Abstract
The motile-sessile transition is critical for bacterial survival and growth. Cyclic-di-GMP (c-di-GMP) plays a central role in controlling this transition and regulating biofilm formation via various effectors. As an effector of c-di-GMP in Escherichia coli and related species, the PilZ domain-containing protein YcgR responds to elevated c-di-GMP concentrations and acts on the flagellar motor to suppress bacterial motility in a brakelike fashion, which promotes bacterial surface attachment. To date, several target proteins within the motor, MotA, FliG, and FliM, along with different regulatory mechanisms have been reported. However, how YcgR acts on these components remains unclear. Here, we report that activated YcgR stably binds to MotA at the MotA-FliG interface and thereby regulates bacterial swimming. Biochemical and structural analyses revealed that c-di-GMP rearranges the PilZ domain configuration, resulting in the formation of a MotA-binding patch consisting of an RXXXR motif and the C-tail helix α3. Moreover, we noted that a conserved region in the YcgR-N domain, which is independent of MotA interaction, is necessary for motility regulation. On the basis of these findings, we infer that the YcgR-N domain is required for activity on other motor proteins. We propose that activated YcgR appends to MotA via its PilZ domain and thereby interrupts the MotA-FliG interaction and simultaneously interacts with other motor proteins via its YcgR-N domain to inhibit flagellar motility. Our findings suggest that the mode of interaction between YcgR and motor proteins may be shared by other PilZ family proteins.
Highlights
The motile-sessile transition is critical for bacterial survival and growth
With the c-di-GMP– bound YcgR, MotAc was eluted at a volume of 12.87 ml, corresponding to an apparent molecular weight of 94.1 kDa and suggesting a MotAc tetramer bound to a single ligand-bound YcgR monomer with an apparent molecular weight of 30.9 kDa (Fig. 1B)
Three independent studies suggested the motor proteins MotA, FliG, and FliM as the YcgR partners [7, 11, 12]. These studies proposed different models where YcgR interacted with different targets (i.e. MotA [7], both FliG and FliM [11], or individual FliG [12]) to regulate motility and suggested that activated YcgR alters the MotA-FliG [7, 11] or FliG-FliM [12] interaction to decrease the flagellar rotation speed and induce CCW motor bias
Summary
The motile-sessile transition is critical for bacterial survival and growth. Cyclic-di-GMP (c-di-GMP) plays a central role in controlling this transition and regulating biofilm formation via various effectors. As an effector of c-di-GMP in Escherichia coli and related species, the PilZ domain– containing protein YcgR responds to elevated c-di-GMP concentrations and acts on the flagellar motor to suppress bacterial motility in a brakelike fashion, which promotes bacterial surface attachment. One PilZ domain protein in E. coli [8, 10], YcgR, responds to the elevated c-di-GMP concentration to regulate bacterial motility and is involved in biofilm formation by facilitating the subsequent surface attachment [4, 7, 11, 12]. They proposed that YcgR binds to the central fragment of FliG where FliM binds, altering FliG-FliM interaction and biasing the flagella rotation [12] These studies make a great improvement for the knowledge of c-di-GMP regulating flagellar motility. The interaction mode between YcgR and motor proteins could be shared by other PilZ family proteins acting on their targets
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