Aemprapa S. and Williams P.A. (1998) The implications
of the xylQ gene of TOL plasmid pWW102 for the evolution of aromatic
catabolic pathways. Microbiology 144: 1387-1396.
ABSTRACT
Pseudomonas putida strain O2C2 is able
to grow on toluene, m-xylene and p-xylene through benzoate and the corresponding
methylbenzoates (toluates). The catabolic genes are encoded on a large
TOL plasmid, pWW102, of 220 kb. The complete catabolic genes were cloned
on four large overlapping restriction fragments covering a total of 28
kb of the plasmid, which was carefully mapped by restriction enzyme analysis.
The presence of the xyl genes on the cloned DNA was confirmed by assay
of representative enzymes of both operons. Virtually all the genes were
located on the cloned DNA by hybridisation of Southern blots with gene-specific
probes from related pathways of other catabolic plasmids. Within the limitations
of available restriction sites, the analysis showed that the genes are
in two blocks. The major block carries the meta pathway operon xylXYZLTEGFJQKIH
with the two regulatory genes xylSR immediately downstream. The
upper pathway operon xylUWCMAB(N) is about 2 -3 kb downstream of
the regulatory genes and transcribed in the same direction as the meta
pathway operon. Within each operon the gene order appears to be identical
to that found in other TOL plasmids, but the relative location of the operons
most closely resembles that found on plasmid pWW53, although there is no
evidence of any xyl duplications on pWW102. The nucleotide sequence
of the xylQ gene for the acetaldehyde dehydrogenase (acylating)(ADA),
together with the 3' end of the upstream xylJ (for 2-oxopent-4-enoate
hydratase) and the 5' end downstream xylK (for 4-hydroxy-2-oxovalerate
aldolase) was determined. The xylQ gene was ligated into expression
vector pTrc99a and high levels of XylQ protein were detected by enzyme
assay and by SDS-PAGE. All three genes xylJQK showed a high degree
of homology with genes encoding isofunctional proteins from other Pseudomonas
meta pathways, the highest being with the naphthalene catabolic genes
nahLOM
from the plasmid of Pseudomonas sp. NCIB9816. The implications of
the sequence homologies to the evolution of these pathways is discussed.