| Bacillus thuringiensis is a Gram-positive, spore-forming bacterium which forms the parasporal crystals at the onset of the sporulation growth phase. This is the main feature which distinguishes it from most other spore-forming bacilli. In a few cases, crystals are produced inside the exosporium and associated with the spores after sporulation. This special phenotype, which is named as spore crystal association (SCA), typically occurs in B. thuringiensis subsp. finitimus. The SCA phenotype was identified a half century ago. To date, the gene(s) required for conferring such localization has not yet been reported. The aim of this study was to identify genes determining the SCA phenotype in B. thuringiensis subsp. finitimus strain YBT-020.1. The genome sequence of the B. thuringiensis strain YBT-020 was determined at the Beijing Genomics Institute (BGI; Shenzhen, China) with a strategy of Solexa paried end sequencing technology. The complete genome of B. thuringiensis strain YBT-020 is comprised of a circular chromosome (5,355,490 bp) encoding 5.477 putative open reading frames (ORFs) and two circular plasmids, named pBMB26 (187,880 bp) and pBMB28 (139,013 bp), which encoding 200 and 149 putative ORFs, respectively.2. Plasmid conjugation experiments indicated that the SCA phenotype in strain YBT-020 was closely related to two large plasmids (pBMB26 and pBMB28). A shuttle bacterial artificial chromosome (BAC) library of strain YBT-020 was constructed, screened from which six fragments from BAC clones were discovered to cover the full length of pBMB26, and four others cover pBMB28. Using fragment complementation testing, two fragments with a length of approximately 35 kb located on pBMB26 and pBMB28 were observed to recover the SCA phenotype in an acrystalliferous mutant, B. thuringiensis strain BMB171. Furthermore, deletion analysis indicated that the crystal protein gene cry26Aa from pBMB26, along with five genes from pBMB28, were indispensable for the SCA phenotype. The orfl and orf2 encoded the putative peptides proteins showed low similarity with the crystal proteins NT40KD and NT32KD; The genes orf3, orf4, and orf5 are similar to the genes in an operon that encode germination complex, and are especially similar to the spore germination. 3. To confirm that the six genes were involved in determining the SCA phenotype in strain YBT-020, three mutant strains were constructed and characterized. Strain BMBJ1 was a cry26Aa gene disruption mutant from strain YBT-020. Microscopic observation revealed that BMBJ1 had lost the ability to form crystals. After the plasmid pBMB0617 harboring the cry26Aa gene was transferred into BMBJI, the crystals were absorbed and the SCA phenotype was regained. It confirmed that the crystal protein gene, cry26Aa, was essential for SCA; The operon containing orf1 and orf2 genes was disrupted to create mutant strain BMBJA. We observed that the crystals of this mutant were formed outside the exosporium during sporulation and were free from the spores after sporulation. In complementation experiments, the plasmid pBMB251B5 carrying orfl and orf2 was capable to recover the SCA phenotype in BMBJA. To identify the necessity of orfl and orf2 for SCA phenotype, a frameshift was introduced into orf1 and orf2 to generate plasmids pBMB251B5A and pBMB251B5B respectively, and then these plasmids were transferred into strain BMBJA. No SCA phenotyope been observation in the transformants indicated that both the products of orfl and orf2 are indispensable for SCA phenotyope. The operon containing orf3, orf4, and orf5 genes was disrupted, resulting in mutant strain BMBJB. Microscopic observation revealed that the SCA phenotype remaining after sporulation, however, this SCA phenotype was not as stable as that of strain YBT-020. After growing for 100 h, the crystals were separated from spores and no SCA phenotype could be observed. These observations suggested that the products of orf3, orf4, and orf5 were only essential in sustaining SCA phenotype, but not involved in the formation of that in strain YBT-020. In complementation experiments, the plasmid pBMB251B3 carrying intact orf3, orf4, and orf5 genes was demonstrated to be capable of restoring the stability of the SCA phenotype in BMBJB.4. BLAST analysis showed that there is no Rep-like gene located on pBMB26. However, orf69 gene encoding a putative protein showed identity to DNA polymerase III containing gamma and tau subunits, and orf70 gene encoding a putative protein showed identity to bacterial PcrA protein. They were both likely related to plasmid replication. A 12-kb EcoRI fragment was capable of supporting replication when cloned on a replication probe vector. Deletion and frame-shift mutation analysis showed that a 4.1-kb region encompassing two putative ORFs was essential for the replication of the miniplasmid in B thuringiensis, orf70 gene encoding a 49.8-kDa protein (named Rep26) with a helix-turn-helix motif showed no homology with known replication proteins and orf69 gene encoding a protein of 82.6-kDa showed homology to bacterial PcrA protein. GST-tagged ORF70 protein binding to the putative origin of replication (Ori) region which located in the region of orf70 gene was shown by gel retardation. Our results revealed that the replicon of p26 was widely distributed and ORF70 protein might be defined as a novel family of plasmid-encoded initiator proteins in megaplasmids among the B. cereus group5. Blast analyses revealed that ORF32 is similar to replicase Ori44 with 93% identity of amino acides sequence. Therefore, it is supposed that ORF32 and regions nearby form the replicon of p28. A 4.1 kb DNA fragment containing ORF32 was cloned by replication clone vector pHT304E, and it was demonstrated to replicate in plasmid curing strain 4Q7. Deletion analyses showed that a 3-kb region was required for replication and its plasmid replication stability was above 71%. Additionally, blast analyses revealed that pBAC721 containing the conjugative elements of pBMB28, Biparental mating assays demonstrated that pBAC721 was self-transferable, which favors the suggestion above. |
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