A New Class of TOL Plasmid Deletion Mutants in Pseudomonas putida MT15 and Their Reversion by Tandem Gene Amplification

Abstract

~~~Pseudomonasputida MT15 contains a large plasmid, pWW15, of about 250 kbp, which encodes the genes for toluene and xylene catabolism. Growth on benzoate selects strongly against the wild-type and results in the segregation of three phenotypically distinguishable mutant types. (1) B1 mutants, which have lost the complete plasmid.(2) B3 mutants, in which the plasmid has undergone a large deletion of about 90 kbp which appears to affect the regulation of the catabolic enzymes; these mutants retain the ability to grow on m-xylene and toluene (Mxy+ Tln+) but no longer grow on the metabolite of m-xylene, m-toluate (Mtol-). (3) A novel class not previously described, the B5 mutants, which still grow well on toluene but grow very poorly on m-xylene and do not grow on m-toluate (Mxy-Tln+ Mtol-). The B5 mutants appear to share the regulatory lesion of the B3 mutants but in addition do not express the xylF and xylG gene products, 2-hydroxymuconic semialdehyde hydrolase and 2-hydroxymuconic semialdehyde dehydrogenase. The plasmids in the B5 mutants have also undergone a deletion of about 90 kbp similar to, but distinguishable from, that in the B3 mutants.Both B3 and B5 mutants can revert to growth on m-toluate. The revertants all show elevated constitutive levels of catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase and 2-hydroxymuconic semialdehyde hydrolase which are not further induced by m-toluate. The reversion is accompanied by the tandem amplification of a region of 23-28 kbp on either side of the original deletion. As a result of Southern hybridizations, it was shown that the amplified region contains the structural genes of some of the enzymes which metabolize m-toluate but not the enzymes which convert m-xylene to m-toluate.

Loss of Tdn catabolic genes by deletion from and curing of plasmid pTDN1 in Pseudomonas putida: rate and mode of loss are substrate and pH dependent

“…It seems reasonable to assume that benzoate can be metabolized via the plasmid-encoded pathway in strain UCC22. MT14, MT15 and MT20 produce several classes of phenotypically distinguishable TOL-deletion mutants Pickup & Williams, 1982;Keil & Williams, 1985) and it has been suggested that such TOL plasmids have regions of homology which act as 'hotspots' for recombinational deletion events (Williams et al, 1988). In the case of pWWO growth of the host on benzoate was found to highlight two effects, either complete plasmid loss or deletion of a specific 39 kbp region bearing all the catabolic genes (Bayley et al, 1977).…”

“…functional, cells growing by the chromosomal pathway become amplified due to faster growth rate on the substrate (Nakazawa & Yokota, 1973;Williams & Murray, 1974). Such amplified cells are plasmid 'cured' or have suffered plasmid deletions (Williams & Worsey, 1976;Kunz & Chapman, 1981 ;Pickup &Williams, 1982;Pickup et al, 1983;Keil & Williams, 1985). The period of amplification varies considerably and occurs faster with TOL plasmids present in Pseudornonas MT14, MT15 and MT20 than with the archetypal TOL plasmid pWWO of Pseudornonas putida mt-2 (Paw 1) (Williams & Worsey, 1976).…”

Duplication of both xyl catabolic operons on TOL plasmid pWW15

“…Cells of Pseudomonas were grown in induced and uninduced conditions as described in Keil & Williams (1985) and cells of E. coli were grown overnight in Luria broth with addition of an appropriate antibiotic. All enzyme assays were carried out on sonic extracts of cells using previously reported methods : benzyl alcohol dehydrogenase (BADH) and benzaldehyde dehydrogenase (BZDH) (Worsey & Williams, 1975); catechol 2,3-dioxygenase (C230), 2-hydroxymuconic semialdehyde dehydrogenase (HMSD), 2-hydroxymuconic semialdehyde hydrolase (HMSH), 4-oxalocrotonate tautomerase (OT) and 4-oxalocrotonate decarboxylase (OD) (Sala-Trepat & Evans, 197 1); 2-oxopent-4-enoate hydratase (OEH) (Collinsworth et al, 1973).…”

“…pWW 15 is one of three large, structurally related TOL plasmids, p W W 14, p W W 1 5 and pWW20, which have the characteristic property of being strongly counterselected during growth of their natural hosts in benzoate minimal media (Williams & Worsey, 1976). After about 20 generations no strains with the typical TOL phenotype can be detected in the population, which consists either of plasmid-free cells or of cells which have an altered, defective catabolic phenotype (Williams & Worsey, 1976;Pickup & Williams, 1982;Pickup et al, 1983;Keil & Williams, 1985) and which have suffered large deletions of plasmid DNA. In this paper we report the existence of two upper pathway operons and two meta pathway operons on pWW 15.…”

“…(b) The presence or absence of the relevant HindIII and XhoI fragments were examined in a number of the mutants containing large deletions in pWW 15 which we had obtained by growth of MT15 on benzoate (Keil & Williams, 1985): for example, in one such plasmid both H F and XB are absent but HJ and XA are present, thus implicating H F and XB as overlapping. .xylCAB and the operator-promoter (OPI) are as determined by Keil et al (1987~).…”