Cloning, sequence analysis, and characterization of the astA gene encoding an arylsulfate sulfotransferase from Citrobacter freundii.

Arylsulfate sulfotransferase (ASST) transfers a sulfate group from a phenolic sulfate ester to a phenolic acceptor substrate. In the present study, the gene encoding ASST was cloned from a genomic library copy of Citrobacter freundii, subcloned into the vector pGEM3Zf(-) and sequenced. Sequencing revealed two contiguous open reading frames (ORF1 and ORF2) on the same strand and based on amino acid sequence homology, they were designated as astA and dsbA, respectively. The amino acid sequence of astA deduced from C. freundii was highly similar to that of the Salmonella typhimurium, Enterobacter amnigenus, Klebsiella, Pseudomonas putida, and Campylobacter jejuni, encoded by the astA genes. However, the ASST activity assay revealed different acceptor specificities. Using p-nitrophenyl sulfate (PNS) as a donor substrate, alpha-naphthol was found to be the best acceptor substrate, followed by phenol, resorcinol, p-acetaminophen, tyramine and tyrosine.

Kinetic mechanism and identification of the active site tyrosine residue in Enterobacter amnigenus arylsulfate sulfotransferase.

Bacterial arylsulfate sulfotransferase (ASST) catalyzes the transfer of a sulfate group from a phenyl sulfate ester to a phenolic acceptor. The kinetic mechanism of Enterobacter amnigenus ASST was determined. Plots of 1/v versus 1/[substrate (A)] at different fixed substrate (B) concentrations gave a series of parallel lines. One of the reaction products, p-nitrophenol, inhibited the enzyme noncompetitively with respect to p-nitrophenyl sulfate, but competitively to alpha-naphthol. These results correspond to a ping pong bi bi mechanism. By site-directed mutagenesis, we substituted each conserved tyrosine residue with phenylalanine. Among the mutants, Y123F showed severely reduced catalytic activity. We conclude that Tyr 123 is an essential active site residue. A mechanistic hypothesis is presented to account for these observations.

Cloning and sequencing of the astA gene encoding arylsulfate sulfotransferase from Salmonella typhimurium.

Arylsulfate sulfotransferase (ASST) transfers a sulfate group from a phenolic sulfate ester to a phenolic acceptor substrate. In the present study, the gene encoding ASST was cloned from a genomic library of Salmonella typhimurium. The gene was subcloned into the vector pKF3 and was sequenced. A recombinant clone harboring the gene was directly identified using a fluorescent assay. Sequencing revealed two contiguous open reading frames (ORFs) on the same strand. Based on amino acid sequence homology, ORF1 and ORF2 are designated as astA and dsbA, respectively. The deduced amino acid sequence of astA from S. typhimurium was highly similar to those of the Enterobacter amnigenus, Klebsiella, and Campylobacter jejuni ASSTs, encoded by the astA genes. However, an ASST activity assay revealed a different acceptor specificity. Using p-nitrophenyl sulfate (PNS) as a donor substrate, phenol is the best acceptor substrate, followed by alpha-naphthol, resorcinol, tyramine, acetaminophen, and tyrosine.

Bacterial arylsulfate sulfotransferase as a reporter system.

To investigate whether the arylsulfate sulfotransferase (ASST) is suitable as a reporter system for monitoring gene expression, a reporter vector carrying the fragments of the astA coding region without the promoter region was constructed and designated as pSY815. As a test of the ASST reporter system's suitability, the regulatory regions of ermC and lacZ were inserted upstream of the coding region of the reporter gene to generate pSY815-EC and pSY815-LZ, respectively. In the absence of the inserted regulatory regions, the plasmids displayed very low background activities in Bacillus subtilis and Escherichia coli. The ASST activity under the control of the ermC regulatory region was increased 4.4-fold in B. subtilis when induced by 0.1 microgml(-1) of erythromycin. These results were consistent with a lacZ reporter gene assay of the ermC regulatory region. Furthermore, we confirmed that the lacZ promoter in E. coli was strongly induced to a 17.9-fold increase by 0.05 mM of isopropyl-beta-D-thiogalactopyranoside (IPTG) in this reporter system. These results indicate that the ASST is a suitable reporter system. The lack of endogenous activity, the simple detection of enzyme activity in the living cell, the commercially available non-toxic substrates, and the high sensitivity make ASST a useful genetic reporter system for monitoring gene expression and understanding gene regulation.

Molecular cloning of the arylsulfate sulfotransferase gene and characterization of its product from Enterobacter amnigenus AR-37.

The gene encoding the Enterobacter amnigenus AR-37 arylsulfate sulfotransferase (ASST) was cloned, sequenced, and expressed in Escherichia coli NM522. Sequencing led to the identification of three contiguous open reading frames (ORFs) on the same strand. Based on amino acid sequence homology, ORF1, ORF2, and ORF3 are designated astA, dsbA, and dsbB, respectively. A multiple sequence alignment revealed conserved regions in ASST. An N-terminal amino acid sequence analysis of the purified ASST from E. coli NM522 (pEAST72) showed that it is subject to N-terminal processing. The specific activity of purified ASST is 436.5 U/mg of protein. The enzyme is a monomeric protein with a molecular mass of 64 kDa. Using phenol as an acceptor substrate, 4-methylumbelliferyl sulfate is the best donor substrate, followed by beta-naphthyl sulfate, p-nitrophenyl sulfate (PNS), and alpha-naphthyl sulfate. For PNS, alpha-naphthol is the best acceptor substrate, followed by phenol, resorcinol, p-acetaminophen, tyramine, and tyrosine. The enzyme has a different acceptor specificity than the enzyme purified from Eubacterium A-44. It is similar to Klebsiella K-36 and Haemophilus K-12. The apparent K(m) values for PNS using phenol as an acceptor and for phenol using PNS as a donor are 0.163 and 0.314 mM, respectively. The pI and optimum pH are 6.1 and 9.0, respectively.

Induction of ermAMR from a clinical strain of Enterococcus faecalis by 16-membered-ring macrolide antibiotics.

We cloned the MLSB resistance determinant by PCR from a clinical isolate of Enterococcus faecalis 373, which is induced more strongly by a 16-membered-ring macrolide, tylosin, than by erythromycin. To elucidate the molecular basis of resistance of E. faecalis 373, we analyzed the cloned gene, designated ermAMR, by site-directed mutagenesis and reporter gene assay. Our results showed that an arginine-to-cysteine change in the seventh codon of the putative leader peptide endowed tylosin with resistance inducibility and that TAAA duplication enabled the control region to express the downstream methylase gene at a drastically increased level.

Distribution of bacteria with the arylsulfate sulfotransferase activity.

This study is to predict the possible roles of the arylsulfate sulfotransferase (ASST) in the microorganism. At first we studied the spectrum of a distribution of the ASST enzyme through about 1,300 bacteria and the several selected strains were compared with Klebsiella K-36 previously reported in the level of DNA homology using the Southern blot method. From this study, we could predict that this enzyme would not exist in specific bacteria and it might not be a critical enzyme for the life of bacteria.