Role of the sigmaD-dependent autolysins in Bacillus subtilis population heterogeneity

Abstract

Exponentially growing populations of Bacillus subtilis contain two morphologically and functionally distinct cell types: motile individuals and nonmotile multicellular chains. Motility differentiation arises because RNA polymerase and the alternative sigma factor sigma(D) activate expression of flagellin in a subpopulation of cells. Here we demonstrate that the peptidoglycan-remodeling autolysins under sigma(D) control, LytC, LytD, and LytF, are expressed in the same subpopulation of cells that complete flagellar synthesis. Morphological heterogeneity is explained by the expression of LytF that is necessary and sufficient for cell separation. Moreover, LytC is required for motility but not at the level of cell separation or flagellum biosynthesis. Rather, LytC appears to be important for flagellar function, and motility was restored to a LytC mutant by mutation of either lonA, encoding the LonA protease, or a gene encoding a previously unannotated swarming motility inhibitor, SmiA. We conclude that heterogeneous activation of sigma(D)-dependent gene expression is sufficient to explain both the morphological heterogeneity and functional heterogeneity present in vegetative B. subtilis populations.

FIG. 1.

The LytF autolysin and flagellin are expressed in the same subpopulation of cells. Fluorescence microscopy images were obtained for cells of laboratory strain PY79 (DS3032) (A), wild-type undomesticated strain 3610 (DS2856) (B), and a sigD null mutant (DS3894) (C) that were grown to mid-log phase (OD₆₀₀, 0.5) and stained to reveal membranes (FM4-64; false color, red). Each strain also contained the promoter of the lytF (autolysin) gene fused to CFP expression (P_lytF-CFP; false color, blue) and the promoter of the hag (flagellin) gene fused to YFP expression (P_hag-YFP; false color, green). Arrowheads indicate the location of a chain. Scale bar = 2 μm.

FIG. 2.

The lytF mutant has a severe defect in cell separation. Phase-contrast microscopy was performed with cells having the genotypes indicated grown to mid-log phase (OD₆₀₀, 0.5). Scale bar = 5 μm. The bar graph shows a quantitative measure of chaining. Cells were stained with FM4-64, visualized by fluorescence microscopy, and manually counted, and the proportions of the populations that grew in chains (>4 connected cells) (black bars) and not in chains (≤4 connected cells) (gray bars) were determined. More than 800 cells were counted for each strain. The following strains were used in the experiment: PY79 (swrA), 3610 (wild type), DS3819 (lytC), DS108 (lytD), DS3821 (lytC lytD), DS1447 (lytF), DS1499 (lytD lytF), DS3820 (lytC lytF), DS3822 (lytC lytD lytF), and DS323 (sigD).

FIG. 3.

Expression of lytF is sufficient to dissolve chains. (A) Phase-contrast microscopy of cells grown to mid-log phase (OD₆₀₀, 0.5) in the presence of 1 mM IPTG. sigD was mutated in all cells. The strains indicated also contained IPTG-inducible copies of either lytC (DS3519), lytD (DS3521), or lytF (DS3520) fused to the P_hyspank promoter. Scale bar = 5 μm. (B) Phase-contrast microscopy of the sigD P_hyspank-lytF strain (DS3520) grown to mid-log phase in the presence of the amounts of IPTG indicated.

FIG. 4.

Cells in which lytC is mutated have a swarming motility defect. Quantitative swarm expansion assays were performed with single (A), double (B), and triple (C) mutants with mutations in the lytC, lytD, and lytF autolysin genes. Each symbol indicates the average of measurements from three experiments. The following strains were used in the experiment: 3610 (wild type), DS3819 (lytC), DS108 (lytD), DS3821 (lytC lytD), DS1447 (lytF), DS1499 (lytD lytF), DS3820 (lytC lytF), and DS3822 (lytC lytD lytF).

FIG. 5.

Cells with simultaneous lytC, lytD, and lytF mutations are proficient for flagellar assembly. The images are fluorescence microscopy images of cells grown to mid-log phase (OD₆₀₀, 0.5) and stained with FM4-64 (membrane; false color, red) and Alexa Fluor C₅ 488 maleimide (flagella; false color, green). Each strain expresses the modified flagellar filament protein Hag(T209C). The following strains were used in the experiment: (A) wild-type strain DS1916, (B) lytC lytD lytF mutant DS3824, (C) sigD mutant DS3159, and (D) swrA mutant DS1895. Scale bar = 2 μm.

FIG. 6.

Mutations in lonA and smiA restore motility to a lytC lytD double mutant. (A) lonA genetic region and lonA complementation construct. The large open arrows indicate open reading frames. The bent arrows indicate promoters. The solid triangles indicate sites of transposon insertions that restored motility to the lytC lytD double mutant. (B) Quantitative swarm expansion assays for lytC lytD mutant DS3961, lytC lytD lonA mutant DS5300, and lytC lytD lonA (P_lonA-lonA) mutant DS5318. Each symbol indicates the average of measurements from three experiments. (C) smiA genetic region and smiA complementation constructs. The large open arrows indicate open reading frames. The bent arrows indicate promoters. The solid triangles indicate sites of transposon insertions that restored motility to the lytC lytD double mutant. (D) Quantitative swarm expansion assays for lytC lytD mutant DS3961 and lytC lytD smiA (amyE::P_hyspank-smiA) mutant DS5036 in the presence (+IPTG) and absence (−IPTG) of 1 mM IPTG. Each symbol indicates the average of measurements from three experiments. (E) The bypass suppression model proposes that the motility defect of the lytC lytD double mutant is due to the uncontrolled activity of the motility inhibitors LonA and SmiA. The compensatory suppression model proposes that swarming motility is reduced in the absence of LytC and LytD, but swarming motility may be improved by mutating either of the swarming motility inhibitors LonA and SmiA. (F) Quantitative swarm expansion assays for wild-type strain 3610, ΔsmiA mutant DS4987, and ΔlonA mutant DS5286. Each symbol indicates the average of measurements from three experiments.