Abstract: Sequence analysis of a virulence-attenuated Bacillus thuringiensis signature tagged mutant 6F8 led to the identification of an 18 182 bp locus encoding 29 potential protein-coding ORFs. Thirteen of the 29 putative ORFs were found to share extensive homology with genes on plasmid pE33L466 of the pathogenic Bacillus cereus E33L strain. Nine ORFs were not only found in a cluster, but also in the same gene order, in both organisms. A number of mobile elements, including a transposon Tn4430, a novel IS231 family insertion sequence ISBth4 and various phage-related proteins, were found flanking this conserved gene cluster. These features of the 6F8 locus suggested that it might have undergone several DNA insertions from different sources by horizontal gene transfer. Transcriptional analyses of the 6F8 locus revealed that ORFs 1-23 were cotranscribed as a single transcript. Null mutants were constructed to investigate the function of the sequences flanking the signature-tagged mutagenesis insertion sites. Competition assays performed with the wild-type and null mutants demonstrated that the Tn4430 transposon element plays an important role in the full virulence of B. thuringiensis during Manduca sexta infection. This study provides the first experimental evidence that a Tn4430 family transposon is directly associated with B. thuringiensis virulence.

Abstract: In this report, we investigate the link between nutrient limitation, RelA-mediated (p)ppGpp production, and virulence in the phytopathogen Erwinia carotovora subsp. atroseptica. A relA null mutant (JWC7) was constructed by allelic exchange, and we confirmed that, unlike the wild-type progenitor, this mutant did not produce elevated levels of (p)ppGpp upon nutrient downshift. However, (p)ppGpp production could be restored in strain JWC7 during nutrient limitation by supplying relA in trans. During growth on exoenzyme-inducing minimal medium, the relA mutant showed a diminution in secreted pectate lyase and protease activities and a severe defect in motility. The relA mutant was also impaired in its ability to cause rot in potato tubers. In the presence of serine hydroxamate (a competitive inhibitor of seryl tRNA synthase and a potent inducer of the stringent response in wild-type E. carotovora subsp. atroseptica), exoenzyme production was essentially abolished in JWC7 but could be restored in the presence of plasmid-borne relA. The inhibition of exoenzyme production in JWC7 caused by serine hydroxamate could not be overcome by addition of the quorum-sensing signal molecule, N-3-oxohexanoyl-l-homoserine lactone. Quantitative reverse transcription-PCR analysis of selected RNA species confirmed that the effects of relA on secreted pectate lyase activity and motility could be attributed to a reduction in transcription of the corresponding genes. We conclude that nutrient limitation is a potent environmental cue that triggers (p)ppGpp-dependent exoenzyme production in E. carotovora subsp. atroseptica. Furthermore, our data suggest that nutrient limitation [or rather, (p)ppGpp accumulation] is a prerequisite for effective quorum-sensing-dependent activation of exoenzyme production.

Abstract: Transposon Tn917 was used to identify Bacillus thuringiensis genes required for virulence and survival in a Manduca sexta (tobacco hornworm) septicaemia model. Uniquely tagged transposons, n = 72, were constructed and used to generate 1152 insertion mutants. Sixteen pools of 72 mutants were screened in the infection model, and 12 virulence-attenuated mutants were unable to survive the infection. Analysis of the mutated DNA sequences implicated an arsR family transcriptional regulator, a histone-like DNA-binding protein, a transposon, and several sequences of unknown function in B. thuringiensis pathogenesis.

Abstract: Bt L-7601 is a UV resistant wild-type strain, which belongs to Bacillus thuringiensis subsp. dendrolimus serotype H4a4b. It was isolated from nature, and produced a dark brown pigment during the exponential phase of growth. Bt L-7601 had the ability to produce pigment in a general nutrition-abundant medium, which had no L-tyrosine. The pigment was identified as melanin based on chemical testing, its light absorbance, and FT-IR analysis. Bt L-7601 has a strong resistance to UV light. After 30 min irradiation its survival rate was 17 times higher than that of the strain B. thuringiensis subsp. colmeri 15A3, which had no pigment. Results of the bioassays of residual insecticidal activity of Bt formulation with and without pigment produced by Bt L-7601 against larvae of Helicoverpa armigera and Spodoptera exigua after exposure to UV irradiation showed that the pigment is an excellent UV protective agent for the insecticidal proteins.

Abstract: Bacillus thuringiensis isolates from different ecological regions and sources of China were analyzed to study the distribution and diversity of cry genes and to detect the presence of novel cry genes. Strains containing cry1-type genes were the most abundant and represent 237 of the 310 B. thuringiensis isolates (76.5%). About 70 and 15.5% of the isolates contained a cry2 gene or cry9 gene, respectively, while 10.0% of the strains did not contain a cry1, cry2, or cry9 gene. Among the cry1 containing isolates, cry1A (67.7%), cry1I (60.6%), cry1C (43.9%), and cry1D (39.4%) genes were the most abundant. Forty-three different cry1 gene profiles were detected in this collection. Several cry1 genes were associated at a high frequency, such as the cry1C-cry1D and cry1A-cry1I gene combination. The cry1A and cry2 amplicons were digested with selected restriction enzymes to examine sequence diversity. Based on this RFLP analysis, one novel cry1A-type gene was observed.

Abstract: Bacillus thuringiensis wild type strain 15A3 belongs to subspecies colmeri serotype H-21. RFLP and PCR analysis show that it contains six types of ICP genes: cry1Aa, cry1Ac, cry1Ca, cry1D, cry1I and cry. The sequence of the 1.45 kb N-terminal fragment of cry1Aa differed from that of published. SDS-PAGE showed that the crystal consists of proteins with molecular weight about 130, 79, 70, 65, 51 and 45 kD. Strain 15A3 didn't sysnthesize heat-stable beta-exotoxins according to test of house fly aberration. The 1.2 tons fermentative production exhibited high toxicity against three lepidopteran pests: H. armigera, S. exigua and H. cunea. It was proved that wild type strain can produce a broad specturm of ICP.

Abstract: A new strain of Bacillus thurinigiensis Bt with high toxicity against noctuidae larvae has been screened for many generations since isolated from larvae candle of Aphomia gularis in Yiyang County, Jiangxi Province, in China. By comparison and analysis of results of physiological and biochemical test, flagella antigen identification and esterase type, the strain is identified as H4a-4c Bt serovar. kenyae. Since its crystal protein type and plasmid type are different from those of Bt serovar. kenyae's type 023. The strain is assigned a novel strain: Btken-Ag. Btken-Ag's parasporal crystals are multi-morphorous: bipyramid, cube, small irregular sphere and embedded. After UMT dissolve, PAGE and SDS-PAGE separation preparation and analysis, it is found that its major insecticide component to Heliothis armigera is 61kD toxic protein. By ELISA homology analysis, it is found that this toxic protein has high homology with crystal protein of 023 and 7501 (H4a-4c), partly homology with that of HD-1(H3a-3b), but no homology with that of Bti(H14) and (Bacillus sphaericus) Ts-1 strain. In bioassay with larvae from Culex pipiens, Pseudaletia unipuncta and Heliothis armigera, together with other ten Bt strains, Btken-Ag is toxic to 3 star larvae of Culex pipiens; two isolates of Btken-Ag (b1-4 and H4-1) show higher toxicity than type strain 023 and HD-1 do to Pseudaletia unipuncta and Heliothis armigera.