A 12 × 12 genetic interaction matrix to validate GIANT-coli. A. A representative 1536-format, M9-glycerol plate showing the double mutants resulting from crossing pseudo-Hfr pal::kan with 12 Cm^R ASKA recipients arrayed in boxes of 16×8=128 replicas. The red box is a sterility control, since no recipients are arrayed in this spot. B. Quantification of (A). Error bars depict standard deviations (n > 240). C–D. Heat maps representing 12 × 12 crosses in LB (C) and M9-glycerol (D) based on the combined data from the 384 and 1536 plate formats and averaged results of reciprocal genetic interactions. The gray lines indicate that no results were extracted in M9-glycerol from surA::kan and surA::cat, as these clones grew very poorly in this medium. The color-coded bar ranges from a minimum size score (MIN) to a maximum (MAX) calculated for each dataset separately. E. Scatter plot of averaged normalized colony-size scores comparing growth in M9-glycerol versus LB for the 65 pairs (of 78 total) that grew in both media. Double mutants with substantially different growth in the two media are identified by name. The differential phenotype of the pal⁻ompA⁻ double mutant is further analyzed in (F–H). F–H. The pal⁻ompA⁻ double mutant was reconstructed by P1 transduction and the conditional interaction identified by GIANT-coli was recapitulated by lethality on M9-glycerol plates (F), smaller colony size in LB plates (G), and longer lag-phase and slightly slower growth rate in LB medium (H). Doubling times of wildtype, ompA::cat, pal::kan and ompA::cat pal::kan in (H) are approximately 28.5’, 30’, 31.5’ and 38’, respectively.

Genome-wide screens using GIANT-coli. A, B. Cross of pseudo-Hfr pal::cat ASKA mutant with the entire F⁻ Kan^R Keio collection (3985 mutants) arrayed in 1536 format (1536 colonies per plate); each Keio mutant is present twice as adjacent duplicates (768 unique recipients per plate). A representative image of one M9 plate (out of six total) is shown in panel (A). Interactions identified in the 12 × 12 matrix recapitulated here are marked with differently colored boxes. Black: the self-mating pair, pal::cat pal::kan; yellow: the synthetic lethal pair, pal::cat ompA::kan; red: pal::cat degP::kan, a neutral interaction in minimal media. The distribution of all interaction scores of pal::cat with the Keio collection is shown as a histogram in (B). In the histogram the number of Keio mutants within a bin of 0.25 are plotted against the interaction scores; the mean of the distribution (red dotted line) and 1 and 2 standard deviations (green dotted lines) are shown. C. Linkage biases of Hfr mating in LB (green) and M9 (blue). Median interaction scores, extracted using a variation of the E-MAP analysis software18, are aggregated into 5 kb bins sliding with 1 kb steps and are plotted as a function of chromosomal distance in kbs between genes. The analysis is based on 14 genome-wide screens in M9 glycerol and 9 in LB (data not shown). D. Suppression analysis of yraP::cat lethality in 3% SDS. Pseudo-Hfr yraP::cat was mated with the Keio collection as in (A) above except that double mutants were selected on both antibiotics in the presence of 3% SDS. A representative image of one plate is shown; complete data are shown in Supplementary Table 2B.

A flowchart depicting the different steps used in GIANT-coli. An Hfr donor (male) strain carrying a selectable marker (kan) replacing an ORF is mated on agar plates with arrayed F⁻ recipients (females; 1536 per plate) carrying a different selectable marker (cat) replacing another ORF (Step1). Following mating, cells are subjected to an intermediate selection on the bacteriocidal antibiotic kanamycin (Step 2) and then to a final selection for double mutants using both antibiotics (Step 3). Images of two representative plates used for generating a mating plate are shown below the cartoon.

A toolkit that facilitates the use of GIANT-coli in genome-wide analyses. A. High-throughput conversion of an entire single-gene knockout F⁻ library to an Hfr donor library. A double male strain is crossed with the Keio deletion library and a male (Hfr) Keio library is isolated by selecting on Amp-Kan or Gen-Kan plates (depending on whether the pseudo-Hfr locus is linked to gen or to bla). The entire process is carried out on agar plates. Transfer capabilities of a number of the newly generated pseudo-Hfr’s have been validated. B. Targeted integration of F-transfer functions at different chromosomal loci. Conditionally-replicating CIP vectors contain oriRγ (red circle), a ~40 kb BamH1 fragment of F (blue) including its 33 kb transfer region (dark blue) and oriT (blue circle), aadA, conferring streptomycin and spectinomycin resistance (violet) and ~300 bp of chromosomal homology (green). For pNTM3, the chromosomal region is from rhaM. CIPs are carried in a strain expressing the Π protein, which allows the plasmid to replicate from oriRγ. Upon mating to the F⁻ Keio or ASKA deletion mutants, which lack Π, CIPs are unable to replicate; selection for streptomycin and/or spectinomycin resistance results in chromosomal integration dictated by the particular homology region present on the CIP.