Correlation between serovars of Bacillus thuringiensis and type I beta-exotoxin production

beta-Exotoxin is a thermostable metabolite produced by some strains of Bacillus thuringiensis. Because of vertebrate toxicity, most commercial preparations of B. thuringiensis are prepared from isolates that do not produce beta-exotoxin. The aim of the present study was to find out the possible relationship between serovars of B. thuringiensis and beta-exotoxin production. A specific HPLC assay for type I beta-exotoxin has been used to detect this exotoxin in supernatants from final whole cultures of 100 strains belonging to four serovars of B. thuringiensis: thuringiensis, kurstaki, aizawai, and morrisoni. For each serovar, 25 strains randomly chosen from two Spanish collections were analyzed. Frequency of beta-exotoxin production was higher in B. thuringiensis serovar thuringiensis, whereas only two strains from serovar kurstaki showed beta-exotoxin production. None of the 25 strains belonging to serovars aizawai and morrisoni was found to produce this compound. Along with data from other studies, serovars can be classified as "common," "seldom," or "rare" beta-exotoxin producers. The serovar-dependent beta-exotoxin production is discussed in relation to the evolutionary process of serovar differentiation, the plasmid compatibility and limited plasmid exchange between serovars, and with the serovar-dependent regulation of plasmid-encoded genes.     

Diversity of Bacillus thuringiensis strains from Colombia with insecticidal activity against Spodoptera frugiperda (Lepidoptera:Noctuidae)

AIMS: To identify and characterize Bacillus thuringiensis strains highly toxic to Spodoptera frugiperda, and to explore the genetic diversity of such strains. METHODS AND RESULTS: The insecticidal activity of 1100 strains of B. thuringiensis from Colombian soil samples was assayed against first instar S. frugiperda larvae, and 32 active strains were found. After a second bioassay evaluation, the eight most potent strains were selected for further characterization, which included crystal protein profiles determined by polyacrylamide gel electrophoresis, plasmid profile, plasmid restriction patterns, cry gene composition, qualitative determination of beta-exotoxin production, random amplified polymorphic DNA, serotyping, and toxicity to S. frugiperda. All Colombian strains contained cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C and cry1D genes. However, PCR profiles of the Colombian strains suggested the presence of variants of the cry1 genes. Serotyping indicated that these strains belong to the kurstaki, thuringiensis, canadiensis and indiana subspecies. Interestingly, three strains belonging to different serotypes and subspecies were found in the same soil sample, and toxicity ranged between 11 and 976 ng cm(-2) of diet. CONCLUSIONS: It has been shown that B. thuringiensis strains belonging to different serotypes and displaying variable potency to S. frugiperda larvae can be found in the same soil sample. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained indicate that some of the B. thuringiensis strains studied could be of interest for further development for S. frugiperda control programmes.     

Larval susceptibility of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), to Bacillus thuringiensis H serovars isolated in Japan

A total of 1700 Japanese strains of Bacillus thuringiensis, belonging to at least 47 H serogroups, were examined for insecticidal activity against larvae of the diamondback moth, Plutella xylostella. The high-level toxicity was associated with 612 isolates (36.0%). Of these, 608 isolates (99.3%) fell into 13 H serogroups belonging to the low-numbered H serotypes, H1-H10. Conversely, most isolates belonging to the high-numbered serotypes (>H10) had little or no larvicidal activity; only one isolate of the serovar japonensis H23 was active. P xylostella larvae were susceptible to 89.8% of the serovar morrisoni H8a:8b strains and 85.7% of galleriae H5a:5b strains. High values of 60-80% were also obtained in six serovars (thuringiensis H1, alesti H3a:3c, kurstaki H3a:3b:3c, kenyae H4a:4c, aizawai H7, and tolworhi H9), while relatively low values of <60% in two other common serovars, sotto H4a:4b and darmstadiensis H10a:10b. Five selected isolates, belonging to H serovars other than kurstaki and aizawai, were 10-60 times less toxic than the reference strain HD-1 (serovar kurstaki). Parasporal inclusion proteins of these strains were immunologically unrelated to those of the strain HD-1 and the aizawai type strain.     

