Study On Antibacterial Mechanism Of Xenorhabdus Nematophila Regulated By PH

The metabolites of symbiotic bacteria associated with entomopathogenic nematodes own much bioactivity such as antifungal, antibacterial, insecticidal, antivirus and anticancer in the field of medicine and agriculture, which make it become a kind of novel bioresource with high potential for development and application. p H as an important exogenous signals, had great influence to the growth, metabolism and antibacteria activity of the symbiotic bacteria. But how symbiotic bacteria induction environment p H change and to respond, how to regulate their growth, metabolism and the production of antimicrobial substances through complex metabolic regulation and control network, was rarely reported. In order to clear the relationship bewteen p H, Cpx RA and antibacterial substances produce, find clues to regulate symbiotic bacteria metabolites and isolate hidden natural products, we had studied the difference of antibacterial activity, metabolic product, genes transcription and expression of x. nematophila YL001 strain which was early isolated and identified having high antibacterial activity, under the condition of different initial p H. The main results and conclusions of the thesis are as follows:1. The antimicrobial activities of extracellular metabolic products from p H4.5 to p H8.5 of YL001, were tested in vitro and in vivo.The bioassay results indicated that the antimicrobial activities of supernatant fermentation, ethylacetate extractes, methanol extracts were all improved with the increasing of the initial p H. The bioassay results indicated that the antimicrobial activities of supernatant fermentation, ethylacetate extractes, methanol extracts were all improved with the increasing of the initial p H. It especially could markedly inhibit the mycelia growth to M. grisea, P. capsici, B. cinerea, P. piricola, A. solani and A. brassicae of cell-free filtrate. At p H 8.5, the inhibition rate to these was more than 90%. The inhibition diameter to B. subtilis was 21.00 mm. Tissue experiment showed that the therapeutic of cell-free filtrate were 44.81%, was lower than the control（83.22%）; The protective effect of cell-free filtrate were 89.54%, higher than the control（78.69%）; at p H8.5, the inhibition diameter to Bacillus subtilis was 14.65 mm of ethylacetate extractes.Tissue experiment showed that the therapeutic of ethylacetate extractes were 39.94% at p H8.5, which was lower than the control（56.63%）; The protective effect of ethylacetate extractes was 61.22%, was lower than the control（72.86%）; at p H8.5, the inhibition diameter to B. subtilis was 16.50 mm of methanol extracts. Tissue experiment showed that the therapeutic was 12.50% 27.55%, was lower than the control（59.18%）; The protective effect of methanol extracts was 62.57%, was lower than the control（73.17%）.2. Using RT-PCR and q RT-PCR analysis the main antibacterial substances biosynthesis gene and the main transcriptional regulation genes related to antibacterial activity under the condition of p H5.5, 6.5, 7.0, 7.5, 8.5. RT-PCR and q RT-PCR analysis shown that the main antibacterial substances biosynthesis gene relatively expression of genes xcn A^L, isn A^C, pax A^T, XNC₂713 and antiplasmodial activity biosynthesis gene xab B^E was increased with the increase of p H; Main transcriptional regulatory genes cpx R, omp R, lrp, rpo E, rpo S; Cpx RA system induction gene cpx A and responsible for XCN deactivation gene xcn M, xcn N was decreased with the increase of p H. The expression of ack A, Lrh A, pax A, pax B, pax C, xab B, xab C, xab D, xab E, xnd reduced after rising first: on the whole, the expression quantity was increased. Compared with the condition at p H5.5, the expression of genes xcn A, isn A, isn B, isn C, pax A, pax B, pax C, pax T, xnd, nil R, rpo B, Lrh A and ack A was increased 1.46, 0.53, 2.18, 2.71, 0.88, 3.89, 3.68, 1.64, 8.65, 1.16, 4.07, 2.35 and 0.64 fold at p H8.5, respectively; The expression of genes cpx R, omp R, xcn M, xcn N, lrp, rpo E, rpo S, cpx A and cpx P was decreaced 0.64, 0.71, 0.4, 0.81, 0.87, 0.98, 0.998, 0.76, 0.92 fold at p H8.5, respectively.3. Using HPLC and LC-MS analysis metabolic profiling and the active substances relative content of ethylacetate extracts and methanol extracts under the condition of different p H had obvious differences. HPLC and LC-MS analysis showed that active substances content of ethylacetate extracts under the condition of different p H was different, especially to the content of Nematophin. At p H8.5, the content of Nematophin was more 2.04 fold than at p H5.5. LC-MS analysis showed that the main active substances XCN1 of the methanol extracts under the condition of different p H was also different. At p H8.5, the content of XCN1 was 15.37 fold more than that at p H5.5.In conclusion, with the increasing of p H, the activities of YL001 metabolites increasing was the cause of the rise of antimicrobial substances, biosynthesis gene expression, active substance content iand active material types increased.