Establishment Of The Protein Interaction Networks Related To Spore-Crystal Association In Bacillus Thuringiensis YBT-020

Bacillus thuringiensis is a gram-positive bacteria which can form spores and crystals during its sporulation. The parasporal crystals are usually separated from the spores after mother cell lysis for the majority of Bacillus thuringiensis. However, a few Bacillus thuringiensis, such as serovar finitimus, its parasporal crystals formed inside the exosporium, and won’t separated from the spores. This special phenotype is called Spore Crystal Association(SCA for short), and the crystals are called associated crystals.There are few international studies about the molecular mechanism of SCA, and it mainly focuses on the Bacillus thuringiensis serovar finitimus YBT-020 which is collected in our research group. Now we have confirmed that the associated crystals locate inside the exosporium cap. In strain YBT-020, the gene scaA and scaB on plasmid pBMB28 determine the location of associated crystals, and gene sca C, scaD and scaE which also on plasmid pBMB28 maintain the stability of SCA. Moreover, Cry26 Aa is the main component of associated crystals, whose encoding gene is on plasmid pBMB26. It has been proved that the SCA is still formed only at the present of exosporium cap, but complete exosporium is necessary for SCA. Nevertheless, how Sca A and ScaB make the associated crystals which mainly is Cry26 Aa proteins locate inside the exosporium is still unknown. We speculated that it is mainly determined by the interactions between Cry26 Aa and ScaA or ScaB. Rather to our surprise, no apparent interactions were detected among them by the Yeast two-hybrid and Dot blot experiment in our previous study. This work continued on the work on the protein interaction in large scale.1. We confirmed the interactions between Cry26 Aa and ScaA or ScaB by GST-Pull Down, Ligand Blot and Bacterial two-hybrid. It provides a significant evidence for explaining how ScaA and ScaB determine the location of the associated crystals.2. The protein interaction network related to the SCA was constructed. We firstly constructed the Bacterial two-hybrid target proteins screening library, which contains 87 of genes concerning all predicted exosporium and spore coat proteins genes as well as scaA, scaB, scaC, scaD and sca E. 26, 23 and 5 proteins were found directly interact with Cry26 Aa and ScaA or ScaB, respectively, based on the library screening. Then we used those proteins as bait to screen from the library and get 1236 interacting proteins. At last, we establish the protein interactions network.3. Based on the protein interactions network and the functions of proteins, we speculated BclA, BclB, ExsFB, BxpB, CotY, ExsY, ExsB, CotE, YaaH, InH2 and Yckk2 are the key proteins for the SCA. Then BclA2-2, Yckk2, BclB and YaaH were confirmed by knocking out the genes.4. Based on the protein interactions network, validation of the key proteins directing the SCA and the conformed interaction between Cry26 Aa and ScaA or ScaB, we speculate the location model of SCA formation. First, CotY directs the formation of cap framework, then BclA, BxpB, ExsFB and BclB assemble into the cap. In this process, ScaA and Cry26 Aa gather inside the cap by the interaction with BclA, then Cry26 Aa forms the crystal nucleus with the help of ScaA and ScaB. It offers important information for revealing the dynamic process of SCA formation. At the same time, the protein interactions network shows the relationship on exosporium, spore coat and crystals at the molecular level, which lay foundation for studying the development mechanism of exosporium and spore coat, and also the spore and crystals’ co-development.Furthermore, we presented the new way for seeking factors that determines location of associated crystals in different SCA stains. We can screen the proteins directly interact with both those spore structural proteins and crystal proteins among which contain potentially factors determining the location of associated crystals. It will expedite the studying of the formation mechanism of SCA, and also benefit for solving the common mechanism of SCA formation, which will help to develop and produce sporangium-like insecticide.