SwrD (YlzI) Promotes Swarming in Bacillus subtilis by Increasing Power to Flagellar Motors

ABSTRACT

The bacterium Bacillus subtilis is capable of two kinds of flagellum-mediated motility: swimming, which occurs in liquid, and swarming, which occurs on a surface. Swarming is distinct from swimming in that it requires secretion of a surfactant, an increase in flagellar density, and perhaps additional factors. Here we report a new gene, swrD, located within the 32 gene fla-che operon dedicated to flagellar biosynthesis and chemotaxis, which when mutated abolished swarming motility. SwrD was not required for surfactant production, flagellar gene expression, or an increase in flagellar number. Instead, SwrD was required to increase flagellar power. Mutation of swrD reduced swimming speed and torque of tethered flagella, and all swrD-related phenotypes were restored when the stator subunits MotA and MotB were overexpressed either by spontaneous suppressor mutations or by artificial induction. We conclude that swarming motility requires flagellar power in excess of that which is needed to swim.

IMPORTANCE Bacteria swim in liquid and swarm over surfaces by rotating flagella, but the difference between swimming and swarming is poorly understood. Here we report that SwrD of Bacillus subtilis is necessary for swarming because it increases flagellar torque and cells mutated for swrD swim with reduced speed. How flagellar motors generate power is primarily studied in Escherichia coli, and SwrD likely increases power in other organisms, like the Firmicutes, Clostridia, Spirochaetes, and the Deltaproteobacteria.

INTRODUCTION

Bacterial flagella drive two forms of active movement called swimming and swarming motility. During swimming motility, cells rotate helical flagella that act like propellers to push individuals through a three-dimensional liquid environment (1). Swarming motility also requires flagellar rotation, but swarming cells move as multicellular groups across a surface, within films of water approximately the depth of a single cell (2, 3). In the Gram-positive bacterium Bacillus subtilis, swarming is distinct from swimming in that it has additional physiological requirements, including the secretion of a surfactant and an increase in flagellar density on the surface of the cell (4, 5). The surfactant acts to reduce surface tension and create the thin layer of water within which to swarm. The reason that cells require an increase in flagellar density is unknown, but it may be necessary to increase the total amount of thrust generated by the cell.

Flagella rotate when consumption of the proton motive force powers a change in the interaction between the flagellar rotor and stator proteins. The rotor is a single gear-like structure (called the C-ring) made from many subunits of the proteins FliG, FliM, and FliN and sits on the cytoplasmic face of each membrane-anchored flagellum (6–9). The stators are complexes of membrane-bound proton channels made from two subunits of MotB, which conducts protons, and four subunits of MotA, which interacts with the C-ring protein FliG (10, 11). When protons flow through MotB, a conformational change occurs in MotA that is thought to electrostatically push on FliG and create the torque for flagellar rotation (8, 10, 12–16). Stator number is dynamic, and torque is porportional to the number of stators associated with the flagellum (17–21). More torque-generating units are added commensurate with rotational load to create proportional power, and thus the flagellum operates as a constant torque motor (17, 22, 23). The biophysics of flagellar rotation has been studied primarily in Escherichia coli and Salmonella enterica, and while B. subtilis encodes similar motor proteins, its motor properties are less well understood.

Here we characterize ylzI, a gene of unknown function located within the large fla-che operon of flagellar and chemotaxis genes. We rename the ylzI gene and its gene product swrD and SwrD, respectively, as mutation of swrD abolished swarming but not swimming motility. The swrD mutant was not defective in surfactant production or involved in an increase in flagellar number, as found in other swarming mutants. Instead, the swarming defect was correlated with a decrease in swimming speed and a decrease in motor torque. Moreover, all phenotypes of the swrD mutant were bypassed by overexpression of MotA and MotB, suggesting that SwrD increased power via the flagellar stators. The swrD gene is coexpressed with the motA and motB genes in many organisms, including the spirochetes, which may require increased stator power to rotate their cell bodies in viscous environments.

RESULTS

SwrD is required for swarming motility.

The gene swrD (ylzI) is located within the 32-gene, 27-kb fla-che operon and is predicted to encode SwrD, a 71-amino-acid protein of unknown function (Fig. 1). Cells mutated for SwrD are severely defective in swarming motility, a flagellum-dependent form of migration across a 0.7% agar surface (2, 24). To determine whether SwrD was required for swimming motility, in which flagella power movement through a loose agar matrix, cells were inoculated in the center of a 0.3% lysogeny broth (LB) agar plate and incubated for 12 h. Wild-type cells created a large zone of colonization by consuming nutrients locally and swimming up the resulting nutrient gradient by chemotaxis (Fig. 2) (25, 26). The zone of colonization was dramatically reduced when cells were mutated for either flagellar biosynthesis (hag) or chemotaxis (cheA) (Fig. 2). The swrD mutant exhibited a zone of colonization smaller than that of the wild-type cells, but the zone was larger than that made by the aflagellate and nonchemotactic mutants (Fig. 2). Finally, swrD mutant cells swam in liquid when observed via wet-mount phase-contrast microscopy. We conclude that SwrD is not strictly required for swimming motility.

FIG 1.

Genetic context of the swrD gene and suppressor of swrD (sod) mutations. The swrD gene (green arrow), formerly annotated as ylzI, is located within the fla-che operon (top row of arrows). Open arrows indicate open reading frames, bent arrows indicate promoters. Blue carets indicate the location of suppressor of swrD (sod) mutant classes in the second flagellar cluster containing σ^D-dependent genes unlinked to the fla-che operon. Red (flgM) and yellow (fliS) arrows indicate genes discussed further in the text. The diagram is not to scale.

FIG 2.

Cells mutated for swrD are modestly reduced for swimming motility. Circles are top views of 0.3% agar LB plates containing 1 mM IPTG, centrally inoculated with the indicated strain, grown for 12 h at 37°C, and filmed against a black background such that zones of colonization appear white and uncolonized agar appears black. In addition to the indicated background, each strain is also mutated for surfactin (to abolish swarming over the surface) and extracellular polysaccharide (to abolish sliding over the surface). The genotype is indicated above the corresponding plate. +++ indicates that the motA and motB genes were overexpressed by induction with 1 mM IPTG. The following strains were used to generate the figure: DK374 (wild type [WT]), DK2203 (cheA), DK378 (hag), DK1405 (swrD), DK5314 (motAB⁺⁺⁺), and DK5315 [swrD (motAB⁺⁺⁺)]. Scale bar, 1 cm.

