A pair of compounds found abundantly in soybeans can coax one of the world’s most important antibiotic-producing bacteria into shedding its protective cell wall, researchers report. When exposed to the plant flavonoids daidzein and genistein while under intense osmotic stress, Streptomyces coelicolor transformed from branching filaments into rounded, wall-deficient cells—some of which became capable of dividing. The discovery identifies plant chemicals as direct triggers of an unusual bacterial survival and growth state, offering a new window into how soil microbes respond to their chemical surroundings and potentially creating a controllable route for biotechnology.

The finding is striking because Streptomyces bacteria are not ordinary single-cell organisms. They grow as branching, fungus-like hyphae and pass through a complex multicellular life cycle that includes differentiation, programmed cell death and the production of spores. Their filamentous networks are also biochemical powerhouses: members of the group produce more than two-thirds of clinically used antibiotics, along with anticancer, immunosuppressive and agricultural compounds. Much of this biology depends on the cell’s peptidoglycan wall, a mesh-like polymer that provides mechanical strength and prevents the membrane from bursting. Yet Streptomyces can sometimes abandon this structure and adopt alternative forms known as wall-deficient cells.

The researchers distinguish two important versions of this unusual state. S-cells are non-dividing, stress-induced cells that lack a rigid wall but remain metabolically active and retain the machinery needed to rebuild filamentous growth. L-forms, by contrast, are wall-deficient cells that can reproduce autonomously. Both are fundamentally different from extracellular vesicles, which are membrane-bound particles released by bacteria and generally cannot replicate. S-cells may look superficially similar to vesicles because they are rounded and wall-less, but they are living cells rather than inert packages. L-forms can divide without the conventional FtsZ-based divisome, the protein machinery that normally organizes bacterial cytokinesis and directs the construction of a new septum.

Previous experiments had shown that wall-deficient cells could appear in S. coelicolor after specific genetic changes, particularly when the bacteria were exposed to high concentrations of sucrose. But osmotic protection alone was not enough. At 0.3 molar sucrose, the medium could prevent wall-less cells from bursting, yet it did not reliably initiate their formation. A stronger treatment, 0.64 molar sucrose, was required, and even then the wild-type bacterium did not produce these forms in conventional glucose-yeast extract-malt extract medium. The new study began with an unexpected observation: the same strain readily formed rounded cells in soya flour-mannitol medium under the stronger osmotic challenge. That suggested that a soybean-derived molecule, rather than nutrients in general, might be acting as a morphogenetic signal.

Soybeans are rich in isoflavones, a class of flavonoids that includes daidzein and genistein. These molecules are already known to influence bacterial physiology, alter membrane properties and participate in plant-microbe communication. In legume roots, for example, they help activate gene expression in symbiotic rhizobia, bacteria that invade plant tissues and establish nitrogen-fixing partnerships. The researchers estimated that the soya flour medium contained roughly 21 micrograms per millilitre of daidzein and 22.2 micrograms per millilitre of genistein. They therefore added purified versions of the two compounds to flavonoid-free laboratory medium and tested whether they could reproduce the soybean effect.

The results were dose-dependent and dramatic. At 6.25 micrograms per millilitre, either flavonoid produced small but detectable numbers of rounded cells. Combining the compounds strengthened the response, generating cells with an average area of 5.9 square micrometres. At 15 micrograms per millilitre, the formation of these structures increased sharply. At 25 micrograms per millilitre—close to the estimated concentration in the soybean medium—the bacteria produced much larger S-cells. When daidzein and genistein were combined at this concentration, the average cell area reached 17.6 square micrometres, and individual structures grew to as much as 35 square micrometres. Under the most effective conditions, nearly all of the stained cellular material adopted the rounded S-cell morphology, leaving virtually no recognizable filamentous hyphae.

Microscopy confirmed that the transformation was not simply a swelling of ordinary hyphae. The team used FM5-95, a fluorescent membrane dye, and wheat germ agglutinin linked to Alexa Fluor 488, a probe that binds the N-acetylglucosamine and N-acetylmuramic acid residues characteristic of peptidoglycan. The induced structures were surrounded by membranes but showed no detectable cell-wall staining, demonstrating that they were genuinely wall-deficient. Some cells retained thin wall fragments or discrete patches of peptidoglycan, possibly marking remnants of the original wall or locations where wall synthesis and degradation were still occurring. A subset also displayed unusually strong internal membrane staining, a feature associated with L-form biology in other bacteria.

