E. coli cells race to rebuild their proteins in the first hours of starvation

Starvation is the default condition of bacterial life. Although laboratory microbiology is built around the spectacle of exponentially dividing cultures, in the wild bacteria spend most of their existence without food, slowly drawing down internal reserves while waiting for better times. A new study published in Molecular Systems Biology by Théo Gervais, Bjoern Kscheschinski and colleagues at the Biozentrum of the University of Basel and the Swiss Institute of Bioinformatics now provides the most detailed single-cell portrait yet of what happens inside Escherichia coli in the hours and days after its food disappears. The answer is striking: rather than simply powering down, the bacterium launches a tightly choreographed remodeling of its entire protein inventory, and the work it does in the first few hours determines how well it will survive everything that comes afterward.

The team combined a microfluidic device known as the dual input mother machine with time-lapse fluorescence microscopy and automated image analysis to follow thousands of individual cells through an abrupt switch from glucose-rich growth medium to a carbon-free version of the same medium. Each strain carried a transcriptional fluorescent reporter for one of 22 different promoters, spanning native genes and synthetic constitutive promoters chosen to cover a wide range of expression levels and regulatory behaviors. This multiplexed setup allowed the researchers to quantify, for every single cell, its size, growth rate and the rate at which it produced a fluorescent protein from each promoter, corrected for the confounding effects of photobleaching, fluorescent protein maturation and, crucially, protein degradation.

The first finding is a matter of minutes. Upon removal of the carbon source, every cell stopped growing essentially immediately, within the three-minute resolution of the microscopy. Contrary to any picture of the stationary phase as a mixture of growing and dying cells, the population entered growth arrest as one. Most remarkably, after growth had already ceased, roughly 38 percent of cells went on to divide during the following ten hours, undergoing what biologists call reductive division. These divisions shrank the average cell size of the population and occurred preferentially in longer cells, likely those that had already progressed furthest in their cell cycle, perhaps having started or completed DNA replication. Even after 60 hours without food, more than 90 percent of cells were still capable of regrowing when nutrients returned.

With growth arrested, attention turned to gene expression. Here the diversity across promoters was dramatic. Ribosomal protein promoters such as rplN, which drive the cell’s protein synthesis machinery during exponential growth, were shut down almost completely within an hour of the switch. Other promoters, like hupA, showed a sharp initial drop followed by a slower, sustained trickle of production. A third class, exemplified by hslV and the RpoS-regulated promoter bolA, did something counterintuitive: production briefly increased, sometimes above its level during growth, before decaying exponentially over the following hours. By late starvation, the relative production rates across the 22 promoters spanned more than two orders of magnitude, from about 20 percent down to 0.1 percent of their growth-phase values, underscoring how profoundly the regulatory network is rewired when nutrients vanish.

What makes these dynamics especially noteworthy is their consistency across cells. The researchers had anticipated that individual bacteria might respond to starvation in idiosyncratic ways, as has been observed for other stresses such as the SOS response to DNA damage, where the population-level induction conceals almost no coordination between cells. Instead, during early starvation, single cells followed nearly identical trajectories. For promoters showing a burst of expression, more than half of all the variance observed across cells and time was captured simply by the population average, a degree of coordination rarely seen in bacterial gene regulation. This homogeneity suggests that entry into starvation triggers a tightly controlled program rather than a stochastic drift into dormancy.

Production rates, however, are only half the story; protein concentrations are what determine physiology. Because growth arrest halts the dilution of existing proteins while degradation continues, and because production does not stop instantly, many proteins accumulated substantially even as their synthesis slowed. The researchers measured the degradation rate of fluorescent proteins directly and found that it too decays exponentially during starvation, dropping from initial values around 0.02 to 0.03 per hour. This slowdown makes mechanistic sense: most protein degradation in E. coli is carried out by ATP-dependent proteases, and ATP levels plummet when carbon is gone. Integrating production, dilution and degradation in a simple mathematical model, the team showed that promoter concentrations either fell by as much as half or rose by up to twelvefold, with nearly all of these changes occurring within the first five to ten hours. After that, protein levels were essentially frozen, locking in a phenotype that persisted for days.

The model also revealed an elegant set of principles governing the final proteome. The concentration fold-change for each gene depends on just three effective ratios: the total degradation capacity, the balance between the fold-change in production and the fold-change in degradation relative to growth rate, and the relative time scales over which production and degradation decay. Even without a burst of expression, a promoter whose production decays gradually over about five hours can achieve a fourfold increase in protein concentration, simply because dilution has stopped while synthesis continues. For RpoS-regulated promoters such as bolA, an initial burst pushes the increase further, to around sevenfold. Experiments with an rpoS deletion strain confirmed the central role of this alternative sigma factor, although not all starvation-induced promoters depended on it, pointing to additional regulators still to be identified.

Importantly, the response did not require cells to anticipate starvation in advance. In a separate set of experiments, the researchers let a batch culture exhaust its glucose gradually while flowing the same medium through the microfluidic chip, so that cells inside the chip experienced a slower, roughly 45-minute transition into growth arrest instead of an abrupt five-minute switch. The gene expression dynamics and the resulting protein concentration changes were remarkably similar between the gradual and abrupt transitions, suggesting that the program is triggered by the cessation of growth itself rather than by sensing dwindling nutrients. Even when protein synthesis was temporarily blocked with chloramphenicol during the first five hours of starvation, the expression program simply resumed once the drug was removed, implying that the eventual decline in production reflects exhaustion of internal resources rather than a specific transcriptional shutoff.

The most consequential result concerns what this early remodeling is for. When the researchers subjected starving cells to a strong oxidative stress with hydrogen peroxide at the 20-hour mark, nearly 98 percent of normally starved cells survived. But when translation was inhibited from the very start of starvation, survival fell to 19 percent, whereas inhibiting protein production only after the first five hours was far less damaging, allowing 65 percent of cells to survive. The protective early expression tracked the accumulation of RpoS-regulated reporters, and a strain lacking rpoS altogether fared even worse than translation-inhibited wild-type cells, with survival dropping almost to zero. Together, these experiments demonstrate that the protein production of the first five hours is what arms the cells against stresses arriving much later, when their capacity to respond has essentially vanished.

The findings also suggest how starving cells manage to keep producing proteins at all. With ribosomal gene expression shut off, the reduced translation capacity is redirected away from growth machinery toward protective proteins, effectively raising the relative output of many genes even as total synthesis falls. The energy likely comes from endogenous metabolism, the controlled self-digestion of abundant cellular constituents: the team’s degradation measurements imply that roughly 40 percent of the proteome may be broken down within the first day of starvation, releasing metabolites and fueling ATP production. In this light, the first hours of starvation emerge as a resource-limited but precisely coordinated investment phase, in which a bacterium cannibalizes part of itself to build the molecular toolkit it will need to endure an uncertain and potentially hostile future. For a organism long caricatured as a simple growth machine, E. coli turns out to be a far more deliberate survivor than its reputation suggests.

Subject of Research: Proteome remodeling and gene expression dynamics in single E. coli cells during carbon starvation

Article Title: E. coli prepares for starvation by dramatically remodeling its proteome in the first hours after loss of nutrients

Article References: E. coli prepares for starvation by dramatically remodeling its proteome in the first hours after loss of nutrients. (n.d.). https://doi.org/10.1038/s44320-026-00226-5

Image Credits: AI Generated

DOI: 10.1038/s44320-026-00226-5

Keywords: E. coli, carbon starvation, proteome remodeling, gene expression, microfluidics, single-cell microscopy, RpoS, stationary phase, protein degradation, stress tolerance, bacterial survival, Molecular Systems Biology