The swarming defect of the swrD mutant could have been due to a polar effect on downstream genes, as swrD sits in the middle of the fla-che operon and the 3′ end of the swrD open reading frame overlaps with the 5′ end of the fliL gene located immediately downstream. To determine whether the absence of SwrD was responsible for the motility defect, a complementation construct was generated such that the swrD open reading frame was fused downstream of the native promoter of the fla-che operon, P_fla-che, and integrated at an ectopic locus (amyE::P_fla-che-swrD). The swrD mutant displayed a severe swarming defect, as previously reported, and a transparent watery ring surrounded the nonswarming colony, which indicated that surfactin was still being synthesized (4, 27) (Fig. 3A). When the swrD complementation construct was ectopically integrated in the swrD mutant, swarming motility was restored to wild-type levels (Fig. 3A). We conclude that the swarming defect of the swrD mutant is due to the absence of SwrD protein and not to polar effects on genes downstream in the fla-che operon. SwrD was named swarming motility protein D because it was required for swarming but not swimming, the swarming defect was not due to a lack of surfactant production, and it was the fourth protein with the Swr prefix in B. subtilis (28).

FIG 3.

Cells mutated for swrD do not swarm, and swarming can be rescued by increasing the expression of motAB. (A to F) Quantitative swarm expansion assays. The relevant genotype of each strain is indicated within the graph. Gene names in parentheses indicate genes introduced at an ectopic locus. + indicates that genes are expressed from their native promoter. +++ indicates that the genes indicated have been overexpressed by the addition of 1 mM IPTG. Wild type (WT) data were reproduced in panels A to D and separately in panels E and F to match the experimental strains in the corresponding data set. Each data point is the average result of three replicates. The following strains were used to generate the data: in panel A, 3610 (WT), DS6657 (swrD), and DS7550 [swrD (swrD⁺)]; in panel B, 3610 (WT), DK1597 [swrD (swrA⁺⁺⁺)], and DS860 (swrA); in panel C, 3610 (WT), DS6698 (swrD sod2), and DK1839 [swrD sod2 (flgM⁺)]; in panel D, 3610 (WT), DS7527 (swrD sod4), and DK48 (swrD sod48); in panel E, 3610 (WT), DS222 (motAB), and DK801 [motAB (motAB⁺⁺⁺)]; in panel F, 3610 (WT), DS6657 (swrD), and DK4651 [swrD (motAB⁺⁺⁺)].

SwrD is not required for flagellar biosynthesis.

One reason cells fail to swarm is because of the synthesis of an insufficient number of flagellar basal bodies, as found in cells mutated for the master activator of flagellar biosynthesis, SwrA (5, 29, 30). To determine whether the swrD mutant was defective in basal body synthesis, a swrD mutation was introduced into a strain in which the flagellar basal body could be detected as fluorescent puncta due to a fluorescent protein fused to the flagellar C-ring component protein FliM (31). Whereas cells mutated for swrA had a reduced number of FliM-green fluorescent protein (GFP) fluorescent puncta per cell, cells mutated for swrD had a number of puncta comparable to those of the wild-type swimming cells (Fig. 4A and B). Cells of the swrD mutant also had the capacity to increase flagellar basal body number, as basal body number increased when SwrA was overexpressed (Fig. 4B). Whereas overexpression of SwrA creates constitutively hyperflagellated cells (5, 31) and abolishes the swarming lag period in the wild type (29), SwrA overexpression did not restore swarming to the swrD mutant (Fig. 3B). We conclude that the swrD mutant has a defect in swarming motility unrelated to an increase in flagellar basal body number.

FIG 4.

Cells mutated for swrD are not defective in flagellar assembly or flagellar number. (A) Fluorescence micrographs of wild-type (DS8521), swrA mutant (DS8600), and swrD mutant (DK1358) cells expressing the FliM-GFP fusion that indicates the location of flagellar basal bodies. (B) Three-dimensional structured illumination microscopy (3D SIM) was used to count the flagellar basal bodies (FliM-GFP puncta) per cell length in the wild type (DS8521), swrD mutant (DK1358), and swrD swrA⁺⁺⁺ mutant containing a P_hyspank-swrA construct grown in the presence of 1 mM IPTG (DK4616). (C) Fluorescence micrographs of wild-type (DS7673), swrB mutant (DK478), and swrD mutant (DK1359) cells expressing the FlgE^T123C allele that indicates the location of flagellar hooks when stained with a fluorescent maleimide dye. (D) 3D SIM was used to count the flagellar hooks (FlgE^T123C puncta) per cell length in the wild type (DS7673), swrD mutant (DK1359), and swrD mutant containing a P_hyspank-swrA construct (swrA⁺⁺⁺) grown in the presence of 1 mM IPTG (DK4724). (E) Fluorescence micrographs of wild-type (DS1916), swrB mutant (DS9319), and swrD mutant (DS8816) cells expressing the Hag^T209C allele that indicates the location of flagellar filaments when stained with a fluorescent maleimide dye.

Another reason cells fail to swarm is because of the synthesis of an insufficient number of flagellar hooks, as found in cells mutated for the activator of flagellar type III secretion, SwrB (32). To determine whether swrD mutants were defective in hook synthesis, a swrD mutant was introduced into a strain with an allele for the FlgE hook structural protein FlgE^T123C that could be fluorescently labeled by the addition of a fluorescent maleimide stain (33). Whereas cells mutated for SwrB had a reduced number of fluorescent hooks per cell, cells mutated for SwrD had a number of fluorescent hooks comparable to that of the wild type (Fig. 4C and D). Cells mutated for SwrD also had the capacity to increase the flagellar hook number, as hook number increased when SwrA was overexpressed (Fig. 4D), again despite being unable to restore swarming motility (Fig. 3B). We conclude that the swrD mutant has a defect in swarming motility unrelated to an increase in flagellar hook number.

