15,19,26,34,35,39). TOL plasmids from different strains are diverse with regard to molecular sizes, fragmentation patterns by restriction endonucleases, selftransmissibility, rate of dissimilation ofp-methyl-substituted substrates, and formation of different classes of deletion mutants after growth on benzoate (7,17,27,35,37,38).Although the archetype TOL plasmid pWWO (34) has a single xylE gene coding for the ring cleavage dioxygenase catechol 2,3-oxygenase (C230), it was recently demonstrated in this laboratory that the 250-kilobase (kb) plasmid pWW15 carries two nonhomologous genes which specify the apparently unrelated enzymes C2301 and C2301I (16). To investigate the evolutionary relationship between the catabolic genes on different TOL plasmids, we cloned xylE genes from five TOL plasmids found in Pseudomonas strains isolated from soil samples of diverse geographical origins. Like pWW15, two of the plasmids reported here also carried two nonhomologous C230 genes. Unexpectedly, the other plasmids in this study each possessed two closely related C230 genes which exhibited strong homology with each other and with the xylE gene of pWWO. MATERIALS AND METHODSBacterial strains and plasmids. Pseudomonas strains and plasmids used in this study are described in Table 1. Strains MT74, MT84, and MT88 were isolated by selective enrichment on m-toluate minimal medium during this study; MT5 and MT14-26 have been described previously (7,26,35).

Duplication of both xyl catabolic operons on TOL plasmid pWW15

“…The evidence presented here further confirms our earlier results showing that many TOL plasmids carry duplications of catabolic DNA (Keil et al, 19856;Chatfield & Williams, 1986;Osborne et al, 1988) and extends the scope of the duplications revealed. The duplications previously reported involved only the genes of the meta pathway, in particular C230, whereas here we report the first identification of a duplicated upper pathway operon.…”

“…There is an increasing amount of evidence which suggests that the genetic organization of the xyl catabolic genes found on pWW0, namely single operons for the upper pathway (Harayama et a/., 1989) and the meta pathway genes (Harayama & Rekik, 1990), is not typical of many of these other plasmids. Chatfield & Williams (1986) showed that newly isolated plasmids contained either two homologous genes or two nonhomologous genes for the ring-cleavage dioxygenase, catechol 2,3-dioxygenase (C230, xylE). Plasmid p w w 5 3 falls into the former group, carrying two homologous xylE genes as part of a complete duplication of the meta pathway operon (Osborne et a/., 1988).…”

Physical mapping and characterization of a catabolic plasmid from the deep-subsurface bacterium Sphingomonas sp. strain F199

“…Analysis of the 180-kb plasmid pNL1 identified C23O genes in two regions of the plasmid, but the regulation and substrate specificity of these two C23O genes on pNL1 are currently unknown. Multiple copies of homologous or nonhomologous C23O structural genes have previously been identified on TOL plasmids in Pseudomonas strains isolated from soil (5). Also, two nonhomologous C23O genes were present on plasmid pWW15 in Pseudomonas putida MT15 (12); the substrate specificities of the two C23O genes were distinct, as was their regulation.…”

A Comparison of the Multiple Alleles of xylS Carried by TOL Plasmids pWW53 and pDK1 and its Implications for their Evolutionary Relationship

“…This evidence suggests that the genetic organization of the xyl catabolic genes found on pWW0 , namely single operons for the upper pathway (Harayama et al, 1989), and the meta pathway genes (Harayama & Rekik, 1990), is not typical of other TOL plasmids. Not only are the xyl operons arranged in different orders and orientations but there is increasing evidence that many carry more than one copy either of whole operons or of individual genes (Keil et al, 1985b;Chatfield & Williams, 1986;O'Donnell & Williams, 1991). In particular we have shown that the 107 kbp plasmid pWW53 carries two homologous, but distinguishable, direct repeats of the meta pathway operon xylXYZLTEGFJQlyIH both of which are functional (Osborne et al, 1988).…”