Genome-wide screening for trait conferring genes using DNA microarrays

Abstract

We report a DNA microarray-based method for genome-wide monitoring of competitively grown transformants to identify genes whose overexpression confers a specific cellular phenotype. Whereas transcriptional profiling identifies differentially expressed genes that are correlated with particular aspects of the cellular phenotype, this functional genomics approach determines genes that result in a specific physiology. This parallel gene-trait mapping method consists of transforming a strain with a genomic library, enriching the cell population in transformants containing the trait conferring gene(s), and finally using DNA microarrays to simultaneously isolate and identify the enriched gene inserts. Various methods of enrichment can be used; here, genes conferring low-level antibiotic resistance were identified by growth in selective media. We demonstrated the method by transforming Escherichia coli cells with a genomic E. coli library and selecting for transformants exhibiting a growth advantage in the presence of the anti-microbial agent Pine-Sol. Genes conferring Pine-Sol tolerance (19 genes) or sensitivity (27 genes) were identified by hybridizing, on DNA microarrays containing 1,160 E. coli gene probes, extra-chromosomal DNA isolated from transformed cells grown in the presence of various levels of Pine-Sol. Results were further validated by plating and sequencing of individual colonies, and also by assessing the Pine-Sol resistance of cells transformed with enriched plasmid library or individual resistance genes identified by the microarrays. Applications of this method beyond antibiotic resistance include identification of genes resulting in resistance to chemotherapeutic agents, genes yielding resistance to toxic products (recombinant proteins, chemical feedstocks) in industrial fermentations, genes providing enhanced growth in cell culture or high cell density fermentations, genes facilitating growth on unconventional substrates, and others.

Figure 1

PGTM. E. coli MG1655 genomic DNA was fragmented, size selected, repaired, and ligated into TOPO-TA cloning vector. E. coli DH5α were transformed with the ligation reaction, successful transformants were harvested, and their plasmids were purified. This library was used as the starting pool for all subsequent transformations. To perform the dynamic screen, chemically competent DH5α were transformed with the library and grown immediately in LB + Ampicillin (LBA) until an OD₆₀₀ of 0.1–0.2 (≈5 h), at which point they were inoculated (at 1% vol/vol) into selective (0.1–2.5% vol/vol Pine-Sol) and nonselective LBA. Plasmid samples were obtained throughout growth, fragmented by sonication, labeled with Cy3, and mixed with genomic E. coli DNA labeled with Cy5. The mixture was hybridized to an E. coli DNA microarray containing 1,160 gene probes. Differences among Cy3 intensities reflect the enrichment for specific transformant subpopulations out of the heterogeneous transformant library. Transformants that contain inserts that provide a growth advantage (green) will become the majority members of the total plasmid population at the expense of transformants that contain inserts that either do not provide a growth advantage (yellow) or that provide a growth disadvantage (red). In the example bar charts, the doubling times corresponding to each transformant type were 45 min (green), 60 min (yellow), and 85 min (red). Starting from an equal distribution of transformants (33% each), transformants with green inserts will make-up close to 90% of the total cell populations after 10 doublings, whereas those with red inserts will be diluted to ≈1% of the total number of cells.

Figure 2

(A) DNA microarray images from plasmid populations throughout growth in selective and nonselective media (Pine-Sol; PS). The green signals correspond to plasmid samples labeled with Cy3-dUTP, and the red signals correspond to the control genomic DNA labeled with Cy5-dUTP and added in equal proportions to each array. Plasmids with gene inserts, identified by the large green spots on the array, were reproducibly observed in antibiotic-containing cultures and were not enriched in the antibiotic-free cultures. (B) The subarrays shown are representative blocks from the full eight-block (≈1,160 gene probes) array. The patterns of high intensity Cy3 spots in the 0.4% arrays reveal the presence of a selected subpopulation of clones not apparent in the 0.0% samples. Similar patterns of selection were observed in the remaining five blocks. The samples shown are from the last two time points indicated in A.

Figure 3

Quantified intensity values for the stationary phase arrays displayed in Fig. 2B. Cy3 intensity values (green Fig. 2) are located along the y axis and the Cy5 intensity values (red of Fig. 2) are represented along the x axis. The scales were set to be equal in both figures to emphasize the similar distribution of Cy5 signals (control genomic DNA) and the dramatically different distribution of Cy3 intensities. The genes with the largest Cy3 intensity correspond to genes that were selected for in the presence of Pine-Sol but were clearly not selected for in identical growth conditions without the presence of Pine-Sol. These genes were designated Pine-Sol resistance genes.