Swarming motility likely requires an increase in the number of flagellar filaments, which depends on the activity of both SwrA (to increase basal bodies) and SwrB (to increase hooks). To determine whether the swrD mutant was specifically defective in flagellar filament synthesis, a swrD mutation was introduced into a strain with an allele for the Hag filament structural protein, Hag^T209C, that could be fluorescently labeled with a maleimide stain (34–36). Compared to the reduced number of flagella found in cells mutated for SwrB, cells mutated for SwrD appeared to have a number of fluorescent filaments qualitatively comparable to that of the wild type (Fig. 4E). With the caveat that we are currently unable to obtain an accurate count of the flagellar filaments per cell, swrD mutant cells do not appear to have a defect in flagellar assembly or flagellar number. We further conclude that cells mutated for SwrD are defective in swarming motility for reasons other than those found in cells mutated for either SwrA or SwrB. We infer that SwrD promotes an as-yet-unknown requirement for swarming motility in B. subtilis.

Enhanced σ^D activity restores swarming to cells that lack SwrD.

To determine the mechanism by which SwrD activates swarming motility, spontaneous suppressors that restored swarming motility to a swrD mutant were isolated. Whereas a swrD mutant was severely defective for swarming motility and grew as a tight colony in the center of a swarm agar plate, upon prolonged incubation (∼24 h), spontaneous mutations that restored swarming and emerged as flares of cells arose. In total, 21 suppressor of swrD (sod) mutants were independently isolated, and all but two were identified by classical transposon-assisted, SPP1-mediated generalized transduction genetic linkage mapping, followed by directed gene sequencing. All identified sod suppressor mutations genetically mapped to the second flagellar cluster of motility genes and, based on their genetic locations, were grouped into three different classes (Fig. 1; Table 1).

TABLE 1.

Suppressor of swrD (sod) alleles^a

Class and suppressor	Genotype	Strain
Class I (FlgM loss-of-function mutations)
sod1	flgMQ31^FS	DS6697
sod2	flgMQ64*	DS6698
sod3	flgMQ31^FS	DS6699
sod5	flgMN17^FS	DS7528
sod6	flgMV72*	DS7529
sod7	flgMQ24*	DS7530
sod8	flgMQ64*	DS8791
sod39	flgMQ9*	DK39
sod40	flgMQ64*	DK40
sod41	flgMQ64*	DK41
sod42	flgMQ64*	DK42
sod43	flgMQ64*	DK43
sod44	flgMT26^FS	DK44
sod46	flgMN80^FS	DK46
sod47	flgMQ64*	DK47
sod51	flgMQ64*	DK615
sod52	flgMQ64*	DK616
Class II (PfliD promoter-down mutation): sod4	PfliD TGTAAT→CGTAAT	DS7527
Class III (FliS translation-down mutation): sod48	fliSRBS GGAGG→AGAGG	DK48
Unidentified
sod38	Unknown	DK38
sod50	Unknown	DK614

^aEach sod mutation was independently isolated from a separate swarm plate, each inoculated with a separate colony of the DS6657 ΔswrD parent. Thus, although some of the mutations were identical, they are not siblings and represent independent genetic events. ^FS, frameshift after the indicated codon; *, stop codon. Bold letters indicate the nucleotide position changed by the mutation. Underlined letters indicate the −35 element of the P_fliD σ^D-dependent promoter. RBS, ribosome binding site or Shine-Dalgarno sequence.

sod class I: FlgM loss-of-function mutations.

The sod class I suppressor mutations restored partial swarming to the swrD mutant (Fig. 3C). Linkage mapping and sequencing revealed that 17 out of 19 sod mutations (e.g., sod2) were either frameshift or nonsense truncations within the flgM gene, likely leading to FlgM loss of function (Fig. 1, sod class I). Consistent with a FlgM loss-of-function phenotype, introduction of an ectopically integrated complementation construct in which the wild-type flgM open reading frame was expressed under the control of its own promoter (amyE::P_flgM-flgM), restored a swarming defect to a swrD sod class I background (Fig. 3C). FlgM is the anti-sigma factor that inhibits σ^D-dependent gene expression (37–39). Consistent with loss of anti-sigma factor activity, expression increased from a series of β-galactosidase (lacZ) reporters fused to the σ^D-dependent promoters P_hag, P_motA, and P_flgM but not from the σ^A-dependent promoter P_fla-che in the swrD sod class I mutant background (40–43) (Fig. 5A). We conclude that one way to restore swarming to a swrD mutant is to abolish FlgM and increase σ^D activity. We note, however, that mutation of swrD alone did not decrease the expression magnitude from the σ^D-dependent LacZ reporters nor did it decrease the expression frequency from a σ^D-dependent GFP reporter (Fig. 5A; see Fig. S1 in the supplemental material). We infer that SwrD does not act to generally increase σ^D activity and that the sod mutations that disrupt FlgM are likely compensatory rather than bypass suppressors.

FIG 5.

Suppressors of swrD (sod) increase σ^D-dependent gene expression. (A) Expression of various lacZ reporters fused to the promoter regions indicated along the x axis was measured in swrD and swrD sod mutant backgrounds. To generate each bar, the expression of each reporter in the various backgrounds was divided by the expression of the reporter in a wild-type background and expressed as a fold change. The raw data and strains used to generate this panel are presented in Table S3 in the supplemental material. (B) Expression of either P_fliD or P_fliD^sod4 fused to lacZ in wild-type (WT) and swrD mutant backgrounds. Error bars are the standard deviation of results of three biological replicates.

sod class II: P_fliD promoter-down mutation.

