sal Genes determining
the catabolism of salicylate esters are part of a supraoperonic cluster
of catabolic genes in Acinetobacter sp. strain ADP1
Rheinallt M. Jones, Vassilis
Pagmantidis & Peter A. Williams
J.Bacteriol.(2000) 182: 2018-2025
April.
ABSTRACT
A 5-kbp region upstream of the are-ben-cat genes was cloned from
Acinetobacter sp. strain ADP1, extending the supraoperonic cluster
of
catabolic genes to 30 kbp. Four open reading frames, salA, salR,
salE, and salD, were identified from the nucleotide sequence.
Reverse
transcription-PCR studies suggested that these open reading frames
are organized into two convergent transcription units, salAR and
salDE.
The salE gene, encoding a protein of 239 residues, was ligated into
expression vector pET5a. Its product, SalE, was shown to have esterase
activity against short-chain alkyl esters of 4-nitrophenol but was
also able to hydrolyze ethyl salicylate to ethanol and salicylic acid.
A mutant of
ADP1 with a Kmr cassette introduced into salE had lost the ability
to utilize only ethyl and methyl salicylates of the esters tested as sole
carbon sources, and no esterase activity against ethyl salicylate could
be detected in cell extracts. SalE was induced during growth on ethyl
salicylate but not during growth on salicylate itself. salD
encoded a protein of undetermined function with homologies to the Escherichia
coli
FadL membrane protein, which is involved in facilitating fatty acid
transport, and a number of other proteins detected during aromatic catabolism,
which may also function in hydrocarbon transport or uptake processes.
A Kmr cassette insertion in salD deleteriously affected cell growth
and
viability. The salA and salR gene products closely resemble
two Pseudomonas proteins, NahG and NahR, respectively encoding salicylate
hydroxylase and the LysR family regulator of both salicylate and naphthalene
catabolism. salA was cloned into pUC18 together with salR
andsalE, and its gene product showed salicylate-inducible hydroxylase
activity against a range of substituted salicylates, with the same relative
specific activities as found in wild-type ADP1 grown on salicylate.
Mutations involving insertion of Kmr cassettes into salA and salR
eliminated
expression of salicylate hydroxylase activity and the ability to grow
on either salicylate or ethyl salicylate. Studies of mutants with disruptions
of
genes of the beta-ketoadipate pathway with or without an additional
salE mutation confirmed that ethyl salicylate and salicylate were
channeled into the beta -ketoadipate pathway at the level of catechol and
thence dissimilated by the cat gene products. SalR appeared to regulate
expression of salA but not salE.