Preliminary Identification of a New Toxin Against Caenorhabditis Elegans Produced by Bacillus Thuringiensis Subsp. Chinensis and Its Metabolic Relationship with Thuringiensin

Bacillus thuringiensis（Bt for short） is a well-known gram-positive and spore-forming soil bacterium that produces many small molecular metabolites which exhibit high toxicity against many pests. For example, thuringiensin（Thu for short） and Zwittermicin A（ZwA for short） et al.. So it has broad application prospects.A high-yielding wild strain of thuringiensin, B. thuringiensis subsp.chinensis strain CT-43, was isolated by our lab. The thuringiensin was extracted from the supernatant of CT43 late-exponential phase culture using organic solvent extraction method. Two main peaks were detected using HPLC（Higher pressure liquid chromatogram）. The peak with the retention time of 7 minutes was identified as the characteristic absorption peak of thuringiensin by HPLC-MS（HPLC- Mass spectrometry）. The other peak with the retention time of 6 minutes represent a unknown metabolite. Its molecular weight was 677 by HPLC-MS. We deduced that the unknown metabolite was a new kind of compound by comparing with other reported metabolite produced by Bt. It was named Tox-x in this study. The result of bioassay showed that Tox-x was highly toxic against Caenorhaditis elegans with as high as 90% lethal death. The dilution times of the Tox-x which was used for bioassay was 6.25.The optimal parameters for thuringiensin and Tox-x production respectively were obtained. The CT-43 was cultured in G medium for 20 h, then fed with sodium citrate and glucose at final concentration of 2.0 g L^-1 and 16 g L^-1 respectively, and then continued cultured for additional 32 h. Then the thuringiensin was extracted from the supernatant using organic solvent extraction method. The result of HPLC showed that the peak area of thuringiensin was 74.84% of the total peak area, while that of thuringiensin was 29.85% using the initial condition. The optimal medium LB-1 which contain 18 g L^-1 peptone, 15 g L^-1 yeast extract and 10 g L^-1 NaCl for Tox-x production was got by changing the concentration of the nutrient element of LB. Tox-x was extracted from supernatant of CT-43 culture and the peak area of Tox-x was 64.18% of the total peak area while that of Tox-x was 31.76% using the initial condition.Meanwhile, six CT-43 plasmid curing mutants were conducted for Thu and Tox-x detection. The results showed that the mutant CT-43-7 could synthesize highly pure thuringiensin only, but not Tox-x; and the mutant CT-43-55 could synthesize highly pure Tox-x only, but not thuringiensin. So we can extract thuringiensin and Tox-x using matching mutants.