The sod class II suppressor mutation sod4 restored partial swarming to the swrD mutant (Fig. 3D). Linkage mapping and sequencing indicated that a single sod (sod4) was a mutation upstream of the P_fliD promoter (Fig. 1, sod class II). The mutation changed a T to a C 2 bp upstream of the −35 region of the σ^D-dependent promoter (TGTAAT to CGTAAT; −35 element sequence underlined) (44). To determine the consequence of the sod class II mutation, P_fliD reporters were generated by fusing either the wild type or the sod4 mutant allele promoter region upstream of lacZ. Unlike other σ^D-dependent promoters, expression from P_fliD was reduced in the swrD mutant relative to the wild type, albeit only 2-fold (Fig. 5B). The swarming defect in a swrD mutant was not due to a specific reduction in P_fliD activity, however, as the sod4 allele reduced expression from P_fliD another 10-fold relative to the wild type (Fig. 5B). We conclude that the P_fliD^sod4 allele is a promoter-down mutation, and we infer that the 2-fold reduction of P_fliD activity in a swrD mutant may be a consequence, rather than a cause, of the swrD swarming motility defect. Finally, the swrD sod4 background showed an increase in the P_hag, P_motA, and P_flgM σ^D-dependent transcriptional reporters (Fig. 5A). Thus, the sod class II P_fliD promoter-down mutation restored swarming by increasing generalized σ^D activity like the sod class I mutations.

sod class III: FliS translation-down mutation.

The sod class III suppressor mutation (sod48) restored partial swarming to the swrD mutant (Fig. 3E). Linkage mapping and sequencing identified a mutation upstream of the fliS gene encoding the chaperone for flagellin secretion, FliS (45–47). The mutation was in the putative Shine-Dalgarno ribosome binding sequence, changing a G to an A (GGAGGA to AGAGGA) and moving it away from consensus (48). To determine the effect of the mutation on FliS expression, Western blot analysis was performed using anti-FliS as a primary antibody. FliS was present in the wild type and absent in a fliS deletion mutant. In the swrD null mutant, FliS levels appeared to be slightly higher than in the wild type (Fig. 6A). The swrD sod48 mutation reduced FliS levels, supporting the idea that it had impaired the Shine-Dalgarno sequence and, thus, translation (Fig. 6A). FliS levels were not correlated with a rescue of swarming, however, as the swrD sod4 (class II) and swrD sod2 (class I) mutants showed reduced and elevated FliS levels relative to the swrD mutant, respectively (Fig. 6A). Finally, the swrD sod48 background showed an increase in the P_hag, P_motA, and P_flgM σ^D-dependent transcriptional reporters (Fig. 5A). We conclude that the restoration of swarming by the sod class III FliS translation-down mutation was not directly due to a reduced level of FliS but rather to an indirect increase in generalized σ^D activity.

FIG 6.

Rescue of swarming to a swrD mutant is correlated with elevated levels of MotA. (A) Western analysis of cell lysates resolved by SDS-PAGE and probed with anti-FliS and anti-SigA antibody (to serve as a loading control), respectively. The same samples were used to make each panel, but 10-fold less lysate was loaded in each lane for the anti-SigA Western blot. The following strains were used to generate the panel: 3610 (WT), DS7792 (fliS), DS6657 (swrD), DS6698 (swrD sod2), DS7527 (swrD sod4), and DK48 (swrD sod48). (B) Western analysis of cell lysates resolved by SDS-PAGE and probed with anti-MotA and anti-SigA antibody (to serve as a loading control), respectively. The same samples were used to make each panel, but 10-fold less lysate was loaded in each lane for the anti-SigA Western blot. The following strains were used to generate the panel: 3610 (WT), DS7498 (motA mutant), DS6657 (swrD), swrD P_hyspank-motAB mutant grown in the presence of 1 mM IPTG (motAB⁺⁺⁺) (DK4651), DS6698 (swrD sod2), DS7527 (swrD sod4), and DK48 (swrD sod48).

In summary, we conclude that all three sod suppressor classes were effectively similar in that they increased σ^D activity, albeit by different mechanisms. Class I mutations abolished activity of the anti-sigma factor FlgM directly, whereas class II and class III mutations reduced the synthesis of the flagellin secretion chaperone FliS at the transcriptional and translational levels, respectively. FlgM is antagonized by export through the flagellar secretion system, and reduced synthesis of FliS may reduce flagellin export, perhaps reducing substrate competition, thereby increasing FlgM secretion, and thus increasing free σ^D protein (24, 46, 47). Since there did not appear to be reduced expression from σ^D-dependent promoters in the swrD parental background, however, we infer that the sod suppressor mutants increase the expression of one or more σ^D-dependent genes that compensate for the lack of SwrD.

Cells lacking SwrD have a defect in flagellar power.

There are many genes under the control of σ^D, and enhanced expression of one or more of them could compensate for the absence of SwrD (29, 49). We decided to focus our attention on the σ^D-dependent motAB operon, encoding the proton-conducting stator units MotA and MotB that power flagellar rotation, for the following reasons. First, colonies of a swrD mutant were mucoid in a manner dependent on PgsB, a protein required for the synthesis of secreted poly-γ-glutamate, and phenocopied colonies of cells mutated for motA, motB, or other genetic constructs that inhibited flagellar rotation (50, 51) (Fig. 7A). Second, in many bacteria, including Clostridia, Spirochaetes, and Thermotogales, genes encoding homologs of SwrD are located immediately upstream of the genes encoding MotA and MotB (Fig. 7B). Third, levels of the MotA protein appeared to be elevated in each of the sod suppressor classes in Western blot analysis (Fig. 6B). SwrD did not appear to be involved in regulating MotA levels, however, as MotA levels did not change in the swrD mutant. Thus, we hypothesized that the function of SwrD was related to flagellar stator activity in a way that could be bypassed by an excess of stators in the membrane.

FIG 7.

Colonies of a swrD mutant are mucoid. (A) Pictures of colonies of the indicated strain grown overnight at 37°C on a 1.5% LB agar plate. A plus sign in the lower right corner of the image indicates whether the colony was mucoid such that when touched with a toothpick, a sticky strand of poly-γ-glutamate was extracted. The following strains were used to generate this panel: 3610 (WT), DS6657 (swrD), DK3213 (swrD pgsB), DS222 (motAB), and DK3214 (motAB pgsB). (B) Cartoons of genetic neighborhoods with swrD homologs, as indicated at microbesonline.org. Arrows represent the indicated open reading frames, with swrD homologs colored green and motA or motB homologs colored orange.