Time-lapse imaging revealed that most of the stress-induced cells behaved as non-dividing S-cells, but a small minority crossed into the L-form state. Among 137 cells tracked over 17 hours, nine—about 6 percent—showed pronounced deformation and movement. Three cells, approximately 2.2 percent of the total, moved and divided. The dividing L-forms appeared to establish transient membranous bridges between daughter cells, structures that resemble midbody-like connections described during cytokinesis in animal cells and during L-form division in Listeria monocytogenes. Because wall-deficient division does not depend on the standard septum-building apparatus, such observations may help researchers understand how membranes themselves can generate new cells when the rigid bacterial wall is absent.

The molecular explanation remains unresolved, but the experiments point toward an interaction between flavonoid activity and the machinery that remodels peptidoglycan. The researchers examined mutants affecting four proteins: the N-acetyltransferase SCO0954, the D-alanyl-D-alanine carboxypeptidase SCO4439, the GOLPH3-like protein SCO4440 and the EngA GTPase SCO1758. These proteins are involved directly or indirectly in cell-wall remodeling, and mutations in their genes can promote S-cell and L-form formation under hyperosmotic stress. In soybean medium, the mutant carrying a disruption affecting sco1758 produced significantly smaller and fewer wall-deficient cells, while the sco0954 knockout and a strain overexpressing sco0954 approached complete conversion to S-cells. The pattern suggests that flavonoids do not act through a single isolated switch but may lower the physiological barrier to wall removal while the bacterium’s peptidoglycan and membrane systems determine how far the transformation proceeds.

One possibility is that daidzein and genistein alter membrane fluidity, permeability or redox balance, indirectly destabilizing the coordination between membrane growth and peptidoglycan synthesis. Flavonoids are often discussed as antimicrobial compounds because, at sufficiently high concentrations, they can damage bacterial membranes and disrupt cellular homeostasis. But the new results suggest that their effects can be more nuanced than simple toxicity. At the concentrations tested—roughly 60 to 100 micromolar—the compounds may function as environmental signals in combination with severe osmotic stress, prompting a reversible change in cell architecture. The study examined only two flavonoids, so it is not yet clear whether the response is specific to soybean isoflavones or represents a broader reaction to membrane-active plant phenolics.

The ecological implications are tantalizing but still speculative. Daidzein and genistein are released into the rhizosphere by legume roots, where concentrations can vary sharply across microscopic distances and may become locally elevated at the plant surface. A wall-less state could potentially help soil-dwelling actinomycetes negotiate crowded, chemically complex plant-associated habitats. Wall-deficient bacteria are flexible and deformable, and L-forms in other species have been linked to stress tolerance, antibiotic persistence and resistance to certain bacteriophages. There is also a natural precedent for wall-less bacteria living inside plants: phytoplasmas lack peptidoglycan entirely and inhabit phloem tissues, moving through narrow sieve pores. None of this demonstrates that Streptomyces S-cells colonize plants in nature, however. Establishing that connection will require experiments in soil, on living roots and in other streptomycete species.

The work could also matter for biotechnology. Streptomyces already supplies a vast catalogue of medically valuable molecules, while bacterial extracellular vesicles are being explored as delivery vehicles for proteins, nucleic acids and therapeutics. S-cells and L-forms are not vesicles, but their membrane-rich, wall-deficient architecture could provide a complementary platform for studying or engineering the movement of biological cargo. The flavonoid trigger offers something that has been missing: a defined chemical means of inducing the state without relying solely on genetic mutations or poorly characterized soybean extracts. Before such applications become realistic, researchers will need to determine whether induced cells can be recovered efficiently, how reliably they return to normal filamentous growth, what they contain, and whether the process can be controlled without compromising viability. For now, the central message is that a molecule produced by a plant can push a major bacterial antibiotic producer into a radically different form—revealing just how responsive microbial cell architecture can be to the chemistry of its environment.

The PIE technology is built upon prime editing, a groundbreaking method that allows for precise changes to the DNA sequence without causing unintended double-strand breaks. Geneticists have faced challenges in manipulating larger genomic regions due to inherent limitations in existing methodologies. The newly developed PIE system addresses these challenges, offering a more refined approach to achieve substantial modifications across vast stretches of the genome. This potential for large-scale engineering could lead to breakthroughs in understanding genetic disorders and developing therapies.

The first version of PIE, dubbed PIEv1, utilizes a pair of prime-editing guide RNAs (pegRNAs). While effective, it produces one imprecise junction—an outcome that may limit its applicability in setting ambitious genetic engineering goals. Recognizing this limitation, researchers expanded the PIE approach. PIEv2 and subsequently PIEv3 introduced an additional pair of pegRNAs, aiming to enhance the accuracy and overall efficiency of the inversions being performed. This iterative refinement highlights the rapid pace of innovation within the field of genetic editing.

