Identification of Proteins Possibly Involved in Glucosinolate Metabolism in L. agilis R16 and E. coli VL8
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This study was aimed to identify sinigrin-induced bacterial proteins potentially involved in the metabolism of glucosinolate in two glucosinolate-metabolising bacteria Lactobacillus agilis R16 and Escherichia coli VL8. Sinigrin (2 mM) was used to induce the proteins in both bacteria under anaerobic incubation for 8 h at 30 °C for L. agilis R16 and 37 °C for E. coli VL8 and the controls without sinigrin were performed. Allyl isothiocyanate and allyl nitrile as two degradation products of sinigrin were detected in sinigrin-induced cultures of L. agilis R16 (27 % total products) and E. coli VL8 (38 % total products) from a complete sinigrin degradation in 8 h for both bacteria. 2D gel electrophoresis was conducted to identify induced proteins with at least twofold increased abundance. Sinigrin-induced L. agilis R16 and the control produced 1561 and 1543 protein spots, respectively. For E. coli VL8, 1363 spots were detected in sinigrin-induced and 1354 spots in the control. A combination of distinct proteins and upregulated proteins of 32 and 35 spots in L. agilis R16 and E. coli VL8, respectively were detected upon sinigrin induction. Of these, 12 and 16 spots from each bacterium respectively were identified by LC–MS/MS. In both bacteria most of the identified proteins are involved in carbohydrate metabolism, oxidoreduction system and sugar transport while the minority belong to purine metabolism, hydrolysis, and proteolysis. This indicated that sinigrin induction led to the expressions of proteins with similar functions in both bacteria and these proteins may play a role in bacterial glucosinolate metabolism.
KeywordsGlucosinolate Gut bacteria Myrosinase Isothiocyanate Two-dimensional electrophoresis
L. agilis R16
E. coli VL8
Liquid chromatography mass spectrometry
The authors thank Llanos-Palop et al. (1995) for L. agilis R16 strain used in this work. The study was supported in part by a strategic programme grant to Institute of Food Research (Norwich) from the UK Biotechnology and Biological Sciences Research Council (BB/J004545/1).
Conflict of interest
The authors have declared no conflict of interest.
- 3.Beecher CWW (1994) Cancer preventive properties of varieties of Brassica oleracea: a review. Am J Clin Nutr 59:1166S–1170SGoogle Scholar
- 12.Kelleher MO, McMahon M, Eggleston IM, Dixon MJ, Taguchi K, Yamamoto M, Hayes JD (2009) 1-Cyano-2,3-epithiopropane is a novel plant-derived chemopreventive agent which induces cytoprotective genes that afford resistance against the genotoxic alpha, beta-unsaturated aldehyde acrolein. Carcinogenesis 30:1754–1762CrossRefGoogle Scholar
- 17.Saha S, Hollands W, Teucher B, Needs PW, Narbad A, Ortori CA, Barrett DA, Rossiter JT, Mithen RF, Kroon PA (2012) Isothiocyanate concentrations and interconversion of sulforaphane to erucin in human subjects after consumption of commercial frozen broccoli compared to fresh broccoli. Mol Nutr Food Res 56:1906–1916CrossRefGoogle Scholar
- 25.Luang-In V (2013) Influence of human gut microbiota on the metabolic fate of glucosinolates. PhD thesis, Imperial College LondonGoogle Scholar
- 26.Klose J (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues—novel approach to testing for induced point mutations in mammals. Humangenetik 26:231–243Google Scholar
- 30.Bergmeyer HU, Gawehn K, Grassl M (1974) In: Bergmeyer HU (ed) Methods of enzymatic analysis, 2nd edn. Academic Press Inc, New YorkGoogle Scholar
- 32.Bouwman F, Renes J, Mariman E (2004) A combination of protein profiling and isotopomer analysis using matrix-assisted laser desorption/ionization-time of flight mass spectrometry reveals an active metabolism of the extracellular matrix of 3T3-L1 adipocytes. Proteomics 4:3855–3863CrossRefGoogle Scholar
- 38.Delahunty C, Yates JR (2006) Proteomics: a shotgun approach without two-dimensional gels. Encyclopedia of Life Sciences (ELS). John Wiley & Sons, Ltd, Chichester. doi: 10.1038/npg.els.0006197
- 40.Gower WR, Carr MC, Ives DH (1979) Deoxyguanosine kinase—distinct molecular-forms in mitochondria and cytosol. J Biol Chem 254:2180–2183Google Scholar
- 42.Jakobsen TH, Bragason SK, Phipps RK, Christensen LD, van Gennip M, Alhede M, Skindersoe M, Larsen TO, Hoiby N, Bjarnsholt T, Givskov M (2012) Food as a source for quorum sensing inhibitors: iberin from horseradish revealed as a quorum sensing inhibitor of Pseudomonas aeruginosa. Appl Environ Microbiol 78:2410–2421CrossRefGoogle Scholar
- 49.Witt E, Frank R, Hengstenberg W (1993) 6-Phospho-beta-galactosidases of gram-positive and 6-phospho-beta-glucosidase-b of gram-negative bacteria—comparison of structure and function by kinetic and immunological methods and mutagenesis of the lacG gene of Staphylococcus aureus. Protein Eng 6:913–920CrossRefGoogle Scholar