To determine whether overexpression of motA and motB might be responsible for rescuing swarming motility to the swrD mutant in the various sod suppressors, both genes were cloned downstream of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible P_hyspank promoter and inserted at an ectopic site in the chromosome (amyE::P_hyspank-motAB). When the inducible construct was introduced into a motAB mutant, swarming motility was rescued in the presence but not the absence of 1 mM IPTG, indicating that the construct was expressed and capable of producing functional stators (Fig. 3E). When the inducible construct was introduced into a swrD mutant, both swarming motility and swimming motility were restored to wild-type levels in the presence of 1 mM IPTG and elevated levels of MotA were observed by Western blotting (Fig. 2, 3F, and 6B). We conclude that overexpression of flagellar stators is sufficient to compensate for the absence of SwrD and to rescue swarming motility. We infer that overexpression of motA and motB likely accounts for the swarming rescue of the various sod mutants.

We next wanted to determine whether the swrD mutant has a phenotype consistent with a defect in the flagellar stators. As MotA and MotB are also required for swimming motility, we monitored swimming speeds in various genetic backgrounds by phase-contrast microscopy and cell tracking software. Whereas wild-type cells swam with an average velocity of approximately 30 μm/s, swrD mutant cells swam 2-fold slower, with an average velocity of 15 μm/s (Fig. 8A). Overexpression of motA and motB increased the swimming speed of the swrD mutant to wild-type levels and did not increase the swimming speed further in the wild type (Fig. 8A). Swimming speed is the product of the approximately 17 flagella per cell (Fig. 4) (5). We next measured the effect of SwrD on individual flagella by observing cells rotating around a single tethered flagellum and calculating flagellar torque. Flagella of the swrD mutant generated 6-fold-less torque than the wild type, and wild-type torque values were restored to the swrD mutant when motA and motB were overexpressed (Fig. 8B). We conclude that SwrD is required for swarming because it is required to increase flagellar power in a manner that can be compensated for by extra copies of MotA and MotB in the membrane.

FIG 8.

Cells mutated for SwrD have reduced swimming speed and flagellar torque. (A) Swim speed analysis of wild-type (DK4987) and swrD mutant (DK5022) cells unmodified (gray bars) or wild-type (DK5029) and swrD mutant (DK5030) cells in which the motA and motB genes were overexpressed by induction with 1 mM IPTG (black bars). Swimming velocity was calculated for 100 cells, and the average and standard deviation are reported. (B) Torque values of singly tethered wild-type (DK4987) and swrD mutant (DK5022) cells (gray bars) or wild-type (DK5029) and swrD mutant (DK5030) cells in which the motA and motB genes were overexpressed by induction with 1 mM IPTG (black bars). Torque values were calculated for 40 cells tethered by a single flagellum.

DISCUSSION

Swarming motility is a flagellum-mediated form of surface movement, and in B. subtilis, swarming requires an increase in flagellar density above a strict threshold (5). Motile, liquid-grown cells introduced to a surface experience a lag of approximately 1 to 2 h prior to swarming, during which the flagellar density on the cell surface doubles (5). At least two regulators are required for the surface-dependent increase in flagellar number: SwrA activates transcription of the fla-che operon to increase the number of flagellar basal bodies (29, 31), and SwrB activates flagellar type III secretion to increase the number of flagellar hooks (32). Precisely why an increase in flagellar density is required to swarm is unknown, but each additional flagellum adds to the total thrust of the cell, and perhaps a threshold amount of power is necessary to overcome surface forces. Here we report and characterize another protein required for swarming, SwrD, that is not impaired for flagellar number but is impaired in the torque generated by each flagellum.

SwrD is a 71-amino-acid protein encoded within the B. subtilis fla-che operon, and mutation of SwrD results in a 6-fold reduction in flagellar torque that may be overcome by specifically overexpressing the flagellar stator components MotA and MotB. The mechanism by which SwrD increases torque is unknown save that MotA protein levels were not impaired in a swrD mutant and thus SwrD appears to act at the level of MotAB activity. The MotAB stator complexes in E. coli dynamically associate with the basal body, where torque increases and decreases with stator association and dissociation (17, 18). If stators are also dynamic in B. subtilis in the absence of SwrD, then MotAB overexpression may increase the probability or duration (duty ratio) of stator-rotor interaction (52, 53). Thus, SwrD could be a tether that retains MotAB at the basal bodies to decrease stator dynamism and increase stator association in the wild type. Perhaps consistent with stator association, SwrD activity appears related to stator-rotor stoichiometry, as the swrD phenotype was overcome by increasing stators relative to rotors but was not overcome by increasing expression of all flagellar genes at the same ratio (e.g., SwrA overexpression). Although results with overexpression of MotA and MotB point to stator dynamism, we have not been able to generate a functional fluorescent fusion to either protein in B. subtilis, and thus stator dynamism is not directly testable. Whether SwrD interacts with MotAB, whether it interacts with other basal body components, or whether it functions intracellularly or extracellularly is currently unknown.

In B. subtilis, the gene encoding SwrD is located immediately upstream of, and may be translationally coupled with, the gene that encodes FliL. Primarily studied in Proteobacteria that lack SwrD, FliL associates with the basal body and deletion of FliL reduces swimming speed and motor torque (54–58). Further, FliL has been implicated in the regulation of swarming motility in both Proteus mirabilis and Salmonella enterica (57, 59–61). In B. subtilis, however, disruption of FliL results in a swarming motility defect that is less severe than that caused by disruption of SwrD, and unlike cells mutated for swrD, cells mutated for fliL did not experience improved swarming when MotA and MotB were overexpressed (see Fig. S2 in the supplemental material). Thus, while FliL and SwrD both increase flagellar power, we infer that they may do so by different mechanisms.

