CoChat
Sign Up
Cited by 143Bacterial Genetics and BiotechnologyBacterial biofilms and quorum sensingBiochemical and Structural Characterization

The DOI matches its Crossref record; 98 of the 98 references it cites were found in a registry or an index.

A mechanical signal transmitted by the flagellum controls signalling in Bacillus subtilis

Open access
Cairns, Lynne S., Marlow, Victoria L., Bissett, Emma, Ostrowski, Adam, Stanley‐Wall, Nicola R.·2013·Molecular Microbiology
DOI 10.1111/mmi.12342PMC PMC3963450
Open this paper in CoChat — free accountOpen PDF

Abstract

Summary: In the natural environment bacteria predominantly live adhered to a surface as part of a biofilm. While many of the components needed for biofilm assembly are known, the mechanism by which microbes sense and respond to contact with a surface is poorly understood. B acillus subtilis is a G ram‐positive model for biofilm formation. The DegS – DegU two‐component system controls several multicellular behaviours in B . subtilis , including biofilm formation. Here we identify the B . subtilis flagellum as a mechanosensor that activates the DegS – DegU regulatory pathway. Inhibition of flagellar rotation by deletion or mutation of the flagellar stator gene, motB , results in an increase in both degU transcription and DegU ∼ P driven processes, namely exoprotease production and poly‐γ‐ dl ‐glutamic acid biosynthesis. Similarly, inhibition of flagellar rotation by engaging the flagellar clutch or by tethering the flagella with antibodies also promotes an increase in degU transcription that is reflective of increased DegU ∼ P levels in the cell. Collectively, these findings strongly indicate that inhibition of flagellar rotation acts as a mechanical trigger to activate the DegS – DegU two‐component signal transduction system. We postulate that inhibition of flagellar rotation could function as a mechanical trigger to activate bacterial signal transduction cascades in many motile bacteria upon contact with a surface.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1111/mmi.12342

