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Applied and Environmental Microbiology, May 2007, p. 3061-3068, Vol. 73, No. 9
0099-2240/07/$08.00+0     doi:10.1128/AEM.02296-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Dynamics of Genomic-Library Enrichment and Identification of Solvent Tolerance Genes for Clostridium acetobutylicum{triangledown} ,{dagger}

Jacob R. Borden and Eleftherios Terry Papoutsakis*

Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208

Received 28 September 2006/ Accepted 22 February 2007

A Clostridium acetobutylicum ATCC 824 genomic library was constructed using randomly sheared DNA. Library inserts conferring increased tolerance to 1-butanol were isolated using two protocols. Protocol I utilized a single round of butanol challenges in batch culture, while protocol II, which gave clearly superior outcomes, was based on the serial transfer of stationary-phase cultures into progressively higher butanol concentrations. DNA microarray analysis made a high-resolution assessment of the dynamic process of library enrichment possible for the first time. Protocol I yielded a library insert containing the entire coding region of the gene CAC0003 (which codes for a protein of unknown function) but also several DNA fragments containing promoter regions. Protocol II enabled the successful identification of DNA fragments containing several intact genes conferring preferential growth under conditions of butanol stress. Since expression using the employed library is possible only from natural promoters, among the enriched genes, we identified 16 genes that constitute the first cistron of a transcriptional unit. These genes include four transcriptional regulators (CAC0977, CAC1463, CAC1869, and CAC2495). After subcloning plasmids carrying the CAC0003 and CAC1869 genes, strains 824(pCAC0003) and 824(pCAC1869) exhibited 13% and an 81% increases, respectively, in butanol tolerance relative to the plasmid control strain. 824(pCAC1869) consistently grew to higher cell densities in challenged and unchallenged cultures and exhibited prolonged metabolism. Our serial enrichment approach provided a more detailed understanding of the dynamic process of library enrichment under conditions of selective growth. Further characterization of the genes identified in this study will likely enhance our understanding of the complex phenotype of solvent tolerance.

* Corresponding author. Mailing address: Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208. Phone: (847) 491-7455. Fax: (847) 491-3728. E-mail: e-paps{at}northwestern.edu

{triangledown} Published ahead of print on 2 March 2007.

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

Applied and Environmental Microbiology, May 2007, p. 3061-3068, Vol. 73, No. 9
0099-2240/07/$08.00+0     doi:10.1128/AEM.02296-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Connor, M. R., Liao, J. C. (2008). Engineering of an Escherichia coli Strain for the Production of 3-Methyl-1-Butanol. Appl. Environ. Microbiol. 74: 5769-5775 [Abstract] [Full Text]  


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