Intrinsic timekeeping in Escherichia coli

A. A. Crofts; A. Bates; J. S. Takahashi; L. V. Hooper

doi:10.64898/2026.11.03.761655

AI summary · DOI: 10.64898/2026.11.03.761655 · bioRxiv: Microbiology

Intrinsic timekeeping in Escherichia coli

A. A. Crofts, A. Bates, J. S. Takahashi, L. V. Hooper

Original paper Published on 04 Nov. 2026 bioRxiv preprint License: CC BY 4.0 Also in: fr

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The essentials

Temperature and blue-light cycles synchronize E. coli biofilm rhythms, and the authors identify the Rcs phosphorelay as necessary for autonomous curli rhythmicity.

Question

Can E. coli anticipate recurring environmental cycles through intrinsic timekeeping, and which signaling pathways support the rhythms? The study examines whether temperature and light organize opposing curli and flagellar programs and whether the Rcs and BluFR systems contribute.

Method

The authors tracked Congo-red curli staining and promoter-driven chromoprotein reporters in biofilms of commensal strain MP1 and laboratory strain BW25113, using 12-hour cycles between 25.5°C and 28.5°C followed by constant-temperature conditions. They tested Rcs deletion mutants and blue- or red-light cycles, quantified spatial patterns as time series or endpoint radial profiles, and assessed rhythms with JTK_CYCLE. They also compared structural models of RcsC and RcsD with cyanobacterial clock-associated proteins using Foldseek and pairwise structural alignment.

Findings

Result Temperature-entrained biofilms showed curli rhythms that persisted after release to constant temperature, with free-running periods near 24 hours in both strains.
See the passage in the paper
This free-running phenotype was also observed in the laboratory strain BW25113 (Fig. 2F,H; Fig. S2B), with both strains exhibiting free-running periods of ∼24 hours (Fig. 2D,F).
Result Curli production and fliC promoter activity progressively separated in phase, with their peaks offset by about 10 hours by day three.
See the passage in the paper
Over subsequent cycles, however, the two outputs diverged and by day three, their peaks were offset by ∼10 hours (Fig. 3D,E; Fig. S3).
Result Deleting rcsD or rcsB abolished significant free-running curli rhythmicity, while ΔrcsF biofilms showed a longer-period rhythm with incomplete penetrance.
See the passage in the paper
However, deletion of rcsD or rcsB eliminated free-running curli rhythmicity after release to constant temperature, whereas ΔrcsF biofilms exhibited a longer, ∼30-hour rhythm with incomplete penetrance (Fig. 4B; Fig. S4A,B).
Result Blue-light cycles, but not red-light cycles, produced spatially separated curli rings; the authors could not directly establish periodicity in this assay.
See the passage in the paper
Blue light, but not red light, induced spatially separated curli rings (Fig. 5I,J; Fig. S5B).
Authors’ interpretation The authors interpret the genetic and structural evidence as indicating that autonomous timing resides within or is tightly coupled to Rcs, while emphasizing that structural resemblance does not establish a shared mechanism or direct evolutionary homology.
See the passage in the paper
These structural relationships do not imply direct evolutionary homology or imply that the Rcs phosphorelay operates through the same molecular mechanism as the Kai oscillator.

Limitations

The light-cycle assay used endpoint imaging, so radial position could not be directly translated into elapsed time and periodicity could not be directly assessed.
See the passage in the paper
Because biofilms were imaged only at the experimental endpoint to preserve controlled light conditions, radial position cannot be directly converted into elapsed time.
The proposed role of essential IgaA in the Rcs timing system could not be explored in this biofilm-level system.
See the passage in the paper
However, IgaA is an essential protein in E. coli, which makes exploring its role in our biofilm-level system challenging.
The study's timing evidence is based on biofilm assays under particular laboratory conditions, so it does not establish how broadly the mechanism operates across environments or bacterial species.

Why it matters

The authors argue that this work establishes genetically defined intrinsic timekeeping in E. coli and provides an entry point for investigating autonomous rhythms in bacteria. They propose that the findings may prompt searches for timekeeping in other bacteria with complex signaling systems, while presenting the Rcs–cyanobacterial-clock parallels as organizational convergence rather than proof of a shared mechanism.

Written by AI from the paper. Passages are checked word for word against the text we analysed; the rest may contain errors.

