Figure 1.

DD-endopeptidases other than MepS, MepM, and MepH can also function in cell wall assembly. (A) Spot dilution assay on LB agar for MG1655 (wild type) and HC611 (P_ara::mepS, ΔmepM) strains harboring plasmids that overexpress various DD-endopeptidase genes: pHC800 (empty vector), pTK1(mepM), pTK2(mepS), pTK3(mepH), pTK4(mepA), pTKD1(dacB), pTKD4(pbpG), pTKD5(ampH), or pWJ20(mepK). The strains were grown overnight in M9-arabinose medium and normalized for cell density to an OD₆₀₀ of 2. The normalized cultures were serially diluted 10-fold in LB and 5 μl of each dilution (10⁻¹–10⁻⁶) was spotted onto LB agar lacking arabinose but containing 1 mM IPTG to deplete mepS expression and induce the expression of various DD-endopeptidases from the tac promoter. The plates were incubated at 30°C and photographed after 22 h. (B) Phase contrast images of the same strains after growth in LB. Overnight cultures were washed with LB and diluted to an OD₆₀₀ of 0.01 in LB containing 1 mM IPTG. Growth was then continued at 37°C with agitation. After incubation for 4 h, samples of the cells were chemically fixed and the cells were imaged using phase contrast optics. The bars equal 3 μM.