Location Of Spore-Crystal Association Critical Factor In Bacillus Thuringiensis Subsp. Finitimus YBT-020

Bacillus thuringiensis is a sort of spore-producing Gram-positive bacteria, which can form crystals during sporulation. In most of B. thuringiensis species, the crystals are formed outside the exosporium and are departed from the spores after mother cell lysis. While, the special phenotype that crystals are produced inside the exosporium and associated with the spore after sporulation exists in a few strains, for example, the finitimus strain YBT-020, which is denominated "spore crystal association"(SCA).Through screening BAC library of strain YBT-020and fragments complementation testing,6key genes including, a crystal protein encoding gene cry26Aa, two crystal-like gene scaA and scaB, three genes, scaC-E, involved in spore germination were identified sufficient for the producing of SCA phenotype. Previously, a hypothesis was supposed to explain the mechanism of SCA that Cry26Aa is the major component of crystal, ScaA and ScaB as the important factors can directly or indirectly anchor crystal protein to spore coat. Then, the Cry26Aa was packaged by the forming exosporium.To prove the proposed hypothesis, serious experiments were employed as fowllowing:1. To observe the position relationship between spores and crystal in different developmental stages of spores, samples from constant cultures of wild type YBT-020and the mutant JA were examined under transmission electron microscope. The results indicate that the crystal cannot be observed until the complete formation of cap structure of exosporium for both YBT-020and JA. The crystal was produced inside of cap structure in YBT-020but outside of it in SCA mutant JA, which can be thought as the early character of SCA. In addition, the formation of lamellar spore coat starts after the formation of the crystal is accomplished in both YBT-020and JA, suggested that the produce of SCA does not related with the spore coat or its components.2. A vector harboring the cry26-gfp fused genes, cry26-gfp-pHT304, was constructed and transformed into the gene cry26deletion mutant J1, resulting the recombinant strain BMB1904. The specific temporal and spatial expression of protein Cry26Aa was confirmed by observing under the scanning confocal microscope. The results show that Cry26Aa expresses after the stage of forespore septum, distributed in the cell and grows during the stage of forming incipient forespore, a mature crystal protein forms at the stage of exosporium. Finally, the GFP labeled Cry26located inside the exosporium after mother cell lysis.3. The vectors, scaA-gfp-pHT304and scaA-scaB-gfp-pHT304containing gfp gene fused scaA and scaA-B, were constructed and transformed into the scaA-B mutant JA and resulting the recombinant strain BMB1902and BMB1903. The fluorescence can be detected from the early stage of exosporium, for both BMB1902and BMB1903strains. The detected fluorescence signal in ScaA-B-GFP strain focused near the cap structure of exosporium, which indicated that they expressed at the time early or around the time that cap forming, significantly, earlier than spore coat forming. Therefore, the key factor ScaA and ScaB are related to the cap structure of exosporium but not to spore coat.4. As ScaA and ScaB show low amino acid identity of30%and22%respectively to crystal proteins NT40KD and NT32KD, these two proteins were supposed potentially to form crystals at high level expression of their genes. When their promoters were replaced as strong promoters, the two proteins were over-expressed, but no crystal can be detected under microscopy, which indicated that ScaA and ScaB had no potential to form crystals.5. Plasmid pEMB0557, usually called BAC60, was constructed to clone large DNA fragment as a shuttle tool vector, and was conserved in this lab. Under the condition of free selective press, the genetic stability of it was low as50%plasmids would be losed after100generations. Previously, the relativity between the size of fragment that pEMB0557vectored and its stability was investigated and indicated that in most cases, as the increasing of the size of the fragment that the plasmid vectored, the lower that the stability of it did. Howener, two exceptional large fragments,625and721respectively with the size of30kb and48.8kb, originated from pBMB28in YBT-020exhibited high stability of100%and87%both after100generations, respectively. To find out minimal factor(s) in these fragments to confer the high stability of pEMB0557, truncated experiment was employed to the fragment625, which conferred higher stability of100%to the plasmid pEMB0557.By subclone the BamHI fragments of625into pEMB0557, a10.3kb fragments can confer the equal stability as625to pEMB0557was screened. Through sequence analysis, the fragment was divided by a transposase encoding gene. The genes encoding a putative bacteriocin gene cluster upstream of it, and a remained4kb was located downstream the transposase gene. We supposed that the stable factor was more possible located at the4kb fragment. This fragment was cloned into pEMB0557by PCR and ligase, and conferred the full stability to pEMB0557. Further cloning fragment containing different ORFs in this region, determined the stability factors is localized at a2023bp fragment with an orf9. However, the orf9encoding a SprT-like family protein was not related to the genes involved in plasmid stability. After precisely scan,5repeat unit, TAATTGAATAA, with homology to par sites were found. It suggested that these cis-units could complement the defected Ia type active partition system in pEMB0557by supporting more repeat units.