Fuenmayor S.L., Wild M., Boyes A.L. & Williams P.A. (1998).
A gene cluster encoding steps in the conversion of naphthalene to gentisate
in Pseudomonas sp. U2. J.Bacteriol.
180:
2522-2530
ABSTRACT
Pseudomonas sp. U2 was isolated from oil contaminated
soil in Venezuela by selective enrichment on naphthalene as sole carbon
source. The genes for naphthalene dioxygenase were cloned from the plasmid
DNA of strain U2 on an 8.3-kb BamHI fragment. nagAa (for
the ferredoxin reductase), nagAb (for the ferredoxin) and nagAc,
nagAd (for the large and small subunits of the dioxygenase) were located
by Southern blot hybridizations and by nucleotide sequencing. The genes
for naphthalene cis-dihydrodiol dehydrogenase (nagB ) and
for salicylaldehyde dehydrogenase (nagF) were identified from subclones
by their activity and phenotypic effect. Between nagAa and nagAb
were
two open reading frames, homologs of which have also been identified in
a similar location in two nitrotoluene-utilizing strains (J.V.Parales,
A.Kumar, R.E.Parales, and D.T.Gibson, Gene 181:57-61,1996; W.-C.Suen, B.Haigler,
and J.C.Spain, J.Bacteriol. 178:4926-4934, 1996) and a naphthalene-utilizing
strain (G.J.Zylstra, E.Kim, and A.K.Goyal, 1997. pp.257-269, In Genetic
Engineering, Vol.19, J.K.Setlow (ed.), Plenum Press, New York). Recombinant
E.coli
strains with plasmids carrying this region were able to convert salicylate
to gentisate, which was identified by a combination of GLC/MS and NMR.
The first open reading frame, designated nagG, encodes a protein
with characteristics of a Rieske-type iron-sulfur center homologous to
the large subunits of dihydroxylating dioxygenases and the second, designated
nagH,
encodes a protein with limited homology to the small subunits of the same
dioxygenases. Cloned together in E.coli, nagGHAb were able to convert
salicylate into gentisate (2,5-dihydroxybenzoate) and therefore encode
a salicylate 5-hydroxylase activity. Single gene knockouts of both
nagH
and nagAb demonstrated their functional role in the formation of
gentisate. It is proposed that NagGH are structural subunits of salicylate
5-hydroxylase linked to an electron transport chain consisting of NagAb
and NagAa, although E.coli appears to be able to partially substitute
for the latter. This constitutes a novel mechanism for monohydroxylation
of the aromatic ring. The absence of salicylate hydroxylase and catechol
2,3-dioxygenase in strain U2 was demonstrated by enzyme assay and by Southern
hybridization. However growth on both naphthalene and salicylate caused
induction of gentisate 1,2-dioxygenase, confirming this route for salicylate
catabolism in strain U2. Sequence comparisons suggest that the novel gene
order nagAaGHAbAcAdBF represents the archetype for naphthalene strains
which use the gentisate pathway instead of the meta cleavage of
catechol .
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