A 10 kbp region upstream of the ben-cat supraoperonic cluster of catabolic genes was cloned from Acinetobacter sp. strain AD Pl. This region contains genes that encode the catabolism of aromatic esters, converting them to the corresponding aromatic carboxylic acids by the sequential action of inducible enzymes. A set of genes for three inducible enzymes: areA-encoding an esterase, areB-encoding a benzyl alcohol dehydrogenase, and areC-encoding a benzaldehyde dehydrogenase, were found to be located 3.5kbp upstream of the ben genes. Individual disruptions of the areA, areB and areC genes on the chromosome by insertion of a Km-resistance cassette, resulted in strains which were severely reduced in the ability to utilise aryl esters as sole carbon growth substrates as compared to the wild-type strain. Each are gene was individually expressed to high specific activity in Escherichia coli. The relative activities against different substrates of the cloned enzymes were, within experimental error, identical to that of wild-type Acinetobacter sp. ADPl grown on benzyl acetate, benzyl alcohol or 4-hydroxybenzyl alcohol as the carbon source. The substrate preferences of all three enzymes were broad, encompassing a range of substituted aromatic compounds and in the case of the AreA esterase, different carboxylic acids. Mutants blocked in genes of the ~-ketoadipate pathway with or without an additional areA mutation confirmed that benzyl acetate, and 2-and 4-hydroxybenzyl acetates were channelled into the ~-ketoadipate pathway at the level of benzoate, catechol and protocatechuate respective! y. Studies of the expression of areCBA by RT-PCR, by biochemical assays, and by are::lacZ chromosomal gene fusions found that the genes are co-transcribed on the same mRNA and are induced as an operon by benzyl acetate, benzyl alcohol or benzaldehyde. Adjacent to areC is the gene areR that encodes for a protein that has amino acid sequence homology to members of the 054-dependent NtrC family of transcriptional regulators. Sequence analysis of the promoter region upstream of areC showed that it contained all the elements typical of a54-dependent gene expression. A disruption of areR on the chromosome by insertion of a Km-resistance cassette resulted in a mutant that was unable to induce AreA, B, or C activity. Sequence analysis of the region adjacent to areA identified four open reading frames: salA, salR, salE and salD, which are organised in two convergent transcription units, salAR and salDE, as was confirmed by RT-PCR. salD encodes a protein with undetermined function but which shows homologies with membrane proteins thought to be involved in hydrocarbon uptake. A Kmr insertion in salD deleteriously affected cell growth and viability. salE was cloned into expression vector pET5a and its product was shown to have esterase activity against short-chain alkyl esters of 4-nitrophenol. SalE was also shown to have hydrolytic activity against ethyl salicylate, producing ethanol and salicylic acid. The insertional inactivation of salE with a Kmr cassette resulted in a mutant that lost the ability to utilise 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 on salicylate itself. The SalA and SalR gene products are homologous to two Pseudomonas proteins NahG and NahR, respectively encoding salicylate hydroxylase and the positive LysR-family regulator of both salicylate and naphthalene catabolism. salA was cloned into pUC18 together with salR and salE and showed salicylate-inducible hydroxylase activity against a range of substituted salicylates, with the same relative specific activity as found in wild type ADPl grown on salicylate. Knockout mutations with Kmr cassettes of salA and salR respectively, resulted in mutants that were unable to grow on salicylate or ethyl salicylate, or to express salicylate hydroxylase activity. Mutants blocked in genes of the ~-ketoadipate pathway with or without an additional salE mutation confirmed that ethyl salicylate and salicylate were channelled into the ~-ketoadipate pathway at the level of catechol and thence dissimulated by the cat genes products. The function of the are and sal genes is to catabolise a range of aryl esters and alkyl salicylate into carboxylic acids, which are further catabolised to central metabolites by the adjacent hen and cat genes. This contributes to the metabolic diversity of ADPl and enables it to grow on substrates that are likely to be found as natural breakdown products of plant compounds.