AI-generated keywords: Escherichia coli biofilms circadian rhythms Rcs phosphorelay environmental entrainment

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Comprehensive Summary
Layman's Summary
Blog article
Original abstract

This bioRxiv preprint asks whether *Escherichia coli* can keep time internally, rather than simply responding to immediate changes in its environment. The authors investigate biofilm curli production, which supports adhesion, and activity of the flagellar gene *fliC*, which is associated with motility. They test whether daily environmental cycles can synchronize these processes, whether rhythms persist after the cycles stop, and which signaling pathways may be involved. The researchers grew *E. coli* biofilms on Congo red-containing agar, which stains curli, and exposed them to alternating 12-hour periods at 25.5°C and 28.5°C. As the biofilms expanded, successive periods of curli production formed rings, providing a spatial record of activity over time. During the first cycle, curli staining increased after warming and decreased after cooling, consistent with an immediate temperature response. With continued cycling, the peak shifted to about six hours before the next temperature change, which the authors interpret as anticipatory entrainment. Curli rhythms were detected in all three independent biofilms assessed, with 24-hour rhythmicity statistically significant in each (P < 5 × 10⁻¹⁷). To test for an internal rhythm, the researchers cycled biofilms for three days and then maintained them at a constant 25.5°C. Curli production continued to oscillate for at least three days after the cycles ended. The reported average free-running periods were 24.6 hours for the commensal strain MP1 and 24.3 hours for the laboratory strain BW25113. Biofilms maintained at a constant temperature throughout did not show significant curli oscillations. The authors also measured activity from an *fliC* promoter using a chromoprotein reporter. Reporter activity cycled under temperature shifts and showed modest rhythms after the biofilms were returned to constant conditions. Curli and *fliC* activity began largely in phase but diverged over successive cycles; by about day three, their peaks were roughly 10 hours apart. This suggests that temperature entrainment can place adhesion- and motility-associated outputs in opposing phases. However, the reporter measures promoter activity, not flagellar movement itself. Genetic tests implicated the Rcs phosphorelay, a signaling system that regulates both curli and flagellar pathways. Deleting *rcsB* or *rcsD* abolished significant free-running curli rhythms, although the mutants remained rhythmic during temperature cycling. Deleting *rcsF* did not prevent responses to cycling or progressive phase shifts, but produced a longer free-running rhythm of about 30 hours that was not observed in all cases. The authors distinguish environmental entrainment from autonomous rhythmicity and propose that autonomous timing resides within, or is closely coupled to, the Rcs system. The researchers also tested the BluFR pathway, which senses blue light and low temperature and can signal to Rcs. A *ycgZ* promoter reporter tracked temperature cycles but did not free-run or show the progressive phase adjustment observed in curli. At constant temperature, blue-light cycles—but not red-light cycles—produced spatially separated curli rings. Their apparent persistence after the lighting cycles ended is consistent with entrainment, although this assay could not directly establish a free-running period. Structural comparisons revealed similarities between regions of RcsC and RcsD and cyanobacterial clock-associated proteins, despite low sequence identity. The authors present these as architectural parallels, not evidence of direct homology or a shared mechanism. Important limitations include the small number of independently grown biofilms in several experiments, reliance on spatial staining and reporter proxies, and the inability to quantify light-cycle rhythms directly over time. The proposed oscillator mechanism is inferred from mutant behavior and structural comparisons rather than demonstrated biochemically.

Can E. coli keep time?

Bacteria constantly adjust to changes around them: temperature shifts, light, and other environmental cues can all alter gene activity. But can a bacterium do more than react in the moment? Can it keep track of time internally and prepare for changes before they happen?

In “Intrinsic timekeeping in Escherichia coli,” A. A. Crofts, A. Bates, J. S. Takahashi, and L. V. Hooper investigate whether E. coli has an internal timing system. They focus on two behaviors with contrasting roles: production of curli, fibers that help biofilms attach to surfaces, and activity of fliC, a gene associated with motility. Their results suggest that daily environmental cycles can coordinate these processes—and that some rhythms continue even after the cycles stop.

Reading time in a growing biofilm

To track curli production, the researchers grew E. coli biofilms on agar containing Congo red, a dye that stains curli. They alternated the temperature between 25.5°C and 28.5°C every 12 hours. As a biofilm spread across the plate, periods of stronger and weaker curli production appeared as bands, creating a spatial record of activity over time.

