Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2017

Improved Cell Surface Protein Recovery in Lactobacillus casei GCRL163 under Heat Adaptations (#203)

kayode Titus ADU 1 , John Bowman 1 , Anthony Baker 1 , Margaret Britz 1 2
  1. University of Tasmania, Hobart, TASMANIA, Australia
  2. Faculty of Veterinary and Agricultural Science, University of Melbourne, Melbourne, Victoria, Australia

Lactobacillus casei GCRL163 possess fewer cell surface proteins in comparison to other lactobacilli. Procedures for consistent surface protein extraction from such bacteria are poorly documented. We have used successfully ethanol precipitation as a more rapid, efficient and affordable protocol to recover cell surface proteins rather than the conventional desalting and osmotic-based concentrating methods following LiCl treatments. Degree of cell lysis was determined by quantifying DNA release using fragment analyser and membrane integrity probed by flow cytometry. Our results revealed a statistically validated list of 699 proteins identified using label-free quantitative proteomics in the LiCl extracts suggesting cell lysis at 35oC (28.85%) and 45oC (39%). Supplementing LiCl with sucrose reduced cell lysis by 7.95% at 35oC. Extracting the surface-associated proteins by Tris-trypsin shaving resulted in minimal cell lysis. Further, we demonstrated that irrespective of the methods used or culturing temperatures, cell lysis occurred in untreated cells at mid-log phase. However cells were more susceptible to lysis at higher temperature with or without treatment.

This study provides insights into identification of surface proteins and strategies of extracting surface proteins with minimal artefact contaminants from the intracellular proteome of lactobacilli.