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Updated: Feb 20, 2026
Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG
Published on: May 16, 2017
The parasporal crystals of Bacillus pumilus strain 15.1: a potential virulence factor?
Diana C Garcia-Ramon1, Colin Berry2, Carmen Tse2
1Institute of Biotechnology, Campus Fuentenueva, University of Granada, Granada, Spain.
Abstract:
Bacillus pumilus strain 15.1 was previously found to cause larval mortality in the Med-fly Ceratitis capitata and was shown to produce crystals in association with the spore. As parasporal crystals are well-known as invertebrate-active toxins in entomopathogenic bacteria such as Bacillus thuringiensis (Cry and Cyt toxins) and Lysinibacillus sphaericus (Bin and Cry toxins), the B. pumilus crystals were characterized. The crystals were composed of a 45 kDa protein that was identified as an oxalate decarboxylase by peptide mass fingerprinting, N-terminal sequencing and by comparison with the genome sequence of strain 15.1. Synthesis of crystals by a plasmid-cured derivative of strain 15.1 (produced using a novel curing strategy), demonstrated that the oxalate decarboxylase was encoded chromosomally. Crystals spontaneously solubilized when kept at low temperatures, and the protein produced was resistant to trypsin treatment. The insoluble crystals produced by B. pumilus 15.1 did not show significant toxicity when bioassayed against C. capitata larvae, but once the OxdD protein was solubilized, an increase of toxicity was observed. We also demonstrate that the OxdD present in the crystals has oxalate decarboxylate activity as the formation of formate was detected, which suggests a possible mechanism for B. pumilus 15.1 activity. To our knowledge, the characterization of the B. pumilus crystals as oxalate decarboxylase is the first report of the natural production of parasporal inclusions of an enzyme.
Insights
Bacillus pumilus strain 15.1 produces unique parasporal crystals. These crystals contain oxalate decarboxylase (OxdD), an enzyme whose solubilized form shows toxicity to Med-fly larvae.
Area of Science:
- Microbiology
- Biochemistry
- Insect Pathology
Background:
- Bacillus pumilus strain 15.1 induces mortality in Med-fly (Ceratitis capitata) larvae.
- Parasporal crystals from entomopathogenic bacteria often contain insecticidal toxins.
- The protein composition of B. pumilus 15.1 crystals was previously uncharacterized.
Purpose of the Study:
- To characterize the parasporal crystals produced by Bacillus pumilus strain 15.1.
- To identify the protein component of the crystals and determine its function.
- To investigate the potential insecticidal activity of the crystal protein.
Main Methods:
- Peptide mass fingerprinting and N-terminal sequencing were used to identify the crystal protein.
- Genome sequencing of B. pumilus strain 15.1 was performed for comparison.
- A plasmid-cured derivative was used to confirm chromosomal encoding of the protein.
- Bioassays were conducted against C. capitata larvae with both insoluble crystals and solubilized protein.
Main Results:
- The crystals were composed of a 45 kDa protein identified as oxalate decarboxylase (OxdD).
- The oxalate decarboxylase gene is chromosomally encoded.
- Solubilized OxdD showed increased toxicity to C. capitata larvae compared to insoluble crystals.
- OxdD exhibited oxalate decarboxylase activity, producing formate.
Conclusions:
- Bacillus pumilus 15.1 naturally produces parasporal inclusions of oxalate decarboxylase (OxdD).
- Solubilized OxdD exhibits toxicity towards Med-fly larvae, suggesting a novel mechanism for bacterial insecticidal activity.
- This is the first report of parasporal enzyme production by Bacillus pumilus.
