Jean-Charles Côté

Discrimination among Bacillus thuringiensis H serotypes, serovars and strains based on 16S rRNA, gyrB and aroE gene sequence analyses.

Our aim was to investigate the capability of each of three genes, 16S rRNA, gyrB and aroE, to discriminate, first, among Bacillus thuringiensis H serotypes; second, among B. thuringiensis serovars from the same H serotype; and third, among B. thuringiensis strains from the same serovar. The 16S rRNA, gyrB and aroE genes were amplified from 21 B. thuringiensis H serotypes and their nucleotide sequences determined. Additional strains from four B. cereus sensu lato species were included for comparison purposes. These sequences were pair-wise compared and phylogenetic relationships were revealed. Each of the three genes under study could discriminate among B. thuringiensis H serotypes. The gyrB and aroE genes showed a discriminatory power among B. thuringiensis H serotypes up to nine fold greater than that of the 16S rRNA gene. The gyrB gene was retained for subsequent analyses to discriminate B. thuringiensis serovars from the same H serotype and to discriminate strains from same serovar. A total of 42 B. thuringiensis strains, which encompassed 25 serovars from 12 H serotypes, were analyzed. The gyrB gene nucleotide sequences were different enough as to be sufficient to discriminate among B. thuringiensis serovars from the same H serotype and among B. thuringiensis strains from the same serovar.

Isolation and characterization of a novel Bacillus thuringiensis strain expressing a novel crystal protein with cytocidal activity against human cancer cells

Aims:  To characterize a novel, unusual, Bacillus thuringiensis strain, to clone its cry gene and determine the spectrum of action of the encoded Cry protein.

Pathogenicity of six isolates of Beauveria bassiana (Balsamo) Vuillemin (Deuteromycotina, Hyphomycetes) to Perillus bioculatus (F) (Hem., Pentatomidae)

Abstract:  The two‐spotted stink bug, Perillus bioculatus (F) (Hem., Pentatomidae) is considered an important predator of Colorado potato beetle egg masses and small larvae. The susceptibility of P. bioculatus second instar nymphs to six isolates of Beauveria bassiana (Balsamo) Vuillemin, which are highly pathogenic to Colorado potato beetle, was tested in the laboratory. Five isolates were highly pathogenic and caused more than 77% mortality after 8 days at a concentration of 106 conidia/ml. However, isolate IPP46 showed low pathogenicity and caused only 11% mortality of nymphs. The pathogenicity of isolates was independent of their host, source or country of origin. Isolate IPP46 was tested at six different concentrations. A linear relationship between the concentration of B. bassiana and the mortality of nymphs was observed. The 50% lethal concentration (LC50) value was 3.4 × 107 conidia/ml. The LT50 decreased with increase of conidia concentration used. The present results suggest that isolate IPP46 has good potential as a biological control agent within an integrated pest management programme.

Bacillus thuringiensis Serovars bolivia, vazensis and navarrensis Meet the Description of Bacillus weihenstephanensis

The Bacilluscereus sensu lato group comprises six related species: B. cereus, B. anthracis, B. thuringiensis, B. mycoides, B. pseudomycoides and B. weihenstephanensis. Bacillus thuringiensis is a mesophile. It is distinguished from other members of the B. cereus group by the apparition of an inclusion body upon sporulation. B. weihenstephanensis, however, is a psychrotolerant. It grows at 7°C but not at 43°C. It is further characterised by the presence of specific signature sequences on two genes, the 16S rRNA gene (the small subunit ribosomal RNA gene) and cspA (encoding the major cold shock protein). Five B. thuringiensis serovars selected from previous studies, bolivia, vazensis, navarrensis, azorensis and asturiensis were studied here for their capability to grow at 7°C but not at 43°C. Next, their 16S rRNA and cspA genes were analysed for the presence of B. weihenstephanensis-specific signature sequences. Bacillus thuringiensis serovars bolivia, vazensis and navarrensis met the description of B. weihenstephanensis.

Parasporin-2 from a New Bacillus thuringiensis 4R2 Strain Induces Caspases Activation and Apoptosis in Human Cancer Cells.

In previous studies, parasporin-2Aa1, originally isolated from Bacillus thuringiensis strain A1547, was shown to be cytotoxic against specific human cancer cells but the mechanisms of action were not studied. In the present study, we found that proteinase K activated parasporin-2Aa1 protein isolated from a novel B. thuringiensis strain, 4R2, was specifically cytotoxic to endometrial, colon, liver, cervix, breast and prostate cancer. It showed no toxicity against normal cells. Upon treatment with proteinase K-activated parasporin-2Aa1, morphological changes were observed and western blot analysis revealed the cleavage of poly (ADP-Ribose) polymerase, caspase-3 and caspase-9 in cancer cell lines exclusively, indicative of programmed cell death, apoptosis. Flow cytometry analyses,using propidium iodide and annexin V, as well as a caspases 3/7 assay confirmed apoptosis induction. Further analyses were performed to study survival pathways, including AKT, XIAP, ERK1/2 and PAR-4, a known inducer of apoptosis. These results indicate that parasporin-2Aa1 is a selective cytotoxic protein that induces apoptosis in various human cancer cell lines from diverse tissues.

Bacillus weihenstephanensis characteristics are present in Bacillus cereus and Bacillus mycoides strains.

