Detection of bacterial sulfatase activity through liquid- and solid-phase colony-based assays

Bacterial arylsulfatases are crucial to biosynthesis in many microorganisms, as bacteria often utilize aryl sulfates as a source of sulfur. The bacterial sulfatases are associated with pathogenesis and are applied in many areas such as industry and agriculture. We developed an activity-based probe 1 for detection of bacterial sulfatase activity through liquid- and solid-phase colony-based assays. Probe 1 is hydrolyzed by sulfatase to generate fluorescent N-methyl isoindole, which is polymerized to form colored precipitates. These fluorescent and colorimetric properties of probe 1 induced upon treatment of sulfatases were successfully utilized for liquid-phase sulfatase activity assays for colonies and lysates of Klebsiella aerogenes, Mycobacterium avium and Mycobacterium smegmatis. In addition, probe 1 allowed solid-phase colony-based assays of K. aerogenes through the formation of insoluble colored precipitates, thus enabling accurate staining of target colonies under heterogeneous conditions. Electronic supplementary material The online version of this article (doi:10.1186/s13568-017-0449-3) contains supplementary material, which is available to authorized users.

Compound 4: To a suspension of lithium aluminum hydride (250 mg, 2 equiv.) in anhydrous THF was added 3 (900 mg, 4.3 mmol) dissolved in anhydrous THF at 0℃ slowly under N 2 . The reaction mixture was stirred at room temperature for 5 h. Then, the solution was cooled down to 0℃ and quenched by 1 M aqueous NaOH solution. The mixture was dried with Na 2 SO 4 and filtered through a pad of Celite. The filtrate was concentrated in vacuo. The reduction product was used for the next synthetic step without further purification.

Compound 6:
To a solution of 4 (540 mg, 2.98 mmol) in MeOH was added 2M solution of methylamine in THF (4 ml, 2 equiv.). The resulting mixture was stirred at room temperature overnight, cooled down to 0℃, and treated with sodium borohydride (570 mg, 5 equiv). The mixture was stirred for 1 h and then, quenched by water. After methanol was removed in vacuo, the residue was dissolved in ethyl acetate and washed with aqueous NaHCO 3 and brine 3 times, dried over Na 2 SO 4 and concentrated in vacuo. Product 5 was used for the next synthetic step without further purification (219 mg).

2-2. Lysis
The fully-grown bacterial culture was centrifuged at 3300 rpm for 10 minutes. The collected pellets were washed three times with distilled water and then re-suspended in 50 mM Tris-buffer (pH 7.4) with a final OD 600 of 3.0. To obtain lysate of the bacteria, 300 µl of the re-suspension was added to a 1.5 ml sonication tube and then sonicated for 5 minutes. After the sonication, centrifuge was applied and the supernatant was transferred to a new tube. The Bradford method was used to determine the protein concentration of the lysate.

3-1. Optical response of probe 1 by purified sulfatases
Biochemical activity assays with H. Pomatia sulfatase (Sigma, S9226) and A. aerogenes sulfatase (Sigma, S1629) were carried out in 96-well plates with the total volume of each plate being 250 μl at 37℃ and pH 7.4. Stock solution of H. pomatia sulfatases was prepared to be 0.625 mg/ml in 50 mM Tris buffer (500 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , pH 7.4) and stock solution of probe 1 was prepared to be 100 mM in DMSO. Then, 1 mM probe and various amounts of sulfatase in 50 mM Tris buffer were used. Fluorescence intensity (λ ex =327 nm, λ em =415 nm) was measured in a time-dependent manner (0 -165 min.). For the UV absorbance studies, 1 mM probe 1 with various amounts of sulfatase was incubated at 37℃. Stock solution of A. aerogenes sulfatase was prepared to be 0.05 mg/ml in 50 mM Tris buffer (500 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , pH 7.4).

3-2. Enzyme kinetics and limits of detection
Kinetic experiments of probe 1 with A. aerogenes (AA) sulfatase were carried out at 37 °C in 50 mM Tris buffer (pH 7.48). The fluorescence intensities of a series of different concentrations of probe 1 (0, 10, 20, 50, 100, 200, 500, 1000 and 2000 μM) with AA sulfatase (0.02 mg/mL) in a 96-well black bottom plate were measured in a time-dependent manner. The rate of increase in fluorescence intensity at 415 nm was used to determine the kinetic parameters of enzyme hydrolysis. The kinetic parameters (K M and V max ) were determined from triple-reciprocal plots of hydrolysis rates versus substrate concentration (Michaelis-Menten plot).