Evidence of Bacillus thuringiensis intra-serovar diversity revealed by Bacillus cereus group-specific repetitive extragenic palindromic sequence-based PCR genomic fingerprinting

Bacillus thuringiensis is classified into serovars on the basis of H-flagellar antigens. Several alternative typing methods have been described. Among them, a B. cereus group-specific repetitive extragenic palindromic (Rep)-PCR fingerprinting technique was shown to be discriminative and able to identify B. thuringiensis serovars. The aim of this study was to investigate the genomic diversity and relationship among B. thuringiensis strains collected from different Argentinean ecosystems. Thirty-seven B. thuringiensis reference strains and 131 Argentinean isolates were analyzed using a B. cereus group-specific Rep-PCR. Fourteen different patterns were identified among the Argentinean isolates. Eight could not be associated to any pattern obtained from a reference strain. The pattern identical to the serovar kurstaki HD-1 strain was the most frequently identified in 68 native isolates. The profiles allowed tracing a single dendrogram with two groups and eight main lineages. Some strains showed distinctive patterns despite belonging to the same serovar. An intraspecific diversity resulted from this analysis that was highlighted by this technique since strains from a given serovar showed distinct profiles. This study may help to establish a system of B. thuringiensis classification with a higher discrimination level than established by the H antigen serotyping.     

Fingerprinting of Bacillus thuringiensis type strains and isolates by using Bacillus cereus group-specific repetitive extragenic palindromic sequence-based PCR analysis

A total of 119 Bacillus thuringiensis strains (83 type strains and 26 native isolates), as well as five B. cereus group species, were analyzed by repetitive extragenic palindromic sequence-based PCR analysis (Rep-PCR) fingerprinting. Primers Bc-REP-1 and Bc-REP-2 were specifically designed according to an extragenic 26-bp repeated sequence found in the six B. cereus group genomes reported. A total of 47 polymorphic bands were detected, and the patterns varied from 5 to 13 bands in number and from 0.2 to 3.8 kb in size. Virtually each type strain showed a distinctive B. cereus (Bc)-Rep-PCR pattern, except for B. thuringiensis serovars dakota (H serotype 15 [H15]) and sotto (H4a,4b), as well as serovars amagiensis (H29) and seoulensis (H35), which shared the same patterns. As expected, serovar entomocidus (H6) and its biovar subtoxicus showed an identical pattern; similarly, serovars sumiyoshiensis (H3a,3d) and fukuokaensis (H3a,3d,3e), which share two antigenic determinants, also showed identical Bc-Rep-PCR patterns. Interestingly, serovars israelensis (H14) and malaysiensis (H36), which share several phenotypic attributes, also showed identical Bc-Rep-PCR patterns. Native, coleopteran-active strains, including the self-agglutinated LBIT-74 strain, showed Bc-Rep-PCR patterns identical or very similar to that of the tenebrionis strain. Likewise, native mosquitocidal strains (including some self-agglutinated strains) also showed patterns identical or very similar to that of the serovar israelensis IPS-82 strain. Additionally, native beta-exotoxin-producing strains from serovar thuringiensis showed patterns identical to that of the B. thuringiensis type strain. The B. cereus group-specific Bc-Rep-PCR fingerprinting technique was shown to be highly discriminative, fast, easy, and able to identify B. thuringiensis serotypes, including nonflagellar and self-agglutinated strains.     

Ribotypes and plasmid contents of Vibrio anguillarum strains in relation to serovar.

