Prophages are essentially low-key until they’re not.   

But bacterial cell lysis is not the only possible outcome in these high-stakes moments. Scientists are discovering that when multiple prophages exist within a bacterial host, the ways in which they interact and compete may provide unique (albeit temporary) advantages to the stressed host cell. These inter-prophage dynamics ultimately have important implications for bacteria, phages and the organisms and environments they both call home.

Phages exhibit 2 life cycles: lytic and lysogenic. Lytic phages lyse the host cell after producing new viral particles. Lysogeny involves the integration of phage DNA into the host genome. During prophage induction, the genome is excised from the chromosome and used to generate new phage particles, leading to cell lysis.

Source: Silpe, J.E., et al/PLoS Pathogens, 2023 via a CC BY 4.0 license

Prophages Help Bacteria Until They Kill Them

Most bacteria are hauling around a prophage or 2 (or more) in their genome. One analysis of over 33,600 prophages from 13,713 prokaryotic genomes identified prophages in 75% of the genomes studied, highlighting their ubiquity. This value might even be an underestimation—as researchers discover new types of phages, they may get better at predicting the presence or prophages in bacterial genomes.  

Although forced genome invasion sounds harmful, prophages can enhance the fitness and survival of bacteria by adding “bonus” genes/functions to the cell. 

For example, prophages harbor an abundant repertoire of antibiotic resistance genes, which promote cell survival under antibiotic stress, as well as secretion systems, toxins and immune evasion genes that boost the virulence potential of bacterial pathogens. The nasty effects of cholera, diphtheria and botulism can all be traced to prophage-associated virulence factors.  

Prophages also encode anti-phage defense systems that inhibit invading phages from successfully infecting the host. This protects the host cell and, as a result, the resident prophages.    

May the Best Prophage Win Salmonella is often found in macrophages during infection.

Source: Flickr/NIH

But the prophages face a problem: the resources in a bacterial cell are finite. If more than 1 type of prophage exists in a bacterium, as is often the case, the prophages must compete with one another to propagate and move on. Which prophages prevail and how they do so doesn’t just determine the fate of the phages, but also the fate of the bacteria in which they reside.

Helaine’s lab has been exploring the mechanisms and dynamics of prophage competition using a strain of the gut pathogen Salmonella Typhimurium that harbors 4 prophages. During infection, Salmonella often resides in macrophages, where it is exposed to immune mediators and responses that damage its DNA. Some cells can grow in these conditions, while others that are super stressed with intense DNA damage form persisters—transiently non-growing bacteria that can resist antibiotics and immune responses, making them tricky to get rid of.

Helaine’s team showed that Salmonella experimentally cured of its prophages survives better in macrophages compared to wild-type bacteria. As such, macrophages are not killing the bacteria alone; the prophages are doing a big part of the job.

Furthermore, actively growing bacteria isolated from macrophages have substantially less prophage activation compared to persister cells, in which all 4 prophages excise from the chromosome. But the prophages in persisters do not replicate equally. One prophage called ST64B is more successful than the others.

“As we understand it, in persisters, but not in growing bacteria, prophages are induced and they start replicating, and [ST64B] has a head start compared to the other prophages,” Helaine noted.

Yet, this head start does not guarantee cell lysis. Experiments using Salmonella strains harboring combinations of each of the 4 prophages revealed that bacteria containing a prophage known as Gifsy-1 (including wild-type cells) lysed the least, whereas those lacking Gifsy-1 but carrying ST64B lysed the most. It appeared as if Gifsy-1 was blocking the lytic cycles of other prophages, especially ST64B.

It turns out that's exactly what was happening.

Gifsy-1 encodes an anti-phage system (ribonuclease effector module with ATPase, inhibitor and nuclease, a.k.a. RemAIN) that cleaves tRNAs in the bacterial cell. TRNA serves as an essential link between mRNA and growing amino acid chains during protein production. RemAIN-mediated fragmentation of tRNAs, including those associated with prophage activation, slows down translation in the cell and, by extension, inhibits prophage replication. Gifsy-1 is unharmed by RemAIN for reasons that are still unclear. What this means, though, is that the system helps Gifsy-1 produce more phage particles than co-resident prophages. It also indicates that anti-phage systems don’t just protect against invading phages; they’re also useful against those that are already settled.

The lab recently identified another Gifsy-1-encoded anti-phage system called HepS with a similar mechanism to RemAIN, pointing to a more generalized phenomenon in the context of prophage competition.

Prophage Competition Protects Persister Cells

While prophage activation generally spells doom for the bacterial host, competition between prophages can also promote bacterial persistence. “These prophages, when they reactivate, can kill some bacteria in the population. But the other thing we realized is that they also reactivate in some bacteria that survive [as persisters], and they actually contribute to that phenotype,” Helaine said.  

The activity of systems like RemAIN and HepS prevents some prophages in the cell from entering their lytic cycle, thus protecting the bacterial host.

Source: Getty Images/wildpixel

Persister cells have more prophage induction compared to normally growing cells. Systems like RemAIN and HepS can prevent a large fraction of prophages from completing their lytic cycle in persisters, meaning there are fewer chances for an explosive cell death. Gifsy-1 does still kill cells, though not all the cells in which it is activated, possibly because of additional prophage control systems that have yet to be identified. Helaine also posits that persister cells survive prophage induction, in part, because they are less sensitive to the slowdown in translation resulting from RemAIN and HepS activity compared to the phage. Additional research is required to test this hypothesis.

“When Salmonella is inside macrophages, it's stressed because the macrophages are attacking the bacteria,” Helaine summarized. “These bacteria experience now another layer of complexity, which is the prophages that are coming up and are trying to also kill the bacteria. But, thanks to this prophage-prophage competition, when the prophages activate, they activate anti-phage defense systems that modify the bacteria and keep them in that state of persistence.” 

These findings are valuable for understanding nuances of bacterial behavior that could help determine how best to treat persistent infections. Data from Helaine’s lab suggest that similar dynamics are at play in bacteria other than Salmonella, like Pseudomonas and Klebsiella.

Explore Prophages in Complex Communities

To that end, the importance of studying prophage-prophage and prophage-bacterial interactions extends beyond the macrophage interior. Bacteria occupy diverse environments by forming complex, multi-species communities. While it is clear prophages impact bacterial physiology, their role in host-associated and environmental microbiomes is still being uncovered.

In a recent analysis of 14,987 bacterial genomes isolated from human body sites, prophage DNA made up an average of 1-5% of each bacterial genome, depending on the isolation site. These prophages can be induced in response to diet, hormones, immune responses and other factors to promote bacterial resilience to environmental stressors or increase virulence or antibiotic resistance, all of which have implications for host health.

In environments like the gut, many different signals can trigger prophage induction, which implications for community structure and function.

Source: Zhang, S., et al./Applied and Environmental Microbiology, 2025

Researchers are also finding that prophages shape biofilm structure and function. For instance, lysis of Streptococcus pneumoniae cells by prophage induction releases extracellular DNA to support formation of the biofilm matrix by the remaining population.  

Keep Looking for the Prophages 

The picture that emerges from these findings depicts prophages as key players in how bacteria live (and die). Helaine noted she had “very happily ignored” prophages until she couldn’t any longer, a sentiment reflecting the largely overlooked role of prophages in varied contexts. But if ongoing research suggests anything it’s that, in the phage world, it’s worth keeping an eye on the quiet ones. 

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Phages are promising tools for combating antimicrobial resistant infections. ASM Health’s Phage Therapy Coordination Initiative is working to unite science, clinical practice and policy to move phage therapy from isolated use to scalable national readiness.