Figure 2. MltG overproduction is lethal in cells defective for PBP1b.

A–D. Cells of TU122/pDY1 were transformed with the multicopy E. coli library and plated on LB agar supplemented with IPTG and Xgal. A typical screening plate is shown in panel A, and close-up images highlighting the different colony phenotypes are shown in B–D. See text for details. E. Diagram of the genomic locus resulting in a multicopy lethal phenotype in ΔponB cells. F. Cells of MG1655 or its ΔponB derivative containing plasmid pRY53 [P_ara::mltG] were grown overnight in M9 minimal maltose medium supplemented with chloramphenicol. Cells in the resulting cultures were harvested, washed twice in an equal volume of M9 salts, and resuspended in M9 salts at an OD₆₀₀ of 1.0. The resulting cell suspensions were subjected to serial dilution, and 5μL of each dilution were spotted onto M9 agar plates containing 0.2% glucose or arabinose as indicated. Plates were incubated at 30°C for 2 days prior to imaging. G. Overnight cultures of the strains in F as well as the same strains carrying an empty vector control were diluted 1:100 in M9 maltose medium with chloramphenicol and grown to mid-log with shaking at 37°C. The cells were harvested, washed as above, and resuspended to an OD₆₀₀ of 0.02 in M9 arabinose medium. Growth at 37°C was then monitored by following culture optical density (OD₆₀₀). H–I. ΔponB cells carrying either an empty vector pCM6 [P_ara::empty] or plasmid pRY53 [P_ara::mltG] were grown on LB glucose plates, and then scraped and resuspended in M9 salts for imaging on agarose pads using DIC optics. Bars equal 4 microns.