How Dead Bacterial Cells Help Build a Biofilm

A bacterium can die while some of its molecules continue doing useful work. In a biofilm, cellular material can become part of the shared structure around neighbouring cells. A recent study adds an unexpected player to that story: R-loops, structures made from DNA and RNA. [1] [2]

The peer-reviewed study was published in Nature Communications on 12 December 2025. Sudarsan Mugunthan and colleagues investigated Pseudomonas aeruginosa, mainly through controlled laboratory biofilms, alongside observations in another bacterial species and clinical airway samples. [1]

Key findings

• DNA and RNA can contribute to a biofilm’s physical scaffold.
• The new work connects persistent R-loops, cellular stress and matrix formation.
• Clinical samples support relevance; the study did not establish a treatment.

Why would a biofilm need DNA outside its cells?

A biofilm is more than a collection of bacteria sitting close together. Its cells occupy a shared extracellular matrix: material outside the cells that helps organize the community. Depending on the organism and conditions, that matrix can include sugars, proteins and nucleic acids. The biological question is therefore also a materials question: what gives this mixture its structure? [5]

DNA’s structural role is not a discovery from 2025. A 2002 Science paper established extracellular DNA as necessary for biofilm formation in the system investigated. The newer question is how these long molecules assemble into networks, and which molecular partners help those networks hold together. [3] [5]

Cell breakdown was already part of the story

In 2016, researchers used live-cell imaging to observe a dramatic route for releasing cellular contents in P. aeruginosa. Some cells lost their integrity and burst, releasing DNA and other material. Membrane fragments could also reorganize into small vesicles. Genetic experiments linked this process to an enzyme that breaks down the cell wall. The work showed that breakdown of individual cells can contribute ingredients to a larger bacterial structure. [4]

That history matters when interpreting the new headline. The 2025 advance is not simply that dead cells leave DNA behind. It concerns the molecular form of some released nucleic acids and how that form helps determine the matrix’s behaviour. Connecting those two scales—cell physiology and extracellular material—is the interesting step. [1] [2]

The clue that RNA was doing structural work

A related study published on 27 November 2023 found RNA associated with extracellular DNA fibres in P. aeruginosa biofilms. Certain messenger RNAs were enriched in the matrix, rather than appearing merely in proportion to their abundance inside cells. Removing associated RNA disrupted DNA fibres and their material properties. This suggested that RNA could help build a scaffold as well as carry messages. [5]

The researchers’ original account explains an important detection problem: RNA bound within DNA-rich fibres could be difficult for probes to reach. Making it more accessible allowed its location to be observed. One abundant RNA, called lasB, was useful as a marker, but losing that RNA did not abolish the DNA network. A conspicuous component is not automatically the only essential component. [6]

What exactly is an R-loop?

Normally, DNA’s two strands pair with one another. In an R-loop, an RNA strand pairs with one DNA strand, while the other DNA strand is displaced. The result involves three strands: a DNA–RNA hybrid plus single-stranded DNA. It is not three strands wound together into a conventional triple helix. [1]

The 2025 work connects persistent versions of these structures inside cells with a role outside them. The matrix is described as viscoelastic: its response combines liquid-like flow and solid-like elasticity. This describes how a material deforms under force; it does not mean that a biofilm is an impenetrable shell. [2]

How the team tested a structural role

The team combined microscopy, sequencing, genetic perturbations and measurements of material behaviour. These approaches ask different questions: where are the structures, which molecules are involved, what changes when cellular pathways are altered, and does the matrix’s physical response change? Seeing a molecule and showing that it contributes to a material are separate tasks. [1] [2]

As the authors explain, selectively disrupting either the RNA–DNA hybrid or the displaced DNA affected the matrix. Disrupting both components had a much stronger effect on its viscoelastic properties. That functional experiment strengthens the case that the structures contribute to the material, rather than simply being debris trapped within it. [2]

From genomic stress to a shared scaffold

The authors propose a pathway in which stress responses and the strand-exchange protein RecA promote R-loop formation. Persistent genomic R-loops contribute to instability in some cells; cell breakdown then releases structures that become part of the extracellular network. This is the study’s mechanistic interpretation, supported by multiple experiments, rather than a claim that bacteria consciously sacrifice themselves. [2]

It is also not a universal explanation for every extracellular DNA molecule. A 2012 study of early Enterococcus faecalis biofilms found DNA-rich extracellular structures without evidence of substantial cell lysis under those conditions. The observations were consistent with release by viable cells. Species, developmental stage and growth conditions can change the route by which DNA reaches a matrix. [7]

What the finding does—and does not—mean

Related R-loop structures were detected in Staphylococcus epidermidis biofilms and in clinical sputum specimens from people with bronchiectasis and high P. aeruginosa loads. Those observations extend the story beyond one laboratory model. They do not demonstrate that disrupting the structures improves a patient’s infection or is safe in an airway. [1] [2]

Another distinction matters: biofilm structure is not synonymous with inherited antibiotic resistance. A 2019 consensus statement distinguishes resistance, which permits growth at higher antibiotic concentrations, from tolerance, which concerns longer survival during exposure. Persistence describes a particularly surviving subpopulation. A change in matrix mechanics therefore cannot, on its own, establish that resistance has been reversed. [8]

A realistic research implication is to test whether these structures offer a useful point of intervention. That would require evidence about delivery, specificity, safety and meaningful outcomes in more complex settings. For example, researchers would need to distinguish reducing an isolated material’s elasticity from reducing viable bacterial burden. These are proposed next questions, not benefits demonstrated by the study.

The memorable idea is that a molecule’s role depends on its setting. DNA and RNA are familiar as carriers of biological information. In these biofilms, their interactions also help shape a physical material. Understanding that change of role makes the community easier to explain—and gives researchers a more precise question to investigate. [5] [6]

Sources and references

1. Mugunthan S, Dong Z, Chotirmall SH, Kjelleberg S, Seviour T. Stress-induced toxic genomic R-loops support biofilm extracellular matrix formation. Nature Communications 16, 11451 (2025). Published 12 December 2025.
2. Mugunthan S, Seviour T, Kjelleberg S. From genomic instability to matrix integrity: Extracellular R-loops shape the biofilm scaffold. Springer Nature Research Communities, Behind the Paper (13 January 2026). Original authors’ explanation, not a separate research study.
3. Whitchurch CB, Tolker-Nielsen T, Ragas PC, Mattick JS. Extracellular DNA required for bacterial biofilm formation. Science 295(5559), 1487 (2002).
4. Turnbull L et al. Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms. Nature Communications 7, 11220 (2016).
5. Mugunthan S et al. RNA is a key component of extracellular DNA networks in Pseudomonas aeruginosa biofilms. Nature Communications 14, 7772 (2023). Published 27 November 2023.
6. Mugunthan S. Extracellular RNA is directly involved in building Pseudomonas aeruginosa biofilm matrix. Springer Nature Research Communities, Behind the Paper (6 December 2023). Original author’s explanation.
7. Barnes AMT, Ballering KS, Leibman RS, Wells CL, Dunny GM. Enterococcus faecalis Produces Abundant Extracellular Structures Containing DNA in the Absence of Cell Lysis during Early Biofilm Formation. mBio 3(4), e00193-12 (2012).
8. Balaban NQ et al. Definitions and guidelines for research on antibiotic persistence. Nature Reviews Microbiology 17, 441–448 (2019). Consensus Statement.