Purification and characterization of the arylsulfatase synthesized by Pseudomonas aeruginosa PAO during growth in sulfate-free medium and cloning of the arylsulfatase gene (atsA).
An arylsulfatase (EC 3.1.6.1) was extracted from Pseudomonas aeruginosa PAO1 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 SDS/PAGE to be 57 kDa, and the enzyme was presumed to be a monomer after gel filtration chromatography. The arylsulfatase showed maximal activity at 57 degrees C and pH 8.9, and a Km of 105 microM for 4-nitrocatecholsulfate. Despite previous reports that both inducible and derepressible forms of arylsulfatase exist in P. 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 P. 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 P. aeruginosa arylsulfatase is neither periplasmic nor membrane-associated, unlike other known arylsulfatases. The atsA gene was located at 15-17' on the P. aeruginosa genome by Southern hybridization. Only a single copy was observed under moderate stringency conditions.
Beil
S
S
Institute of Microbiology, Swiss Federal Institute of Technology, Zürich.
Kehrli
H
H
James
P
P
Staudenmann
W
W
Cook
A M
AM
Leisinger
T
T
Kertesz
M A
MA
D000595:Amino Acid Sequence; D001192:Arylsulfatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D001192:Arylsulfatases