The advances made in PIEv3b are particularly noteworthy. This version bolstered the design of the accompanying plasmid, fundamentally improving the coupling mechanism required for precise inversions. Additional enhancements allowed PIEv3b to achieve inversion efficiencies nearing 61.7% for segments up to 1 megabase (Mb) and 14.2% for larger segments reaching 50 Mb. These impressive figures represent a significant leap forward when juxtaposed against prior twin prime editing techniques using integrase, which produced considerably lower efficiencies.

Furthermore, a head-to-head comparison with traditional nuclease-based methods underscored PIEv3b’s advantages both in terms of efficiency and precision. As existing genetic engineering strategies often invoke risk by introducing double-strand breaks—adding a layer of potential for unintended mutations or genomic instability—the PIE system stands out. It appears as a more controlled and reliable alternative for researchers aiming to explore the genetic landscape without compromising the integrity of cellular DNA.

One particularly eye-catching application of PIE involves the transformation of human chromosomes from metacentric—where centromeres sit at or around the chromosome’s center—to telocentric configurations, where the centromere is located at one end of the chromosome. By inverting extensive chromosomal segments, such as 30 Mb and even 100 Mb, the research opens doors to redefining our fundamental understanding of human genetics and chromosomal architecture.

The broader implications of such capabilities cannot be overstated. From the potential to discover new genetic pathways linked to diseases to innovative approaches for gene therapy, PIE represents an arsenal at the disposal of genetic researchers and clinicians alike. It could pave the way for tailored treatments, where genetic disorders are corrected at their source, rather than merely managing symptoms.

Additionally, while the immediate focus is on applications within mammalian cells, the long-term vision extends to model organisms, agricultural biotechnology, and potentially even synthetic biology. By adapting PIE for various systems, researchers could harness this technology to create new traits in crops or engineered organisms that could lead to sustainable solutions to global challenges.

The potential for commercialization also exists, with biotech companies eyeing the implications of PIE for developing gene editing services. As research continues to evolve, aligning these technological advances with practical applications will be essential. This will require collaboration across sectors, ensuring that PIE finds its way not only into laboratories but also into clinics and ultimately into the wider community.

In conclusion, the advent of PIE based on prime editing presents an exciting chapter within the field of genetic engineering. Its high inversion efficiency combined with enhanced precision signifies a paradigm shift that could redefine how structural variations are approached in genomic studies. By unlocking the ability to manipulate chromosomes at an unprecedented scale, PIE can offer valuable insights into the intricate tapestry of genetics, holding implications that reach far beyond single-gene editing.

Researchers and practitioners dedicated to genomic innovation should closely follow the developments surrounding PIE. As this technology matures, it promises to usher in a new era of possibilities across a range of scientific and medical disciplines. The journey of exploring and implementing PIE technologies will be crucial in shaping the future landscape of genetics and its applications, making it a topic worthy of continued exploration and excitement within the scientific community.

Subject of Research: Chromosomal inversion using Prime-Editing-Based Inversion with Enhanced Performance (PIE).

Article Title: Efficient and precise inversion of genomic DNA from large to chromosomal scale.

Article References:
Zhang, A., Sun, X., Wu, Y. et al. Efficient and precise inversion of genomic DNA from large to chromosomal scale.
Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-02033-9

Image Credits: AI Generated

DOI: https://doi.org/10.1038/s41589-025-02033-9

Keywords: Chromosomal inversion, prime editing, genetic engineering, genomic integrity, biotech, genetic manipulation, precision medicine, mammalian cells.

For more than a decade, the CRISPR-Cas system has mesmerized scientists as a powerful adaptive immune mechanism employed by bacteria and archaea to fend off viral invaders. These systems, consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, recognize and degrade invading phage nucleic acids with extraordinary precision. In response, many phages have evolved anti-CRISPR (Acr) proteins that inhibit different stages of the CRISPR-Cas immune response, enabling them to escape detection and destruction.

The type III CRISPR systems present a particularly fascinating and complex form of immunity. Unlike the more extensively studied type II systems, type III complexes provide multi-layered defense that targets both DNA and RNA molecules from invading phages. Their ability to simultaneously detect and degrade transcripts while triggering downstream nucleolytic activities makes their inhibition a challenging endeavor for phages. Until now, known type III Acr proteins were scarce, displaying limited effectiveness or operative through poorly understood strategies.

In this context, the discovery of AcrIIIA2 stands out. The research team led by Johnson et al. has uncovered an unexpected mode of CRISPR system neutralization where the phage-encoded AcrIIIA2 hijacks a critical and highly conserved host enzyme—enolase—to disable the immune defense. Enolase, a cornerstone of glycolysis, catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate and is abundant in a wide range of bacterial cells. Intriguingly, this enzyme moonlights beyond metabolism, here serving as a structural cofactor in the phage’s anti-immune arsenal.

