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Investigating the Link between Changes in Surface Characteristics and Protein Expression for Aggregated and Non-Aggregated Bacillus Cereus

Jillian R. A. Newton1, Kevin Eboigbodin1, Alexander F. Routh2, and Catherine A. Biggs1. (1) Chemical and Process Engineering, The University of Sheffield, Mappin Street, Sheffield, United Kingdom, (2) BP Institute, University of Cambridge, Madingley Rise, Madingley Road, Cambridge, United Kingdom

Bacillus cereus is a toxigenic bacterium, which readily forms biofilms and aggregates in solution and as such was used in this study of bacterial aggregation in gram-positive organisms.  The proteomes of both aggregated and non-aggregated cells were analysed and compared to the cell surface charge in order to gain an understanding of the biological and physical properties surrounding bacterial aggregates.  Cultures were grown in Luria-Bertani (LB) media with and without the addition of 0.5% glucose.  Autoaggregation was observed in glucose free cultures at 24h, whereas glucose rich cultures remained in the planktonic state.  The bacterial surface electrokinetic properties from different growth phases for both non-aggregated and aggregated cells were determined by measuring the electrophoretic mobility using a phase amplitude light scattering (PALS) Zeta potential analyser (Brookhaven Zeta PALS, UK).  Changes in electrophoretic mobility during growth phase were found for both aggregated and non aggregated cells.  The two modes of growth followed a similar decrease in magnitude of the electrophoretic mobility during the growth phase until 24 hours (point of observed autoaggregation).  For the aggregated cells this decrease in electrophoretic mobility then remained constant after 24h, however, for the non-aggregated cells, after 24h the mobility increased in magnitude.  To investigate the differences in electrophoretic mobility between aggregated and non aggregated cells further, conventional proteomics using large format 2-D electrophoresis, in triplicate with narrow range (pH 4-7) IEF strips and 10% SDS-PAGE gels was conducted and found to underline characteristic differences between the two modes of growth.  Comparison of the 2DE profiles and examination of staining intensities using PDQuest 7.4.0 gel analysis software showed a semi-quantitative up-regulation of 26 proteins in aggregated cells when compared to the non-aggregated cells, which showed an up-regulation of only 5 proteins.  A selection of the most reproducible and abundant spots were selected for MS fingerprinting and protein sequencing by MS/MS.  Protein identification was performed using the Mascot database and results included an up-regulation of flagellin in aggregated cells and up regulation of several internal (e.g. glyceraldehyde-3-phosphate dehydrogenase) and membrane (e.g phosphoglycerate kinase) glycolytic enzymes in the aggregated cells.  Flagellin is a subunit which polymerises to form the filament of flagella.  The role of flagella in biofilm formation has been linked to initial surface adhesion, generation of force in order to overcome bacterial repulsion and promotion of bacterial spread over surfaces.  Glycolytic enzymes have also been shown to have a secondary function in the adhesion of biofilms.  Therefore there are distinct differences between aggregated and non aggregated cells which have been observed through physico-chemical characteristics such as surface charge, and biochemical processes such as protein expression.  Further work will be directed at finding the biological link between the two processes.