Overview of TagModule design and tagged transposon mutagenesis strategy. (a) Each TagModule contains a unique uptag (blue) and downtag (green) flanked by universal priming sites (arrows). TagModules were cloned into pCR8/GW/TOPO, a Gateway-compatible entry vector and sequence verified with a primer on the pCR8/GW/TOPO backbone. Spc^R indicates spectinomycin resistance; pUCori indicates the origin of replication; attL1 and attL2 are the Gateway recombination sites. (b) To use the TagModules for tagged transposon mutagenesis, first the transposon delivery plasmid must be converted into a Gateway compatible destination vector by addition of an attR1-ccdB-Cm^R-attR2 cassette. Addition of LR clonase to a pool of TagModule entry clones (a colored bar represents a pair of tags) and a transposon destination vector induces recombination at the att sites, replacing the Gateway conversion cassette with the TagModule. The resulting pool of tagged transposons can then be used to mutagenize the organism of interest. TnL and TnR are the ends of the transposon, and each TagModule is represented by a different color rectangle.(c) Tagged mutants can then be pooled and grown competitively in a condition of choice. Strains lacking genes essential for optimal growth in the test condition will grow slower and become underrepresented in the pool; resistant strains grow faster and will be overrepresented. Recovery, amplification and hybridization of the tags to an array will yield information on the abundance of each tag, which can then be converted to a relative fitness value or sensitivity score for each mutant.

Validation of TagModule activity. (a) TagModules were pooled, and uptags and downtags were amplified and hybridized to an Affymetrix TAG4 array. Tags with raw intensity values below a cutoff of 5-fold above median background (red line) were flagged for exclusion (red points). Pearson’s correlation is indicated in the upper left corner, P < 10⁻¹⁶. (b) Independently amplified TagModule pools are highly reproducible even lacking array or tag normalization. Pearson’s correlation is in the upper left. (c) Uptag and downtag PCRs were hybridized to arrays in defined ratios, using a total of 7.5 µl of PCR product (top). (Left) Actual tag ratios are plotted corresponding to their expected ratio, and right, density plots of corresponding color highlights the resolution of the tag ratios. (d) Comparison of calculated uptag and downtag ratios from (c) with colors corresponding to their expected ratio; Pearson’s correlation is indicated in the upper left, P < 10⁻¹⁶.

Validation of TagModule-based transposon mutagenesis in S. oneidensis MR-1. The relative fitness values of 1761 tagged transposon mutants in rich media (LB) was plotted versus their values in (a) minimal media or (b) LB + 300 mM NaCl as determined by pooled competitive growth followed by hybridization to a TAG4 array. A value of 1 corresponds to no growth defect relative to the pool average, whereas a value <1 indicates a growth defect in that condition. The red points in (a) correspond to 38 mutant strains with a specific growth defect in minimal media relative to rich media; the red points in (b) correspond to 25 mutant strains with a growth defect in LB + 300 mM NaCl (see ‘Materials and Methods’ section for selection criteria). (c) Representative single strain growth curves in LB + 300 mM NaCl for mutants with growth defects from (b). Wild type S. oneidensis MR-1 is included as a control (black). (d) Single strain doubling times for all 25 defective mutants from the red points in (b) were compared with the relative fitness values obtained from the pooled growth assay. Pearson correlation is in the upper left (P = 0.0006). Mutant stains with individual growth curves in (c) are indicated. The points represent the average of at least four independent growth curve assays for each strain. Red points represent mutant strains with significantly different doubling times compared to the wild-type S. oneidensis MR-1.

Validation of TagModule-based transposon mutagenesis in C. albicans. (a) A total of 1290 heterozygous disruption strains were grown for 20 generations in 50 μM clotrimazole or 1% DMSO. Increasing log₂ ratio (control versus experimental intensities) reflects increasing sensitivity. The 37 most sensitive strains (red points) were selected for confirmation; the 15 with an enriched GO annotation are marked. (b) Representative single strain growth curves for sensitive mutants from (a). Wild-type C. albicans (BWP17) is included as a control (black). For ERG11, two different disruption events were screened, a 43% and a 100% gene disruption. (c) Pearson’s correlation (upper left) of log₂ ratios obtained from TAG4 array hybridization and log₂ ratios of growth rate (control versus experimental) obtained from (b) for sensitive strains from (a). Wild-type log₂(growth rate) = 0. Genes mapping to GO function phosphatidylinositol metabolic and biosynthetic process are marked *; genes mapping to GO function response to stimulus are marked ‡.