Through a combination of biochemical assays and high-resolution structural analyses, the investigators delineated how AcrIIIA2 forms a ternary complex with the host’s enolase and the Streptococcus thermophilus type III-A CRISPR ribonucleoprotein (Csm) complex. This coordinated assembly obstructs the initial binding of phage RNA substrates to the CRISPR machinery—a critical step required for immune activation. By blocking RNA recognition, AcrIIIA2 effectively halts the cascade of anti-phage responses typically deployed by type III systems.

The data reveal that enolase acts as an essential structural scaffold within this tripartite complex, stabilizing protein-protein interactions that otherwise would be transient or weak. This exploitation of a housekeeping enzyme for immune evasion represents a paradigm shift in understanding phage-host interplay. Rather than targeting Cas proteins directly, the phage commandeers a ubiquitous metabolic enzyme to indirectly incapacitate the immune apparatus, showcasing a highly evolved and stealthy viral strategy.

Importantly, the enolase-chaperoned AcrIIIA2 mechanism prevents the formation of the RNA-bound state of the Csm complex, thereby suppressing subsequent immune activities such as RNA cleavage and collateral nucleic acid degradation. This represents a strategic blockade at the very front line of CRISPR detection, effectively rendering the bacterial immune system blind to phage infiltration.

The study’s findings were supported by detailed structural data obtained via cryo-electron microscopy, which provided atomic-level views of the AcrIIIA2-enolase-Csm assembly. These structures illustrate how AcrIIIA2 interfaces with both enolase and the CRISPR complex, inducing conformational changes that occlude the RNA binding channel. Such molecular insight highlights potential avenues for engineering synthetic inhibitors or modulators of CRISPR systems based on this scaffolding interaction.

Beyond the immediate microbiological implications, this discovery raises fascinating questions about the evolutionary dynamics between phages, their bacterial hosts, and the repurposing of metabolic enzymes. It suggests that metabolic enzymes, far from being passive players, might serve dual roles within the cellular milieu, potentially influencing immune responses under certain circumstances. For phages, co-opting a conserved host protein like enolase ensures a robust and widely applicable method to disable immunity across divergent bacterial strains.

This work also underscores the immense diversity and sophistication of anti-CRISPR strategies employed by phages. While many Acrs target Cas proteins directly, AcrIIIA2’s reliance on a host-derived scaffold illustrates a novel evasion paradigm that could inspire new biotechnological tools. For example, manipulating enolase or AcrIIIA2-like molecules could enable controlled modulation of CRISPR immunity, with applications spanning gene editing fidelity and phage therapy.

Furthermore, the study helps explain previous observations that type III anti-CRISPR activities often appear conditional or partial—by identifying this scaffold-dependent mechanism, the authors provide a molecular basis for context-dependent Acr functionality. This insight may redefine how scientists screen for and characterize Acrs in other bacterial species and viral contexts.

The discovery, published in Nature Microbiology in 2025, involved an interdisciplinary collaboration combining microbiology, structural biology, and enzymology. It stands as a testament to the power of integrative approaches in unraveling complex biological systems, particularly in host-pathogen interactions. The elucidation of AcrIIIA2’s mode of action marks a significant milestone in the expanding field of CRISPR research.

Looking ahead, this research lays the groundwork for exploring whether other phage-encoded Acrs similarly hijack host metabolic enzymes or scaffolds. It also invites investigations into whether bacterial hosts can counter-adapt by modifying enolase or its interactions to resist such viral sabotage. Such evolutionary considerations may reveal layers of complexity in microbial immune conflicts yet to be discovered.

In conclusion, the identification of a phage anti-CRISPR protein that co-opts host enolase to subvert type III CRISPR immunity provides a compelling example of the molecular ingenuity viruses employ to thrive. This novel anti-defence strategy not only expands our understanding of microbial immunity and viral countermeasures but opens exciting possibilities for bioengineering and therapeutic innovation. The convergence of metabolism and immunity in this delicate molecular dance promises to be a fertile ground for future discoveries in microbiology and beyond.

Subject of Research:
Anti-CRISPR protein AcrIIIA2 from Streptococcus thermophilus phages inhibiting type III-A CRISPR immunity by co-opting host enolase.

Article Title:
A phage-encoded anti-CRISPR protein co-opts host enolase to prevent type III CRISPR immunity.

Article References:
Johnson, K.A., Goswami, H.N., Catchpole, R.J. et al. A phage-encoded anti-CRISPR protein co-opts host enolase to prevent type III CRISPR immunity. Nat Microbiol (2025). https://doi.org/10.1038/s41564-025-02178-2

Image Credits: AI Generated

DOI: https://doi.org/10.1038/s41564-025-02178-2