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