The work builds on a well-known fact of microbial life: microorganisms in nature rarely live alone. In the human gut, the Human Microbiome Project identified roughly 3,000 bacterial species coexisting in a single ecological niche, and their stability depends on constant chemical conversation. When antibiotics or dietary shifts disturb this balance, cross-feeding structures and nutrient competition shift with them, sometimes with severe health consequences. Understanding and eventually engineering such communication is therefore not just an academic curiosity — it is a prerequisite for designing stable synthetic microbial communities for industry, agriculture, and medicine.

Previous efforts had already pushed the boundaries of artificial cross-species signaling. In 2013, researchers showed that Escherichia coli could be made to talk to Bacillus megaterium using quorum-sensing peptides borrowed from Staphylococcus aureus. More recently, the Dresden group demonstrated communication in the opposite direction: yeast engineered to produce bacterial quorum-sensing peptides CSF and PhrF successfully activated reporter responses in B. subtilis. What remained missing was the reverse channel — bacteria signaling to yeast — and that is precisely what the new study delivers.

The engineering strategy exploited one of the most thoroughly characterized signaling systems in eukaryotic biology: the yeast mating pheromone pathway. Haploid Saccharomyces cerevisiae cells exist as one of two mating types, a or α, and each secretes a small peptide pheromone that binds to a specific receptor on cells of the opposite type. Receptor activation triggers a signaling cascade that induces morphological changes, cell-cycle arrest, and the expression of mating-specific genes. Among these, the FIG1 gene is switched on roughly 100-fold within just 20 minutes of receptor activation — a speed and dynamic range that makes it an ideal handle for synthetic biologists.

To turn B. subtilis into a yeast-language speaker, the team built on a signal peptide toolbox they had developed previously. The codon-optimized α-factor gene was inserted into an evaluation vector, preceded by the secretion signal peptide of the bacterial protein YfkD to route the peptide through B. subtilis’s native Sec secretion system. Expression was placed under the control of P_liaI, a promoter inducible by the antibiotic bacitracin. The final construct was integrated into the amyE locus of the protease-deficient strain WB800N, yielding the producer strain TMB6428. When induced with bacitracin, this strain secreted α-pheromone at concentrations of up to 8 nanomolar — modest by industrial standards, but consistent and robust, and sufficient for the task at hand.

On the receiving end, the researchers used a haploid yeast strain carrying deletions in BAR1 and FAR1. BAR1 encodes an aspartyl protease that degrades and inactivates α-factor, so its removal prevents the secreted signal from being destroyed before it can act. FAR1 encodes a cyclin-dependent kinase inhibitor responsible for the G1 growth arrest that normally follows pheromone exposure; deleting it keeps the sensor cells alive and dividing rather than frozen in mating mode. Into this background, the team integrated a single genomic copy of the fluorescent protein mNeongreen under control of the α-inducible FIG1 promoter. Genomic integration was necessary because B. subtilis cannot grow properly in yeast minimal medium, which would have made plasmid selection by auxotrophy impractical. The choice of mNeongreen over standard GFP was deliberate: originally discovered in the lancelet Branchiostoma lanceolatum, mNeongreen is the brightest green fluorescent protein known, resists photobleaching better than GFP, and matures faster — all advantages when the expected signal is weak.

Getting the two organisms to grow together posed its own challenges. B. subtilis doubles roughly every 30 minutes, while haploid yeast needs about 90 minutes in rich medium, so the team had to balance starting densities carefully. They settled on a ratio of approximately 1:6, with yeast at an OD600 of 0.1 and bacteria at 0.6 — a bias toward the bacterial producer that compensates for the fact that the engineered strain secretes only a single copy of α-factor, whereas yeast’s native MFα gene encodes four tandem copies processed into four separate peptides. The cultivation medium also required compromise: standard LB broth supplemented with 2% glucose satisfied the nutritional needs of both organisms at a shared incubation temperature of 31.5 °C.

The proof of principle came when mid-exponential co-cultures were induced with bacitracin. Within two hours, Western blot analysis detected mNeongreen protein in co-cultures containing the α-factor-producing bacteria, and the signal intensified at four and twenty-two hours as pheromone accumulated. Flow cytometry of 20,000 cells per sample confirmed statistically significant fluorescence in induced co-cultures, while negative controls — uninduced cultures and co-cultures with wild-type B. subtilis W168, which cannot produce α-factor — showed no detectable signal. The positive control, yeast treated with 250 nanomolar synthetic α-factor, produced brighter fluorescence than the co-culture, consistent with the bacterial secretion rate being lower than the bolus concentration used artificially. Under physiological conditions, 100 nanomolar synthetic α-factor is sufficient to induce mating in BAR1-deleted yeast, so the bacterial output, though modest, falls within a biologically meaningful range.

Perhaps the most visually striking evidence came from epifluorescence microscopy. Beyond the green glow of mNeongreen, the microscope revealed yeast cells undergoing shmoo formation — the characteristic pear-shaped morphological change that haploid cells adopt in response to authentic mating pheromone. Shmoo formation appeared about four hours after induction and increased over the following eighteen hours, demonstrating that the α-factor secreted by B. subtilis was not merely recognized at the receptor level but was fully functional, capable of driving the complete physiological differentiation program of yeast mating. The bacterial cells, meanwhile, showed no morphological changes, confirming that the signal flowed in one direction only.

The study also revealed a manageable imperfection: the P_liaI promoter exhibits slight leakiness, producing faint fluorescence in uninduced co-cultures after four and twenty-two hours. The researchers attribute this to known intrinsic transition-state induction of the LiaRS system, and note that the yeast-only positive control did not show this time-dependent increase because synthetic α-factor was added only once at the start rather than continuously. Looking ahead, the team envisions a synthetic sensor-actuator circuit in which engineered B. subtilis cells detect environmental signals — antibiotics, for example — and respond by secreting yeast pheromones that trigger the production of degradation enzymes in yeast. Since engineered S. cerevisiae capable of degrading β-lactam antibiotics in response to α-pheromone has already been described, the two pieces of such a self-regulating loop already exist. With accumulation of degradation end products potentially serving as a built-in negative feedback to shut the circuit down, the prospect of autonomous, self-contained microbial consortia — communicating across kingdoms to clean up pollutants or produce chemicals sustainably — has moved measurably closer to reality.

Subject of Research: Engineered interkingdom pheromone signaling between Bacillus subtilis and Saccharomyces cerevisiae

Article Title: Interkingdom communication 2.0: pheromone-based control between Bacillus subtilis and Saccharomyces cerevisiae

Article References: Interkingdom communication 2.0: pheromone-based control between Bacillus subtilis and Saccharomyces cerevisiae. (n.d.). https://doi.org/10.1007/s00253-026-14069-5

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14069-5

Keywords: interkingdom communication, Bacillus subtilis, Saccharomyces cerevisiae, pheromone signaling, synthetic biology, quorum sensing, mNeongreen, co-culture, FIG1 promoter, microbial consortia, biotechnology, signal transduction