Bacteria are often thought of as simple, single-celled organisms, yet they have evolved remarkably sophisticated strategies to survive and thrive in challenging environments. One of the most striking of these strategies is the formation of biofilms: dense communities of bacteria embedded in a self-produced matrix. Biofilms protect cells from environmental stress, nutrient fluctuations, and predation, and they can make bacterial populations more resilient in ways that single cells cannot achieve. A key component of these matrices is extracellular DNA (eDNA), which has long been recognized as providing structural support. But how exactly do bacteria transform their genetic material into a viscoelastic scaffold capable of supporting an entire community?
Our study explored a surprising answer: R-loops. These triple-stranded nucleic acid structures, consisting of a DNA:RNA hybrid and a displaced single DNA strand, were traditionally considered unusual or even problematic intermediates in cellular processes. However, we found that extracellular R-loops are not merely by-products; they appear to play an active role in shaping biofilm architecture. We first noticed that mature Pseudomonas aeruginosa biofilms contained R-loops intertwined with eDNA fibres. Using immunofluorescence microscopy, we observed that these structures were abundant and increasingly prevalent as the biofilm matured. Interestingly, R-loops were largely absent in planktonic cells, suggesting that their presence is a specific feature of the biofilm lifestyle. This hinted at a functional, rather than incidental, role in biofilm formation.
The idea that extracellular R-loops might influence biofilm mechanics led us to explore their contribution to viscoelasticity. When we selectively degraded RNA:DNA hybrids or the displaced single-stranded DNA, the biofilm’s viscoelasticity was only partially affected. However, removing both components almost entirely dissolved the structural properties of the biofilm. This indicates that R-loops, together with eDNA, form a cooperative framework that houses and protects to the bacterial community. Further evidence came from studying bacterial mutants. Cells lacking enzymes that normally resolve R-loops during replication produced more robust biofilms, suggesting that controlled accumulation of R-loops can enhance matrix formation. We also observed R-loops in other species, including Staphylococcus epidermidis, and in clinical airway samples from patients with chronic P. aeruginosa infections. This suggests that the structural role of R-loops may feature across other – and potentially diverse – bacterial species and environments, including those relevant to human health.
A particularly striking aspect of R-loops is that their formation appears decoupled from ongoing transcription. Our sequencing analysis showed that the RNA component of extracellular R-loops does not simply reflect the most actively transcribed genes. Instead, these “in trans” R-loops can form at genomic locations distant from the RNA’s transcription site. This distinguishes them from the conventional, “in cis” R-loops associated with transcription or DNA repair and suggests a regulated, functional role for extracellular R-loops in the biofilm matrix.
Central to the formation of these R-loops is the RecA protein, well known for its role in the bacterial SOS response and DNA repair. RecA is capable of assembling nucleoprotein filaments that insert RNA strands into DNA, forming R-loops. We observed that biofilms lacking RecA had reduced biomass, fewer R-loops, and diminished viscoelasticity, confirming the link between this protein, R-loop formation, and biofilm structure. We then found that the activation of RecA is connected to the stringent stress response, a system triggered by nutrient limitation and other environmental stresses, which results in elevated levels of the alarmone ppGpp. Nutrient deprivation, particularly amino acid limitation, promotes the formation of extracellular R-loops, highlighting how environmental cues can shape biofilm architecture through a coordinated stress response.
Interestingly, R-loops also intersect with programmed cell death pathways. The AlpA-mediated lysis pathway is required for the release of extracellular R-loops. Cells undergoing lysis contribute their nucleic acids to the biofilm matrix, effectively sacrificing themselves to enhance community resilience. This dual role – mediating cell death while simultaneously strengthening the biofilm – demonstrates a remarkable form of bacterial altruism. The released R-loops appear to act as crosslinking agents within the eDNA network, providing structural stability and allowing surviving cells to thrive under stress. These findings suggest that R-loops represent a conserved strategy for bacteria to respond to environmental stress by transforming genomic instability into a functional advantage. Rather than being random or deleterious, extracellular R-loops are deliberate, regulated components that reinforce biofilm structure, contributing to community survival under challenging conditions. The implications of this are wide-ranging.
From a fundamental perspective, our work challenges the traditional view of R-loops as merely aberrant or toxic intermediates. They are instead active, functional molecules that bacteria can deploy to build resilient, viscoelastic structures. In clinical and environmental contexts, this raises new considerations for biofilm control. Strategies that stress bacteria – such as certain antibiotics or nutrient limitations – might inadvertently stimulate R-loops formation and strengthen biofilms, potentially complicating treatment or containment efforts. Understanding the molecular underpinnings of R-loop-mediated biofilm stability may therefore inform approaches to prevent or disrupt biofilm formation, whether in chronic infections, industrial systems, or environmental settings. Moreover, the observation that R-loops are found in multiple species and in human-relevant clinical samples points to a broadly conserved mechanism. Environmental stressors, nutrient fluctuations, and interspecies interactions could all influence R-loop formation, offering a new lens through which to understand biofilm dynamics in natural and host-associated microbial communities.
In summary, our study highlights a surprising capacity of bacteria to turn environmental stress into structural strength. Extracellular R-loops, formed in response to stringent and SOS stress responses, exemplify how what might seem like genomic chaos can be harnessed to create constructive order. By sacrificing a fraction of the population and repurposing nucleic acids, bacteria construct a viscoelastic matrix that benefits the community as a whole. This work not only reveals a new functional role for R-loops but also underscores the sophisticated ways in which bacteria adapt to their environment, with implications for both microbial ecology, industrial applications and human health.