Extracellular proteolytic activity plays a central role in swarming motility in Bacillus subtilis

Abstract

Natural isolates of Bacillus subtilis exhibit a robust multicellular behavior known as swarming. A form of motility, swarming is characterized by a rapid, coordinated progression of a bacterial population across a surface. As a collective bacterial process, swarming is often associated with biofilm formation and has been linked to virulence factor expression in pathogenic bacteria. While the swarming phenotype has been well documented for Bacillus species, an understanding of the molecular mechanisms responsible remains largely isolated to gram-negative bacteria. To better understand how swarming is controlled in members of the genus Bacillus, we investigated the effect of a series of gene deletions on swarm motility. Our analysis revealed that a strain deficient for the production of surfactin and extracellular proteolytic activity did not swarm or form biofilm. While it is known that surfactin, a lipoprotein surfactant, functions in swarming motility by reducing surface tension, this is the first report demonstrating that general extracellular protease activity also has an important function. These results not only help to define the factors involved in eliciting swarm migration but support the idea that swarming and biofilm formation may have overlapping control mechanisms.

FIG. 1.

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.

FIG. 2.

Exogenous protease rescues swarming motility in a nonswarming, protease-deficient strain. Swarming behavior of A164Δ10 untreated control (A) or with 0.065 (B), 0.65 (C), or 6.5 (D) U of proteinase K is shown. Swarm plates were incubated at 37°C for 24 h. Swarming motility was also rescued by treatment with subtilisin and dispase I treatment (data not shown) at all concentrations except the lowest (see Materials and Methods for concentrations tested and inoculation method).

FIG. 3.

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.

FIG. 4.

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.

FIG. 5.

Extracellular protease activity is required for biofilm formation. A164 deletion strains were analyzed for their ability to form biofilms. While mutants defective in flagellar formation (A164Δhag), sporulation (A164Δ1), or surfactin production (A164Δsfp and MBin6) retained the ability to produce biofilm, a mutant (A164Δ10) deficient in both surfactin and extracellular protease activity, as well as sporulation, did not form a biofilm.