Measuring β‐Galactosidase Activity in Gram‐Positive Bacteria Using a Whole‐Cell Assay with MUG as a Fluorescent Reporter

Norman H. L. Chiu1, Amanda L. Watson2

1 Joint School of Nanoscience and Nanoengineering, University of North Carolina at Greensboro, Greensboro, 2 Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.44
DOI:  10.1002/cptx.35
Online Posting Date:  November, 2017
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Abstract

The use of β‐galactosidase enzyme as a biomarker has the potential to determine activity levels of the microbiome of a variety of organisms due to its common presence in both eukaryotes and prokaryotes. Completing the assay in a whole‐cell format facilitates the monitoring of β‐galactosidase activity in its actual cellular environment. This unit describes an optimized fluorescent assay for β‐galactosidase that has enough sensitivity to detect the enzymatic activity despite the thick gram‐positive bacterial cellular membrane. The use of a smaller fluorometric substrate, namely 4‐methylumbelliferyl β‐D‐galactopyranoside (MUG), has facilitated its penetration into the cells as well as its direct detection without any extra steps. This assay provides an improved technique for measuring a well‐studied reporter enzyme and offers new avenues for using β‐galactosidase as a biomarker. © 2017 by John Wiley & Sons, Inc.

Keywords: fluorescence; gram‐positive; MUG; whole‐cell assay; β‐galactosidase

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of β‐Galactosidase in Lactobacillus Bacteria Using 4‐Methylumbelliferyl β‐D‐Galactopyranoside (MUG) in a 96‐Well Plate
  • Support Protocol 1: Creating an Overnight Culture of Lactobacillus from Bacterial Stock
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Detection of β‐Galactosidase in Lactobacillus Bacteria Using 4‐Methylumbelliferyl β‐D‐Galactopyranoside (MUG) in a 96‐Well Plate

  Materials
  • Overnight culture of Lactobacillus helveticus (see protocol 2Support Protocol)
  • De Man, Rogosa, and Sharpe (MRS) medium (Microbiological grade; see recipe)
  • De Man, Rogosa, and Sharpe (MRS) medium with D‐lactose (see recipe)
  • D‐Lactose monohydrate >98% (HPLC grade)
  • L‐Cysteine, optional (Bioreagent for cell culture)
  • Z‐buffer (see recipe)
  • 2‐Mercaptoethanol (Molecular biology grade)
  • 4‐Methylumbelliferyl β‐D‐galactopyranoside (MUG), >99%
  • Dimethyl sulfoxide (DMSO)
  • 70% (v/v) ethanol solution
  • Autoclave
  • 13 × 100–mm glass culture tubes with lids (Pyrex)
  • Incubator with CO 2 regulation
  • CO 2 gas
  • 0.2‐micron filters
  • Visible spectrophotometer
  • Sterile micropipet tips, various sizes
  • Micropipets, various volumes
  • Sterile 1.5‐ml microcentrifuge tubes
  • Sterile 15.0‐ml centrifuge tubes
  • Benchtop fixed angle centrifuge, equipped for 1.5‐ and 15‐ml tubes
  • Fluorescence compatible microplate reader (POLARstar OPTIMA, BMG Labtech)
  • White polystyrene flat bottom microtiter plate, 96‐well format
  • Multichannel micropipet (optional)

Support Protocol 1: Creating an Overnight Culture of Lactobacillus from Bacterial Stock

  Additional Materials (also see protocol 1Basic Protocol)
  • Lactobacillus bacterial stock, frozen –70°C (ATCC)
  • Growing Lactobacillus bacteria
  • Sterile inoculating tool (optional)
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Figures

Videos

Literature Cited

Literature Cited
  Alam, J., & Cook, J. L. (1990). Reporter genes: Application to the study of mammalian gene transcription. Analytical Biochemistry, 188, 245–254. doi: 10.1016/0003‐2697(90)90601‐5.
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  Grange, J. M., & Clark, K. (1977). Use of umbelliferone derivatives in the study of enzyme activities of mycobacteria. Journal of Clinical Pathology, 30, 151–153. doi: 10.1136/jcp.30.2.151.
  Griffith, K. L., & Wolf, Jr R. E. (2002). Measuring β‐galactosidase activity in bacteria: Cell growth, permeabilization, and enzyme assays in 96‐well arrays. Biochemical and Biophysical Research Communications, 290, 397–402. doi: 10.1006/bbrc.2001.6152.
  Juers, D. H., Matthews, B. W., & Huber, R. E. (2012). LacZ β‐galactosidase: Structure and function of an enzyme of historical and molecular biological importance. Protein Science, 21, 1792–1807. doi: 10.1002/pro.2165.
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  Vidal‐Aroca, F., Giannattasio, M., Brunelli, E., Vezzoli, A., Plevani, P., Muzi‐Falconi, M., & Bertoni, G. (2006). One‐step high‐throughput assay for quantitative detection of β‐galactosidase activity in intact Gram‐negative bacteria, yeast, and mammalian cells. BioTechniques, 40, 433–440. doi: 10.2144/000112145.
  Watson, A. L., & Chiu, N. H. (2016). Fluorometric cell‐based assay for β‐galactosidase activity in probiotic gram‐positive bacterial cells. Lactobacillus Helveticus, 128, 58–60. doi: 10.1016/j.mimet.2016.06.030.
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