Advertisement

Determining the Localization of Carbohydrate Active Enzymes Within Gram-Negative Bacteria

  • Richard McLean
  • G. Douglas Inglis
  • Steven C. Mosimann
  • Richard R. E. Uwiera
  • D. Wade Abbott
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1588)

Abstract

Investigating the subcellular location of secreted proteins is valuable for illuminating their biological function. Although several bioinformatics programs currently exist to predict the destination of a trafficked protein using its signal peptide sequence, these programs have limited accuracy and often require experimental validation. Here, we present a systematic method to fractionate gram-negative cells and characterize the subcellular localization of secreted carbohydrate active enzymes (CAZymes). This method involves four parallel approaches that reveal the relative abundance of protein within the cytoplasm, periplasm, outer membrane, and extracellular environment. Cytoplasmic and periplasmic proteins are fractionated by lysis and osmotic shock, respectively. Outer membrane bound proteins are determined by comparing cells before and after exoproteolytic digestion. Extracellularly secreted proteins are collected from the media and concentrated. These four different fractionations can then be probed for the presence and quantity of target proteins using immunochemical methods such as Western blots and ELISAs, or enzyme activity assays.

Key words

Signal peptideSecretion Subcellular localization Cell fractionation Osmotic shock Whole cell dot blot Gram-negative bacteria 

Notes

Acknowledgments

This work was supported by an Alberta Innovates BioSolutions grant #BIOFS026 and Agriculture and Agri-Food Canada.

References

  1. 1.
    Pfeffer SR, Rothman JE (1987) Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem 56(1):829–852CrossRefPubMedGoogle Scholar
  2. 2.
    Bernstein HD, Poritz MA, Strub K, Hoben PJ, Brenner S, Walter P (1989) Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle. Nature 340(6233):482–486CrossRefPubMedGoogle Scholar
  3. 3.
    Bernstein HD, Zopf D, Freymann DM, Walter P (1993) Functional substitution of the signal recognition particle 54-kDa subunit by its Escherichia coli homolog. Proc Natl Acad Sci 90(11):5229–5233CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Ivankov DN, Payne SH, Galperin MY, Bonissone S, Pevzner PA, Frishman D (2013) How many signal peptides are there in bacteria? Environ Microbiol 15(4):983–990CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8(10):785–786CrossRefPubMedGoogle Scholar
  6. 6.
    Thein M, Sauer G, Paramasivam N, Grin I, Linke D (2010) Efficient subfractionation of gram-negative bacteria for proteomics studies. J Proteome Res 9(12):6135–6147CrossRefPubMedGoogle Scholar
  7. 7.
    Feliu JX, Villaverde A (1994) An optimized ultrasonication protocol for bacterial cell disruption and recovery of β-galactosidase fusion proteins. Biotechnol Tech 8(7):509–514CrossRefGoogle Scholar
  8. 8.
    Jaschke PR, Drake I, Beatty JT (2009) Modification of a French pressure cell to improve microbial cell disruption. Photosynth Res 102(1):95–97CrossRefPubMedGoogle Scholar
  9. 9.
    Neu HC, Heppel LA (1965) The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem 240(9):3685–3692PubMedGoogle Scholar
  10. 10.
    Nossal NG, Heppel LA (1966) The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem 241(13):3055–3062PubMedGoogle Scholar
  11. 11.
    Sharp JA, Echague CG, Hair PS, Ward MD, Nyalwidhe JO, Geoghegan JA, Foster TJ, Cunnion KM (2012) Staphylococcus aureus surface protein SdrE binds complement regulator factor H as an immune evasion tactic. PLoS One 7(5):e38407CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Cuskin F, Lowe EC, Temple MJ, Zhu Y, Cameron EA, Pudlo NA et al (2015) Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism. Nature 517(7533):165–169CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Rogowski A, Briggs JA, Mortimer JC, Tryfona T, Terrapon N, Lowe EC et al (2015) Glycan complexity dictates microbial resource allocation in the large intestine. Nat Commun 6(7481)Google Scholar
  14. 14.
    Schein CH (1989) Production of soluble recombinant proteins in bacteria. Nat Biotechnol 7(11):1141–1149CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Richard McLean
    • 1
    • 2
  • G. Douglas Inglis
    • 2
  • Steven C. Mosimann
    • 2
  • Richard R. E. Uwiera
    • 3
  • D. Wade Abbott
    • 1
    • 2
  1. 1.Functional Genomics of Complex Carbohydrate Utilization, Lethbridge Research and Development CentreAgriculture and Agri-Food CanadaLethbridgeCanada
  2. 2.Department of Chemistry and BiochemistryUniversity of LethbridgeLethbridgeCanada
  3. 3.Department of Agricultural, Food and Nutritional ScienceUniversity of AlbertaEdmontonCanada

Personalised recommendations