An arylsulfatase (EC 3.1.6.1) was extracted from Pseudomonas aeruginosa PA01 and purified 2700-fold to homogeneity. Synthesis of this enzyme was repressed when sulfate, cysteine or thiocyanate was supplied as the sole sulfur source for growth, but derepressed with all other sulfur sources tested. The apparent molecular mass was determined by SDSPAGE to be 57 kDa, and the enzyme was presumed to be a monomer after gel filtration chromatography. The arylsulfatase showed maximal activity at 57°C and pH 8.9, and a K , of 105 pM for 4-nitrocatecholsulfate. Despite previous reports that both inducible and derepressible forms of arylsulfatase exist in f? aeruginosa, we found only one enzyme under a variety of growth conditions: a sulfate-repressed enzyme with a native isoelectric point of 4.76. The gene encoding this enzyme (atsA) was isolated by complementation of a Tn5-751 mutant of I? aeruginosa PAO1.Sequencing revealed a 1602-bp reading frame encoding a 534-amino-acid protein with sequence similarity to known bacterial and eukaryotic arylsulfatases (30-40% and 25-30 % identity, respectively), but lacking the signal peptide which is present in all known sequences. The lack of this signal peptide suggests that the I? aeruginosa arylsulfatase is neither periplasmic nor membrane-associated, unlike other known arylsulfatases. The atsA gene was located at 15-17' on the I? aeruginosa genome by Southern hybridization. Only a single copy was observed under moderate stringency conditions.

The bacterium, Burkholderia (previously Pseudomonus) sp. strain PS12, reported earlier to degrade 1,2,4-trichlorobenzene is shown here to utilize also 1,2,4,S-tetrachlorobenzene (CL-benzene) as a growth substrate. To investigate the possibility that this organism attacks C1,-benzene with a chlorobenzene dioxygenase which concomitantly causes dehalogenation, and to analyze the substrate range of the initial enzyme, a 5503-bp DNA fragment from PS12, exhibiting high similarity to genes coding for class IIB dioxygenases, was cloned and expressed in Escherichia coli. The sequence includes the tec genes coding for the cr-subunit and /I-subunit of a terminal dioxygenase, a ferredoxin and a reductase. E. coli cells producing these proteins were able to dioxygenolytically attack a range of aromatic compounds including chlorinated benzenes and toluene, and also dinuclear aromatics such as biphenyl and dibenzo-p-dioxin. The enzyme was shown by "O0, incorporation experiments to dioxygenolytically attack a chlorosubstituted carbon atom of C1,-benzene, thereby forming an unstable diol intermediate which spontaneously rearomatizes with concomitant chloride elimination to the corresponding 3,4,6-trichlorocatechol (CLcatechol).

Pseudomonas putida S-313 (= DSM 6884) grew in sulfate-free medium when the sole sulfur source supplied was one of several arylsulfonates involved in the synthesis, application, or biodegradation of linear alkylbenzenesulfonate (LAS) surfactants. 2-(4-Sulfophenyl)butyric acid, 4-n-butyl-1-methyl-6-sulfotetralin, and 4-toluenesulfonic acid were each completely utilized during growth, as were the model LAS 1-(4-sulfophenyl) octane and the arylsulfonate dyestuff Orange II. The product in each case was the corresponding phenol, which was identified by gas chromatography-mass spectrometry or 'H nuclear magnetic resonance. Stoichiometric conversion of 4-toluenesulfonic acid to 4-cresol was observed. The molar growth yields observed were 2.4 to 2.8 kg of protein per mol of S, which were comparable to the yield for sulfate. Commercial LAS disappeared from growth medium inoculated with strain S-313, but negligible growth occurred; digestion of cells in alkali led to recovery of the LAS mixture, which seemingly sorbed to the cells. However, mixed culture L6 was readily obtained from batch enrichment cultures containing commercial LAS as a sole sulfur source and an inoculum from domestic sewage. Culture L6 desulfonated components of the LAS surfactant to the corresponding phenols, which were identified by gas chromatography-mass spectrometry. Compounds with shorter alkyl chains were desulfonated preferentially, as were the centrally substituted isomers. In the presence of200 ,uM sulfate, culture L6 grew well and LAS disappeared, although this was due purely to sorption, as shown by digestion of the cells in alkali. Thus, under sulfate-limited conditions, LAS can be desulfonated directly.

An arylsulfatase (EC 3.1.6.1) was extracted from Pseudomonas aeruginosa PA01 and purified 2700-fold to homogeneity. Synthesis of this enzyme was repressed when sulfate, cysteine or thiocyanate was supplied as the sole sulfur source for growth, but derepressed with all other sulfur sources tested. The apparent molecular mass was determined by SDSPAGE to be 57 kDa, and the enzyme was presumed to be a monomer after gel filtration chromatography. The arylsulfatase showed maximal activity at 57°C and pH 8.9, and a K , of 105 pM for 4-nitrocatecholsulfate. Despite previous reports that both inducible and derepressible forms of arylsulfatase exist in f? aeruginosa, we found only one enzyme under a variety of growth conditions: a sulfate-repressed enzyme with a native isoelectric point of 4.76. The gene encoding this enzyme (atsA) was isolated by complementation of a Tn5-751 mutant of I? aeruginosa PAO1.Sequencing revealed a 1602-bp reading frame encoding a 534-amino-acid protein with sequence similarity to known bacterial and eukaryotic arylsulfatases (30-40% and 25-30 % identity, respectively), but lacking the signal peptide which is present in all known sequences. The lack of this signal peptide suggests that the I? aeruginosa arylsulfatase is neither periplasmic nor membrane-associated, unlike other known arylsulfatases. The atsA gene was located at 15-17' on the I? aeruginosa genome by Southern hybridization. Only a single copy was observed under moderate stringency conditions.

Conditions were optimized for the batch growth of Pseudomonas putida 5-313 under sulfur-limited conditions. P. putida grew exponentially with sulfate as the sole source of sulfur, and growth was concomitant with the utilization of sulfate until it was exhausted. A further 20% of protein was synthesized after the apparent disappearance of sulfate. A mass balance for the utilized sulfate in cell material was calculated, given the observed molar growth yield of about 3.6 kg protein (mol S)-' and a sulfur content of 0.41 O/ O S in dry matter. Similar data were obtained for growth with cysteine and thiocyanate. The organism also grew exponentially with 4-toluenesulfonate (TS) as sulfur source, essentially as observed with sulfate, except that negligible protein formation after exhaustion of TS was observed. Similar data were also obtained with 4-nitrocatecholsulfate (NCS) and ethanesulfonate. Any substrate pair selected from sulfate, cysteine and thiocyanate was utilized simultaneously, and although one of the pair of substrates was always preferred, growth continued at the same rate when only one substrate remained. Growth after substrate exhaustion was observed. Any substrate pair selected from TS, NCS and ethanesulfonate gave similar data, but with less growth after exhaustion of the sulfur sources. If a mixed substrate pair was chosen from the two groups, the sulfur source from the first-named group was initially used exclusively, and the second source of sulfur was utilized subsequently, after a lag phase. The data are considered to reflect the control of scavenging for sulfur and of distribution of sulfur in the cell exerted by the sulfate-starvation-induced stimulon [Kertesz, Leisinger & Cook, J Bacferiol (1993) 175,1187