Characterization of an acid-inducible sulfatase in Salmonella enterica serovar typhimurium

Abstract

Sulfatases of enteric bacteria can provide access to heavily sulfated mucosal glycans. In this study, we show that aslA (STM0084) of Salmonella enterica serovar Typhimurium LT2 encodes a sulfatase that requires mildly acidic pH for its expression and activity. AslA is not regulated by sulfur compounds or tyramine but requires the EnvZ-OmpR and PhoPQ regulatory systems, which play an important role in pathogenesis.

Fig 1

(A) Sulfatase activity of S. Typhimurium (ST) and E. coli (EC) as determined by hydrolysis of X-sulfate (blue coloring) on MOPS minimal media adjusted to pH 5.5 or pH 7.0. S. Typhimurium shows activity at pH 5.5, but not at pH 7.0. No activity was detected in E. coli. (B) Sulfatase activity was detected only in cells grown at pH 5.5 and assayed at pH 5.5. The enzyme activity was determined by hydrolysis of p-nitrophenyl sulfate using whole cells permeabilized with chloroform and is expressed as Miller units (1 unit = 1,000 × A₄₂₀ − (1.75 × A₅₅₀)/(T × V × A₆₀₀), where T is the time of reaction and V is the volume of the culture assayed (27). The results are the means ± standard deviations of at least 3 independent experiments.

Fig 2

AslA is regulated by the PhoPQ and EnvZ-OmpR regulatory systems. (A) The sulfatase activity was reduced 2-fold in mutants lacking phoPQ and was reduced 50-fold in mutants lacking envZ-ompR compared to the wild type. Mutants affected in cadC, adiY, and rpoS had activity similar to the wild type. (B) Complementation by plasmid-based ompR resulted in restoration of sulfatase activity in the ompR mutant but was significantly less in the envZ, phoP, and phoQ mutants. *, P < 0.01; **, P < 0.001. Sulfatase was assayed by quantifying the release of p-nitrophenol from p-nitrophenyl sulfate using whole cells permeabilized with chloroform and expressed as Miller units (27). The results are the means ± standard deviations of at least 3 independent experiments. No activity was detected in the ompR mutant containing only the plasmid vector (VC).

Fig 3

Analysis of aslA expression by RT-PCR. aslA was not expressed in cells grown at pH 7.0 (lane 1). The expression was induced when cells were shifted to pH 5.5 (lane 2). No expression was detected in the ompR mutant (lane 3). Addition of chloramphenicol after shifting the wild-type cells to pH 5.5 led to inhibition of aslA expression (lane 4). Expression of recA was used as a control.