Research ArticleBiofilms

The extracellular matrix protein TasA is a developmental cue that maintains a motile subpopulation within Bacillus subtilis biofilms

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Science Signaling  19 May 2020:
Vol. 13, Issue 632, eaaw8905
DOI: 10.1126/scisignal.aaw8905
  • Fig. 1 Flagellar motility promotes B. subtilis colony expansion and engulfment of foreign objects.

    (A) Time-lapse images of the engulfment of cellulose acetate or filter paper discs placed 0.3 cm from the inoculation point by wild-type (WT) and Δhag colonies, both harboring the flagellin reporter Phag-GFP. Images show colony growth at the indicated time points after inoculation. For each strain, representative bright field and GFP fluorescence images are shown, as well as a magnified image of GFP fluorescence at the 36-hour time point. n = 9 for each strain. Scale bars, 2 mm. (B) Higher magnification representative images showing cells covering a filter paper disc at the indicated time points for a wild-type colony carrying both the Phag-GFP (motility, green) and PtapA-mKate (matrix, red) reporters. Three colonies per strain were examined, with at least 10 representative fields recorded per colony. Scale bar, 1 μm. (C) Quantification of filter paper disc engulfment by the parental wild-type and indicated mutant strains. Discs were placed at distances of 0.3 and 0.5 cm from the inoculation point. At the indicated times, the extent of engulfment was determined as the percentage of the disc circumference covered by bacterial cells. Disc coverage by at least 12 colonies per strain per condition is shown. The boxes indicate the lower and upper quartiles, and the central line indicates the median. Whiskers above and below the box indicate the 90th and 10th percentile. Outliers are shown as filled circles (•). The differences between all the mutants and WT and between ΔepsH and Δsrf strains and Δhag strain were significant. P < 0.05, as determined by analysis of variance (ANOVA) test, followed by Tukey’s honestly significant difference (HSD).

  • Fig. 2 TasA induces expression of flagellar genes in biofilms.

    (A) Time-lapse images of the engulfment of cellulose acetate or filter paper discs placed 0.3 cm from the inoculation point, by wild-type and ΔtasA colonies, both harboring the Phag-GFP reporter. Images show colony growth at the indicated time points after inoculation. For each strain, representative brightfield and GFP fluorescence images of one colony are shown, as well as a magnified image of GFP fluorescence at the 36-hour time. n = 9 colonies for each strain. Scale bars, 2 mm. (B) Representative bright field and fluorescence images of colonies of wild-type and the indicated ECM mutants harboring the Phag-GFP reporter, grown for 48 hours. Scale bar, 2 mm. (C) Flow cytometry analysis of cells from colonies in (B). Nonfluorescent colonies (gray) were used as a control (gray). The dashed vertical line indicates the autofluorescence signal used for gating. The solid line indicates the median of the gated population. Percentage of gated cells ± SD of three colonies is shown. ***P ≤ 0.001, compared to wild type, as determined by ANOVA, followed by Tukey’s HSD. n = 9 colonies for each strain. AU, arbitrary units. (D) Growth curves for wild-type and the indicated ECM mutants harboring the Phag-GFP reporter in shaking liquid cultures. Averages and SDs of six cultures for each strain are shown. (E) Phag-GFP fluorescence of the cultures in (D) at 10 hours of growth normalized to OD600. Fluorescence of six cultures at 10 hours of growth normalized to OD600 is shown. **P ≤ 0.01, as compared to wild type, as determined by ANOVA, followed by Tukey’s HSD.

  • Fig. 3 Secreted TasA maintains hag expression.

    (A) Time-lapse images of a strain carrying PtapA-tapA-sipW-tasA-mCherry, taken using the CellASIC microfluidic platform. Images were taken at the fixed time intervals after loading as indicated. Red, mCherry. Images are representative of nine independent experiments. Scale bars, 10 μm. (B) Time-lapse images a strain carrying PtapA-tapA-sipW-tasA-mCherry and Phag-GFP, taken using the CellASIC microfluidic platform. Images were taken at the fixed time intervals after loading as indicated. Red, mCherry; yellow, GFP. White arrowheads indicate progeny of the cell marked with an arrow. Images represent one of three independent experiments. Scale bars, 10 μm. (C) Normalized Phag-GFP fluorescence in cultures treated with the indicated compounds as a function of each compound’s osmolarity or viscosity. Fluorescence at 10 hours of growth was normalized to the OD600 and then normalized to the fluorescence of a nontreated sample in the same experiment. Each point represents the average of at least three cultures; bars represent SD. (D) A representative colony of wild-type and the indicated mutants harboring the Phag-GFP reporter, grown for 24 hours. Representative brightfield and GFP fluorescence images are shown. n = 5 colonies for each strain. Scale bar, 2 mm. (E) Flow cytometry measurements of colonies in (B) and floating biofilms (pellicles) of the indicated strains. Data for two representative colonies (of seven analyzed) for each strain are shown. The dashed vertical line indicates the autofluorescence used for gating. (F) Flow cytometry analysis of colonies of the indicated strains, grown for 24 hours. Percentage of gated GFP-positive cells ± SD of nine colonies is shown. ***P ≤ 0.001 as compared to wild type or ΔtasA, as determined by Dunnett’s test.

