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. Author manuscript; available in PMC: 2018 Apr 3.
Published in final edited form as: Nat Microbiol. 2017 Oct 3;2(12):1648–1657. doi: 10.1038/s41564-017-0029-y

Fig. 5. Plots showing HADA incorporation in the PG of prey E. coli mutants upon B. bacteriovorus predation and showing the damage by osmotic shock to bdelloplasts formed by B. bacteriovorus Ldt mutants.

Fig. 5

a, Chart of mean HADA fluorescent signal of prey strains preyed on by B. bacteriovorus (+Bd), and pulsed with HADA at 35–45 min post-mixing (the time point of maximal HADA incorporation for E. coli S17-1). Controls were in Ca/HEPES buffer without B. bacteriovorus predation, but pulsed with HADA at the same time point. Measurements are total mean background-corrected fluorescent signal of prey cells, reported in relative fluorescent units measured by MicrobeJ. Prey cells lacking all six L,D-transpeptidases (A6LDT) accumulated more HADA fluorescence following predation by B. bacteriovorus. Control samples without B. bacteriovorus predation accumulated considerably less HADA fluorescence. Controls of A6LDT prey cells without Bdellovibrio predation accumulated negligible HADA fluorescence. Data are from two (for the controls) or three independent repeats. Error bars are s.e.m. WT: E. coli BW25113 wild-type strain YB7421; A6LDT: E. coli BW25113 A6LDT strain deficient in all six L,D-transpeptidases; ΔdacA: E. coli BW25113 strain YB7423 deficient in dacΔ; Δ6LDTΔdacA: E. coli BW25113 Δ6LDTΔdacA strain YB7439 deficient in all six L,D-transpeptidases and dacA. NS, not significant; all other comparisons were significant P<0.0001, with the one exception shown, by the Mann-Whitney test. b, CPRG β-galactosidase assay measuring cytoplasmic leakage of shocked E. coli bdelloplasts formed by wild-type (E. coli + Bd WT) or bdelloplasts formed by Δ2ldt mutant B. bacteriovorus (E. coli + Bd Δ2ldt) with controls of uninvaded E. coli prey cells (E. coli alone) or B. bacteriovorus cells alone (Bd WT alone). Red colour from positive CPRG reaction was measured by spectrophotometry at 574nm and readings were normalized to each experiment. Bdelloplasts were harvested by centrifugation and shocked by resuspension in Ca/HEPES buffer for no shock, except centrifugation alone (Buffer), Ca/HEPES buffer supplemented with 750 mM NaCl (Upshock) or upshock followed by further centrifugation and resuspension in water (Downshock). Error bars are s.e.m. Statistical significance was determined by Student’s t-test (two-tailed) *P<0.05, **P< 0.01, ***P< 0.001. Data are the mean of seven independent repeats.