Viscous drag on the flagellum activates Bacillus subtilis entry into the K-state

Abstract

Bacillus subtilis flagella are not only required for locomotion but also act as sensors that monitor environmental changes. Although how the signal transmission takes place is poorly understood, it has been shown that flagella play an important role in surface sensing by transmitting a mechanical signal to control the DegS-DegU two-component system. Here we report a role for flagella in the regulation of the K-state, which enables transformability and antibiotic tolerance (persistence). Mutations impairing flagellar synthesis are inferred to increase DegU-P, which inhibits the expression of ComK, the master regulator for the K-state, and reduces transformability. Tellingly, both deletion of the flagellin gene and straight filament (hag^A233V ) mutations increased DegU phosphorylation despite the fact that both mutants had wild type numbers of basal bodies and the flagellar motors were functional. We propose that higher viscous loads on flagellar motors result in lower DegU-P levels through an unknown signaling mechanism. This flagellar-load based mechanism ensures that cells in the motile subpopulation have a tenfold enhanced likelihood of entering the K-state and taking up DNA from the environment. Further, our results suggest that the developmental states of motility and competence are related and most commonly occur in the same epigenetic cell type.

Figure 1

Low comK expression in the absence of flagella synthesis. (A) The rate of comK expression is inhibited in the fla/che deletion strain (BD7763) compared to its isogenic wild-type equivalent (BD6439). (B) Dose-dependent suppression of comK expression by flagellar synthesis. Transcription rates from the PcomK-luc reporter in a strain with an IPTG inducible fla/che operon in the native locus. Strain BD7695 (Physp-fla/che-operon PcomK-luc) was grown in the absence and presence of different concentrations of IPTG (as indicated). PcomK-luc expression in the wild-type (BD6439) is shown for comparison. All strains used for this experiment were built in the undomesticated background NCIB 3610. The vertical arrows in each panel point to the time of entry into stationary phase (T₀), determined from optical density measurements during growth in the plate reader.

Figure 2

Deletion of hag and motB lower the expression of comK and a Δhag mutation lowers the frequency of entry to the K-state. The effects of hag and motB knockouts on PcomK-luc expression in the presence (A) or absence (B) of ComK are shown. The following strains were used in these experiments: wild-type PcomK-luc (BD4773), Δhag PcomK-luc (BD7636), ΔmotB PcomK-luc (BD7466), ΔcomK PcomK-luc (BD4893), Δhag ΔcomK PcomK-luc (BD7261) and ΔmotB ΔcomK PcomK-luc (BD7488). The expression profiles in A and B are plotted on different scales. The vertical arrows in panels A and B point to T₀. (C) Single-cell expression and microscopic enumeration of PcomG-yfp expressing cells in Δhag (BD7262) and wild-type (BD5783) populations. The indicated strains were grown to the time of maximum K-state expression (T₂) and samples were taken for microscopy. Representative images are shown. In the upper right corner are the average percentages of K-state cells determined by counting at least 1000 cells for each strain, done in duplicate. Scale bar is 5μm.

Figure 3

DegU mediates the increase in aprE transcription caused by deletion of hag. (A) Increased expression of the PaprE-luc reporter in the hag and motB knock out strains. (B) aprE expression can be completely suppressed by a degU deletion in the hag background. The strains used were as follows: wild-type PaprE-luc (BD6904), Δhag PaprE-luc (BD7639), ΔmotB PaprE-luc (BD7719), ΔdegU PaprE-luc (BD7084) and Δhag ΔdegU PaprE-luc (BD7371). The vertical arrows in each panel point to T₀.

Figure 4

DegU phosphorylation represses the basal expression of comK in motB and hag knockouts. The expression curves show PcomK-luc expression in a comK deletion background. The degU^D56N allele as well as the degS deletion bypass Δhag (A) and ΔmotB (B) for expression from PcomK. Strains used were as follows: ΔcomK PcomK-luc (BD4893), Δhag ΔcomK PcomK-luc (BD7261), ΔmotB ΔcomK PcomK-luc (BD7488), Δhag ΔdegS ΔcomK PcomK-luc (BD7491), ΔmotB ΔdegS ΔcomK PcomK-luc (BD7490), Δhag degU^D56NΔ comK PcomK-luc (BD7475) and ΔmotB degU^D56N ΔcomK PcomK-luc (BD7492). The vertical arrows point to T₀.

