Bacillus toyonensis biovar Thuringiensis: an overlooked entomopathogen?

This paper highlights the emerging recognition of *Bacillus toyonensis* biovar *Thuringiensis* as an overlooked entomopathogen capable of producing insecticidal parasporal crystals via horizontally transferred megaplasmids, underscoring the necessity for genome-based taxonomic reassessment within the *Bacillus cereus* group to correct historical misclassifications.

The Gist

Imagine the bacterial world as a massive, bustling city called the Bacillus neighborhood. For a long time, scientists thought they had a very clear map of this city. They knew that one specific family, the Bacillus thuringiensis (let's call them the "Bug-Busters"), were the only ones who carried a special weapon: a crystal-shaped toxin that could kill insect pests. They were the famous pest control agents of the agricultural world.

Then, there was another family in the neighborhood called Bacillus toyonensis. They were known as the "Friendly Neighbors." For decades, they were used as probiotics (good bacteria) in animal feed to help pigs, chickens, and cows stay healthy. Nobody thought they had any weapons; they were just the nice guys.

The Big Discovery: The "Friendly Neighbor" with a Secret Weapon

This paper is like a detective story where scientists realized that some of the "Friendly Neighbors" have been hiding a secret.

1. The Mix-Up: Scientists found some bacteria that looked and acted exactly like the famous "Bug-Busters." They had the same crystal weapons and killed insects just as well. But when they looked at the bacteria's "ID card" (their DNA), they were shocked. These weren't Bacillus thuringiensis at all. They were actually Bacillus toyonensis!
2. The New Name: Because these bacteria are genetically toyonensis but act like thuringiensis, the authors suggest a new name: Bacillus toyonensis biovar Thuringiensis. Think of it like finding a Golden Retriever that has learned to herd sheep like a Border Collie. It's still a Golden Retriever by blood, but it has the "herding" trait.

How Did This Happen? The "Bacterial File Transfer"

You might wonder, "How did the friendly bacteria get the bug-killing weapon?"

The answer is Horizontal Gene Transfer (HGT). Imagine bacteria as people in a city who can swap USB drives.

• Usually, bacteria pass traits to their children (vertical transfer).
• But in this case, a "Friendly Neighbor" (B. toyonensis) picked up a USB drive (a plasmid) from a "Bug-Buster" (B. thuringiensis).
• This USB drive contained the blueprints for the crystal weapon.
• Once the friendly bacteria plugged in that USB drive, it suddenly had the ability to kill insects, even though its main body (chromosome) was still a friendly toyonensis.

The "Imposter" Problem

The paper also points out that the opposite has happened. Some bacteria that were labeled as "Bug-Busters" in scientific databases are actually just "Friendly Neighbors" who picked up the weapon.

• Strain MC28 and Strain Rock 1-3 were thought to be B. thuringiensis.
• But when scientists ran a high-tech DNA check (like a super-accurate fingerprint scanner), they realized these were actually B. toyonensis that had stolen the weapon.
• The authors are saying, "We need to update our address books! These aren't Bug-Busters; they are Friendly Neighbors with a secret arsenal."

Why Does This Matter?

1. Better Pest Control: If we know that "Friendly Neighbors" can also kill pests, we have a whole new group of bacteria to use for organic farming. We don't have to rely only on the traditional Bug-Busters.
2. Accurate Science: For a long time, scientists classified bacteria just by what they looked like (did they have crystals?). This paper says, "Stop looking at the costume; look at the DNA." We need to reclassify these bacteria so our maps are correct.
3. Evolution in Action: It shows how fast bacteria can change. They don't just evolve slowly over millions of years; they can swap tools and weapons instantly, turning a harmless probiotic into a powerful insect killer overnight.

In a Nutshell

This paper is a wake-up call for scientists. It reveals that the line between "harmless probiotic" and "deadly insect killer" is blurrier than we thought. Through a process of swapping genetic "USB drives," the friendly Bacillus toyonensis has quietly become a major player in insect control, and it's time we gave it the credit (and the new name) it deserves.

Technical Summary

1. Problem Statement

The Bacillus cereus group comprises closely related species, including Bacillus cereus, Bacillus thuringiensis, and Bacillus toyonensis. Traditionally, B. thuringiensis has been distinguished from other members of the group by its ability to produce parasporal crystals (containing Cry and Cyt $\delta$-endotoxins) during sporulation, which confer insecticidal properties. However, recent genomic advancements reveal that B. thuringiensis and B. cereus are nearly identical at the chromosomal level, with the insecticidal traits often encoded on mobile megaplasmids rather than the core chromosome.

The central problem addressed in this study is the taxonomic misclassification of certain bacterial strains. Specifically, strains that possess the phenotypic traits of B. thuringiensis (parasporal crystals and insecticidal activity) but are genomically classified as B. toyonensis (or previously misidentified as B. thuringiensis despite being B. toyonensis) have been overlooked. The authors argue that current phenotype-based identification is insufficient and that the emergence of "B. thuringiensis-like" phenotypes in B. toyonensis via Horizontal Gene Transfer (HGT) necessitates a genome-based taxonomic reassessment.