In summary, we conclude that a net increase in flagellar thrust is needed for B. subtilis to swarm. One way to increase power is to increase the flagellar density, controlled, at least in part, by SwrA and SwrB. Another way to increase total power is to increase the torque of each flagellum, controlled, at least in part, by SwrD. Neither strategy is sufficient to swarm, however, and both flagellar number and flagellar torque must be increased. Whereas SwrA and SwrB are narrowly conserved, phylogenetic analysis suggests that SwrD is found in a broad distribution of bacteria and is often encoded upstream of MotA and MotB (Fig. S3). We note that one phylum of bacteria that encodes SwrD is the Spirochaetes. Pathogenic spirochetes navigate viscous environments and can move between tight junctions of eukaryotic cells during infections, two behaviors that may require increased flagellar power. We suspect that SwrD may also increase torque to spirochete endoflagella and may be required for virulence.

MATERIALS AND METHODS

Strains and growth conditions.

B. subtilis strains were grown in lysogeny broth (LB) (10 g tryptone, 5 g yeast extract, 5 g NaCl [per liter]) broth or on LB plates fortified with 1.5% Bacto agar at 37°C. When appropriate, antibiotics were included at the following concentrations: 10 μg/ml tetracycline, 100 μg/ml spectinomycin, 5 μg/ml chloramphenicol, 5 μg/ml kanamycin, and 1 μg/ml erythromycin plus 25 μg/ml lincomycin (mls). Isopropyl-β-d-thiogalactopyranoside (IPTG; Sigma) was added to the medium at the indicated concentration when appropriate.

Swarm expansion assay.

Cells were grown to mid-log phase at 37°C in LB broth and resuspended to an optical density at 600 nm (OD₆₀₀) of 10 in pH 8.0 phosphate-buffered saline (PBS) buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 2 mM KH₂PO₄) containing 0.5% India ink (Higgins). Freshly prepared LB containing 0.7% Bacto agar (25 ml/plate) was dried for 10 min in a laminar flow hood, centrally inoculated with 10 μl of the cell suspension, dried for another 10 min, and incubated at 37°C (4). The India ink demarks the origin of the colony, and the swarm radius was measured relative to the origin. For consistency, an axis was drawn on the back of the plate and swarm radius measurements were taken along this transect. For experiments including IPTG, cells were propagated in broth in the presence of IPTG, and IPTG was included in the swarm agar plates. All experiments containing IPTG in this study were performed with a concentration of 1 mM IPTG.

Swim motility assay.

For swim videos, cells were grown to mid-log phase (∼0.6 OD₆₀₀) and a hanging drop wet mount was prepared. Video was captured with a Nikon 80i microscope with a Nikon Plan Apo 100× phase-contrast objective using MetaMorph software. For swim plate assays, cells were toothpick inoculated into LB containing 0.3% Bacto agar (25 ml/plate).

Strain construction.

All PCR products were amplified from B. subtilis genomic DNA from the indicated strains. Constructs built were either introduced into the domesticated B. subtilis strain PY79 or the ancestral, competent cured plasmid strain DS2569 and transferred to the 3610 background via SPP1-mediated phage transduction or transformed directly into DK1042 (3610 comI^Q12L) (62). All strains used in this study are listed in Table 2. All primers used to build strains for this study are listed in Table S1 in the supplemental material, and all plasmids are listed in Table S2 in the supplemental material.

TABLE 2.

Bacillus subtilis strains

Strain	Genotype (reference)
3610	Wild type
DK38	ΔswrD sod38
DK39	ΔswrD sod39 (flgMQ9*)
DK40	ΔswrD sod40 (flgMQ64*)
DK41	ΔswrD sod41 (flgMQ64*)
DK42	ΔswrD sod42 (flgMQ64*)
DK43	ΔswrD sod43 (flgMQ64*)
DK44	ΔswrD sod44 (flgMT26^FS)
DK46	ΔswrD sod46 (flgMN80^FS)
DK47	ΔswrD sod47 (flgMQ64*)
DK48	ΔswrD sod48 (fliSRBS GGAGG→AGAGG)
DK67	ΔswrD amyE::Pfla-che-lacZ cat
DK68	ΔswrD amyE::Phag-lacZ cat
DK374	srfAC::Tn10 spec epsH::tet (45)
DK378	Δhag srfAC::Tn10 spec epsH::tet (45)
DK478	swrB::tet ΔflgE amyE::Pfla-che-flgET123C cat (32)
DK614	ΔswrD sod50
DK615	ΔswrD sod51 (flgMQ64*)
DK616	ΔswrD sod52 (flgMQ64*)
DK801	motAB::tet amyE::Physpank-motAB spec
DK1042	comIQ12L (62)
DK1358	ΔswrD ΔfliM amyE::Pfla-che-fliM-GFP spec
DK1359	ΔswrD ΔflgE amyE::Pfla-che-flgET123C cat
DK1405	ΔswrD srfAC::Tn10 spec epsH::tet
DK1597	ΔswrD amyE::Physpank-swrA spec
DK1839	ΔswrD sod2 amyE::PflgM-flgM cat
DK2203	ΔcheA srfAC::Tn10 spec epsH::tet
DK2907	ΔswrD sod48 amyE::PmotA-lacZ cat
DK2919	ΔswrD sod4 amyE::PmotA-lacZ cat
DK2925	ΔswrD amyE::PflgM-lacZ cat
DK2926	ΔswrD sod2 amyE::PflgM-lacZ cat
DK2927	ΔswrD sod4 amyE::PflgM-lacZ cat
DK2929	ΔswrD sod48 amyE::PflgM-lacZ cat
DK2968	ΔswrD sod4 amyE::Phag-lacZ cat
DK2970	ΔswrD sod48 amyE::Phag-lacZ cat
DK3046	ΔswrD amyE::PmotA-lacZ cat
DK3187	ΔswrD sod4 amyE::Pfla-che-lacZ cat
DK3188	ΔswrD sod48 amyE::Pfla-che-lacZ cat
DK3213	ΔswrD pgsB::Tn10 spec
DK3214	motAB::tet pgsB::Tn10 spec
DK4616	ΔswrD ΔfliM amyE::Pfla-che-fliM-GFP spec thrC::Physpank-swrA mls
DK4651	ΔswrD amyE::Physpank-motAB spec
DK4724	ΔswrD ΔflgE amyE::Pfla-che-flgET123C cat thrC::Physpank-swrA mls
DK4757	ΔswrD sod2 amyE::Phag-lacZ cat
DK4758	ΔswrD sod2 amyE::Pfla-che-lacZ cat
DK4759	ΔswrD sod2 amyE::PmotA-lacZ cat
DK4987	ΔcheB comIQ12L
DK4698	amyE::PfliD-lacZ cat
DK4699	ΔswrD amyE::PfliD-lacZ cat
DK4805	amyE::PfliDsod4-lacZ cat
DK4812	ΔswrD amyE::PfliDsod4-lacZ cat
DK5022	ΔswrD ΔcheB comIQ12L
DK5029	ΔswrD ΔcheB amyE::Physpank-motAB spec comIQ12L
DK5030	ΔcheB amyE::Physpank-motAB spec comIQ12L
DK5113	ΔfliL amyE::Physpank-motAB spec
DK5314	epsH::tet srfAA::mls amyE::Physpank-motAB spec
DS5315	ΔswrD epsH::tet srfAA::mls amyE::Physpank-motAB spec
DS222	motAB::tet (51)
DS791	amyE::Pfla-che-lacZ cat (29)
DS793	amyE::Phag-lacZ cat (29)
DS811	amyE::flgM-lacZ cat (29)
DS860	amyE::Physpank-swrA spec (29)
DS908	amyE::Phag-GFP cat (29)
DS1849	amyE::PmotA-lacZ cat (43)
DS1916	amyE::Phag-hagT209C spec (36)
DS6540	ΔfliL (24)
DS6657	ΔswrD (24)
DS6697	ΔswrD sod1 (flgMQ31^FS)
DS6698	ΔswrD sod2 (flgMQ64*)
DS6699	ΔswrD sod3 (flgMQ31^FS)
DS7498	ΔmotA (51)
DS7527	ΔswrD sod4 (PfliD TGTAAT→CGTAAT)
DS7528	ΔswrD sod5 (flgMN17^FS)
DS7529	ΔswrD sod6 (flgMV72*)
DS7530	ΔswrD sod7 (flgMQ64*)
DS7550	ΔswrD amyE::Pfla-che-swrD cat
DS7673	ΔflgE amyE::Pfla-che-flgET123C cat (33)
DS7696	ΔfliL amyE::Phag-GFP cat (24)
DS7792	ΔfliS (45)
DS8521	ΔfliM amyE::Pfla-che-fliM-GFP spec (31)
DS8600	ΔswrA ΔfliM amyE::Pfla-che-fliM-GFP spec (31)
DS8791	ΔswrD sod8 (flgMQ64*)
DS8816	ΔswrD amyE::Phag-hagT209C spec
DS9319	ΔswrB amyE::Phag-hagT209C spec
PY79	sfp0 swrA

