Molecular Mechanism Of Spore Formation That Bacillus Nematode B16 Induced By Nematodes

Bacillus nematocida B16 is a new species of Bacillus sp.with nematicidal activity,which was isolated from the forest soil of Yunnan.Previous studies found that B16 used volatile substances to attract and lure nematodes to devour them,then they were colonized in the gut of the nematodes,and secreted the proteases to hydrolyze the intestinal epithelial tissue of the nematodes,which destroyed the intestinal structure and function of nematodes and eventually led to the death of nematodes.In the process of B16 infection,the spores produced by the bacteria were essencial structure of B16 infection.The mortality of nematodes was very low when they were infected with vegetative cells of B16,but most of them died rapidly when they were tested with spores of the bacteria.So what is the relationship between B16 spore formation and nematodes? Is the infection of B16 to nematodes accidental,or is there an inevitable connection? Can bacteria B16 sense the presence of nematodes and take corresponding measures? It is still unclear.In this study,Bacillus nematocida B16 and Caenohibditis elegans were selected as research objects to study how bacteria B16 perceive signal substances and further regulate the corresponding gene expression,so as to reveal the molecular mechanism of nematodes induced Bacillus B16 to produce spores.The main results of this study are as follows:1.It was found that the B16 induced by nematodes began to form spores at 18 h,while the control groups began to form spores at 24 h.At 32 h,there was the greatest difference in the number of spores between the two groups,and the number of spores in the experimental group was 66.8 times of that in the control group.2.The volatile signals released by nematodes sensing the presence of B16 were detected by SPME-GC-MS as 2,6-Bis(1,1-dimethylethyl)-4-(1-oxopropyl)phenol,Morpholine,and Undecanoic acid propyl ester.The results showed that B16 induced by morpholine began to form spores at 18 h,while the control group began to form spores at 24 h.Neither 2,6-Bis(1,1-dimethylethyl)-4-(1-oxopropyl)phenol nor Undecanoic acid propyl ester could induce B16 to form spores in advance.3.The results of transcriptome analysis were as follows: The control group at 32 h was compared with the same group at 18 h,the expression of 27 sporulation-related genes in B16 was significantly increased,and the difference was more than 2 times.Compared the experimental group in 18 hours with the control group in the same time,the expression of 9 sporulation-related genes in B16 was significantly up-regulated,and the difference of gene expression was more than 2 times.Compared the experimental group in 32 hours with the control group in the same time,the expression of 9 sporulation-related genes in B16 was significantly up-regulated,and the difference was more than 2 times.Compared the experimental group in 18 hours with the control group in the same time,the phosphatase gene spo0 E of B16 was down-regulated more than twice.Compared the experimental group in 32 hours with the control group in the same time,the phosphatase gene rapA of B16 was also down-regulated more than twice.The expression levels of spo0 E and rapA genes in B16 induced by nematode at 18 h and 32 h were confirmed by real-time fluorescence quantitative PCR(qPCR),so did with the B16 group induced by morpholine at 18 h and 24 h.The qPCR results showed that compared with the B16 group without nematodes,the expression of spo0 E and rapA in the B16 group induced by nematode 32 hours,decreased 58.33 times and 47.64 times,respectively;the expression of spo0 E and rapA gene of B16 induced by morpholine 24 hours was down-regulated by 9.32 and 2.72 times,respectively.4.The spo0E-B16 and rapA-B16 homologous overexpression strains with high expression of spo0 E and rapA genes in B16 respectively were constructed and verified by qPCR.The results showed that compared with original B16 strain,the expression of spo0 E and rapA genes in overexpressing strains increased separately 102 and 28.554 times.Through the test of spore formation capabilities,it was found that the overexpressed strains lost the ability to produce spores,and the overexpression strains also failed to form spores when induced by morpholine and nematodes.Based on the above results,We can conclude that the molecular mechanism of the nematodes inducing Bacillus nematocida B16 forming spore is as follows: B16 perceives the existence of nematodes by recognizing morpholine molecules released by nematodes,and morpholine can enter B16 cells,inhibit the expression of spo0 E and rapA genes,and promotes B16 to form spores in advance.