The challenge facing researchers working with phages of Gram-positive bacteria is structural. The cell wall of organisms such as G. stearothermophilus is a dense lattice of peptidoglycan and associated polymers that blocks the uptake of large DNA molecules, including entire phage genomes. Conventional phage engineering relies on homologous recombination, recombineering, CRISPR-Cas systems, or yeast-based assembly, but these approaches are effective mainly for mesophilic phages and their hosts. One successful workaround has been the use of L-form bacteria, cell-wall-deficient variants that retain metabolic activity and can be transfected with synthetic phage genomes. The new work takes a related but distinct route: enzymatic removal of the cell wall to generate protoplasts, followed by polyethylene glycol-mediated delivery of purified phage genomic DNA directly into these osmotically stabilized cells.

Protoplast transformation itself is not new. Classical studies on Bacillus subtilis in the late 1970s showed that protoplasts treated with polyethylene glycol could efficiently take up circular plasmid DNA, and the method was later adapted to thermophilic bacilli, including what are now classified as Geobacillus and Parageobacillus. However, nearly all published protocols describe the introduction of plasmid DNA or short fragments intended for homologous recombination. The literature contained essentially no reports of routinely introducing complete bacteriophage genomes into these bacteria. The Polish research team, led by Ireneusz Sobolewski and Agnieszka Zylicz-Stachula with Piotr Skowron, set out to close that gap, demonstrating that protoplasts can serve as a functional platform for phage research independent of natural viral infection.

The optimization process was systematic and granular. The researchers evaluated eight parameters affecting the recovery of infectious TP-84 particles after transfection: the number of protoplast washing steps, lysozyme concentration across a tenfold range, incubation time with the wall-digesting enzyme, the physiological state of the donor culture, the number of bacterial cells used, the amount of genomic DNA, the volume of the DNA solution, and the molecular weight and concentration of polyethylene glycol. Each variable was tested across meaningful ranges, and the results revealed sharp optima. Too many bacteria or too much lysozyme drastically reduced the yield of viable phages, while two washing steps proved essential to remove residual lysozyme that would otherwise inhibit bacterial growth on recovery plates.

Several findings stand out for their practical implications. Culture density, measured as optical density at 600 nanometers, had no effect on outcome as long as the same number of viable bacteria was used, which means researchers should quantify colony-forming units rather than rely on optical measurements alone. The optimal bacterial input fell between 7.5 x 10^8 and 1.5 x 10^9 colony-forming units, with lysozyme at 0.02 to 0.08 milligrams per milliliter. The highest transfection efficiency came from a 40 percent solution of PEG 4000, a lower molecular weight polymer than the PEG 8000 used in the original protocol, combined with 0.2 to 1 microgram of phage DNA delivered in a volume not exceeding ten percent of the protoplast suspension.

The payoff was substantial. Under the optimized conditions, the yield of infectious TP-84 particles increased approximately 130-fold compared with the team’s earlier protocol, which had already been used to construct the first thermophilic phage display system. Importantly, the procedure was not limited to the original host strain. The researchers successfully applied it to Parageobacillus genomosp. 1 NUB3621, a related thermophile, confirming that protoplasts from that strain could also be formed and transfected, although they caution that strain-specific optimization will likely be required when adapting the method to other bacteria.

Because the goal of the procedure is to reboot infectious phage particles rather than to generate bacterial transformants, conventional transformation efficiency metrics do not apply. Instead, performance was assessed by the reproducible recovery of plaques following transfection, a direct readout of successful genome delivery and viral replication. The final protocol is streamlined for daily laboratory use: bacterial pellets are frozen without cryoprotectant and stored at minus 80 degrees Celsius, then thawed, converted to protoplasts with lysozyme, washed, mixed with phage DNA and PEG, and incubated at 55 degrees Celsius before plating with fresh host cells in soft agar. Plaques appear overnight, giving researchers a rapid visual confirmation that the engineered genome has launched a complete viral replication and assembly program.

