Dynamics of genomic-library enrichment and identification of solvent tolerance genes for Clostridium acetobutylicum

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.

FIG. 1.

Enrichment of tolerance gene fragments in (A) biological replicate 1 and (B) biological replicate 2 of stationary-phase transfer challenges (protocol II). Each lane contains the products of 20 PCR cycles using 0.1 μl of C. acetobutylicum pLib1 minipreps of the indicated challenge culture. Lanes 1 and 7, φX174-HaeIII standard; lanes 2, 3, 4, 5, and 6, PCR products from the 1st, 4th, 7th, 10th, and 13th stationary-phase transfers of the indicated replicate challenge flask.

FIG. 2.

Physical map of the C. acetobutylicum genome color-coded with the degree of fragment enrichment as determined by DNA microarray analysis for (A) biological replicate 1 and (B) biological replicate 2 of stationary-phase transfer challenges (protocol II). Plasmid library insert DNA isolated from the initial challenge inoculum was PCR amplified, Cy3 labeled, and hybridized against Cy5-labeled, amplified library insert DNA from the 4th, 7th, 10th, and 13th challenge transfers. After subtraction of background and nonspecific hybridization signals and averaging of replicate spots, gene signal intensities were ranked within a given challenge transfer. Concentric circles represent (starting with the innermost circle) the ranks of individual genes from the initial inoculum and 4th, 7th, 10th, and 13th transfers, respectively. Genes were color-coded according to their percentile rank in a given transfer, such that the top 5% are red, 5 to 33% are green, and 33 to 100% are gray. Genes that did not generate a signal-to-noise ratio greater than 3 on a given array are white.

FIG. 3.

MIC assays of strains 824(pCAC1869) (), 824(pIMP1) (), and 824(pCAC0003) (□). Growth measurements were used to calculate the percent tolerance at each challenge level for 824(pCAC1869) (□), 824(pIMP1) (○), and 824(pCAC0003) (▵). (A) Inoculation of a dilution series of butanol in CGM. (B) Butanol challenge of actively growing cultures (A₆₀₀ of ∼1.0). Growth data for 12 h postchallenge are displayed. Inset graphs show the percent relative tolerance (% RT) to butanol for library strains relative to the pIMP1 control strain. See the text for details.