pGIL01, a linear tectiviral plasmid prophage originating from Bacillus thuringiensis serovar israelensis

Abstract

Bacillus thuringiensis serovar israelensis harbours, in addition to several circular plasmids, a small linear molecule of about 15 kb. Sequence analysis of this molecule, named pGIL01, showed the presence of at least 30 ORFs, five of which displayed similarity with proteins involved in phage systems: a B-type family DNA polymerase, a LexA-like repressor, two potential muramidases and a DNA-packaging protein (distantly related to the P9 protein of the tectiviral phage PRD1). Experimental evidence confirmed that pGIL01 indeed corresponds to the linear prophage of a temperate phage. This bacteriophage, named GIL01, produces small turbid plaques and is sensitive to organic solvents, which suggests the presence of lipid components in its capsid. Experiments using proteases and exonucleases also revealed that proteins are linked to the genomes of both pGIL01 prophage and GIL01 phage at their 59 extremities. Altogether, these features are reminiscent of those of phages found in the Tectiviridae family, and more specifically of those of PRD1, a broad-host-range phage of Gram-negative bacteria. Dot-blot hybridization, PFGE, PCR and RFLP analyses also showed the presence of pGIL01 variants in the Bacillus cereus group. INTRODUCTIONBacillus thuringiensis, the most widely used entomopathogenic bacterium, belongs to the Bacillus cereus sensu lato group. This cluster also includes B. cereus sensu stricto, an opportunist organism implicated in food poisoning (Granum & Lund, 1997), Bacillus anthracis, a human and animal pathogen, Bacillus mycoides and Bacillus pseudomycoides, characterized by their rhizoid growth (Nakamura, 1998), and the psychrotolerant Bacillus weihenstephanensis (Lechner et al., 1998). Despite their broad virulence, these bacteria are genetically closely related (their 16S RNA gene sequences share more than 99 % identity) and could be regarded as pertaining to a single species (Ash et al., 1991;Helgason et al., 2000).B. thuringiensis strains produce, during their sporulation, crystal toxins (delta-endotoxins) that are highly toxic to a number of insect larvae belonging to the orders Lepidoptera, Diptera and Coleoptera, but harmless to vertebrates. Classically, the numerous entomopathogenic B. thuringiensis strains, which have their own specific insecticidal activity, have been classified in different serotypes on the basis of their flagellar antigens. B. thuringiensis serovar (sv.) israelensis is active against dipteran species and is therefore one of the bioinsecticides of choice to control black flies and mosquitoes, both important vectors of human and animal diseases (for a recent review, see Glare & O'Callaghan, 2000).B. thuringiensis sv. israelensis strain H14 has been reported to contain at least eight DNA molecules, including three small (5?4, 6?7 and 7?6 kb) and four large (128, 145, 240 and 350 kb) circular plasmids, and one linear molecule (G. Jensen & L. Andrup, unpublished results). The pathogenicity of this strain only depends on the presence of the 128 kb plasmid which encodes the Cry and Cyt...

GIL16, a New Gram-Positive Tectiviral Phage Related to theBacillus thuringiensisGIL01 and theBacillus cereuspBClin15 Elements

“…A dot blot analysis revealed the presence of molecules related to GIL01 in five strains of the B. cereus group (B16, B23, DBT012, BGSC4D14, and Bt5), and a subsequent PCR analysis using GIL01-specific primers showed that these elements possess different PCR patterns (28). Further experiments performed with the linear molecule harbored by strain B16 showed that it actually corresponds to the prophage form of a temperate phage named GIL16.…”

“…GIL16 is able to produce small turbid plaques on B. thuringiensis GBJ002, which is also the native host for GIL01 (28). In order to avoid any contamination by other bacteriophages potentially present in strain B16, we isolated B. thuringiensis GBJ002 lysogens for GIL16 and confirmed the presence of the pGIL16 prophage by PCR.…”

“…A previous distribution analysis of similar prophages among the B. cereus group had unveiled the presence of a GIL01-related molecule in B. thuringiensis strain B16. PCR and restriction analyses performed with this element, named GIL16, indicated that it differs from GIL01 (28). In order to further analyze this isolate, we sequenced GIL16, and we report its sequence in this study.…”

“…GIL01 is a temperate bacteriophage that was isolated from Bacillus thuringiensis serovar Israelensis whose prophage form, designated pGIL01, resides in the host cell as an autonomous linear plasmid without any apparent integration into the host chromosome. It has been suggested that GIL01 belongs to the family Tectiviridae (28), whose members are characterized by the presence of an internal lipid membrane. While tectiviruses infecting gram-negative bacteria have been extensively studied at the genetic level, those infecting gram-positive bacteria remain less well characterized.…”

Occurrence of Bacillus thuringiensis and their phages in Yemen soil

“…In the Middle East, the natural occurrence of B. thuringiensis in soil environments was reported from Egypt [31] and Jordan [39]. One of the efficient producers of biological insecticides is Bt which is used in industrial application by many Government and Companies [46]. However, sensitivity of Bt to lysis by bacteriophages is the most one of complicated to such industry.…”