Extended Data Fig. 5: ∆camA construction, purified spore analyses, broth culture growth, and sporulation kinetics.
(a) PCR to distinguish between wild-type camA and ∆camA using flanking primers and primers internal to the deletion. PCRs were performed twice independently. (b) Growth curves comparing wild-type camA, ∆camA, ΔcamA-C, and camA/N165A cultures grown in BHIS liquid media. Early stationary-phase cultures were diluted to a starting O.D. of 0.05 in BHIS media and growth was measured over 9 h. Each pair genotype / timepoint correspond to mean of n = 3 independent biological replicates. Error bars correspond to standard deviation. (c) Phase-contrast microscopy analyses of sporulating culture samples prior to and after spore purification on a density gradient. No gross differences in spore morphology were observed between wild type and the MTase mutant. The germination efficiency (G.E.) of purified spores from the indicated strains is shown below. Scale bar represents 5 µm. Microscopy analyses were performed on three independent spore preparations. (d) Chloroform resistance of purified ∆camA spores relative to wild type. Spores were treated with 10 % chloroform for 15 min after which spore viability was measured by plating untreated and chloroform-treated spores on media containing germinant and measuring colony forming units. No significant differences in germination efficiency or chloroform resistance were observed. Data are presented as mean ± standard deviation of four independent biological replicates. (e) Heat-resistance (HRES) efficiencies of sporulating cultures 22 h after sporulation induction were determined relative to wild-type. Data are presented as mean ± standard deviation. Three independent biological replicates per group were used. *** P < 10-3, one-way ANOVA with Tukey’s test.