Downstream of genome rebooting, the team developed a scaled-up solid-state propagation method that yields enough phage material for genomic DNA isolation and sequencing from a single square Petri dish. Building on older observations that phage assays and confluent lysis work on plates without a bottom agar layer, the researchers optimized bacterial input, multiplicity of infection, agar volume, and recovery conditions. Using a concentrated host culture and a multiplicity of infection of 0.3, approximately 1.5 x 10^10 bacteria could be efficiently infected on one plate, producing roughly 60 milliliters of lysate at a titer of 5 x 10^10 plaque-forming units per milliliter, or about 3 x 10^12 infectious particles in total. Notably, the authors report that solid-state propagation preserved the genetic stability of recombinant TP-84 variants better than liquid culture, an important consideration when maintaining engineered phages over multiple generations.

The final component of the workflow is a modified DNA isolation protocol tailored to agar-extracted lysates. Phage particles are precipitated with PEG 8000 and sodium chloride, treated with DNase and RNase to digest contaminating host nucleic acids, stripped of protein with SDS and proteinase K, and purified through a silica column. The method routinely delivers 5 to 20 micrograms of purified TP-84 genomic DNA, verified to be free of infectious phage particles by plaque assays. That quantity is sufficient for DNA sequencing and for a second round of protoplast transfection, closing the engineering loop and enabling iterative cycles of genome design, rebooting, propagation, and analysis within days rather than weeks.

The broader significance of this work extends beyond TP-84 itself. The team has already used the platform to generate 30 stable recombinant phage variants, inserting a functional superfolder green fluorescent protein gene at multiple positions in the TP-84 genome and engineering selected genes with His-tags and S-tags, work that underpins a thermostable phage display system with potential applications in vaccine development, biological drug production, plant protection, and pollutant removal. Unlike approaches that depend on intact phage particles to deliver genetic material, protoplast transfection accepts genomes assembled entirely in vitro, including fully synthetic constructs designed on a computer. The authors position their protocol as an experimentally validated framework rather than a universal recipe: cell-wall architecture, lysozyme susceptibility, osmotic stability, PEG sensitivity, and host defense systems all vary among bacteria, and each new phage-host pairing will demand its own tuning. Still, for a field in which phages infecting Gram-positive thermophiles have long been genetically intractable, the demonstration that complete viral genomes can be routinely introduced, rebooted, propagated, and recovered from Geobacillus protoplasts marks a meaningful expansion of the synthetic biology toolkit for the viral world at high temperatures.

Subject of Research: Protoplast-mediated transfection and genome engineering of the thermophilic bacteriophage TP-84 in Geobacillus stearothermophilus

Article Title: An Integrated System for Geobacillus Protoplast Formation, Transfection, Recombinant Genomes Rebooting, Efficient Propagation, and Genomic DNA Isolation of the Thermophilic Bacteriophage TP‐84

Article References: Sobolewski, I., Adamowicz, K., Chodorski, J., Skowron, P. M., & Zylicz‐Stachula, A. (2026). An Integrated System for Geobacillus Protoplast Formation, Transfection, Recombinant Genomes Rebooting, Efficient Propagation, and Genomic DNA Isolation of the Thermophilic Bacteriophage TP‐84. MicrobiologyOpen, 15(5), Article e70421. https://doi.org/10.1002/mbo3.70421

Image Credits: AI Generated

DOI: 10.1002/mbo3.70421

Keywords: bacteriophage TP-84, Geobacillus stearothermophilus, protoplast transfection, thermophilic phage, phage genome rebooting, PEG-mediated DNA uptake, phage display, solid-state propagation, genomic DNA isolation, Parageobacillus, synthetic biology, Gram-positive bacteria