Eighty-six strains of the 10 major agglutination types of Vibrio anguillarum (serovars O1 to O10) and 6 nontypeable strains of V. anguillarum have been characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by plasmid profile analysis. Forty-four different ribotypes were observed with the restriction enzyme HindIII. Ribotype similarity was compared by using the Dice coefficient (Sd), and three significantly different levels of homogeneity within the V. anguillarum serovars were observed (serovars O1, O3A, O7, and O9, Sds of > 90%; serovars O2B, O4, and O10, Sds of 80 to 90%; serovars O2A, O3B, O5, and O8, Sds between 46 and 70%). None of the ribotype patterns of V. anguillarum strains were observed among 20 other Vibrio strains typed for comparison. By cluster analysis, the V. anguillarum strains were divided into a main cluster containing 83 strains, while all strains of serovar O3B, one strain (each) of serovars O2A, O5, and O8, and a nontypeable strain were separated from this cluster by at least 15% difference in similarity coefficients. Plasmids were demonstrated in only six strains other than serovar O1. In serovar O1, a 67- to 70-kilobase-pair (kb) plasmid molecule was present in 17 of 19 strains tested; of the two remaining strains, one strain harbored two plasmids (45 and 6.5 kb) and one strain had no plasmids.     

Plasmid pC present in Salmonella enterica serovar Enteritidis PT14b strains encodes a restriction modification system

Salmonella enterica serovar Enteritidis (S. Enteritidis) possesses plasmids of different sizes and roles. Besides the serovar-specific virulence plasmid present in most field strains, S. Enteritidis can harbour plasmids of low molecular mass whose biological role is poorly understood. We therefore sequenced plasmid pC present in S. Enteritidis strains belonging to phage type PT14b. The size of plasmid was determined to be 5,269 bp and it was predicted to encode four open reading frames (ORFs). The first two ORFs were found (initial 3,230 bp) to be highly homologous to rom and mbeA genes of ColE1 plasmid of Escherichia coli. Proteins encoded by the other two ORFs were 99% homologous to a restriction methylase and restriction endonuclease encoded by plasmid pECO29 of a field strain of E. coli. Using insertional mutagenesis we confirmed experimentally that the plasmid pC-encoded restriction modification system was functional and could explain the high resistance of S. Enteritidis PT14b strains to phage infection.     

Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis.

To determine the possibility of plasmid transfer occurring between strains of Bacillus thuringiensis in infected lepidopterous larvae, Galleria mellonella and Spodoptera littoralis were infected with two or more strains of B. thuringiensis and the resulting bacteria from the dead insects were examined for plasmid transfer. Transfer rates of plasmids coding for crystal production and tetracycline resistance were high, reaching levels similar to those obtained in laboratory broth cultures. Transfer was higher in G. mellonella than S. littoralis, probably due to the greater ability of B. thuringiensis to colonize the larvae. In broth cultures, B. thuringiensis was also able to transfer plasmids into sporeforming bacteria present in soil samples. The results suggest that plasmid transfer between strains of B. thuringiensis occurs in nature, resulting in the production of new combinations of delta-endotoxins within populations of the bacteria.     

Serotyping of Bacillus thuringiensis environmental isolates by extracellular heat-stable somatic antigens.

A total of 525 Bacillus thuringiensis environmental isolates, belonging to the five flagellar (H) serovars (alesti, sotto, kenyae, aizawai, and morrisoni), were serotyped by extracellular heat-stable somatic antigens (HSSAs). The isolates belonging to a given H serovar were assigned to a single HSSA serogroup at a high frequency, 87-100%. This indicates that the extent of HSSA variation within a single H serovar is small in the field populations of these B. thuringiensis serovars.     

