Mechanism Of Phosphatidylcholine Synthase Gene On The Pathogenicity Of Pseudomonas Syringae Pv.Syringae

Phosphatidylcholine(PC),a characteristically eukaryotic membrane phospholipid,is present in only about 15%bacteria.Phosphatidylcholine in bacteria is crucial for virulence of various plant and animal pathogens.Nevertheless,the detailed molecular mechanism of the role of PC in the virulence of pathogenic bacteria is not fully understood.Pseudomonas syringae pv.syringae van Hall CFCC 1336(Pss 1336)is a common plant-pathogenic bacterium and was found to only use phosphatidylcholine synthase pathway(Pcs pathway)for its PC biosynthesis.Mutation of the pcs gene abolishes bacterial PC synthesis.In this study,we further analyzed the mechanism that phosphatidylcholine impacts the pathogenicity of Pss 1336.The major virulence of Pss 1336 involves two classes of necrosis-inducing lipodepsipeptide phytoxins called as syringomycins and syringopeptins and different effectors(often>25)injected into plant cells through Type-III secretion system(T3SS).To study the relation between PC and the virulence of lipodepsipeptide phytoxins,biological activities of lipodepsipeptide phytoxins were monitored using Chinese date(Ziziphus zizyphus)immature fruits and fungi(Aspergillus niger and Monascus rubber).It was revealed that the Pss 1336pcs-strain cannot induce necrosis of immaturate fruits of Ziziphus zizyphus,and release lipopeptide toxins against fungi,even adding the choline to the media.HPLC analysis also showed that the Pss 1336pcs-mutant markedly reduced its secretion of lipodepsipeptide phytoxins.Analysis of semi-quantitative RT-PCR revealed that PC presence or absence did not affect gene expressions of SyrD,PseABC and PseEF efflux systems at transcriptional leve.However,Western blotting assays found that PseE and PseF present only in the cytoplasmic fractions but undetectable in the membrane extract of the Pss 1336 pcs’ mutant.PC absence obviously interrupted the translocation of two membrane-associated proteins PseE and PseF from cytoplasm to cell membranes to form an intact PseEF efflux system in bacterial membranes.Failure to form PseEF efflux system could be a major factor for less lipopeptide-phytoxin secretion.Our results demonstrate that PC in bacterial membrane phospholipids plays an important role in maintaining physiological functions of PseEF efflux system.The effector proteins secreted by T3SS function as virulence factors for Pss 1336.To study the relation between PC and the secretion of T3SS,25 genes,including regulatory genes and those genes encoding protein components of T3SS,were cloned from the genomic DNA of Pss 1336.19 genes were expressed in E.coli,in which 16 proteins were successfully purified to homogeneous.Analysis by semi-quantitative RT-PCR showed that the transcriptional level of 34 genes including regulatory genes,protein-component genes and effector genes did not exhibit any significant difference(P>0.05)between the Pss 1336 wild type and the Pss 1336 pcs-mutant.Analysis by Western blotting showed that relative quantities of 14 T3SS-associated proteins were similar to those for the Pss 1336 wild type,except the increase of HrpR(P<0.05).Relative quantities of 7 T3SS-associated proteins were similar to those for the Pss 1336 wild type.In contrast,the relative quantity for HrpB,HrpE,HrpF and HrcJ in the membrane of 1336 pcs-mutant decreased approximately 10%as compared to the Pss 1336 wild type(P<0.05).36%protein components simultaneously decreased more than 10%in bacterial membranes,which resulted in forming an imperfect T3SS apparatus.Clearly,PC plays an important role in the assembly of T3SS apparatus in Pss1336.Our studies have already demonstrated the role of PC in the relationship of bacterial virulence and the dysfunction of secretion systems.