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Applied and Environmental Microbiology, December 2008, p. 7497-7506, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01626-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Development and Application of Flow-Cytometric Techniques for Analyzing and Sorting Endospore-Forming Clostridia{triangledown} ,{dagger}

Bryan P. Tracy,1 Stefan M. Gaida,2 and Eleftherios T. Papoutsakis3,4*

Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208-3120,1 Department of Bioprocess Engineering, Institute of Biotechnology, Technical University of Berlin, Berlin, Germany,2 Department of Chemical Engineering, Colburn Laboratory, University of Delaware, Newark, Delaware 19716,3 Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 197114

Received 15 July 2008/ Accepted 7 October 2008

The study of microbial heterogeneity at the single-cell level is a rapidly growing area of research in microbiology and biotechnology due to its significance in pathogenesis, environmental biology, and industrial biotechnologies. However, the tools available for efficiently and precisely probing such heterogeneity are limited for most bacteria. Here we describe the development and application of flow-cytometric (FC) and fluorescence-assisted cell-sorting techniques for the study of endospore-forming bacteria. We show that by combining FC light scattering (LS) with nucleic acid staining, we can discriminate, quantify, and enrich all sporulation-associated morphologies exhibited by the endospore-forming anaerobe Clostridium acetobutylicum. Using FC LS analysis, we quantitatively show that clostridial cultures commonly perform multiple rounds of sporulation and that sporulation is induced earlier by the overexpression of Spo0A, the master regulator of endospore formers. To further demonstrate the power of our approach, we employed FC LS analysis to generate compelling evidence to challenge the long-accepted view in the field that the clostridial cell form is the solvent-forming phenotype.

* Corresponding author. Mailing address: 15 Innovation Way, Newark, DE 19711. Phone: (302) 831-8376. Fax: (302) 831-7090. E-mail: Papoutsakis{at}dbi.udel.edu

{triangledown} Published ahead of print on 17 October 2008.

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

Applied and Environmental Microbiology, December 2008, p. 7497-7506, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01626-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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