Inverse regulatory coordination of motility and curli-mediated adhesion in Escherichia coli

Abstract

During the transition from post-exponential to stationary phase, Escherichia coli changes from the motile-planktonic to the adhesive-sedentary "lifestyle." We demonstrate this transition to be controlled by mutual inhibition of the FlhDC/motility and sigma(S)/adhesion control cascades at two distinct hierarchical levels. At the top level, motility gene expression and the general stress response are inversely coordinated by sigma(70)/sigma(FliA)/sigma(S) competition for core RNA polymerase and the FlhDC-controlled FliZ protein acting as a sigma(S) inhibitor. At a lower level, the signaling molecule bis-(3'-5')-cyclic-diguanosine monophosphate (c-di-GMP) reduces flagellar activity and stimulates transcription of csgD, which encodes an essential activator of adhesive curli fimbriae expression. This c-di-GMP is antagonistically controlled by sigma(S)-regulated GGDEF proteins (mainly YegE) and YhjH, an EAL protein and c-di-GMP phosphodiesterase under FlhDC/FliA control. The switch from motility-based foraging to the general stress response and curli expression requires sigma(S)-modulated down-regulation of expression of the flagellar regulatory cascade as well as proteolysis of the flagellar master regulator FlhDC. Control of YhjH by FlhDC and of YegE by sigma(S) produces a fine-tuned checkpoint system that "unlocks" curli expression only after down-regulation of flagellar gene expression. In summary, these data reveal the logic and sequence of molecular events underlying the motile-to-adhesive "lifestyle" switch in E. coli.

Figure 1.

FliZ is an inhibitor of σ^S activity. (A) FliZ does not affect σ^S levels, but strongly down-regulates the σ^S-controlled curli regulator CsgD. Derivatives of strain MC4100 carrying either pFlhDC, pFliZ, or the vector (pCAB18) alone were grown in the presence or absence of IPTG as indicated. Samples were taken for immunoblot analysis of σ^S and CsgD at an OD₅₇₈ of 4.0 and after overnight growth (ON). Densitometric quantification is shown below the blots. (B) FliZ reduces the expression of σ^S-controlled genes. Expression patterns of the σ^S-dependent genes csgB, mlrA, ydaM, yciR, osmY, and gadB were recorded using appropriate single-copy reporter fusions in strain MC4100 carrying either pFliZ (squares) or the vector (pCAB18; circles). Cells were grown without IPTG (as even low levels of FliZ are sufficient to interfere with the expression of the genes shown), and OD₅₇₈ (open symbols) and specific β-galactosidase activities (closed symbols) were determined along the growth curve.

Figure 2.

Mutations in fliZ affect the timing of induction of σ^S-dependent genes. Two directly σ^S-controlled genes encoding MlrA (A) and YdaM (B) (i.e., the factors essential for the expression of the curli activator CsgD), as well as a construct with a synthetic promoter that responds exclusively to σ^S, synp9 (C), were assayed. Derivatives of strain W3110 carrying single-copy lacZ reporter fusions to mlrA, ydaM, and synp9 and the corresponding fliZ knockout mutants were grown in LB (for symbols, see the figure). OD₅₇₈ (open symbols) and specific β-galactosidase activities (closed symbols) were determined.

Figure 3.

A complex c-di-GMP control module interferes with motility. (A) Knockout mutations in the genes for the GGDEF proteins YegE and YedQ (but not YdaM) and the c-di-GMP effector YcgR suppress the nonmotile phenotype of strains defective for the EAL domain protein YhjH. Single, double, and triple mutants defective in the genes indicated were tested for motility on swim plates incubated at 28°C. (B) YhjH is a c-di-GMP phosphodiesterase (PDE). Purified YhjH [whose EAL^(48–50) domain actually features an ELL sequence] and YhjH^E48A were tested for PDE activity in vitro using radiolabeled c-di-GMP. Cleavage products are indicated.

Figure 4.

Expression patterns of genes relevant for the motility-to-adhesion switch. Derivatives of strain W3110 carrying single-copy lacZ reporter fusions in yegE and yedQ (A), flhDC (B; fusion inserted early in flhC), fliA and fliAZ (C; the latter fusion inserted early in fliZ), and yhjH (D) as well as knockout mutations in the genes indicated in the boxes above the single graphs, were used for the determination of OD₅₇₈ (open symbols) and specific β-galactosidase activities (closed symbols) along the growth curve.

Figure 5.

The Clp protease plays a key role in down-regulating YhjH, which is a prerequisite for inducing curli expression during entry into stationary phase. (A) A clpP derivative of W3110 (carrying csgB∷lacZ as a reporter) does not induce curli expression, and this defect is suppressed by secondary mutations in flhDC and yhjH, but not in fliZ (for symbols, see figure). (B) Under the same conditions, the expression of a single-copy yhjH∷lacZ fusion was tested in clpP⁺ and clpP mutant backgrounds. OD₅₇₈ (open symbols) and specific β-galactosidase activities (closed symbols) were determined along the growth curve of LB-grown cells.

Figure 6.

The GGDEF/EAL proteins YegE and YhjH affect curli expression by controlling csgD transcription, but in this process do not act via the c-di-GMP-binding protein YcgR. (A–C) csgB∷lacZ expression was assayed in derivatives of strain W3110 carrying mutations in yegE, yhjH, ycgR, ydaM, or yciR as indicated (for symbols, see figure). OD₅₇₈ (open symbols) and specific β-galactosidase activities (closed symbols) were determined along the growth curve. (D) σ^S and CsgD levels were determined by immunblotting of strain W3110 (lanes 1,5,9) and its derivatives with mutations in yegE (lanes 2,6,10), yegE and yhjH (lanes 3,7,11), and yhjH (lanes 4,8,12). Samples were taken 4.5, 9, and 12 h after inocculation (corresponding to an OD₅₇₈ of 1.0, 3.0, and 4.0, respectively). (E) csgD mRNA levels were determined by Northern analysis in W3110 and its mutant derivatives as indicated, with samples taken at an OD₅₇₈ of 4.0.

Figure 7.

A model summarizing the communication network between the FlhDC/motility and σ^S/curli regulatory cascades and the role of different c-di-GMP control modules. At the top of the network, motility and the entire general stress response are inversely coordinated by competition of σ⁷⁰, σ^FliA, and σ^S for RNAP core, with FliZ being a “promotility” component that interferes with σ^S activity. At lower levels of the network hierarchy, motility and adhesion (via curli fimbriae) are inversely coordinated by two separately acting c-di-GMP control modules involving GGDEF/EAL proteins: YegE(+YedQ)/YhjH down-regulates flagellar activity and positively modulates csgD transcription, whereas YdaM/YciR acts specifically in and is essential for csgD transcription only (for more details, see Discussion). Also included is a third GGDEF/EAL module (i.e., YaiC/YoaD) that is expressed under σ^S control later during entry into stationary phase (Weber et al. 2006; our unpublished data) and regulates the activity of cellulose synthase (Römling et al. 2005; Brombacher et al. 2006). Negative effects of the FlhDC/motility system onto the σ^S/curli system are highlighted in red, effects by which the σ^S/curli system down-regulates the FlhDC/motility system are in green. Triangles indicate additional signal input not further specified.