The research, led by Jinfeng Qi and Jianqiang Wu of the Kunming Institute of Botany at the Chinese Academy of Sciences, addresses a stubborn bottleneck in plant functional genomics. Terpene synthases, or TPSs, are the enzymes that convert linear prenyl diphosphate substrates into the enormous diversity of terpenoid structures found across the plant kingdom. The traditional approach to determining what a given TPS produces involves expressing the gene in bacteria such as Escherichia coli and analyzing the products. But bacterial systems come with well-known liabilities: they cannot perform the eukaryotic post-translational modifications that may be essential for enzyme activity, high-level expression often drives misfolded proteins into insoluble inclusion bodies, and the prokaryotic cellular environment lacks the chaperones and folding machinery needed to keep some plant enzymes soluble and active. Generating stable transgenic maize lines to bypass these problems is expensive and slow, given the crop’s recalcitrance to transformation.

Protoplasts—plant cells stripped of their walls by enzymatic digestion—offer an attractive alternative. Exogenous DNA can be delivered into protoplasts by polyethylene glycol-mediated transfection, driving transient expression of genes of interest within 24 to 48 hours. Maize protoplasts have already proven useful for promoter analysis, protein interaction studies, and investigating nonvolatile defensive metabolites such as benzoxazinoids. But nobody had shown whether they could support the study of volatile terpene biosynthesis, which poses a distinct analytical challenge: the products evaporate, making detection and quantification far more delicate than measuring water-soluble compounds. The new work demonstrates that, with the right combination of growth conditions, culture parameters, and extraction chemistry, maize protoplasts can indeed become miniature terpene factories whose output is readily captured and measured.

The first obstacle the team faced was a genuine biological paradox. Protocols for isolating stable maize protoplasts typically call for etiolated seedlings grown in complete darkness, because the large central vacuoles characteristic of skotomorphogenic cells maintain high turgor pressure and help the naked cells survive the mechanical stresses of centrifugation and pipetting. However, light is a critical environmental cue for terpenoid metabolism: it drives plastid development and the accumulation of farnesyl diphosphate, the C15 precursor of sesquiterpenes. When the researchers compared seedlings grown in darkness with those given one, two, three, or eight days of dim light at 4.25 micromoles per square meter per second, they found that the partially greened seedlings accumulated dramatically more precursor and product. After methyl jasmonate treatment, the greenish seedlings released 2.7-fold more (E)-α-bergamotene and 3.8-fold more (E)-β-farnesene than etiolated controls, and farnesyl diphosphate levels rose up to 5.9-fold with eight days of light exposure. The compromise—eight days of dim light—balanced cellular robustness with metabolic competence, resolving what the authors call the light paradox.

Leaf anatomy added a second layer of optimization. The maize leaf is a developmental gradient running from the division-active basal meristem to the fully mature tip. Protoplasts isolated from the base and middle of the second leaf showed excellent integrity and transfection efficiencies above 90 percent with an enhanced green fluorescent protein reporter, but a substantial fraction of protoplasts from the leaf tips ruptured after transfection. The likely explanation lies in cellular architecture: in mature tip cells, the central vacuole has fragmented into smaller vacuoles and the cytoplasm is packed with chloroplasts, undermining osmotic stability and mechanical strength. The base and middle regions of greenish seedling leaves were therefore selected for all subsequent experiments, underscoring how developmental stage alone can make or break a protoplast protocol.

Detecting the volatile products required its own series of refinements. The team compared direct overnight headspace adsorption with solid-phase microextraction against a strategy in which protoplasts are first lysed and extracted with an organic solvent. Extraction with n-hexane or n-pentane vastly outperformed direct adsorption, and n-pentane was chosen for its lower toxicity and greater volatility. The final workflow concentrates the extract, heats it to 65 degrees Celsius to drive volatiles into the headspace, and captures them on a polydimethylsiloxane solid-phase microextraction fiber before gas chromatographic analysis on a Shimadzu GC-2014 system. Culture conditions mattered as well: a nutrient-rich medium supplemented with sucrose, glucose, and Murashige and Skoog salts supported higher terpene accumulation than the standard W5 solution, sealed containers and darkness outperformed light and open vessels, and larger culture volumes helped—in 50-milliliter headspace tubes, (E)-β-farnesene levels ran 60 percent higher than in 2-milliliter tubes.

