Cloning, sequence analysis, and characterization of theastA gene encoding an Arylsulfate sulfotransferase fromCitrobacter freundii

Abstract

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.

Transcriptional Regulation of the assT - dsbL - dsbI Gene Cluster in Salmonella enterica Serovar Typhi IMSS-1 Depends on LeuO, H-NS, and Specific Growth Conditions

“…Neither promoter exhibited activation in the hns mutant, in accord with the expression values from Table 1. Interestingly, P1-L contains a recognition sequence for 32 , which is involved in heat shock stress (13); however, the promoter was activated in wild-type pFMTrc12 and in STYhns99 backgrounds in the absence of a heat shock induction. On the other hand, 32 induction has been detected inside the macrophage during Salmonella Typhimurium infection (20), a condition that should be explored in future studies.…”

“…However, it cannot be excluded that the assT-dsbL-dsbI gene cluster is also transcribed as an operon in N-minimal medium. P1-L contains a putative 32 promoter sequence, whereas P2-L is very likely a 70 promoter (7,13). In this respect, it is interesting that no heat shock stress was utilized for dsbL expression; however, growth in N-minimal medium could be an artificial stress signal for the dsbL activation under 32 , since rpoH activation has been detected when Salmonella is inside macrophages (20).…”

“…P1-L contains a putative 32 promoter sequence, whereas P2-L is very likely a 70 promoter (7,13). In this respect, it is interesting that no heat shock stress was utilized for dsbL expression; however, growth in N-minimal medium could be an artificial stress signal for the dsbL activation under 32 , since rpoH activation has been detected when Salmonella is inside macrophages (20). N-minimal medium, which contains low concentrations of magnesium and phosphate, has been utilized to activate virulence gene expression in Salmonella pathogenicity island 2 (SPI-2), which is preferentially expressed inside the macrophage when Salmonella replicates in the vacuole (1,16,17,20).…”

“…These sulfotransferases use 3=-phosphoadenosine-5=-phosphosulfate (PAPS) as sulfate donor and are involved in a wide variety of biological processes such as cell communication, growth, development, defense, and detoxification (6,19,43,63,65). Unlike mammals, bacterial sulfotransferases are periplasmic enzymes that require phenolic sulfate esters as donor substrates, and it has been suggested that the intestinal microflora uses this enzyme to detoxify phenolic compounds (31,32,34,35).In Salmonella enterica serovar Typhi, the assT gene is located upstream of dsbL and dsbI, a couple of genes encoding a reduction-oxidation (red-ox) pair of proteins involved in the disulfide bond formation required for AssT activation in the periplasm (25,40,44). The nomenclature was adopted from the orthologous genes previously described in uropathogenic Escherichia coli (UPEC) and Salmonella enterica serovar Typhimurium (25,40).…”

“…These sulfotransferases use 3=-phosphoadenosine-5=-phosphosulfate (PAPS) as sulfate donor and are involved in a wide variety of biological processes such as cell communication, growth, development, defense, and detoxification (6,19,43,63,65). Unlike mammals, bacterial sulfotransferases are periplasmic enzymes that require phenolic sulfate esters as donor substrates, and it has been suggested that the intestinal microflora uses this enzyme to detoxify phenolic compounds (31,32,34,35).…”