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

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.

An in-depth characterization of the entomopathogenic strain Bacillus pumilus 15.1 reveals that it produces inclusion bodies similar to the parasporal crystals of Bacillus thuringiensis.

In the present work, the local isolate Bacillus pumilus 15.1 has been morphologically and biochemically characterized in order to gain a better understanding of this novel entomopathogenic strain active against Ceratitis capitata. This strain could represent an interesting biothechnological tool for the control of this pest. Here, we report on its nutrient preferences, extracellular enzyme production, motility mechanism, biofilm production, antibiotic suceptibility, natural resistance to chemical and physical insults, and morphology of the vegetative cells and spores. The pathogen was found to be ß-hemolytic and susceptible to penicillin, ampicillin, chloramphenicol, gentamicin, kanamycin, rifampicin, tetracycline, and streptomycin. We also report a series of biocide, thermal, and UV treatments that reduce the viability of B. pumilus 15.1 by several orders of magnitude. Heat and chemical treatments kill at least 99.9 % of vegetative cells, but spores were much more resistant. Bleach was the only chemical that was able to completely eliminate B. pumilus 15.1 spores. Compared to the B. subtilis 168 spores, B. pumilus 15.1 spores were between 2.67 and 350 times more resistant to UV radiation while the vegetative cells of B. pumilus 15.1 were almost up to 3 orders of magnitude more resistant than the model strain. We performed electron microscopy for morphological characterization, and we observed geometric structures resembling the parasporal crystal inclusions synthesized by Bacillus thuringiensis. Some of the results obtained here such as the parasporal inclusion bodies produced by B. pumilus 15.1 could potentially represent virulence factors of this novel and potentially interesting strain.

Draft Genome Sequence of the Entomopathogenic Bacterium Bacillus pumilus 15.1, a Strain Highly Toxic to the Mediterranean Fruit Fly Ceratitis capitata.

We present the draft whole-genome sequence of the entomopathogenic Bacillus pumilus 15.1 strain that consists of 3,795,691 bp and 3,776 predicted protein-coding genes. This genome sequence provides the basis for understanding the potential mechanism behind the toxicity and virulence of B. pumilus 15.1 against the Mediterranean fruit fly.

Identification, sequencing and comparative analysis of pBp15.S plasmid from the newly described entomopathogen Bacillus pumilus 15.1.

The Bacillus pumilus 15.1 strain, a recently described entomopathogenic strain active against Ceratitis capitata, contains at least two extrachromosomal elements, pBp15.1S and pBp15.1B. Given that B. pumilus is not a typical entomopathogenic bacterium, the acquisition of this extrachromosomal DNA may explain why B. pumilus 15.1 is toxic to an insect. One of the plasmids present in the strain, the pBp15.1S plasmid, was sub-cloned, sequenced and analyzed using bioinformatics to identify any potential virulence factor. The pBp15.1S plasmid was found to be 7785 bp in size with a GC content of 35.7% and 11 putative ORFs. A replication module typical of a small rolling circle plasmid and a sensing and regulatory system specific for plasmids was found in pBp15.1S. Additionally, we demonstrated the existence of ssDNA in plasmid preparations suggesting that pBp15.1S replicates by the small rolling circle mechanism. A gene cluster present in plasmid pPZZ84 from a distantly isolated B. pumilus strain was also present in pBp15.1S. The plasmid copy number of pBp15.1S in exponentially growing B. pumilus cells was determined to be 33 copies per chromosome. After an extensive plasmid characterization, no known virulence factor was found so a search in the other extrachromosomal elements of the bacteria is needed.