2. Methodology

The study employed a combination of microbiological isolation, genomic sequencing, and bioinformatic analysis:

• Sample Collection & Isolation: Soil samples were collected from Jujuy and Córdoba provinces in Argentina. Spore-forming bacteria were isolated using heat shock (80°C for 30 min) to select for spores, followed by plating on nutrient agar.
• Phenotypic Characterization:
  • Colonies resembling B. thuringiensis (matte-white, irregular margins) were selected.
  • Sporulation was induced, and cells were stained with Coomassie Blue to visualize parasporal crystals under light microscopy.
  • Scanning Electron Microscopy (SEM) confirmed the presence of crystals.
• Genomic Sequencing & Assembly:
  • Genomic DNA (chromosomal + plasmid) was extracted from strains Bto_UNVM-42, Bto_UNVM-100, and previously studied Bto_UNVM-94.
  • Illumina high-throughput sequencing was performed.
  • Reads were trimmed and assembled into contigs using the Velvet algorithm (Geneious R11).
• Bioinformatic Analysis:
  • Annotation: Performed using NCBI Prokaryotic Genome Annotation Pipeline and RAST.
  • Gene Search: Contigs were analyzed via BLAST against a custom database of insecticidal proteins.
  • Taxonomic Delimitation: Species identification was conducted using:
    • TYGS (Type (Strain) Genome Server): For genome-genome hybridization analysis.
    • FastANI: To calculate Average Nucleotide Identity (ANI).
    • PubMLST: For Multi-Locus Sequence Typing (MLST) support.
  • Comparative Genomics: The study compared novel isolates against reference strains, including B. thuringiensis MC28, B. cereus Rock 1-3, and the B. toyonensis type strain P18.

3. Key Contributions

• Proposal of New Nomenclature: The authors propose the designation "Bacillus toyonensis biovar Thuringiensis" for B. toyonensis strains that have acquired insecticidal crystal-forming capabilities. This aligns with the nomenclature system proposed by Carroll et al. (2020).
• Identification of Misclassified Strains: The study identifies specific strains in public databases (GenBank/RefSeq) that are likely misclassified:
  • B. thuringiensis MC28 and B. cereus Rock 1-3 are shown to cluster phylogenetically with B. toyonensis rather than their current taxonomic assignments.
  • Novel isolates Bto_UNVM-42 and Bto_UNVM-100 are confirmed as B. toyonensis but possess B. thuringiensis-like traits.
• Hypothesis on Evolutionary Mechanism: The paper provides strong evidence that Horizontal Gene Transfer (HGT) is the primary driver for the acquisition of insecticidal genes (e.g., cry, cyt, vip, tpp) by B. toyonensis. These genes are located on mobile genetic elements (plasmids, insertion sequences), allowing them to transfer across species boundaries within the B. cereus group.

4. Results

• Phenotypic Confirmation: Strains Bto_UNVM-42 and Bto_UNVM-100 were confirmed to produce parasporal crystals and harbor genes encoding insecticidal proteins (Cry, Cyt, and others) toxic to lepidopteran and dipteran insects.
• Genomic Reclassification:
  • ANI Analysis: Strains Bto_UNVM-42 and Bto_UNVM-100 showed >98% ANI with the B. toyonensis type strain P18, confirming their species identity despite their B. thuringiensis-like phenotype.
  • Phylogenetic Clustering: Genome-based phylogenetic trees placed strains MC28 and Rock 1-3 within the B. toyonensis clade, suggesting they should be reclassified as B. toyonensis biovar Thuringiensis.
• Gene Content: The study confirmed that these B. toyonensis strains harbor a diverse repertoire of pesticidal genes, including cry, cyt, vpb, app, mpp, and tpp genes.
• Re-evaluation of Strain GM18: The study suggests that strain GM18, previously identified as B. toyonensis, should also be designated as B. toyonensis biovar Thuringiensis due to the presence of parasporal inclusions and cytotoxic activity against human cancer cells, despite earlier reports of a lack of insecticidal activity.

5. Significance

• Taxonomic Resolution: The findings underscore the limitations of phenotype-based taxonomy in the B. cereus group. The study advocates for a shift toward genome-based taxonomy to accurately define species boundaries, acknowledging that accessory gene content (plasmids) can drastically alter phenotype without changing the core chromosomal species identity.
• Biotechnological Potential: By identifying B. toyonensis as a reservoir for insecticidal genes, the study expands the potential pool of biological control agents. These strains could offer new avenues for pest management and potentially novel biopesticides.
• Evolutionary Insight: The research highlights the dynamic nature of the B. cereus group, where HGT facilitates rapid adaptation and the emergence of new pathogenic or entomopathogenic traits across different species.
• Database Accuracy: The study calls for the correction of public genomic databases (e.g., GenBank) to reflect the true taxonomic status of these strains, preventing future misidentification in research and commercial applications.

In conclusion, the paper demonstrates that Bacillus toyonensis is an overlooked entomopathogen capable of mimicking B. thuringiensis through the acquisition of mobile insecticidal gene clusters, necessitating a revision of current classification frameworks to include the "biovar Thuringiensis" designation.