Promoter choice and cofactor supply further sharpened the system’s sensitivity. When the maize gene ZmTPS10 was expressed from the maize ubiquitin promoter, protoplasts accumulated more (E)-α-bergamotene and (E)-β-farnesene than when the cauliflower mosaic virus 35S promoter drove the same gene. Spraying the mother plants with methyl jasmonate eight hours before protoplast isolation—an approach the authors describe as in planta priming—boosted terpene accumulation in subsequently transfected protoplasts more than tenfold compared with untreated controls, exploiting the intact plant’s metabolic machinery to enrich precursors rather than adding expensive, hydrolysis-prone farnesyl diphosphate directly to the medium. Because all terpene synthases carry a conserved DDxxD domain that binds magnesium, the researchers also tested whether adding 10 millimolar magnesium ions to the culture medium would help. It did: bergamotene rose 2.7-fold and farnesene 2.0-fold, confirming that cofactor availability limits ZmTPS10 activity inside the transfected cells.

To show that the platform extends beyond single-enzyme biochemistry, the team used it to interrogate transcriptional regulation. A previous study had identified ZmMYC2a, a basic helix-loop-helix transcription factor in the jasmonate signaling pathway, as a positive regulator of (E)-α-bergamotene and (E)-β-farnesene biosynthesis. Overexpressing ZmMYC2a in maize protoplasts significantly elevated both volatiles relative to a green fluorescent protein control, demonstrating that the system can resolve regulatory layers as well as catalytic function. The authors argue that the high efficiency of multi-plasmid co-transfection opens the door to reconstructing entire defense signaling cascades—receptor to kinase to transcription factor to structural gene—inside a single batch of cells. Because maize protoplasts provide a homologous cellular environment with the correct cofactors, membranes, and post-translational modification machinery, the resulting data should carry higher physiological fidelity than results from heterologous bacterial or yeast systems.

The implications reach well beyond one crop. Graminaceous staples such as wheat and sorghum share maize’s stubborn resistance to genetic transformation, and a rapid protoplast-based assay could serve as an efficient shortcut for functional gene studies across these species, with potential extension to other non-model plants through optimization of osmotic regulators, enzyme cocktails, and ionic conditions. Ecologically, the stakes are considerable: maize terpene volatiles recruit parasitoid wasps against fall armyworm and other pests above ground, attract entomopathogenic nematodes against the western corn rootworm below ground, and even directly damage the peritrophic matrix of lepidopteran midguts. By shrinking the timeline for assigning function to the roughly 30 TPS genes in the maize genome—about half of which still lack confirmed products—this system promises to accelerate the discovery of defensive chemistry that could inform breeding programs and crop synthetic biology for years to come.

Subject of Research: A maize protoplast transfection system for analyzing the biosynthesis and regulation of volatile terpenoid defense compounds

Article Title: A maize protoplast transfection system for studying the biosynthesis of volatile terpenoids

Article References: Qi, J., Li, M., Hu, Z., Li, R., Li, J., Zhang, M., Ma, C., & Wu, J. (2026). A maize protoplast transfection system for studying the biosynthesis of volatile terpenoids. Crop Health, 4(1), Article 13. https://doi.org/10.1007/s44297-026-00076-5

Image Credits: AI Generated

DOI: 10.1007/s44297-026-00076-5

Keywords: maize, protoplast transfection, terpene synthases, volatile terpenoids, ZmTPS10, methyl jasmonate, plant defense, ZmMYC2a, solid-phase microextraction, secondary metabolism, crop biotechnology, protoplast