The parasporal crystals of Bacillus pumilus strain 15.1: a potential virulence factor?

摘要

Bacillus pumilus strain 15.1 was previously found to cause larval mortality in the Med-fly\ud27 Ceratitis capitata and was shown to produce crystals in association with the spore. As\ud28 parasporal crystals are well-known as invertebrate-active toxins in entomopathogenic\ud29 bacteria such as Bacillus thuringiensis (Cry and Cyt toxins) and Lysinibacillus sphaericus (Bin\ud30 and Cry toxins), the B. pumilus crystals were characterised. The crystals were composed of a\ud31 45 kDa protein that was identified as an oxalate decarboxylase by peptide mass\ud32 fingerprinting, N-terminal sequencing and by comparison with the genome sequence of strain\ud33 15.1. Synthesis of crystals by a plasmid-cured derivative of strain 15.1 (produced using a\ud34 novel curing strategy), demonstrated that the oxalate decarboxylase was encoded\ud35 chromosomally. Crystals spontaneously solubilized when kept at low temperatures and the\ud36 protein produced was resistant to trypsin treatment. The insoluble crystals produced by\ud37 B. pumilus 15.1 did not show significant toxicity when bioassayed against C. capitata larvae,\ud38 but once the OxdD protein was solubilized, an increase of toxicity was observed. We also\ud39 demonstrate that the OxdD present in the crystals has oxalate decarboxylate activity as the\ud40 formation of formate was detected, which suggests a possible mechanism for B. pumilus 15.1\ud41 activity. To our knowledge, the characterization of the B. pumilus crystals as oxalate\ud42 decarboxylase is the first report of the natural production of parasporal inclusions of an\ud43 enzyme.