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Dynamics of Genomic-Library Enrichment and Identification of Solvent Tolerance Genes for Clostridium acetobutylicum

Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA.
Applied and Environmental Microbiology (Impact Factor: 3.95). 06/2007; 73(9):3061-8. DOI: 10.1128/AEM.02296-06
Source: PubMed

ABSTRACT 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.

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    • "Other growth kinetics parameters, ''percentage of tolerance'' and ''relative tolerance (RT)'', were calculated using Eqs. (4) and (5), respectively (Borden and Papoutsakis 2007). These parameters were calculated using the measured growth after a period of time. "
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    • "The same level of information is not available concerning anaerobic bacteria. Certain species such as Clostridium acetobutylicum and Zymomonas mobilis employed in the production of solvents and biofuels have been studied in detail (10, 45); however, very few data exist regarding the solvent tolerance of other anaerobic species important in CAH bioremediation settings. Because CAH-contaminated environments such as soil and aquifers are often complex and inhabited by a variety of species, it is useful to not only understand how solvents influence the species targeted as the catalyst of remediation but also co-existing species which may modulate bioremediation outcomes through mutualism or competition. "
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    • "The relative growth rates were presented as the cell densities measured at a wavelength of 600 nm by spectrophotometer (GE Healthcare Life Sciences "GeneQuant 1300). Densities of toxin-treated cultures were normalized by the density of their respective toxin-free controls under otherwise same growth conditions [42]. From each tolerance assay, percent tolerance relative to unchallenged cultures was estimated at each challenge level and sample time as follows: "
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