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. 2017 Jul 14;13(7):e1006888. doi: 10.1371/journal.pgen.1006888

Fig 3. Only full-length NlpD properly promotes cell separation.

Fig 3

(A-C) MT50 (ΔSSnlpD ΔenvC) or TB140 (ΔenvC) cells were grown in minimal M9-maltose medium at 37°C to an OD600 of 0.2–0.3 before visualization on 2% agarose pads with DIC optics (A-B) or analyzed by flow cytometry (C). Bar = 10μm. (D-H) Overnight cultures of MT50 harboring the integrated expression constructs (D) attHKMT20 (Plac::nlpDWT), (E) attHKMT102 (Plac::nlpD(1–189)), (F) attHKMT104 (Plac::nlpD(1–115)), (G) attHKMT179 (Plac::ssdsbA-nlpD(250–379)), or (H) attHKMT181 (Plac::ssdsbA-nlpD(189–379)) were diluted in minimal M9-maltose medium and grown at 37°C. Mid-log cultures were then backdiluted into M9-maltose medium with or without the inducer IPTG. Cells were further grown at 37°C to an OD600 of 0.2–0.3 before flow cytometry analysis. Histograms of cultures that were either uninduced (grey) or induced with 150μM (D, G-H, blue) or 1mM (E-F, red) IPTG were overlayed. (I) Cells of MT122 (ΔSSnlpD) and MT123 (ΔSSnlpD ΔamiC) alone or harboring the integrated constructs attHKMT20 (Plac::nlpDWT), attHKMT179 (Plac::ssdsbA-nlpD(250–379)), or attHKMT181 (Plac::ssdsbA-nlpD(189–379)) were grown in LB at 30°C. Following normalization for cell density (OD600 = 0.5), 5 μl of the resulting cultures was spotted on LB agar containing 150μM IPTG and 20 μg/ml CPRG. The plates were incubated at 30°C and photographed after 14 hours.