Single-Phage Profiling Reveals Hidden Viral Individuality During the Lysis–Lysogeny Decision

For more than seventy years, the infection cycle of bacteriophage lambda has served as one of the most influential models in molecular biology. This temperate phage infecting Escherichia coli helped establish fundamental concepts in genetics, gene regulation, and cellular decision-making. Yet despite decades of investigation, one crucial question has remained largely inaccessible to experimental observation: do all phages inside the same infected bacterium behave identically, or does each viral genome make its own independent decisions?

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A new study published in Nature Communications has now provided the clearest answer yet. Using an advanced single-phage transcriptomics approach capable of monitoring individual viral genomes inside living bacterial cells, researchers from the University of Illinois have demonstrated that bacteriophages display a surprising degree of individuality during infection. Their findings reveal that different phages occupying the same bacterial cell can simultaneously pursue distinct developmental programs, fundamentally reshaping our understanding of how viral populations determine cellular fate.

The work focuses on the classic lysis-versus-lysogeny decision of bacteriophage lambda. After entering an E. coli cell, lambda faces two radically different developmental pathways. In the lytic cycle, the phage hijacks bacterial metabolism, replicates its genome, produces new virions, and ultimately destroys the host through cell lysis. Alternatively, during lysogeny, the phage integrates its genome into the bacterial chromosome and enters a dormant state that can persist for many generations.

Historically, researchers have viewed this decision primarily at the level of the infected cell. Measurements typically captured the collective behavior of all phage genomes present within a bacterium, masking potential differences between individual viral particles. The new study overcomes this limitation by applying parallel sequential fluorescence in situ hybridization, or par-seqFISH, to directly quantify transcriptional activity from individual phages during synchronized infections.

The scale of the experiment was remarkable. Researchers monitored the expression of 18 lambda genes representing the major viral regulatory programs, including early genes such as cro and N, lysogenic regulators such as cI and rexAB, and lytic genes including R, A, E, V, and J. Samples were collected at eleven time points spanning the first hour after infection, allowing reconstruction of viral developmental trajectories with unprecedented temporal resolution.

To ensure that both developmental outcomes would occur naturally, infections were performed at an average multiplicity of infection of approximately 0.63 phages per bacterial cell. Under these conditions, most bacteria received either one or two phages, generating a mixed population in which both lytic and lysogenic fates emerged. Around 20% of infected cells contained multiple phages, a proportion sufficient to generate significant numbers of lysogens.

The transcriptional data revealed a highly organized developmental program. Early after infection, expression was dominated by genes transcribed from the PL and PR promoters, including cro, N, cII, cIII, O, P, and Q. As infection progressed, transcription diverged toward either lytic or lysogenic pathways, producing distinct cellular populations that could be separated computationally through principal component analysis. Remarkably, the first two principal components captured more than 60% of the transcriptional variation observed across thousands of infected cells.

The most significant breakthrough came when the researchers pushed beyond the cellular level and reconstructed transcriptional activity for individual phage genomes. To accomplish this, they combined spatial transcriptomics with machine-learning analysis. An XGBoost classifier was trained on more than 11,000 image patches and achieved approximately 96% accuracy during training and 92% accuracy during testing, allowing researchers to infer the positions of individual phage genomes based solely on RNA spatial organization.

This approach revealed that roughly 82% of intracellular phages were transcriptionally active, while approximately 70% simultaneously expressed at least five mRNA molecules and transcribed two or more distinct genes. These observations indicated that individual viral genomes could be reliably characterized at the transcriptional level.

What emerged from the analysis challenged long-standing assumptions.

If all phages within a cell behaved identically, each viral genome would be expected to exhibit a transcriptional profile matching the overall fate of its host bacterium. Instead, researchers discovered substantial disagreement among co-infecting phages. In cells ultimately pursuing the lytic pathway, approximately 24% of phages displayed transcriptional programs that deviated significantly from the consensus, with most of these exhibiting signatures normally associated with lysogeny. In other words, some phages appeared to be attempting dormancy while their neighbors drove the host toward destruction.

The opposite situation was rarely observed. Lysogenic cells displayed striking unanimity. Only about 4% of phages deviated from the collective transcriptional state, and researchers found no lytic-profile phages inside cells successfully entering lysogeny. The average difference in transcriptional state between neighboring phages was only 6.2 degrees in lysogenic cells, compared with 36.4 degrees in lytic cells and 41.5 degrees during early infection.

These observations provide direct experimental support for a hypothesis proposed more than a decade ago. Previous theoretical and experimental work suggested that lysogeny may require unanimous agreement among all phages present within a cell. Under this model, any phage that commits strongly to lytic development effectively overrides the lysogenic preference of its neighbors, forcing the entire infected bacterium toward destruction.

The new single-phage measurements provide the first direct visualization of this phenomenon. Rather than behaving as a perfectly coordinated collective, coinfecting phages appear capable of maintaining distinct developmental identities. Lysogeny emerges only when all viral genomes reach consensus.

The study also sheds light on the role of viral replication in cell-fate determination. Using a replication-deficient lambda mutant carrying a defective P gene, researchers demonstrated that active genome replication is required for robust developmental divergence. In the absence of replication, cells frequently entered incomplete lytic states characterized by insufficient expression of lytic regulatory genes. Instead of committing irreversibly to lysis, many infections stalled and failed to reach a stable developmental outcome.

Beyond lambda biology, the findings have broader implications for virology and cellular decision-making. Traditional models often assume that genetically identical copies of the same genome operating within a shared cytoplasm behave uniformly. The new results suggest that this assumption may not always hold. Individual genetic elements can maintain partially independent regulatory states, even when occupying the same cellular environment.

This concept could extend far beyond bacteriophages. Similar phenomena may influence viral coinfections, prophage induction, chromosome replication dynamics, and potentially even gene regulation in higher organisms. The work highlights how cellular behavior can emerge from interactions among physically distinct copies of the same genetic circuitry rather than from a single unified regulatory network.

For phage biology, the study represents a major technological milestone. By combining spatial transcriptomics, fluorescence microscopy, machine learning, and quantitative systems biology, researchers have achieved something that was previously impossible: observing the behavior of individual phages as they negotiate the fate of their bacterial host.

More than seventy years after lambda first became a model organism, it continues to reveal new layers of biological complexity. What once appeared to be a simple binary decision between lysis and lysogeny is increasingly understood as a collective negotiation among individual viral genomes, each contributing its own voice to the final outcome.

Source : Homaee E., Zhu W., Yao T., Golding I. Single-phage profiling illuminates viral individuality during bacterial cell fate determination. Nature Communications (2026), https://doi.org/10.1038/s41467-026-73867-7