At first, the pattern looked like a straightforward response to temperature: curli staining increased after warming and decreased after cooling. But with repeated cycles, the timing changed. The curli peak shifted to about six hours before the next temperature change. That shift is important because it suggests the cells were not simply reacting to each new condition; their activity had become coordinated with the environmental schedule.

The researchers found statistically significant 24-hour curli rhythms in all three independent biofilms they assessed. The shift in timing is consistent with entrainment—the synchronization of a biological process by a repeating external cue. In this case, temperature cycles appeared to set the timing of curli production.

Rhythms that persist

Entrainment alone does not show that cells have an internal clock. To test for an autonomous rhythm, the researchers cycled biofilms for three days and then held them at a constant 25.5°C. Curli production continued to oscillate for at least three days after the temperature changes ended.

The average free-running period—the time taken for a rhythm to repeat without ongoing environmental cycles—was 24.6 hours in the commensal strain MP1 and 24.3 hours in the laboratory strain BW25113. Biofilms kept at a constant temperature from the start did not show significant curli oscillations. Together, these findings suggest that temperature cycles can set a rhythm that then persists in steady conditions.

Adhesion and motility move out of step

The team also monitored activity from the fliC promoter using a chromoprotein reporter. Reporter activity cycled during temperature shifts and showed modest rhythms after the biofilms returned to constant conditions. Curli and fliC activity began largely in phase, but gradually diverged. By about the third day, their peaks were roughly 10 hours apart.

This growing separation suggests that environmental timing can place adhesion-associated and motility-associated outputs in different phases. That could matter for how bacteria balance sticking to a surface with activities linked to movement. However, the reporter measured promoter activity, not flagellar motion itself, so it does not demonstrate that swimming behavior followed the same rhythm.

A role for the Rcs signaling system

Genetic tests pointed to the Rcs phosphorelay, a signaling system that regulates both curli and flagellar pathways. When the researchers deleted rcsB or rcsD, significant free-running curli rhythms disappeared. Yet these mutants remained rhythmic during temperature cycling. This distinction suggests that the cells could still respond to the external schedule, while the internal rhythm seen under constant conditions depended on Rcs signaling.

Deleting rcsF produced a different result. The mutant still responded to temperature cycles and showed progressive shifts in timing, but in some cases its free-running period was longer—about 30 hours. The authors therefore propose that autonomous timing resides within, or is closely coupled to, the Rcs system. The experiments identify a connection, though they do not establish the biochemical mechanism of an oscillator.

Light cues and structural clues

The researchers also explored BluFR, a pathway that senses blue light and low temperature and can signal to Rcs. A ycgZ promoter reporter followed temperature cycles, but it did not free-run or show the progressive phase adjustment seen in curli. In a separate test, blue-light cycles at constant temperature produced spatially separated curli rings; red-light cycles did not. The pattern is consistent with light entrainment, but the assay could not directly quantify rhythms over time or establish a free-running period.

Finally, structural comparisons showed similarities between regions of RcsC and RcsD and proteins associated with cyanobacterial clocks. These are architectural parallels, not proof of direct homology or a shared timing mechanism.

What the findings do—and don’t—show

The study offers evidence that E. coli can show persistent, clock-like timing in curli production after exposure to daily temperature cycles. It also links that timing to Rcs signaling and suggests that adhesion- and motility-associated outputs can shift into different phases.

There are important limits. Several experiments involved few independently grown biofilms, and the conclusions rely on spatial staining and reporter signals rather than direct measurements of every behavior. The light-cycle results were not tracked quantitatively over time, and the proposed oscillator has not been demonstrated biochemically. Even so, the work raises a compelling possibility: a bacterium may not only respond to changing conditions, but also use recurring cues to organize its activity across time.

Created on 09 Nov. 2026

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Intrinsic timekeeping in Escherichia coli

The essentials

Question

Method

Findings

Limitations

Why it matters

Read further

Definitions

Can E. coli keep time?

Reading time in a growing biofilm

Rhythms that persist

Adhesion and motility move out of step

A role for the Rcs signaling system

Light cues and structural clues

What the findings do—and don’t—show

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