Structure Determined The Spore-Crystal Association Phenotype In Crystal Protein Cry26Aa In Bacillus Thuringiensis

In most cases, the parasporal crystal produced by Bacillus thuringiensis is separated from spore in space after the mother cell lysis. In a few strains, such as Bacillus thuringiensis subsp. finitimus YBT-020, their parasporal crystals are formed within the exosporium and remain with the spore after mother cell lysis, this phenotype is named as spore crystal association (SCA). Until now, we know little about this special phenotype. In this study, we have done some works to explain the phenotype in B. thuringiensis subsp. finitimus strain YBT-020.1. Obtained a pBMB28-cured mutant by using the stratage of plasmid incompatibilityThe strain YBT-020contains two plasmids pBMB26and pBMB28. Previously, we have cloned the6key genes (cry26Aa and gene cluster scaABCDE) which control the formation of SCA phenotype. The crystal protein gene cry26Aa located on the pBMB26and the gene cluster scaABCDE located on the pBMB28decided the formation of SCA phenotype together. Besides, we also found that YBT-020could not form SCA phenotype after curing the plasmid pBMB26. However, we had not obtained a pBMB28-cured mutant previously. In order to verify the relationship between pBMB28and SCA phenotype, in this study, we cured the pBMB28from B. thuringiensis subsp. finitimus strain YBT-020by using the stratage of plasmid incompatibility. The pBMB28-cured mutant was named BMB1602, and we found that BMB1602could not form SCA phenotype. This expreriment provided direct evidence that the pBMB28was related to the formation of SCA phenotype.2. Protein interaction among ScaA, ScaB and Cry26AaWhat’s more, previous research showed the crystal protein gene cry26Aa was the main component of the parasporal crystal, scaA and scaB were related to the location of the parasporal crystal, and the other three genes were related to the stability of SCA phenotype. In order to study the function of ScaA and ScaB during the process of SCA formation, protein interaction had been done. Firstly, three fusion proteins, which were Cry26Aa-His, ScaA-GST and ScaB-GST, were expressed in Escherichia coli BL21and purified. Then, dot hybridization had been done to identify the relationship among Cry26Aa, ScaA and ScaB. The results of dot hybridization showed that there was no interaction among Cry26Aa, ScaA and ScaB in vitro. We speculated that there may have interaction among these three proteins after the proteins were modified in vivo, or the way about how Cry26Aa could be located within the exosporium was not by directly interacting with ScaA and ScaB, maybe need the help of other proteins.3. Especial structure determined the SCA phenotype in Cry26AaPrevious research also showed that only Cry26Aa could be located within the exosporium in YBT-020, and other crystal proteins could not form SCA phenotype with the help of five genes scaABCDE in YBT-020. In order to find and analysis the key sequences of Cry26Aa related to the formation of SCA, some experiments such as large fragment exchange, small fragment deletion and point mutagenesis have been done in this study. Firstly, the N-terminal and C-terminal of Cry26Aa were respectively replaced by corresponding sequences from CrylCa and Cry7Ba. The results showed that the fusion proteins could not form SCA phenotype, indicating the N-terminal or C-terminal of Cry26Aa could not control the special phenotype in YBT-020. Then, we respectively truncated the amino acid residues2-31(N-terminal),1105-1164(C-terminal),661-700(middle area),175-204(Domain I),298-327(Domain II),539-568(Domain III) by missing five or ten amino acid residues. The results showed that each region has certain amino acid fragments which could influence the formation of SCA phenotype. So we concluded that the key sequences of Cry26Aa related to the formation of SCA phenophty scattered throughout the whole amino acid sequences.Besides, when truncated residues2-11sites, the truncated-Cry26Aa could form crystal structure but could not form SCA phenophty. After truncated residues12-21sites, the truncated-Cry26Aa could form crystal structure and some could form SCA phenophty. Then alanine scanning mutagenesis had been done of amino acid residues3-16sites, results showed that14mutants all could form SCA phenotype. Therefore, we concluded that the amino acid residues3-16sites of Cry26Aa influence the SCA phenophty on its secondary or3D structure level, but not existing special amino acid residues.According to the above results, we could conclude that the key sequences which determined the formation of SCA phenotype in Cry26Aa had no discipline, they scattered throughout the whole amino acid sequences. Also, our results indicated that the formation of SCA phenotype of Cry26Aa due to its especial secondary or3D structure.