  • Fig. 4 Transcriptomic analysis of cellular pathways regulated by TasA.

    (A) Volcano plot showing genes differently expressed in ΔtasA relative to wild-type cells. Genes belonging to functional categories identified by DAVID analysis are highlighted. (B) Log2 fold change (FC) in gene expression of the genes highlighted in (A) (marked with asterisk) and their neighboring genes. The genes within each functional category are ordered by their chromosomal location. (C and D) Log2 FC in expression of genes highlighted in (A), in ΔsipW relative to wild-type cells.

  • Fig. 5 TasA stimulates the reversion to motility from the biofilm state.

    (A) Time-lapse images of wild-type cells carrying PtapA-CFP (blue) and Phag-GFP (yellow). Images were acquired at the indicated time after inoculation. Arrows indicate one parental cell, and arrowheads indicate its progeny. n = 3 independent experiments. Representative fields are shown. Scale bars, 10 μm. (B) As in (A), but the wild-type strain was compared to the ΔtasA strain carrying the same reporters. n = 3 independent experiments. Representative fields are shown. Scale bars, 10 μm. (C) Chain length at 4.5 hours after inoculation, in wild-type and ΔtasA strains. At least 1800 chains for each strain were analyzed (at least 18 fields in n = 3 independent experiments). (D) Fraction of chain length expressing PtapA-CFP at 4.5 hours. At least 18 chains for each strain were analyzed from three independent experiments. Data represent the means with SD. ***P ≤ 0.001 as determined by Student’s t test. (E) Length of chains in which PtapA was expressed at 4.5 hours after inoculation, in wild-type and ΔtasA strains. At least 180 chains for each strain were analyzed from three independent experiments. (F) Flow cytometry of colonies of the indicated genotypes harboring the Phag-GFP reporter, grown for 48 hours. Three colonies for each strain are shown, and the same wild-type control is shown in both traces. The dashed vertical line indicated autofluorescence used for gating. (G) Ratio of cells expressing Phag-GFP above autofluorescence between the strains shown in (F); comparing the parental strain with a derivative carrying an additional tasA deletion. Average of gated cells in three colonies per strain is shown; error bars represent SD. *P ≤ 0.05 for the ΔtasA derivative strains compared to the parental strain, as determined by ANOVA test, followed by Tukey’s HSD.

  • Fig. 6 Spontaneous hypermotile mutants in ΔtasA colonies target regulators of the motility-biofilm switch.

    (A) Overview of 25 representative ΔtasA colonies (of 141 colonies) harboring Phag-GFP reporter and grown for 5 days, showing fluorescent protrusions at the colony edge. Enlarged images of one typical ΔtasA colony with a protrusion (arrowhead) are shown. Scale bar, 2 mm. (B) An example of a colony formed by Mut310, one of 80 mutants isolated from protrusions as in (A). Scale bar, 2 mm. (C) Diameter of swimming ring in soft agar after 10 hours for wild-type, ΔtasA, ΔtasAΔeps, and representative mutants isolated from protrusions as in (A). Means ± SD of the indicated strains (four colonies per strain) and representative brightfield images of WT and Mut310 on swimming plates are shown. ***P ≤ 0.001 compared to WT, as determined by ANOVA, followed by Tukey’s HSD. Scale bar, 2 cm. (D) Mutations in the slrR locus that were found in hypermotile mutant strains Mut205, Mut340, Mut310, Mut316, and Mut331 isolated from colony protrusions. Mutations in additional genes were identified in the isolates listed in the table. Mutant strain names are shown in green with the parental strain name noted in parentheses. (E and F) Schematic model of SinR- and SlrR-mediated regulation of biofilm and motility genes. (E) During planktonic growth, the expression of both ECM operons and slrR is repressed by SinR. During the transition to the biofilm state, SinI represses SinR, causing the activation of the ECM operons and slrR. In turn, SlrR binds to SinR causing it to repress the motility genes. (F) In slrR mutants, as in the isolates found in this screen, SinR no longer represses the motility genes, and SinR can repress the ECM operons because it is no longer complexed with SlrR.

  • Fig. 7 The CssR/CssS two-component system represses the action of TasA on the motility-biofilm switch.

