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Applied and Environmental Microbiology, June 2002, p. 2770-2780, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.2770-2780.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Proteomic Analysis Reveals Differential Protein Expression by Bacillus cereus during Biofilm Formation

Marinda C. Oosthuizen,1,2* Bridgitta Steyn,2 Jacques Theron,2 Pascal Cosette,3 Denise Lindsay,1 Alexander von Holy,1 and Volker S. Brözel2

School of Molecular and Cell Biology, University of the Witwatersrand, Wits, 2050,1 Laboratory for Biofilm Physiology, Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, 0001, South Africa,2 UMR 6522 CNRS, Université de Rouen, Faculté des Sciences, 76821 Mont-Saint-Aignan Cedex, France3

Received 3 December 2001/ Accepted 1 March 2002

Bacillus cereus, a dairy-associated toxigenic bacterium, readily forms biofilms on various surfaces and was used to gain a better understanding of biofilm development by gram-positive aerobic rods. B. cereus DL5 was shown to readily adapt to an attached mode of growth, with dense biofilm structures developing within 18 h after inoculation when glass wool was used as a surface. Two-dimensional gel electrophoresis (2DE) revealed distinct and reproducible phenotypic differences between 2- and 18-h-old biofilm and planktonic cells (grown both in the presence and in the absence of glass wool). Whereas the 2-h-old biofilm proteome indicated expression of 15 unique proteins, the 18-h-old biofilm proteome contained 7 uniquely expressed proteins. Differences between the microcolony (2-h) proteome and the more developed biofilm (18-h) proteome were largely due to up- and down-regulation of the expression of a multitude of proteins. Selected protein spots excised from 2DE gels were subjected to N-terminal sequencing and identified with high confidence. Among the proteins were catabolic ornithine carbamoyltransferase and L-lactate dehydrogenase. Interestingly, increased levels of YhbH, a member of the sigma 54 modulation protein family which is strongly induced in response to environmental stresses and energy depletion via both {sigma}B and {sigma}H, could be observed within 2 h in both attached cells and planktonic cultures growing in the presence of glass wool, indicating that this protein plays an important role in regulation of the biofilm phenotype. Distinct band differences were also found between the extracellular proteins of 18-h-old cultures grown in the presence and in the absence of glass wool.

* Corresponding author. Mailing address: Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, 0002, South Africa. Phone: 0027 12 420 4430. Fax: 0027 12 420 3266. E-mail: mcoosthuizen{at}excite.com.

Applied and Environmental Microbiology, June 2002, p. 2770-2780, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.2770-2780.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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