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Second messenger–mediated tactile response by a bacterial rotary motor

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Science  27 Oct 2017:
Vol. 358, Issue 6362, pp. 531-534
DOI: 10.1126/science.aan5353
  • Fig. 1 The flagellar motor is a tactile mechanosensor.

    (A) Schematic representation of the bacterial flagellum. (B) Progeny of mother cells attached to the glass surface in a microfluidic channel were either carried away after release or expressed a holdfast (red) before separating to remain attached downstream of the mother cell. Medium flow and chamber dimensions are indicated by light blue and black arrows, respectively. (C) Newborn cells were observed in microchambers of 0.75 μm height with fluorescent wheat germ agglutinin (WGA) lectin added to the medium in order to determine the time span between daughter cell release and the appearance of the holdfast (red). (D) C. crescentus cell dividing in a flow channel. Newly formed holdfast at the flagellated pole of a dividing cell (arrowheads) and time relative to cell division are indicated. Scale bars, 2 μm. (E) Efficiency of SW cell attachment under constant flow. Data are based on 232 [wild type (wt)], 160 (ΔpilA), 88 (ΔfliFG), 139 (ΔflgDE), 88 (ΔmotA), and 107 (ΔfliL) individual separation events. (F) Microcolonies formed from growth and attachment of single ancestors (arrows) in the flow channel. The area covered by the colony (dashed yellow line) served as a measure for the efficiency of rapid holdfast expression. (G) Areas of microcolonies after 15 hours of growth in a flow channel illustrate the efficiency of daughter cell attachment. Box plots show median (horizontal black lines), lower and upper quartiles (dark and light orange boxes, respectively), and extreme values (whiskers). Values were normalized to the median of the wild-type colony area. (H) Time between release of SW progeny from their mothers to detection of holdfast. The strains were grown as illustrated in (C). Because motile daughters are difficult to reliably track in this assay because of their swimming speed, only nonmotile mutants were used for this analysis. Averages are shown as black lines. Measurements for ΔflgDE ΔpilA are significantly different from measurements for ΔflgDE motBD33N (Students t test, P < 0.001).

  • Fig. 2 The diguanylate cyclase DgcB is essential for surface sensing and rapid attachment.

    Attachment of the strains indicated was scored as outlined in Fig. 1G. The C. crescentus rcdG0 Plac::dgcZ strain lacks all diguanylate cyclases and phosphodiesterases but carries a plasmid-born copy of the Plac-driven dgcZ gene from E. coli. In the rcdG0::dgcB::pleD ppdeH strain, two diguanylate cyclase genes, dgcB and pleD, are retained. In addition, the strain carries a plasmid-born copy of the E. coli pdeH gene, encoding a phosphodiesterase. The dgcBE261A allele carries a point mutation in the active site, rendering its product catalytically inactive. Box plots show the median (horizontal black lines), lower and upper quartiles (dark and light orange boxes, respectively), and the extreme values (whiskers). Values were normalized to the median of the wild-type colony area.

  • Fig. 3 C-di-GMP binding to HfsJ initiates holdfast biosynthesis.

    (A) Binding of c-di-GMP to HfsJ (solid circles) was measured with increasing concentrations of radiolabeled ligand as indicated, revealing a dissociation constant (Kd) of 2.7 μM. (Inset) Binding is specific for c-di-GMP. Radiolabeled c-di-GMP (5 μM) was competed with a 100× excess of the nucleotides indicated, and overall binding was determined. (B) Alignment of the N-termini of HfsJ orthologs from C. crescentus (C.c.) and the following closely related species: Caulobacter henricii (C.h.), Caulobacter sp. K31 (C.sp.), Brevundimonas diminuta (B.d.), Brevundimonas naejangsanensis (B.n.), Asticcacaulis biprosthecium (A.b.), Asticcacaulis benevestitus (A.b.), Deinococcus apachensis (D.a.), Rhodothermus marinus (R.m.), and Lihuaxuella thermophile (L.t.). Conserved residues are highlighted, with arginines in red, negative charges in green, and proline or aromatic amino acids in blue. (C) Binding studies with HfsJ identified R23 and R27 as critical residues for c-di-GMP binding. The highly conserved catalytic residue R247 is not required for c-di-GMP binding. (D) In vivo activity of HfsJ. Cells of C. crescentus wild type (n = 764 cells) and hfsJ mutants (R23Q, n = 1077 cells; R247Q, n = 6 cells) were grown in the presence of Oregon Green 488–labeled WGA lectin, and holdfast biogenesis was measured as relative fluorescence intensities of individual cells. (E) Model of C. crescentus surface sensing. Initial surface adherence is mediated by pili and pili retraction positioning the flagellar pole in close contact with the surface. The physical pressure applied on the cell envelope by the surface affects the function of the flagellar rotor-stator components, generating an unknown signal that is sensed by DgcB and converted into a burst of c-di-GMP (red). The second messenger initiates rapid holdfast biogenesis and permanent attachment by activating the key glycosyltransferase HfsJ.

Supplementary Materials

  • Second messenger–mediated tactile response by a bacterial rotary motor

    Isabelle Hug, Siddharth Deshpande, Kathrin S. Sprecher, Thomas Pfohl, Urs Jenal

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods 
    • Figures S1 to S7 
    • References 
    Tables S1 to S4

    Images, Video, and Other Media

    Movie S1
    C. crescentus cell division in a microfluidic flow channel. Holdfast is visualized using fluorescent WGA lectin. Note that in this representative example, a holdfast structure is visible at the flagellated pole of the SW progeny before separation from its mother cell.
    Movie S2
    High speed imaging of a representative SW cell that fails to attach to the surface after separating from its mother in a microfluidic flow channel. Note that SW cells that initiate rotation before separating from their mother generally do not attach.
    Movie S3
    High speed imaging of a representative SW cell that attaches to the surface after separating from its mother in a microfluidic flow channel. Note that SW cells that remain paralyzed before separating from their mother generally attach immediately downstream of their mother.
    Movie S4
    High speed imaging of a representative SW cell that attaches to the surface after separating from its mother in a microfluidic flow channel. Note that this SW cell stops rotation before separating from its mother and remains surface attached.
    Movie S5
    Cycles of growth and division in a microfluidic flow channel result in the formation of microcolonies from single founder cells
    Movie S6
    Time lapse of a dividing C. crescentus cell grown in a microfluidic flow channel that expressed a DgcB-Venus fusion. Note that DgcB localizes to the flagellated cell pole before and after cell division.

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