NlpI genetically interacts with MepS and affects the enzyme activity of MepS and MepM.Figure 3A. HPLC-based PG digestion assay representing EPase activity. The graph shows the relative percentage of the muropeptide TetraTetra present at the end of the incubation period for each protein as described in materials and methods. MepM and PBP4 were incubated with sacculi, whilst MepS and PBP7 were incubated with soluble muropeptides, both from E. coli MC1061, respectively. Values are mean ± standard deviation of three independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in p-values corrected for multiple testing. Relevant p-values are highlighted directly in the figure (*<0.05; **<0.01, ***<0.001) B. Genetic interactions of nlpI with EPase genes. Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates and incubated for 12 h at 37°C. Each plate contained 384 colonies, 96 from the wildtype, single mutants and double mutants. An example of a 384 plate is shown. Double mutants were made twice, swapping the resistance markers to the two single mutants. Colony integral opacity was quantified as a fitness readout, using the image analysis software Iris (Kritikos et al., 2017). Bar plots shows the averaged values of 2 biological experiments, each having 96 technical replicates (i=2, n=192). The error bars represent the 95% confidence interval. C. nlpI deletion changes the morphology of EPase-mutant strains. The graph shows the cell width of single and double deletions strains (800 < n < 2000 cells). The box has a medium between 25 and 75%. The whiskers with the upper and lower vertical line indicating the 95 and 5%. The dots are individual points outside the 5 and 95% range. Above the graph are representative images of cells lacking MepS or PBP4 in combination with a deletion of NlpI. The scale bar equals 2 µm. Gene encoding protein legend: nlpI encodes NlpI, dacB encodes PBP4, pbpG encode PBP7, mepM encodes MepM, meps encodes MepS. D. Inducible mepS expression system (pBAD30) strongly overproduces MepS. Strains were grown in LB at 30°C and cells were collected at OD600≈0.4. The level of MepS contained in the membrane fraction was detected using purified anti-MepS antibody. E. Visualization of the effect of MepS absence or its overexpression on cell width by phase contrast microscopy. Cultures were grown in LB at 30°C and aliquots of culture were taken at OD600≈0.1. The scale bar equals 5 μm. F. MepS level modulates cell width. The graph shows the distribution of mean cell width for each cell, with n corresponding to the number of cells measured for each strain and the median width for the population being indicated by a dotted line and referred to as w. G. Relative fitness of ΔnlpI, ΔmepS and ΔnlpIΔmepS mutants. Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates supplemented 10% sucrose, or LB agar plates containing 0 mM or 500 mM NaCl. Plates were incubated for 12 h at 37°C. Each plate contained 384 colonies, 96 from the wildtype, single mutants and double mutants. H. Cells of wildtype (WT), ΔnlpI, ΔmepS and ΔnlpIΔmepS containing multicopy plasmids with lacZ were grown onto CPRG indicator agar to assay envelope integrity. CPRG (yellow) cannot penetrate intact Gram-negative envelopes. Its conversion by intracellular β-galactosidase to CPR (red) indicates loss of envelope integrity.