Identification of contemporary plasmid virulence genes in ancestral isolates of Salmonella enteritidis and Salmonella typhimurium

Six epidemiologically distinct ancestral strains of Salmonella enteritidis and 5 of S. typhimurium from the pre-antibiotic era were examined for plasmid content, and for presence of plasmid genes implicated in mouse-virulence. Five sizes of plasmid were detected in S. enteritidis varying from 1 to 60 MDa. Two sizes of plasmid were found in S. typhimurium, 28 and 60 MDa. Plasmids of the same size were not common to both serovars. The HindIII restriction patterns of 3 of the ancestral S. enteritidis plasmids were identical to the modern 38 MDa plasmid, while all contained identical bands of 3.5, 2.7 and 1.9 kb. All the 60-MDa S. typhimurium plasmids, ancestral and contemporary, had an identical restriction pattern. Three different sized S. enteritidis plasmids and one size S. typhimurium plasmid contained a 3.5-kb DNA fragment carrying the virulence locus VirA. The VirB virulence locus was located on a 2.7-kb DNA fragment in S. enteritidis and on a 2.5-kb fragment in S. typhimurium. Both loci were precisely conserved between the ancestral strains and the modern representatives of both serovars.     

Diversity of locations for Bacillus thuringiensis crystal protein genes.

The location of crystal protein genes in 22 crystalliferous Bacillus thuringiensis strains representing 14 subspecies was investigated by hybridization of an intragenic restriction fragment from a cloned crystal protein gene to whole plasmid preparations. Hybridization was found to a single plasmid in eight strains, to more than one plasmid in seven strains, and to one or both of two large, unresolved plasmids in two strains. The sizes of the hybridized plasmids ranged from 33 to over 150 megadaltons. In one additional subspecies, hybridization was only to linear DNA fragments, suggesting a chromosomal crystal protein gene, and for four other subspecies, not reported to be toxic to lepidopteran insects, no hybridization was found to either plasmids or to total cell DNA. Hybridization to restriction digests of plasmids and total cell DNA of several strains of subspecies thuringiensis and kurstaki revealed that all homology to the cloned crystal protein gene was plasmid associated and that several of these strains contained multiple regions of homology, implying the presence of multiple crystal protein genes.     

Engineering Bacillus thuringiensis bioinsecticides with an indigenous site-specific recombination system.

The cry genes of Bacillus thuringiensis encode a diverse group of crystal-forming proteins that exhibit insecticidal activity, particularly against the larvae of lepidopteran, coleopteran, and dipteran insects. The efficacy of B. thuringiensis-based biopesticides may be improved through the genetic manipulation of these genes. A gene transfer system has been developed for the introduction and maintenance of cloned insecticidal cry genes on small plasmids in B. thuringiensis. This vector system combines a B. thuringiensis plasmid replicon and an indigenous site-specific recombination system that allows for the selective removal of ancillary or foreign DNA from the recombinant bacterium after introduction of the Cry-encoding plasmid. The site-specific recombination system is useful for engineering strains with unique combinations of cry genes, resulting in new active ingredients with improved insecticidal properties.     

Identification and characterization of the new bacillus thuringiensis serovars pirenaica (serotype H57) and iberica (serotype H59)

Two new Bacillus thuringiensis strains have been classified by the H antigen of the cells and differentiated by their morphological, biochemical and molecular characteristics. The flagellar agglutination showed that both strains bore specific H antigens which allowed their classification as the new serotypes H57 and H59. The serovar names proposed for the type strains characterized in this work are B. thuringiensis ser. pirenaica, for the H serotype 57, and B. thuringiensis ser. iberica, for the H serotype 59. Further characterization of these strains, by means of SDS-PAGE, Western inmunodetection, plasmid profile and cry -gene identification by polymerase chain reaction, confirmed the originality of the two novel serotypes. Toxicity tests carried out against several insect species, belonging to the orders Lepidoptera, Diptera and Coleoptera, showed no detectable insecticidal activity for either of the B. thuringiensis strains.     

Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis

To determine the possibility of plasmid transfer occurring between strains of Bacillus thuringiensis in infected lepidopterous larvae, Galleria mellonella and Spodoptera littoralis were infected with two or more strains of B. thuringiensis and the resulting bacteria from the dead insects were examined for plasmid transfer. Transfer rates of plasmids coding for crystal production and tetracycline resistance were high, reaching levels similar to those obtained in laboratory broth cultures. Transfer was higher in G. mellonella than S. littoralis, probably due to the greater ability of B. thuringiensis to colonize the larvae. In broth cultures, B. thuringiensis was also able to transfer plasmids into sporeforming bacteria present in soil samples. The results suggest that plasmid transfer between strains of B. thuringiensis occurs in nature, resulting in the production of new combinations of delta-endotoxins within populations of the bacteria.     

Pulsed field gel electrophoresis of chromosomal DNA reveals a clonal population structure to Bacillus thuringiensis that relates in general to crystal protein gene content

Seventy strains of Bacillus thuringiensis representing 21 serovars were allocated to 38 genomic groups using pulsed field gel electrophoresis (PFGE) of restriction enzyme-digested DNA. There was a broad correlation between PFGE type and serotype for serovars darmstadiensis, israelensis, kenyae, kumamotoensis, kurstaki, sotto, thuringiensis, and tolworthi, although some serovars included atypical strains. Serovars canadensis and entomocidus were heterogeneous. Detection of crystal protein genes by polymerase chain reaction indicated an approximate correlation between PFGE type and cry gene complement. For example, cry1 products were amplified from DNA from PFGE type 17 strains of serovar aizawai and from PFGE type 23 strains of serovar tolworthi but not from a PFGE 18 strain of aizawai nor from a PFGE type 24 strain of tolworthi. These data suggest a clonal population structure to B. thuringiensis with some consistency of Cry-plasmid composition within PFGE types.     

Application of the multiplex PCR and PCR-RFLP method in the identification of the Bacillus anthracis

The aim of this study was to apply the multiplex PCR and PCR-RFLP method for the identification of the B. anthracis strains and to distinguish those bacteria from other members of the Bacillus cereus group. The multiplex PCR method enables to detect the virulence factors, i.e. the toxin and the capsule in B. anthracis strains. To do that, the authors have used 5 primer pairs specific for the fragments of lef, cya, pag genes which are present in the pXO1 plasmid and encode the toxin, the cap gene, which is present in the pXO2 plasmid and encodes the capsule, and the Ba813 chromosomal sequence. Among the four B. anthracis strains examined, three contained two plasmids and the Ba813 chromosomal sequence, while the fourth one contained the pXO1 plasmid only, together and the Ba813 chromosomal sequence. Other bacterial species, belonging to the B. cereus group, were also examined: 6 strains of B. cereus, 4 strains of B. thuringiensis and one strain of B. mycoides. The presence of Ba813 chromosomal sequence has been detected in two B. cereus strains. Neither plasmids nor Ba813 chromosomal sequence have been discovered in other B. cereus, B. thuringiensis and B. mycoides strains. The results of the survey indicate that the Ba813 chromosomal sequence does not occur solely in B. anthracis strains. The PCR-RFLP method with the use of SG-749f and SG-749r primers enabled to demonstrate the presence of DNA sequence (SG-749) in B. anthracis, B. cereus, B. thuringiensis and B. mycoides strains. Restriction analysis with enzyme AluI of the SG-749 sequence, has shown the presence of two DNA fragments at the size of about 90 and 660 bp in all B. anthracis strains. The restriction profile obtained was characteristic for B. anthracis strains and it did not occur in other investigated bacterial species belonging to the B. cereus group. It was not observed even in such B. cereus strains in which the presence of Ba813 sequence was discovered and it enabled to differentiate between B. anthracis strains and other closely related species of the B. cereus group.     