Complementation construct.

To generate the amyE::P_fla-che-swrD cat ectopic swrD complementation construct, a PCR product containing swrD was amplified from chromosomal 3610 DNA with primer pair 2462/2463 and digested with BamHI and XhoI, and the P_fla-che promoter was amplified from 3610 DNA with primer pair 1802/2460 and digested with XhoI and EcoRI. The fragments were simultaneously ligated into the BamHI and EcoRI sites of pDG364, which carries a chloramphenicol resistance marker and a polylinker between two arms of the amyE gene, to generate pDP329 (63).

LacZ reporter fusions.

The P_fliD region was amplified separately from 3610 and DS7527 chromosomal DNA with primer pair 609/1510 and digested with HindIII and EcoRI. The fragment was ligated into the HindIII and EcoRI sites of pDG268, which carries a chloramphenicol resistance marker and a polylinker upstream of the lacZ gene between two arms of the amyE gene, to create pANR21 and pANR22, respectively (64).

SPP1 phage transduction.

Serial dilutions of SPP1 phage stock were added to 0.2 ml of dense B. subtilis culture grown in TY broth (LB supplemented with 10 mM MgSO₄ and 100 μM MnSO₄, added after autoclaving), and the mixture was incubated statically at 37°C for 15 min. Three milliliters of TYSA (molten TY broth supplemented with 0.5% agar) was added to each mixture and poured onto fresh TY plates, and the mixture was incubated at 30°C overnight. The top agar from the plate containing clear phage plaques was harvested by scraping it into a 15-ml conical tube, vortexing it, and centrifuging it at 6,500 × g for 10 min. The lysate was treated with 25 μg/ml DNase before being passed through a 0.45-μm syringe and being stored at 4°C.

Recipient cells were grown to stationary phase in 3 ml TY broth at 37°C. Cells (1 ml) were mixed with 9 ml of TY and 15 μl donor SPP1 phage stock (chloramphenicol, kanamycin, and spectinomycin markers) or 5 μl donor SPP1 phage stock (mls and tetracycline reporters). The mixture was incubated at room temperature with gentle rocking for 30 min. The transduction mixture was centrifuged at 6,500 × g for 5 min, the supernatant was discarded, and the pellet was resuspended in the remaining volume. One hundred microliters of the suspension was plated on LB medium fortified with 1.5% agar and the appropriate antibiotic (65).

Microscopy.

Fluorescence microscopy was performed with a Nikon 80i microscope with a Nikon Plan Apo 100× phase-contrast objective and an Excite 120 metal halide lamp. FM 4-64 dye was visualized with a C-FL HYQ Texas Red filter cube (excitation filter, 532 to 587 nm; barrier filter, >590 nm). GFP was visualized using a C-FL HYQ fluorescein isothiocyanate (FITC) filter cube (FITC, excitation filter, 460 to 500 nm; barrier filter, 515 to 550 nm). Images were captured with a Photometrics Coolsnap HQ2 camera in black and white, false colored, and superimposed using MetaMorph image software. Counting of flagellar basal bodies and hooks was performed on an OMX three-dimensional structured illumination microscope (3D SIM) in the Light Microscopy Imaging Center (LMIC), Indiana University, and quantification was performed using the Imaris image analysis software.