Figure 5

K-state cells have low DegU-P. (A) The diagram explains the construct used for panel B. YFP with its own ribosomal binding site was placed downstream of the degS degU operon, so that it is expressed from the three native promoters. The P3 promoter is activated by DegU-P (Ogura & Tsukahara, 2010). This strain (BD8556) also carries PcomK-cfp (blue) to monitor expression of the K-state. (B) The top row shows expression of YFP (yellow) and CFP (blue) in strain BD8556 while the bottom row shows expression from its isogenic equivalent (BD8557) that also carries a hag deletion. In the P_degU-YFP images, the comK expressing cells are indicated by arrows. In the Δhag strain, 39 % of the cells were scored visually as expressing high level of YFP, whereas in the wild-type strain shown in the top row, 3.8 % were high expressers. More than 2000 cells were scored for each strain. Cultures were grown to the time of maximum competence (T₂). Representative images are shown. Scale bar is 5 μm.

Figure 6

Basal body numbers are unaltered in Δhag and hag^A233V mutants. (A) Fluorescence microscopy of strains with the indicated genotypes stained for membranes with FM4-64 (red) and flagellar hooks (green). The following strains were used: wild-type (DK3455), Δhag (BD8396) and hag^A233V (DK3456). (B) Graphical representation of flagellar hooks as determined by counting the number of FlgE^T123C puncta per cell per cell length using Imaris image analysis software. Individual cell data presented as a scatter plot. Average of length and spot number indicated as a larger solid dot with standard deviations that emerge from the solid dot as horizontal and vertical lines. N = 28 cells. Red symbols indicate wild-type (DK3455), blue symbols indicate Δhag (BD8396) and green symbols indicate hag^A233V (DK3456). (C) Overlay of hook scatter plots from (B).

Figure 7

A hag deletant and a straight filament mutant have functional motors. Rotational speeds of individual tethered cells belonging to the wild-type strain (top panel), the Δhag strain (middle panel) and the straight mutant (bottom panel) are indicated. The gray lines indicate raw data and the black curves represent filtered values. A majority of the cells predominantly rotated in the CCW direction (positive speeds). A small fraction (< 10%) of the cells exhibited CW rotation. Strains used for this experiment were: wild-type flgE^T123C (BD8207), Δhag flgE^T123C (BD8263) and hag^A233V-T209C (BD7757).

Figure 8

Cells with straight filaments have increased DegU-P levels. (A) The nonmotile phenotype of a hag^A233V mutant. The wild-type (IS75) and the hag^A233V mutant (BD7599) were centrally inoculated on LB fortified with 0.3% agar. After 18 h incubation at 30°C, the plates were photographed against a black background. (B) Fluorescence microscopy of flagella filaments (Hag^T209C). Hag^T209C was stained with a maleimide reactive dye (green) in the wild-type strain (BD7816, hag^T209C) and the hag^A233V mutant (BD7757, hag^T209C-A233V). Strains were grown to mid log phase in LB. (C) hag^A233V decreases comK expression. Effect of hag^A233V (BD7626) on PcomK-luc expression compared to wild-type (BD4773). (D) degU^D56N suppresses high aprE expression in hag^A233V. Expression from PaprE-luc in hag^A233V is higher than wild-type expression. The degU^D56N allele bypasses hag^A233V for expression from PaprE. Strains used were: wild-type PaprE-luc (BD6904), hag^A233V PaprE-luc (BD8007), degU^D56N PaprE-luc (BD7423), and hag^A233V degU^D56N PaprE-luc (BD8011). The vertical arrows in panel C and D indicate T₀.