    (A) Flow cytometry measurements of colonies of wild-type and ΔcssRS strains harboring the Phag-GFP reporter, grown for 24 hours. Nonfluorescent control, gray; GFP, black (WT) and green (ΔcssRS). The dashed vertical line indicates autofluorescence used for gating. Percentage of gated cells ± SD of three colonies is shown. ***P ≤ 0.001, as determined by Student’s t test. n = 3 independent experiments. (B) As in (A) for WT and ΔcssRS strains harboring the PtapA-GFP reporter. Percentage of gated cells ± SD of three colonies is shown. ***P ≤ 0.001, as determined by Student’s t test. n = 3 independent experiments. (C) Ratio of the fractions of Phag-GFP–expressing cells (above autofluorescence) between the indicated strains, with and without an additional tasA deletion. Means and SD of nine colonies for each strain are shown. ***P ≤ 0.001, as determined by Student’s t test. n = 3 independent experiments. (D) Flow cytometry measurements of colonies (three for each strain) of the indicated genetic background harboring Phag-GFP reporter, grown for 24 hours. Percentage of gated cells ± SD of three colonies is shown in parentheses. ***P ≤ 0.001, compared to Mut328, as determined by ANOVA, followed by Tukey’s HSD. (E) K-means clustering of 401 genes that were differentially expressed in the ΔtasA and ΔcssRS transcriptomes relative to WT. n = 3 colonies for ∆tasA and ∆cssRS and n = 2 colonies for WT, all grown for 24 hours. (F) Log2 FC in gene expression of EPS production and sporulation-related genes in ΔtasA and ΔcssRS versus the WT parental strain. (G) STRING interaction network of genes that show differential expression behavior in the ∆tasA and ∆cssRS mutants. Genes without STRING interactions were omitted.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/632/eaaw8905/DC1

    Fig. S1. Flagellar motility promotes colony expansion and engulfment of foreign objects.

    Fig. S2. Motile cells move forward during engulfment, followed by matrix producers.

    Fig. S3. Expression of the flagellin GFP reporter in colonies over time.

    Fig. S4. Formation of TasA-mCherry fibers in the microfluidics system.

    Fig. S5. Osmotic pressure does not affect growth rate or restore hag expression in the ΔtasA mutant.

    Fig. S6. Complementation of a tasA mutant by tapA-sipW-tasA expression in trans.

    Fig. S7. ECM-expressing cells form chains in ΔtasA.

    Fig. S8. TasA stimulates the reversal to motility by antagonizing tapA expression.

    Fig. S9. TasA stimulates the reversal to motility independently of DegU or KinD.

    Fig. S10. The effects of KinA to KinC on TasA-dependent motility expression.

    Fig. S11. The CssR/CssS two-component system.

    Fig. S12. Genes differentially expressed in both ΔtasA and ΔcssRS.

    Fig. S13. Stress response genes regulated by TasA and CssR/CssS.

    Fig. S14. TasA stimulates the transition from a matrix-producing state back to the motile state.

    Table S1. Bacterial strains.

    Table S2. Primers.

    Movie S1. TasA fiber formation.

    Movie S2. Induction of motility occurs after tasA expression.

    Movie S3. TasA stimulates reversal to motility after entering the biofilm state.

    Data file S1. Gene expression analysis.

  • The PDF file includes:

    • Fig. S1. Flagellar motility promotes colony expansion and engulfment of foreign objects.
    • Fig. S2. Motile cells move forward during engulfment, followed by matrix producers.
    • Fig. S3. Expression of the flagellin GFP reporter in colonies over time.
    • Fig. S4. Formation of TasA-mCherry fibers in the microfluidics system.
    • Fig. S5. Osmotic pressure does not affect growth rate or restore hag expression in the ΔtasA mutant.
    • Fig. S6. Complementation of a tasA mutant by tapA-sipW-tasA expression in trans.
    • Fig. S7. ECM-expressing cells form chains in ΔtasA.
    • Fig. S8. TasA stimulates the reversal to motility by antagonizing tapA expression.
    • Fig. S9. TasA stimulates the reversal to motility independently of DegU or KinD.
    • Fig. S10. The effects of KinA to KinC on TasA-dependent motility expression.
    • Fig. S11. The CssR/CssS two-component system.
    • Fig. S12. Genes differentially expressed in both ΔtasA and ΔcssRS.
    • Fig. S13. Stress response genes regulated by TasA and CssR/CssS.
    • Fig. S14. TasA stimulates the transition from a matrix-producing state back to the motile state.
    • Table S1. Bacterial strains.
    • Table S2. Primers.
    • Legends for movies S1 to S3
    • Legend for data file S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). TasA fiber formation.
    • Movie S2 (.mp4 format). Induction of motility occurs after tasA expression.
    • Movie S3 (.mp4 format). TasA stimulates reversal to motility after entering the biofilm state.
    • Data file S1 (Microsoft Excel format). Gene expression analysis.

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