Cites

98 of 98 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1 Deletion of motB from the chromosome is associated with γ-PGA production.A. Colony morphology of 3610 (wild-type), ΔmotB (NRS3494), ΔmotB + amyE::PIPTG-motB-lacI (NRS3775) grown on LB agar plate in the absence or presence of 50 μM IPTG, ΔmotB + pgsB::spc (NRS3434), ΔpgdS (NRS3347), ΔpgdSΔmotB (NRS3348) and ΔmotB + degS::cml (NRS3398).B. Schematic diagram of the γ-PGA synthesis operon and γ-PGA hydrolase gene. Arrows represent open reading frames (ORF), with the direction of the arrow indicating the direction of the ORF. The bent arrow represents the promoter located before the pgdS gene, which is driven by the alternative sigma factor, σD, as indicated.C. SDS-PAGE of γ-PGA collected from cultures of NCIB3610, ΔmotB (NRS3494) and ΔmotB + pgsB::spc (NRS3434) grown to the onset of stationary phase.D. Reverse-transcription-PCR analysis of pgsB and pgdS. Regions of DNA internal to pgsB and pgdS were amplified from cDNA generated from the wild-type (NCIB3610), ΔmotB (NRS3494) and ΔmotB + amyE::PIPTG-motB-lacI (NRS3775) grown in the absence and presence of 50 μM IPTG. Genomic DNA (gDNA) was used as a positive control for the PCR reaction and the ribosomal 16S rRNA was amplified as an internal control.
Figure 1 Deletion of motB from the chromosome is associated with γ-PGA production.A. Colony morphology of 3610 (wild-type), ΔmotB (NRS3494), ΔmotB + amyE::PIPTG-motB-lacI (NRS3775) grown on LB agar plate in the absence or presence of 50 μM IPTG, ΔmotB + pgsB::spc (NRS3434), ΔpgdS (NRS3347), ΔpgdSΔmotB (NRS3348) and ΔmotB + degS::cml (NRS3398).B. Schematic diagram of the γ-PGA synthesis operon and γ-PGA hydrolase gene. Arrows represent open reading frames (ORF), with the direction of the arrow indicating the direction of the ORF. The bent arrow represents the promoter located before the pgdS gene, which is driven by the alternative sigma factor, σD, as indicated.C. SDS-PAGE of γ-PGA collected from cultures of NCIB3610, ΔmotB (NRS3494) and ΔmotB + pgsB::spc (NRS3434) grown to the onset of stationary phase.D. Reverse-transcription-PCR analysis of pgsB and pgdS. Regions of DNA internal to pgsB and pgdS were amplified from cDNA generated from the wild-type (NCIB3610), ΔmotB (NRS3494) and ΔmotB + amyE::PIPTG-motB-lacI (NRS3775) grown in the absence and presence of 50 μM IPTG. Genomic DNA (gDNA) was used as a positive control for the PCR reaction and the ribosomal 16S rRNA was amplified as an internal control.CC BY · PubMed Central
Figure 2 Increasing DegU∼P levels allows γ-PGA production in NCIB3610.A. Schematic diagram of the construction of the ΔdegU + amyE::PIPTG-degU32hy-lacI strain (NRS1325). Bent arrows represent the promoters located before each gene.B. Colony morphology of the ΔdegU + amyE::PIPTG-degU32hy-lacI (NRS1325) strain without and with induction with 25 μM IPTG.C. SDS-PAGE of γ-PGA collected from cultures of ΔdegU + amyE::PIPTG-degU32hy-lacI (NRS1325) and ΔdegU + amyE::PIPTG-degU32hy-lacI + pgsB::spc (NRS3770) grown to the onset of stationary phase in the absence or presence of 25 μM IPTG.
Figure 2 Increasing DegU∼P levels allows γ-PGA production in NCIB3610.A. Schematic diagram of the construction of the ΔdegU + amyE::PIPTG-degU32hy-lacI strain (NRS1325). Bent arrows represent the promoters located before each gene.B. Colony morphology of the ΔdegU + amyE::PIPTG-degU32hy-lacI (NRS1325) strain without and with induction with 25 μM IPTG.C. SDS-PAGE of γ-PGA collected from cultures of ΔdegU + amyE::PIPTG-degU32hy-lacI (NRS1325) and ΔdegU + amyE::PIPTG-degU32hy-lacI + pgsB::spc (NRS3770) grown to the onset of stationary phase in the absence or presence of 25 μM IPTG.CC BY · PubMed Central
Figure 3 Transcription of degU and aprE is increased alongside total protease activity in ΔmotB.A. β-Galactosidase assays of strains carrying the PdegU–lacZ transcriptional reporter fusion. Strains shown are WT (wild-type NRS4351), ΔmotB (NRS4345), motB + amyE::PIPTG-motB-lacI (NRS4396) grown in the absence or presence of 50 μM IPTG, ΔmotAB (NRS4354) and ΔdegU (NRS4373). All cells were collected at the onset of stationary phase.B. β-Galactosidase assays of strains carrying the PaprE–lacZ transcriptional reporter fusion. Strains shown are WT (wild-type NRS1561), ΔmotB (NRS3440), ΔmotB + amyE::PIPTG-motB-lacI (NRS3858) grown in the absence or presence of 50 μM IPTG and ΔmotAB (NRS4093). All cells were collected at the onset of stationary phase.C. Total protease activity assays performed with the supernatant collected from cells grown in (B).Data in parts (A), (B) and (C) are plotted as the average of at least three independent replicates; in (C) data are represented as a fold change relative to the wild-type strain which was assigned value of 1. Error bars represent standard error of the mean. An asterisk denotes significance as calculated by the Student's t-test where * represents P < 0.05, ** P < 0.01 and *** P < 0.001.
