Fig 2. Missense mutations in spoIIIEΔγ rescue sporulation and chromosome transport in vivo.

A. Suppressor mutations rescue spore formation. Intragenic mutations identified by suppressor selection were remade in a spoIIIEΔγ allele by site-directed mutagenesis and expressed from the spoIIIE promoter at an ectopic locus (ycgO) in a ΔspoIIIE strain. Strains were induced to sporulate for 24–36 h in DSM medium, unsporulated cells were eliminated by heat-kill, and the number of spores was measured by plating for cfu. The number of heat-resistant spores per ml is indicated for strains harboring full-length (“f.l.”) spoIIIE (bKM776), spoIIIEΔγ (BOSE2042), and 11 spoIIIEΔγ mutants: P260L (BOSE2286), S264I (BOSE2540), E310K (BOSE2411), E312A (BOSE2121), Y316D (BOSE2284), P319S (BOSE2321), A343V (BOSE2288), E347G (BOSE2323), P492Q (BOSE2120), H493Y (BOSE2538), and T617A (BOSE2123). Each number is the average of at least 3 replicates. Error bars indicate one standard deviation in each direction. B. Suppressor mutations rescue chromosome transport in vivo. Sporulation was induced by resuspension and DNA transport was evaluated using a previously-established fluorescent microscopy assay [12]. yfp and cfp genes are expressed from a forespore-specific promoter (PspoIIQ). yfp is integrated near the origin (yycR), and its expression indicates that asymmetric septation is complete. cfp is integrated near the terminus (pelB), and its expression indicates that the terminus has been transported into the forespore. Percent of termini in forespores is the percent of YFP+ cells that are also CFP+. Data are shown for full-length (“f.l.”) spoIIIE (bBB128), spoIIIEΔγ (bBB412), and 5 spoIIIEΔγ mutants: P260L (BOSE2331), E312A (BOSE2201), A343V (BOSE2332), P492Q (BOSE2200), and T617A (BOSE2202). Each data point represents the average of ≥ 3 replicates, with ≥ 500 forespores scored for each.