Bilological Functionsof 3-ketoacyl-ACP Reductase In Xanthomonas Campestris Pv.campestris

Xanthomonas campestris pv.campestris(Xcc)is a causal agent of the black rot disease of cruciferous plants,which is one of the most destructive diseases and causes severe economic loss across the world.Although Xcc genome encodes the entire gene cluster for fatty acids biosynthesis,little is known about the fatty acids synthetic mechanism in Xcc,especially the relationship of fatty acids synthase system with quorum sensing(QS)system.So the physiological functions of 3-ketoacyl-ACP reductase were studied in this research.The research results were as follows:Firstly,XCC1018(named as XccfabG1)and was XCC0416(named as XccfabG2)found in the genome of ATCC33913 by homology alignment with E.coli fabG.XccfabG1 and XccfabG2 was cloned to an expression vector and then transferred into Escherichia coli fabG temperature sensitive(TS)strain CL104.The result indicated that XccFabG1 encoded by XccfabG1 had the activity of 3-ketoacyl-ACP reductase.XccfabG2 could not complement CL104 by itself,but with Vibrio harveyi aasS gene(or XccfabH gene)and additional octanoic acid or hexanoic acid,XccfabG2 could complement CL104.Furthermore,the labeled fatty acids profile showed that XccfabG1 complemented strain as well as the double transformed XccfabG2 with additional octanoic acid produced saturated and unsaturated fatty acid.Secondly,XccfabG1 and XccfabG2 gene were cloned to vector pET-28 b,expressed in E.coli BL21(DE3),and purified by Ni-NTA.The activities of XccFabG1 and XccFabG2 were analysed in vitro,and the results showed that XccFabG1 could utilize all 3-ketoacyl-ACPs.However,XccFabG2 showed very low activity to 3-ketobutyryl-ACP,but it could utilize the middle chain and long chain 3-ketoacyl-ACPs.Catalytic activity of XccFabG1 to was 3-ketobutyryl-ACP 14 times higher than XccFabG2.But XccFabG2 had higher activities to 3-ketodecanoly-ACP and 3-ketododecanoly-ACP compared to XccFabG1.Thirdly,it was failed to get the XccfabG1 disrupted mutant,until overexpressed XccfabG2 and additional octanoic acid were added.This proved that XccfabG1 is one key gene for Xcc,while the XccfabG2 mutant was successfully got.XccfabG1 mutant could only grow on the NYG medium with the addition of octanoic acid or octanoic acid or hexanoic acid,while XccfabG2 mutant did not show any difference compared to wild type.The expression level of XccfabG2 was much lower than XccfabG1,which made the XccfabG1 to be an essential gene in Xcc.The fatty acid composition of XccfabG1 and XccfabG2 mutants had changed,but not significantly.Lastly,the production of DSF and BDSF of XccfabG2 mutant declined to 25% and 55%,respectively.The production of complement strain of XccfabG2 mutant increased to the wild type level.This indicated that XccfabG2 was involved in the DSF synthases.The promoter region of XccfabG2 could be bind by the global regulate protein Clp.The expression level of XccfabG2 increased in clp mutant,and declined to wild type level in the complemented strain.In the rpfF rpfG and rpfC mutant stain,the expression level of XccfabG2 also increased.To summaries,XccfabG1 is the essential gene for Xanthomonas campestris pv.Campestris ATCC33913,and plays an important role in the fatty acid synthases system as a 3-ketoacyl-ACP reductase.XccFabG2 is a new kind of 3-ketoacyl-ACP reductase,which is functional in middle chain or long chain 3-ketoacyl-ACP reductase.Although the fatty acid composition of XccfabG2 mutant changed;XccfabG2 is not the major 3-ketoacyl-ACP reductase in ATCC33913.Maybe,XccfabG2 plays a role in the DSF synthase.