Figure 3 Transcription of degU and aprE is increased alongside total protease activity in ΔmotB.A. β-Galactosidase assays of strains carrying the PdegU–lacZ transcriptional reporter fusion. Strains shown are WT (wild-type NRS4351), ΔmotB (NRS4345), motB + amyE::PIPTG-motB-lacI (NRS4396) grown in the absence or presence of 50 μM IPTG, ΔmotAB (NRS4354) and ΔdegU (NRS4373). All cells were collected at the onset of stationary phase.B. β-Galactosidase assays of strains carrying the PaprE–lacZ transcriptional reporter fusion. Strains shown are WT (wild-type NRS1561), ΔmotB (NRS3440), ΔmotB + amyE::PIPTG-motB-lacI (NRS3858) grown in the absence or presence of 50 μM IPTG and ΔmotAB (NRS4093). All cells were collected at the onset of stationary phase.C. Total protease activity assays performed with the supernatant collected from cells grown in (B).Data in parts (A), (B) and (C) are plotted as the average of at least three independent replicates; in (C) data are represented as a fold change relative to the wild-type strain which was assigned value of 1. Error bars represent standard error of the mean. An asterisk denotes significance as calculated by the Student's t-test where * represents P < 0.05, ** P < 0.01 and *** P < 0.001.CC BY · PubMed Central
Figure 4 The motB D24A allele cannot complement ΔmotB.A and B. β-Galactosidase assays of strains carrying the (A) PdegU–lacZ or (B) PaprE–lacZ transcriptional reporter fusion. Strains shown are WT (wild-type NRS4351), ΔmotB (NRS4345), motB + amyE::PIPTG-motB-D24A-lacI (NRS4397) grown in the absence or presence of 50 μM IPTG and ΔdegU (NRS4373).B. β-Galactosidase assays of strains carrying the PaprE–lacZ transcriptional reporter fusion. Strains shown are WT (wild-type NRS1561), ΔmotB (NRS3440), ΔmotB + amyE::PIPTG-motB-D24A-lacI (NRS3870) grown in the absence or presence of 50 μM IPTG. All cells in (A) and (B) were collected at the onset of stationary phase.C. Total protease activity assays performed with supernatants collected from cells grown in (B).Data in (A), (B) and (C) are plotted as the average of at least three independent replicates. In (C) data are represented as a fold change relative to the wild-type strain which was assigned value of 1. Error bars represent standard error of the mean. An asterisk denotes significance as calculated by the Student's t-test where * represents P < 0.05; **P < 0.01; and *** P < 0.001.D. Colony morphology of ΔmotB + amyE::PIPTG-motB-D24A-lacI (NRS3870) grown on LB agar in the absence and presence of 50 μM IPTG. SDS-PAGE analysis of γ-PGA collected from cultures of ΔmotB + amyE::PIPTG-motB-D24A-lacI (NRS3870) grown in the absence or presence of 50 μM IPTG.
Figure 4 The motB D24A allele cannot complement ΔmotB.A and B. β-Galactosidase assays of strains carrying the (A) PdegU–lacZ or (B) PaprE–lacZ transcriptional reporter fusion. Strains shown are WT (wild-type NRS4351), ΔmotB (NRS4345), motB + amyE::PIPTG-motB-D24A-lacI (NRS4397) grown in the absence or presence of 50 μM IPTG and ΔdegU (NRS4373).B. β-Galactosidase assays of strains carrying the PaprE–lacZ transcriptional reporter fusion. Strains shown are WT (wild-type NRS1561), ΔmotB (NRS3440), ΔmotB + amyE::PIPTG-motB-D24A-lacI (NRS3870) grown in the absence or presence of 50 μM IPTG. All cells in (A) and (B) were collected at the onset of stationary phase.C. Total protease activity assays performed with supernatants collected from cells grown in (B).Data in (A), (B) and (C) are plotted as the average of at least three independent replicates. In (C) data are represented as a fold change relative to the wild-type strain which was assigned value of 1. Error bars represent standard error of the mean. An asterisk denotes significance as calculated by the Student's t-test where * represents P < 0.05; **P < 0.01; and *** P < 0.001.D. Colony morphology of ΔmotB + amyE::PIPTG-motB-D24A-lacI (NRS3870) grown on LB agar in the absence and presence of 50 μM IPTG. SDS-PAGE analysis of γ-PGA collected from cultures of ΔmotB + amyE::PIPTG-motB-D24A-lacI (NRS3870) grown in the absence or presence of 50 μM IPTG.CC BY · PubMed Central
Figure 5 Engaging the flagellar clutch causes an increase in DegU∼P levels.A–C. β-Galactosidase assays of strains carrying the (A) PdegU–lacZ, (B) PbslA–lacZ or (C) PaprE–lacZ transcriptional reporter fusion. Cells were grown to 0.5 OD600 and induced with 1 mM IPTG (final concentration). Strains shown are: (A) wild-type (NRS4351), epsE + (PIPTG-epsE-lacI (NRS4374)), epsE D94A + (PIPTG-epsE-D94A-lacI (NRS4392)) and epsE K106E (PIPTG-epsE-K106E-lacI (NRS4394)); (B) Wild-type (NRS2052), PIPTG-epsE-lacI (NRS4405), PIPTG-epsE-D94A-lacI (NRS4406) and PIPTG-epsE-K106E-lacI (NRS4407); (C) Wild-type (NRS1561), PIPTG-epsE-lacI (NRS4345), PIPTG-epsE-D94A-lacI (NRS4393) and PIPTG-epsE-K106E-lacI (NRS4395). Data shown in (A), (B) and (C) are plotted as the average of at least three independent replicates. Error bars represent standard error of the mean.D. Total protease activity assays performed with supernatants collected from cells grown in (C) after 120 min of induction in the absence or presence of 1 mM IPTG. Data are plotted as the average of at least three independent replicates and are represented as a fold change relative to the wild-type strain (WT) which was assigned value of 1. Error bars represent standard error of the mean. An asterisk denotes significance as calculated by the Student's t-test, where ** represents P < 0.01; and *** P < 0.001.
