sal genes determining the catabolism of salicylate esters are part of a supraoperonic cluster of catabolic genes in Acinetobacter sp. strain ADP1.
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 Km(r) 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 Km(r) 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 and salE, 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 Km(r) 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.
Jones
R M
RM
School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd LL57 2UW, Wales, United Kingdom.
Pagmantidis
V
V
Williams
P A
PA
D000150:Acinetobacter / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002396:Catechols / Q000378:metabolism; D003001:Cloning, Molecular; D004950:Esterases / Q000235:genetics / Q000378:metabolism; D004952:Esters / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics* / Q000502:physiology; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000235:genetics; D009876:Operon / Q000235:genetics*; D010641:Phenotype; D012329:RNA, Bacterial / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012459:Salicylates / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002396:Catechols; D004952:Esters; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D012459:Salicylates; D006899:Mixed Function Oxygenases; C022619:salicylate 1-monooxygenase; D004950:Esterases; C034221:catechol