For GFP microscopy, cells were grown in LB medium to an OD₆₀₀ of 0.6 to 1.0. One milliliter was harvested, resuspended in 50 μl of 1× PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 2 mM KH₂PO₄) containing 5 μg/ml FM 4-64, incubated for 3 min at room temperature, pelleted, washed two times with 1 ml of 1× PBS buffer, and finally resuspended to an OD₆₀₀ of 10 in 1× PBS. A 4.5-μl suspension volume was spotted on a glass slide and immobilized with a poly-l-lysine-treated glass coverslip prior to microscopy.

For fluorescence microscopy of flagella and flagellar hooks, 1 ml of broth culture was harvested at an OD₆₀₀ of 0.5 to 1.0 and washed once in 1.0 ml of 1× PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 2 mM KH₂PO₄). The suspension was pelleted, resuspended in 50 μl of PBS buffer containing 5 μg/ml Alexa Fluor 488 C₅ maleimide (Molecular Probes), and incubated for 5 min at room temperature. Cells were then washed once with 1 ml PBS buffer. When appropriate, membranes were stained by resuspension in 50 μl of PBS buffer containing 5 μg/ml FM 4-64 (Molecular Probes) and incubated for 10 min at room temperature. Three microliters of suspension was placed on a microscope slide and immobilized with a poly-l-lysine-treated coverslip.

Western blotting.

Strains were grown to mid-log phase, concentrated to an OD₆₀₀ of 10 in lysis buffer (17.2 mM Tris [pH 7.0], 8.6 mM EDTA [pH 8.0], 1 mg/ml lysozyme, 0.1 mg/ml RNase A, 20 μg/ml DNase I, and 50 μg/ml phenylmethane sulfonyl fluoride) and incubated at 37°C for 30 min. SDS sample buffer (500 mM Tris [pH 6.8], 22% glycerol, 10% SDS, and 0.12% bromophenol blue) was added, and samples were boiled for 5 min. Twelve-microliter volumes of boiled samples were loaded onto 10% polyacrylamide native (with no added SDS) or 15% polyacrylamide denaturing (with 0.1% SDS) gels. Lysates were resolved at 150 V for 1.25 h, transferred onto nitrocellulose membranes, and subsequently probed with a 1:10,000 dilution of anti-FliS, a 1:3,000 dilution of anti-MotA (66), or a 1:80,000 dilution of anti-SigA polyclonal antiserum. Following incubation with the primary antibodies, nitrocellulose membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G. Blots were developed using Pierce ECL substrate (Thermo Fisher Scientific).

FliS antibody preparation.

One milligram of purified FliS protein (45) was sent to Cocalico Biologicals for serial injection into a rabbit host for antibody generation. Anti-FliS serum was mixed with FliS-conjugated Affigel-10 beads and incubated overnight at 4°C. Beads were packed onto a 1-cm column (Bio-Rad) and then washed with 100 mM glycine (pH 25) to release the antibody and immediately neutralized with 2 M Tris base. Purification of the antibody was verified by SDS-PAGE. Purified anti-FliS antibody was dialyzed into 1× PBS (pH 7.4) supplemented with 50% glycerol and stored at −80°C.

Swimming velocity analysis.

Cells were grown to an OD₆₀₀ of ∼0.6 and then resuspended to an OD₆₀₀ of 0.2 in LB medium. Tunnel slides were prepared by placing two coverslips with an ∼2-cm gap in between them on a glass slide, and a third coverslip was placed over them; all the coverslips were secured using nail polish strengthener. The cells were then introduced into the tunnel slides and imaged using a Nikon 80i microscope with a Nikon Plan Apo 40× phase-contrast objective, and videos were recorded using a Photometrics Coolsnap HQ2 camera in black and white for 30 s at 5 frames per second. The videos were then analyzed by MicrobeJ (67) tracking software, and the velocity was determined using the MOTION.Velocity function (100 cells for each strain).

Torque calculation.

Cells were grown to an OD₆₀₀ of ∼0.6 and then resuspended to an OD₆₀₀ of 0.2 in LB medium. The cells were then introduced into the tunnel slides (see “Swimming velocity analysis”), singly tethered cells were then monitored using a Nikon 80i microscope with a Nikon Plan Apo 40× phase-contrast objective, and videos were recorded using a Photometrics Coolsnap HQ2 camera in black and white for 90 s at 3 fps. The angle traveled by the cells in radians as a function of time was calculated by utilizing the theta.ORIENTATION function (40 cells for each strain) in MicrobeJ (67). Torque is calculated using the formula N_r = (C_r + r²C_t)2πf, where r is the distance between the center of rotation and the center of mass of a cell, f is the rotation rate, and C_r and C_t are rotational and translational frictional drag coefficients, respectively (68). With the cell approximated as a prolate ellipsoid, C_r = (8πηa³/3)/(ln 2a/b − 0.5) and C_t = 8πηa/(ln 2a/b + 0.5), where a is cell length divided by 2 and b is cell width divided by 2.

β-Galactosidase assay.

Cells were grown to an OD₆₀₀ of ∼0.7 to 1.3 in LB medium in triplicate. One milliliter of each sample was harvested and resuspended in 1 ml of Z buffer (40 mM HaH₂PO₄, 60 mM Na₂HPO₄, 1 mM MgSO₄, 10 mM KCl, and 38 mM β-mercaptoethanol). Lysozyme was added to each sample to a final concentration of 0.2 mg/ml, and samples were incubated for 15 min at 30°C and thereafter kept on ice. Each sample was diluted appropriately to a final volume of 500 μl in Z buffer, and the reaction was started with 100 μl of 4 mg/ml o-nitrophenyl-β-d-galactopyranoside (in Z buffer) and stopped with 250 μl of 1 M Na₂CO₃. The OD₄₂₀ of the reaction mixtures was recorded, and the β-galactosidase activity was calculated with the following formula [OD₄₂₀/(time in minutes × OD₆₀₀)] × dilution factor × 1,000. All reactions were stopped prior to saturation of yellow color (A₄₂₀ < 1.2). For those reactions with low to no β-galactosidase activity, the reaction was run for a maximum of 1 h before stopping.