Figure 5 Engaging the flagellar clutch causes an increase in DegU∼P levels.A–C. β-Galactosidase assays of strains carrying the (A) PdegU–lacZ, (B) PbslA–lacZ or (C) PaprE–lacZ transcriptional reporter fusion. Cells were grown to 0.5 OD600 and induced with 1 mM IPTG (final concentration). Strains shown are: (A) wild-type (NRS4351), epsE + (PIPTG-epsE-lacI (NRS4374)), epsE D94A + (PIPTG-epsE-D94A-lacI (NRS4392)) and epsE K106E (PIPTG-epsE-K106E-lacI (NRS4394)); (B) Wild-type (NRS2052), PIPTG-epsE-lacI (NRS4405), PIPTG-epsE-D94A-lacI (NRS4406) and PIPTG-epsE-K106E-lacI (NRS4407); (C) Wild-type (NRS1561), PIPTG-epsE-lacI (NRS4345), PIPTG-epsE-D94A-lacI (NRS4393) and PIPTG-epsE-K106E-lacI (NRS4395). Data shown in (A), (B) and (C) are plotted as the average of at least three independent replicates. Error bars represent standard error of the mean.D. Total protease activity assays performed with supernatants collected from cells grown in (C) after 120 min of induction in the absence or presence of 1 mM IPTG. Data are plotted as the average of at least three independent replicates and are represented as a fold change relative to the wild-type strain (WT) which was assigned value of 1. Error bars represent standard error of the mean. An asterisk denotes significance as calculated by the Student's t-test, where ** represents P < 0.01; and *** P < 0.001.CC BY · PubMed Central
Figure 6 Engaging the flagellar clutch triggers an increase in γ-PGA production that requires DegS.A. Colony morphology of PIPTG-epsE-lacI (NRS4085), PIPTG-epsE-D94A-lacI (NRS4388) and PIPTG-epsE-K106E-lacI (NRS4389) in the absence and presence of 1 mM IPTG after growth overnight at 37°C.B. Colony morphology of PIPTG-epsE-lacI + degS::cml (NRS4399), PIPTG-epsE-D94A-lacI + degS::cml (NRS4400) and PIPTG-epsE-K106E-lacI + degS::cml (NRS4401) in the absence and presence of 1 mM IPTG after growth overnight at 37°C.C. SDS-PAGE of γ-PGA collected from cultures of 3610 (wild-type NRS1561), PIPTG-epsE-lacI (NRS4345) and PIPTG-epsE-D94A-lacI (NRS4393) and PIPTG-epsE-K106E-lacI (NRS4395), grown in the absence or presence of 1 mM IPTG, at the onset of stationary phase.
Figure 6 Engaging the flagellar clutch triggers an increase in γ-PGA production that requires DegS.A. Colony morphology of PIPTG-epsE-lacI (NRS4085), PIPTG-epsE-D94A-lacI (NRS4388) and PIPTG-epsE-K106E-lacI (NRS4389) in the absence and presence of 1 mM IPTG after growth overnight at 37°C.B. Colony morphology of PIPTG-epsE-lacI + degS::cml (NRS4399), PIPTG-epsE-D94A-lacI + degS::cml (NRS4400) and PIPTG-epsE-K106E-lacI + degS::cml (NRS4401) in the absence and presence of 1 mM IPTG after growth overnight at 37°C.C. SDS-PAGE of γ-PGA collected from cultures of 3610 (wild-type NRS1561), PIPTG-epsE-lacI (NRS4345) and PIPTG-epsE-D94A-lacI (NRS4393) and PIPTG-epsE-K106E-lacI (NRS4395), grown in the absence or presence of 1 mM IPTG, at the onset of stationary phase.CC BY · PubMed Central
Figure 7 Tethering the flagella triggers an increase in degU transcription.A. Micrographs of cells containing the PdegU–lacZ transcriptional reporter fusion (NRS4351) grown presence or absence of Hag antibody in sera. Micrographs are static images taken from movies filmed 5 min after the addition of antibody to the culture (see Movies S1 and S2). Scale bars represent 100 pixels.B. β-Galactosidase assays of the wild-type strain carrying the PdegU–lacZ transcriptional reporter fusion (NRS4351) grown in the presence of a 1:20 dilution of either pre-immune sera or the Hag-specific antibody. Three independent pre-immune sera were used as controls. Data are plotted as the average of at least three independent replicates. Error bars represent standard error of the mean.
Figure 7 Tethering the flagella triggers an increase in degU transcription.A. Micrographs of cells containing the PdegU–lacZ transcriptional reporter fusion (NRS4351) grown presence or absence of Hag antibody in sera. Micrographs are static images taken from movies filmed 5 min after the addition of antibody to the culture (see Movies S1 and S2). Scale bars represent 100 pixels.B. β-Galactosidase assays of the wild-type strain carrying the PdegU–lacZ transcriptional reporter fusion (NRS4351) grown in the presence of a 1:20 dilution of either pre-immune sera or the Hag-specific antibody. Three independent pre-immune sera were used as controls. Data are plotted as the average of at least three independent replicates. Error bars represent standard error of the mean.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Research works printed but not linked: 1

