Purification and Characterization of the Arylsulfatase Synthesized by Pseudomonas aeruginosa PAO During Growth in Sulfate-Free Medium and Cloning of the Arylsulfatase Gene (atsA)

Abstract

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.

Sulfatases: Structure, Mechanism, Biological Activity, Inhibition, and Synthetic Utility

“…ARS activity has been identified in several species of enterobacteria ( Klebsiella ,63, 165 Salmonella ,166, 167 Proteus ,146, 168 Pseudomonas ,169 and Serrati 170), aquatic bacteria ( Alteromonas 52), pathogenic bacteria ( Mycobacteria 171, 172 and Pseudomonas 173, 174), extremophilic bacteria ( Plectonema 175), and soil bacteria ( Comamonas 176 and Pseudomonas 176). Indicative of their role in scavenging, most of the bacterial ARS enzymes are upregulated during sulfur starvation 3.…”

“…50 Although their preferred substrates are unknown, it is thought they may be sulfated carbohydrates. The most thoroughly characterized ARS enzymes are from Pseudomonas aeruginosa 173 and Klebsiella pneumoniae 63 (formerly classified as Klebsiella aerogenes and Aerobacter aerognes ). Early studies on these enzymes focused on genetic induction under sulfur‐limited conditions, whereas recent studies have probed KARS involvement in sulfate transport 50.…”

On the Biodegradation of β-Peptides Part of the PhD thesis of J.V.S. Dissertation no. 14298, ETH Zürich, 2001.

“…In a preliminary communication we reported growth experiments with defined strains of Pseudomonas aeruginosa (PAO1S9) and P. putida (S313, DSM 688410) that tested their ability to metabolize β ‐amino acids and to cleave β ‐peptides 4. The free β ‐amino acids 1 – 4 , the N ‐acetyl‐ β ‐amino acids 5 – 8 , β ‐dipeptides 9 and 10 , dipeptides 11 and 12 (which consist of a β ‐ and an α ‐amino acid), and for comparison, α ‐dipeptide 13 (see Scheme ) were offered to the bacteria as sole carbon and nitrogen sources.…”

Proteome mapping, mass spectrometric sequencing and reverse transcription‐PCR for characterization of the sulfate starvation‐induced response in Pseudomonas aeruginosa PAO1

“…Induction of the operon encoding the 2‐oxoglutarate‐dependent taurine dioxygenase and a putative taurine‐uptake system [10,11] allows E. coli to metabolize taurine (2‐aminoethanesulfonate) as a sulfur source, whereas the ssuEADCB operon [12] is responsible for utilization of a broader range of alkanesulfonates. More recently [9], we set out to characterize the sulfate‐starvation response in P. aeruginosa , an organism that can be found free living in the soil and possesses numerous pathways for the assimilation of different forms of organic sulfur [13–18]. The N‐terminal sequences were determined for eight sulfate‐starvation‐induced (SSI) proteins from P. aeruginosa , but these partial sequences allowed functional assignment for only one of them, namely Sbp, the periplasmic sulfate‐binding protein [9].…”