DNA Relatedness among Bacillus thuringiensis Serovars

Abstract

The genetic relationships of Bacillus cereus and of the Bacillus thuringiensis serovars were assessed from measurements of DNA reassociation. A study of 8 to 10 strains each of 13 of the most commonly encountered serovars revealed that the levels of intragroup DNA relatedness for most serovars ranged from 90 to 100%. In contrast, B. thuringiensis serovars canadensis and kenyae consisted of two DNA relatedness groups, each of which exhibited levels of intragroup relatedness of 80% or higher and levels of intergroup relatedness of 60 to 70%. Analyses of DNA relatedness performed with all of the serovars revealed that the taxa were segregated into 11 phena differentiated from each other at about the 65% level; within each phenon the level of relatedness was 80% or higher. Three phena contained strains belonging to more than one serovar; B. thuringiensis serovars alesti and dendrolimus clustered in phenon 1, serovars aizawai, kurstaki, galleriae, and morrisoni clustered in phenon 7, and serovar darmstadiensis and some strains of serovar kenyae clustered in phenon 11.The levels of DNA relatedness between B. cereus and B. thuringiensis strains ranged between 65 and 70%. My results suggest that many of the B. thuringiensis serovars are genetically distinct but closely related.Bacillus thuringiensis was first isolated by Ishiwata (17) in 1901 from diseased silkworms, the larvae of the moth Bombyx mori (L.). A subsequent isolation was made by Berliner (4) in 1911 from diseased larvae of the Mediterranean flour moth, Anagasta kuehniella (Zeller). Discovery of the biocidal activity on the larvae of many lepidopterids suggested that B. thuringiensis could be used as a biological control agent for protection of crops and forests from insects.A striking morphological and taxonomically distinguishing characteristic of B. thuringiensis is the formation of parasporal crystals. These parasporal bodies consist of crystallized protein which is responsible for the insecticidal activity of the organism (1, 16). The protein is solubilized at the alkaline pHs that occur in the intestines of susceptible insect larvae and is then cleaved by proteases into peptides which produce the toxic effects (29). Crystal formation is not a stable characteristic (6,30). Synthesis of the paracrystalline protoxin is controlled by large plasmids (11,12,30), the maintenance of which is sensitive to frequency of transfer (6) and incubation conditions, such as temperature (30). Interestingly, to some researchers, strains of B. thurine'ensis are virtually indistinguishable phenotypically from Bacillus cereus except for crystal formation (5, 13, 26), but other workers have observed distinct differences between the two taxa (8, 15).de Barjac and Bonnefoi (7) noted that B. thuringz*ensis strains could be divided into 24 serovars on the basis of the immunological reactions of the flagellum antigen. Numerical analyses of phenotypic characteristics failed to differentiate the serovars from each other and B. cereus from B. thuringiensis (3). Opinions regarding the...

Homoduplex and Heteroduplex Polymorphisms of the Amplified Ribosomal 16S-23S Internal Transcribed Spacers Describe Genetic Relationships in the “ Bacillus cereus Group”

“…Some important B. thuringiensis subspecies known to be genetically related were grouped together by ITS-HHP. For example, strains belonging to the B. thuringiensis subspecies aizawai, galleriae, kurstaki, and tolworthi showed the same ITS-HHP patterns, in agreement with the ribotyping data of Priest et al (49), the M13 DNA fingerprinting data of Miteva et al (40), and the DNA-DNA hybridization data of Nakamura (42). The fact that the ITS-HPP patterns of these subspecies were identical reflected their close DNA relatedness (90%) (42), while the B. thuringiensis subspecies darmstadiensis, israelensis, and sotto, which have relatively low DNA-DNA hybridization levels (below 70%) (42), showed different ITS-HPP types and were assigned to a cluster different from that of B. thuringiensis subsp.…”

“…These species are known to be strictly related phylogenetically, as has been shown by DNA-DNA hybridization studies (35,39,(42)(43)(44)57) and the sequencing of the ribosomal RNA genes (2)(3)(4)39). On the other hand, a marked variability is always observed when large collections of strains are examined by DNA fingerprinting methods that target the whole genome (8-13, 17, 25-29, 36, 58) and/or discrete genes (15,16,38,54,55,61).…”

“…One included B. mycoides, B. weihenstephanensis, and B. pseudomycoides. The second included B. cereus and B. thuringiensis, B. anthracis appeared as a lineage of B. cereus.The "Bacillus cereus group," one of the most homogeneous groups of the genus Bacillus, encompasses six validly described species, B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, and B. weihenstephanensis, all of which have an important impact on human activity (18,39,44).These species are known to be strictly related phylogenetically, as has been shown by DNA-DNA hybridization studies (35,39,(42)(43)(44)57) and the sequencing of the ribosomal RNA genes (2-4, 39). On the other hand, a marked variability is always observed when large collections of strains are examined by DNA fingerprinting methods that target the whole genome (8-13, 17, 25-29, 36, 58) and/or discrete genes (15,16,38,54,55,61).…”

Specificity and Polymorphism of the PlcR-PapR Quorum-Sensing System in theBacillus cereusGroup

“…Several molecular and biochemical methods have been used to examine the diversity of the strains composing the B. cereus group. DNA-DNA hybridization methods do not generally make it possible to distinguish precisely between the six species belonging to the B. cereus group (18). Analysis of amplified fragment length polymorphisms (AFLP) shows that B. anthracis is a monomorphic species (12) but that B. thuringiensis and B. cereus exhibit a high degree of variability (7,10).…”

“…We identified four classes of PlcR-PapR pairs, defining four distinct pherotypes in the B. cereus group. We used these findings to look at the evolution of the PlcR-PapR quorum-sensing system with regard to the phylogeny of the species forming the B. cereus group.The Bacillus cereus group includes six highly related species of gram-positive, spore-forming, AT-rich bacteria: Bacillus anthracis, Bacillus cereus (sensu stricto), Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis (15,18,21). These species were originally identified on the basis of phenotypic traits, such as the production of a capsule and toxins (B. anthracis), the rhyzoid morphology of the colonies (B. mycoides and B. pseudomycoides),…”