The Bacillus cereus group comprises seven bacterial species: Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus cytotoxicus, and Bacillus weihenstephanensis. Bacillus weihenstephanensis is distinguished based on its capability to grow at 7 °C but not at 43 °C, and the presence of specific signature sequences in the 16S rRNA and cspA genes and in several housekeeping genes: glpF, gmK, purH, and tpi. Bacillus weihenstephanensis-specific signature sequences were found in some B. cereus and B. mycoides strains suggesting psychrotolerance. This was confirmed by growth at 7 °C but not at 43 °C. The other B. cereus and B. mycoides strains and all B. anthracis, B. thuringiensis, and B. pseudomycoides harbored the mesophilic signature sequences. The strains tested grew at 43 °C but did not grow at 7 °C. A maximum-likelihood phylogenetic tree was inferred from comparisons of the concatenated nucleotide sequences. Three groups and one branch were revealed. Group I, II, and III comprised the mesophilic B. cereus, some mesophilic B. mycoides, and all B. anthracis and B. thuringiensis strains; the psychrotolerant B. cereus and B. mycoides, and all B. weihenstephanensis strains; and some mesophilic B. mycoides and all B. pseudomycoides strains, respectively. The branch corresponds to the single B. cytotoxicus strain. Based on psychrotolerance and multilocus sequence analysis, further confirmed by comparisons of amino acid sequences, we show that some B. cereus and B. mycoides strains should be reclassified as B. weihenstephanensis.

Multilocus sequence analysis of Bacillus thuringiensis serovars navarrensis, bolivia and vazensis and Bacillus weihenstephanensis reveals a common phylogeny

The Bacilluscereus group sensu lato includes six closely-related bacterial species: Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides and Bacillus weihenstephanensis. B. thuringiensis is distinguished from the other species mainly by the appearance of an inclusion body upon sporulation. B. weihenstephanensis is distinguished based on its psychrotolerance and the presence of specific signature sequences in the 16S rRNA gene and cspA genes. A total of seven housekeeping genes (glpF, gmK, ilvD, pta, purH, pycA and tpi) from different B. thuringiensis serovars and B. weihenstephanensis strains were amplified and their nucleotide sequences determined. A maximum likelihood phylogenetic tree was inferred from comparisons of the concatenated sequences. B. thuringiensis serovars navarrensis, bolivia and vazensis clustered not with the other B. thuringiensis serovars but rather with the B. weihenstephanensis strains, indicative of a common phylogeny. In addition, specific signature sequences and single nucleotide polymorphisms common to B. thuringiensis serovars navarrensis, bolivia and vazensis and the B. weihenstephanensis strains, and absent in the other B. thuringiensis serovars, were identified.

Flagellin (FliC) protein sequence diversity among Bacillus thuringiensis does not correlate with H serotype diversity.

In Escherichia coli, the fliC gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Here, the presence of the flagellin fliC gene was studied in 86 Bacillus thuringiensis strains encompassing 67 H serotypes. Nineteen strains from four additional species in the B. cereus sensu lato group, B. cereus, B. anthracis, B. mycoides, and B. weihenstephanensis, were added for comparison purposes. The fliC genes were amplified, cloned and their nucleotide sequences determined and translated into amino acid sequences. A bootstrapped neighbor-joining tree was generated from the alignment of the translated amino acid sequences of the amplicons. Although most B. thuringiensis H serotypes had different flagellin amino acid sequences, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. In addition, although serovars from the same H serotype were sometimes found clustered together, several serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. No correlations could be established between flagellin (FliC) protein sequence diversity among B. thuringiensis H serotypes and H serotype diversity. These suggest that the B. thuringiensisfliC gene does not code for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. In a previous study, the authors have shown that the B. thuringiensishag gene codes for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. It is proposed that the B. thuringiensisfliC gene studied here be renamed and that the so-called hag gene studied before be renamed fliC, both in accordance with the E. coli nomenclature.

Cloning and nucleotide sequence of a new insertion sequence, IS231N, from a non-serotypable strain of Bacillus thuringiensis.

A new IS231 variant, IS231N, has been isolated from an autoagglutinable, non-serotypable strain of B. thuringiensis. IS231N is 1654 bp in length and is delimited by two incomplete 20-bp inverted repeats (IRL and IRR) with two mismatches. No direct repeats (DRs) were found at the right and left borders of IS231N. Surprisingly, IS231N contains three open reading frames (ORFs) that could code for polypeptides of 329 (ORF1), 118 (ORF2), and 17 (ORF3) amino acids, respectively. IS231N lacks the 5th conserved amino acid domain, called C2, owing to the addition of an adenine residue at nucleotide 1319. IS231N shows the highest nucleotide identity (99%) with IS231M, another insertion sequence previously isolated from the same bacterial strain. IS231N, however, shares only 83% amino acid identity with IS231M because of nucleotide substitutions and additions. The ORF1 of IS231N has five fewer amino acids than ORF1 of IS231M. Furthermore, the ORF2-3 putative fusion product in IS231N contains eight fewer amino acids than ORF2 in IS231M. The dendrogram showing the evolutionary relationship between members of the IS231 family and IS231N indicates that IS231N is phylogenetically more closely related to IS231M (83%), followed by IS231F(74%), and is more distant from IS231V and W(46%).

Distribution analysis of IS231-like sequences among Bacillus thuringiensis serovars inferred from restriction fragment length polymorphisms.

The distribution of IS231 has been analyzed in Bacillus thuringiensis serovars. A 723-bp HaeII conserved fragment from IS231M has been used as a probe against EcoRI-digested B. thuringiensis total DNA to yield serovar-specific hybridization profiles. The approach was useful at revealing the extent of distribution of IS231-like sequences between and within strains. Of the 88 B. thuringiensis strains tested, 70 showed hybridization banding patterns that comprised between one and 20 distinct bands. These 70 B. thuringiensis strains were grouped based on banding pattern similarities. Interestingly, intraserovar strains did not necessarily cluster together.