Two-component System LytSR Regulates Spore Formation

Two-component regulatory systems(TCSs) can respond to environmental changes and regulate some relative gene transcriptions through transfering phosphorylation signal,which play important roles in bacterial adaptation and survival in response to a variety of stimuli. It has been proved that five autophosphorylating histidine kinases and transcription master regulator Spo0 A involve in sporulation initiation in Bacillus subtilis. But whether other TCSs involve in sporulation formation remains unclear. B. cereus and B.thuringiensis which belong to the family of B. cereus group are ubiquitous in nature. B cereus is an opportunistic pathogen that may lead to food poisoning due to the production of diarrhea toxin and vomitoxin. Its endospore is the main factor of food pollution because of its strong resistance to harsh conditions. Due to its safety, high insecticidal activity and environment-friendly, B thuringiensis is widely used as insecticidal micriobe. Insecticidal crystal proteins(ICPs) are synthesized during sporulation and accumulate in the mother cell as crystal inclusion. Thus, it is important to study the spore formation process of B cereus group.The two-component regulatory system Lyt S-Lyt R typically consists of a membranespanning histidine kinase sensor(HK) and a cytoplasmic response regulator(RR), which was firstly identified in the Staphylococcus aureus. As a membrane-spanning histidine kinase, Lyt S can sense electrical potential decreases in cell membrane and initiate the phosphorylation, and activation of the response regulator Lyt R, which is presumed to regulate downstream genes lrg AB. Lrg AB can control murein hydrolase activity, which plays an important role in the process of cell death and cell lysis. Similarly the gene structure lyt SR-lrg AB is also existed and very conserved in B. cereus and B thuringiensis,of which the sequence homology is more than 90%. Thus their functions are supposed to be also similar. This study mainly focuses on the function of Lyt S-Lyt R in the spore formation process.The lyt SR mutant HD△lyt SR and lrg AB mutant HD△lrg AB were constructed by homologous recombination, to examine the role of lyt SR-lrg AB. The growth curve showed that the growth of HD△lyt SR had no significant difference compared with Bt HD73.However the sporulation rate of HD△lyt SR was significantly decreased, while HD△lrg ABhad no difference compared with Bt HD73 either in the growth or sporulation rate. It was surprising that the autolysis rate of HD△lyt SR was decreased compared to that of HD73,not for △lrg AB. The lrg AB promoter fusion with lac Z was constructed to analyze the transcription activity of Plrg AB.. The recombinant plasmids were introduced into B.thuringiensis strain HD73 and HD△lyt SR. The β-galactosidase assay indicated that lrg AB is regulated by Lyt SR, similar in S. aureus.The lyt SR promoter Plyt SR fusion with lac Z was constructed to analyze the transcription activity of lyt SR.. The recombinant plasmids were introduced into B.thuringiensis strain HD73 and HD△sig E, respectively. The β-galactosidase assay showed that Plyt SR is controlled by Sig E in the early stage of sporulation. A confocal microscopy analysis demonstrated that Lyt SR modulated the process of forespore engulfment. The transcription of the sporulation genes spo IID, spo IIM, and spo IIP is under the control of Sig E in Bacillus subtilis. The absence of all three genes does septum formation occur at both poles at a frequency comparable to that observed in the sig E mutant. The bipole septum formation occurred in lyt SR mutant. To study whether lyt SR can effect on the transcriptions of the three sporulation genes spo IID, spo IIM, and spo IIP, we constructed the spo IID, spo IIM and spo IIP promoter fusion with lac Z. The recombinant plasmids were introduced into B. thuringiensis strain HD73 and HD△lyt SR, respectively. The β-galactosidase assay indicated that spo IIP is regulated by Lyt SR. It suggests that Lyt SR modulate the engulfment of spore formation through regulating the spo IIP gene.No previous studies reported that TCS invovled in the process of forespore engulfment. This study provides new insight into relationship between Two-component systems and spore formation.