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Journal of Bacteriology, April 2006, p. 3099-3109, Vol. 188, No. 8
0021-9193/06/$08.00+0     doi:10.1128/JB.188.8.3099-3109.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Effects of Phosphorelay Perturbations on Architecture, Sporulation, and Spore Resistance in Biofilms of Bacillus subtilis{ddagger}

Jan-Willem Veening,1,{dagger} Oscar P. Kuipers,1 Stanley Brul,2,3 Klaas J. Hellingwerf,4 and Remco Kort4,{dagger}*

Molecular Genetics Group, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Kerklaan 30, 9751 NN Haren, The Netherlands,1 Laboratory for Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands,2 Advanced Food Microbiology, Unilever Food and Health Research Institute, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands,3 Laboratory for Microbiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands4

Received 10 November 2005/ Accepted 31 January 2006

The spore-forming bacterium Bacillus subtilis is able to form highly organized multicellular communities called biofilms. This coordinated bacterial behavior is often lost in domesticated or laboratory strains as a result of planktonic growth in rich media for many generations. However, we show here that the laboratory strain B. subtilis 168 is still capable of forming spatially organized multicellular communities on minimal medium agar plates, exemplified by colonies with vein-like structures formed by elevated bundles of cells. In line with the current model for biofilm formation, we demonstrate that overproduction of the phosphorelay components KinA and Spo0A stimulates bundle formation, while overproduction of the transition state regulators AbrB and SinR leads to repression of formation of elevated bundles. Time-lapse fluorescence microscopy studies of B. subtilis green fluorescent protein reporter strains show that bundles are preferential sites for spore formation and that flat structures surrounding the bundles contain vegetative cells. The elevated bundle structures are formed prior to sporulation, in agreement with a genetic developmental program in which these processes are sequentially activated. Perturbations of the phosphorelay by disruption and overexpression of genes that lead to an increased tendency to sporulate result in the segregation of sporulation mutations and decreased heat resistance of spores in biofilms. These results stress the importance of a balanced control of the phosphorelay for biofilm and spore development.

* Corresponding author. Mailing address: Laboratory for Microbiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands. Phone: 31 20 5257062. Fax: 31 20 525 7056. E-mail: rkort{at}science.uva.nl.

{ddagger} Supplemental material for this article may be found at http://jb.asm.org/.

{dagger} Both authors contributed equally to this work.

Journal of Bacteriology, April 2006, p. 3099-3109, Vol. 188, No. 8
0021-9193/06/$08.00+0     doi:10.1128/JB.188.8.3099-3109.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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