A. Phase contrast images of PAO1 [WT], BPA107 [ΔenvC], BPA14 [ΔlytM2], BPA70 [nlpDΔN], BPA57 [ΔenvC ΔlytM2], BPA109 [ΔenvC nlpDΔN], and BPA72 [ΔlytM2 nlpDΔN]. Overnight cultures of cells grown in LB at 30°C were diluted 1:2000 in LB and grown for 4h at 42°C. Cells were then placed on agarose pads and imaged with phase contrast optics. All space bars are 2µm.
B. Viability of PAO1 [WT], BPA107 [ΔenvC], BPA70 [nlpDΔN], and BPA109 [ΔenvC nlpDΔN] cells. Overnight cultures were subcultured 1:10 into LB and allowed to grow for 2h at 30°C. They were then normalized to OD600 of 0.5 and subjected to serial dilution. 5µL aliquots of the dilutions were spotted onto LB plates, which were then incubated either at 42°C or 30°C.
C. Viability of PAO1/pPSV38 [WT], BPA57/pPSV38 [ΔenvC ΔlytM2], BPA109 /pPSV38 [ΔenvC nlpDΔN], and BPA72/pPSV38 [ΔlytM2 nlpDΔN], and BPA204/pPSV38 [ΔenvC ΔlytM2 (attTn7::lacIq) (PTOPLAC::nlpD)]. Overnight cultures grown in LB supplemented with 15µg/mL gentamicin and 1mM IPTG were washed, diluted 1:10 in LB with 1mM IPTG, and allowed to grow for 2h at 30°C. The cells were then washed with fresh LB, normalized to OD600 of 0.5, and subjected to serial dilution, spotting 5µL of each dilution on LB plates either containing or lacking 1mM IPTG. The plates were incubated at 30°C. The plasmid pPSV38 encodes lacIq and was needed for effective depletion of NlpD in strain BPA204/pPSV38 [ΔenvC ΔlytM2 (attTn7::lacIq) (PTOPLAC::nlpD)].