  1. 10.1073/pnas.130639110 · DOI

Cited by

Sorted by citations

See all 143

Related

See all

Metadata

Authors
Cairns, L. S., Marlow, V. L., Bissett, E., Ostrowski, A., & Stanley‐Wall, N. R.
Affiliations
University of Dundee (GB)
Corresponding author
Stanley‐Wall, Nicola R.
ORCID
Venue
Molecular Microbiology
Publisher
Wiley
Published
2013-08-14
Volume
90
Issue
1
Pages
6-21
ISSN
  • 0950-382X (ISSN-L)
  • 1365-2958
DOI
10.1111/mmi.12342
PMC
PMC3963450
OpenAlex
W1908456294
Cited by
143
References
98
Citations by year
2026: 7 · 2025: 8 · 2024: 7 · 2023: 7 · 2022: 9 · 2021: 13 · 2020: 17 · 2019: 12 · 2018: 6 · 2017: 12 · 2016: 13 · 2015: 13 · 2014: 11 · 2013: 8
FWCI
9.95
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Microbiology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Discovery Research Portal (University of Dundee) (submittedVersion)
Funders
  • Wellcome Trust (093714, 097945, 093714/Z/10/Z, 097818/Z/11, 097945/B/11/Z, 097818/Z/11/A)
  • International Seafood Sustainability Foundation
  • Directorate for Biological Sciences
  • Biotechnology and Biological Sciences Research Council (BB/I006915/1, BB/H002340/1, BB/D526161/1)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bacterial biofilms and quorum sensing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
Keywords
Bacillus subtilis, flagella, mechanosensing, biofilm formation, MotB, bacterial signal transduction
MeSH terms
Bacillus subtilis, Bacterial Proteins, Flagella, Locomotion, Signal Transduction, Gene Expression Regulation, Bacterial

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review