FIG. 4.

Effects of l-leucine concentration on spore germination rates of strains GC-GerU* and GC-L5_E336A. Spores of strain GC-L5_E336A (⧫), in which the glutamate residue at position 336 of GerVB is replaced by alanine, germinate at an increased rate (A) and display a lower minimal germinant requirement for leucine (B) than spores carrying the parental GerU* receptor (⋄). Spores were germinated in 96-well plates in a total volume of 300 μl of 5 mM Tris-HCl, pH 7.8, at 30°C with variable leucine concentrations. Maximum rates of germination were calculated from the slope of the linear segment of A₆₀₀ loss over time that follows the initial lag phase. Under these experimental conditions, a 60% loss in A₆₀₀ correlates with 100% spore germination, as determined by loss of heat resistance of strain GC-GerU* after a 60-min incubation in 10 mM glucose. This value was used subsequently to estimate the proportion of germinated spores after incubation for 60 min under the range of leucine concentrations tested. Plotted values are averages of experiments conducted in triplicate (error bars represent standard deviations from the mean).