PMC

Protease activity is not required for swimming motility or hyperflagellation on swarm plates. (A) Swim motility assay at 6 h following inoculation. Swimming motility for all A164 mutants was equivalent to that of A164. The appearance of slightly increased motility for A164 is due to the small amount of swarming, which still occurs at this agar concentration and at a faster rate than swimming. Cells were stained for flagella from mid-log A164 cultures (B), A164 swarm plates (C), A164Δ10 mid-log cultures (D), and A164Δ10 swarm plates (E). The absence of surfactin and protease activity had no effect on flagellar formation in liquid medium or on swarm plates. All cells were collected from swarm plates 3 h after inoculation. Microscopic analysis was performed at a magnification of ×100.

Conditioned medium from A164, but not A164Δ10, rescues swarm motility of A164Δ10. A164Δ10 was inoculated onto swarm plates prepared with conditioned medium and incubated for 24 h at 37°C. Conditioned medium incubated with isopropanol alone had no effect on swarming (data not shown). (A) Conditioned medium obtained from A164 (left) or A164Δ10 (right). (B) Conditioned medium obtained from A164 and preincubated with PMSF at a final concentration of 140 (left) or 280 (right) μg/ml, respectively.

Folding and unfolding pathways of the tandem proteins. All rate constants for the transitions are given in s^− 1. Top: A164–A165; the folding and unfolding pathways are the reverse of each other: the folding intermediate comprises folded A164 and unfolded A165. In parentheses: this rate constant could not be measured directly; thus, we are assuming that unfolded A165 does not affect the stability of A164, and the unfolding rate constant is the same as that for A164 alone. Bottom: Since the rate constants for folding of A168 and A169 are very different, A168 both folds and unfolds faster than A169 in the tandem protein. Thus, the folding and unfolding intermediates are different species.

Secreted proteases are required for swarming motility by B. subtilis A164. Swarm motility of B. subtilis A164 (A) and A164 mutants A164Δ5, MBin7, A164Δ8, and A164Δ10 (clockwise from left) (B) is shown. The absence of surfactin production in A164Δ5 and A164Δ8 results in a less-robust swarming phenotype, and a nonswarming phenotype is exhibited by A164Δ10, which is deficient in both surfactin and protease activity. Although MBin7 appears to have a nonswarming phenotype, swarming is initiated after 48 h for this strain and is not completely inhibited until deletions are made in the remaining extracellular protease, aprE, and sigF (MBin10 strain, data not shown). All strains were inoculated with a toothpick onto a 0.7% agar LB swarm plate and incubated for 20 h at 37°C.