Two δ-Endotoxin Genes, cry9Da and a Novel Related Gene, Commonly Occurring in Lepidoptera-Specific Bacillus thuringiensis Japanese Isolates That Produce Spherical Parasporal Inclusions

Six Lepidoptera-specific Bacillus thuringiensis isolates, which belong to the four H serovars (sotto, fukuokaensis, canadensis, and galleriae) and produce spherical parasporal inclusions, were examined for assignment of the classes of the delta-endotoxin genes. Gene analysis was conducted by PCR technique with primers designed to probe the genes cry9Ca and cry9Da. The data revealed that the delta-endotoxin of a serovar canadensis isolate is encoded by the gene cry9Da, while those of the five other strains are encoded by an undescribed delta-endotoxin gene. DNA fragments from five strains had an identical 1917-bp nucleotide sequence, covering the four conserved regions and a partial sequence of the block 5 region. The deduced amino acid sequence exhibited a 70.6% homology to that of the corresponding region of the Cry9Ea delta-endotoxin protein which is active on the order Lepidoptera, and a 63.1% homology to the Cry9Ca protein highly toxic to the noctuid lepidopterans. The results showed that Japanese isolates of B. thuringiensis producing spherical parasporal inclusions with Lepidoptera-specific activity are categorized into two groups: one produces the class Cry9Da protein and the other a novel delta-endotoxin allied to the class Cry9. It also appeared that heterogeneous multiple H serovars are involved in each group.     

Contents of cry genes and insecticidal toxicity of Bacillus thuringiensis strains from terrestrial and aquatic habitats

AIMS: Two Bacillus thuringiensis collections from terrestrial and aquatic habitats were compared in order to study the possible interrelationships between habitat and biological characteristics (serovar, cry genes content and toxicity). METHODS AND RESULTS: Bacillus thuringiensis strains were characterized by serology, PCR, and one-dose treatment against the noctuids Helicoverpa armigera and Spodoptera exigua, and the dipteran Tipula oleracea. A total of 12 and 10 different serovars were identified within terrestrial and aquatic strains, respectively. The number of non-toxic strains was greater in aquatic (41.6%) than in terrestrial habitats (5.3%). The genes cry1C, cry1D and cry1E were significantly more frequent in the terrestrial habitat. The cry1B gene was very frequent within thuringiensis strains. CONCLUSIONS: A high diversity was found in terms of serovars present and cry genes content in both collections. The relative frequency of individual cry genes was different in both collections, and a serovar-dependent distribution of the cry1B gene was found. Some strains sharing the same set of cry genes differed in their toxicity, suggesting important differences in gene expression. SIGNIFICANCE AND IMPACT OF THE STUDY: The inter-relationships between serology, cry gene content and toxicity may allow a better understanding of B. thuringiensis ecology.     

Molecular characterization of Bacillus thuringiensis strains from Argentina

Bacillus thuringiensis INTA 7-3, INTA 51-3, INTA Mo9-5 and INTA Mo14-4 strains were obtained from Argentina and characterized by determination of serotype, toxicity, plasmid composition, insecticidal gene content ( cry and vip ) and the cloning of the single- vip3A gene of the INTA Mo9-5 strain. The serotype analysis identified the serovars tohokuensis and darmstadiensis for the INTA 51-3 and INTA Mo14-4 strains, respectively, whereas the INTA Mo9-5 strain was classified as "autoagglutinated". In contrast to the plasmid patterns of INTA 7-3, INTA 51-3 and INTA Mo9-5 (which were similar to B. thuringiensis HD-1 strain), strain INTA Mo14-4 showed a unique plasmid array. PCR analysis of the four strains revealed the presence of cry genes and vip3A genes. Interestingly, it was found that B. thuringiensis 4Q7 strain, which is a plasmid cured strain, contained vip3A genes indicating the presence of these insecticidal genes in the chromosome. Bioassays towards various lepidopteran species revealed that B. thuringiensis INTA Mo9-5 and INTA 7-3 strains were highly active. In particular, the mean LC(50) obtained against A. gemmatalis larvae with the INTA Mo9-5 and INTA 7-3 strains were 7 (5.7-8.6) and 6.7 (5.6-8.0) ppm, respectively. The INTA Mo14-4 strain was non-toxic and strain INTA 51-3 showed only a weak larvicidal activity.