Communities were seeded with 2 ul droplets containing indicated strains of Acinetobacter with genomic spect resistance, mixed with genomically cm-resistant E. coli carrying the kan-resistant donor plasmid pBAV1k. Spots were incubated at 30°C overnight (a) or at 37°C for 150 min (b–d) or for the indicated time (e–i). Lower limits on y-axes are the limits of detection. (a) E. coli (cm+kan, green bars), Acinetobacter (spect, orange bars), and newly double-resistant Acinetobacter (spect+kan, blue bars) present after growth either alone or in co-culture. (b) Survival of E. coli after growth alone (-,-) or with wild type (+,-), non-killing hcp (-,+), or both (+,+) strains of Acinetobacter. Spots were seeded with Acinetobacter at optical density (OD) five and E. coli at OD 1. (c) HGT during the experiment shown in b, measured as the proportion of wild type (WT - spect, left bar group) or hcp (tetracycline, right bar group) CFUs that were also resistant to kan. Two-strain cultures (dark bars) correspond to the center two bars in b, while the three-strain cultures (light bars) corresponds to the rightmost bar in b. (d) Transformation efficiency of Acinetobacter as in c, but mixed with purified pBAV1k DNA just before spotting. (e–g) Time course showing CFUs of E. coli (dotted green), total Acinetobacter (solid orange) and double-resistant Acinetobacter (dashed blue) in two-strain cultures (wild-type Acinetobacter in e and hcp Acinetobacter in f). (g) The proportion of wild type (solid line) and hcp (dashed line) Acinetobacter cells that have acquired kan resistance during the experiment in e-f. (h,i) Time course showing HGT complementation by killing in trans. The proportion of double-resistant cells is shown for wild type (solid lines) and hcp (dashed lines) Acinetobacter grown with E. coli either separately in two-strain (h) or together in three-strain (i) cultures. (j) A conceptual model for killing-enhanced HGT to Acinetobacter. Statistical significance levels are: with significance calculated using raw data, and , with significance calculated on log10-transformed data (see Materials and methods for sample sizes and statistical analysis, and see main text for exact p-values).