Affect Of Pta-AckA Pathway On Growth And Metabolism Of Xenorhabdus Nematophila YL001

X. nematophila produce many kinds of secondary metabolites which exhibit differentchemical structure, but also broad bioactivities in medicine and agriculture, such asantibacterial, antifungal, pesticide, nematicide, anti-tumor and antivirus. Therefore, thebacteria could be exploited novel pesticides. As a global regulator, CpxR the responseregulator of two-component signal transduction system CpxA/CpxR (also called CpxRA)negatively regulate antibiotic activity of X nematophila. Pta-AckA pathway is primarymetabolism in bacterium, it interconverts Coenzyme A (CoASH), ATP and acetate withacetyl-Coenzyme A (acCoA), ADP and inorganic phosphate. The intermediate acetylphosphate has been proposed to donate phosphorus to CpxR, so that it could regulateexpression level of some genes.X nematophila YL001was isolated from Yangling Shaanxi province, which showed highantibiotic activity on some phytopathogens. To improve the antibiotic activity of the strainYL001and figure out how Pta-AckA pathway regulate the antibiotic activity, we designed toknockout the ackA (acetyl kinase) and pta (phosphotransacetylase) by homologousrecombinant, which Pta-AckA pathway was broken down. We also analyzed differencebetween gene deletion mutant strain ΔackA-pta and wild-type YL001in phenotype andgrowth by fermentation in flask and fermentation tank, metabolomic profiling by HPLC,antibiotic activity by bioassay. We researched relationship between Pta-AckA pathway andCpxRA by detecting cpxP expression in real-time quantification. The results list as follow:1. Fused fragments of ackA deletion and akcApta deletion was produced by overlap PCR,and ligation into suicide vector pDS132which named pDS132ackA and pDS132ackA-pta.pDS132ackA-pta was firstly transformed into E.coli S17-1by heat-shock transformation.Mutant Δ ackA-pta was constructed by homologous recombination with vectorpDS132ackA-pta, which was transformed into YL001by heat-shock. Wild type YL001changed irreversible after heat-shock, so we appoint heat-shock YL001as a mutant strain(HS-YL001).2. Mutant strain ΔackA-pta and HS-YL001exhibited different cultural characteristicscompared with wild-type YL001. HS-YL001and YL001strains is grey white colony, whilemutant ΔackA-pta is dark red colony and smaller. The mutant ΔackA-pta showed reduced viability compared with HS-YL001and YL001. The pH change state of three strains weresimilar, but the pH of mutant ΔackA-pta increased as a whole,and HS-YL001increasedmore. Wild-type YL001could synthesis reducing sugar, while mutant strains ΔackA-pta andHS-YL001could not or synthesis a bit reducing sugar.3. We determined the antibiotic activity of mutant strains ΔackA-pta, HS-YL001andwild-type YL001on in vitro phytopathogen Botrytis cinerea by methods of mycelia growthrate. The results showed that the antibiotic activity of ΔackA-pta increased1.28-fold ofHS-YL001and1.20-fold of wild-type YL001.4. We detected the ethyl acetate extract of mutant strains ΔackA-pta, HS-YL001andwild-type YL001culture by HPLC. Compared with YL001, the amount of3rd,18th,20th,24thminute retention time had changed in mutant strains ΔackA-pta, HS-YL001. The amount of3rdretention time of mutant strains ΔackA-pta increased in contrast HS-YL001.5. The genes expression of xcnA, xcnM, xcnN and cpxP was detected by qRT-PCRanalysis. The level of cpxP expression of ΔackA-pta is0.43-fold lower than its parent strainHS-YL001expression, which is12.64-fold greater than wild-type YL001. When add glucoseinto medium, The level of cpxP expression increased in strains HS-YL001and YL001, whilechange little in mutant strain ΔackA-pta.