| Gram-negative pathogenic bacteria have evolved various secretion systems,such as the type ?(T2SS),type ?(T3SS)and type ?(T4SS)secretion systems which span both the bacterial inner and outer membranes,to transfer virulence factors into host cells and cause disease.In the periplasmic space between the outer and inner membranes,there is a tight peptidoglycan(PG)layer,which is essential for preserving cell integrity by withstanding the turgor and maintaining a defined cell shape.However,the PG layer can also be a barrier impeding the assembly of the membrane-spanning apparatuses of the secretion systems.Therefore,the PG layer needs to be locally remodeled by PG-lytic enzymes to provide space and acceptor sites for the secretion apparatus assembly.Bacteria possess three main classes of PG lytic enzymes:the glycosidases that cleave the glycan backbone,the amidases that cleave the peptide side-chain,and the peptidases that cleave within the peptide side-chain.It is not clear whether any of the amidases or peptidases is involved in the secretion apparatus assembly.Xanthomonas campestris pv.campestris(Xcc),the causal agent of black rot disease of cruciferous crops,encodes four N-acetylmuramoyl-L-alanine amidases designated as AmiC1,AmiC2,AmpD,and AmiD,based on their sequence homology and structure similarity to E.coli amidases.This study investigated whether any of the amidases has an effect on Xcc cell division and pathogenicity.Mutants defective in amiCl,amiC2,ampD,or amiD were constructed and named amiC1::pK18,AamiC2,AampD,and AamiD,respectively.In addition,a mutant defective in both amiCl and amiC2 was also generated and named amiC 1::pK18amiC2.The mutant strains amiCl::pK18 and amiC1::pK18AamiC2 formed unsegment chained cells,while the strains AampD and AamiD displayed single short rod-shape cells similar to the wild type.The overwhelming majority cells of the mutant strain AamiC2 were similar to the wild type,but some short chains consisting of 3-4 cells were observed by light microscopy.Plant tests showed that the virulence by the mutants AampD,AamiD,and AamiC2 were similar to those produced by the wild type and hypersensitive response(HR)induced by AamiC2 were similar to the wild type;however,the virulence and HR caused by amiC1::pK18 and amiC 1::pK 18 AamiC2 were significantly reduced compared to the wild type.These results indicate that AmiC1 is crucial for Xcc cell division and virulence,and AmiC2 has a minor effect on Xcc cell separation.In this work,a recombinant 6×His-tagged truncated AmiCl protein without signal peptide(named His6-AmiCILN32)was created and produced in E.coli.The ability of His6-AmiC1LN32 to degrade PG in vitro was analyzed.The result showed that His6-AmiC1LN32 protein could only weakly degrade PG compared with the positive control mutanolysin.It indicated that AmiC1 may need an activator to enhance the PG degradation.Previous studies revealed that PG hydrolytic activity of the E.coli N-acetylmuramoyl-L-alanine amidases AmiA,AmiB and AmiC is activated by the LytM-family activators EnvC(AmiA and AmiB)and NlpD(AmiC).Xcc wild type strain 8004 contains 10 genes encoding putative LytM-family proteins and two of them share significant homology with the E.coli LytM factors NlpD and EnvC,respectively;therefor,they were named nlpD and envC,respectively.Observation of bacterial cells under light microscopy found that most cells of the mutants lacking NlpD or EnvC developed as unsegmented chains,while the rest of the mutants displayed single short rod-shape cells similar to the wild type.These data suggest that NlpD and EnvC play a key role in Xcc daughter cell separation.Plant tests showed that the virulence and HR caused by the mutant strain ?nlpD were significantly reduced compared to the wild type,while all of the other mutant strains including AenvC produced wild-type disease symptoms and HR.The facts that all of the mutants amiC1::pK18,?nlpD and ?envC were defective in cell division and that the mutants amiC1::pK18 and AnlpD but not AenvC showed a severe defect in virulence suggest that the loss of virulence for nlpD and amiC1 mutants is not associated with the defect in daughter cell separation.Recombinant 6×His-tagged truncated NlpD and EnvC proteins without signal peptide(named His6-NlpDLN22 and His6-EnvCLN14)were created and produced in E.coli.Both recombinant proteins His6-NlpDLN22 and His6-EnvCLN14 could not degrade PG and His6-NlpDLN22 could interact physically with PG.Further experiments showed that His6-NlpDLN22 but not His6-EnvCLN14 could enhance significantly PG degradation by His6-AmiC1LN32.These results indicate that the activity of Xcc AmiC1 to hydrolyze PG needs the activation of NlpD,similar to E.coli AmiC.The subcellular location of the NlpD protein in Xcc was determined in this study.A recombinant strain was constructed,which expressed NlpD protein with a 3 x Flag tag at the C-terminus by an nlpD gene fused with a 3xFlag tag-encoding sequence at the 3'-terminus in the genome of the wild type strain.The total,periplasmic,cytoplasmic,inner membrane and outer membrane protein fractions of the recombinant strain were analyzed by Western blotting.The result showed that NlpD protein presented in all of the protein fractions except the inner membrane fraction.Previous studies showed that the T3SS is essential for the virulence and HR induction of Xcc,and that the extracellular enzymes such as protease,endoglucanase,and amylase secreted by T2SS are important for full virulence of Xcc.The above results demonstrate that AmiC1 and NlpD are important for the virulence and HR induction of Xcc.To gain insight into the mechanism by which AmiC1 and NlpD influence Xcc virulence and HR induction,the secretion efficiency of the T2SS and T3SS of the mutant strains amiC1::pK18 and AnlpD was determined.The results showed that the activity of extracellular protease,amylase and endoglucanase produced by both of the mutants was similar to that of the wild type,while the type III secretion efficiency of both mutants was severely reduced compared to the wild type,indicating that both AmiC1 and NlpD have an effect on T3SS but not T2SS.Xcc T3SS is encoded by a cluster of hrp genes,which contains six operons(hrpA-hrpF).The expression of all of the hrp operons is directly controlled by an AraC-family transcriptional activator HrpX and the expression of hrpX is regulated by the HpaS/HrpG two-component regulatory system.Analysis by hrpG,hrpX,and hrpB promoter-gusA fusion reporters revealed that the transcriptional level of hrpG,hrpX and hrpF in the mutants amiC1::pK18 and AnlpD was similar to that in the wild type,suggesting that AmiC 1 and NlpD are not involved in the expression of hrp gene cluster and their influence on T3SS is not achieved via affecting the expression of hrp genes.In addition,this work found that deletion of Xcc nlpD could also reduce significantly the production of extracellular polysaccharide and cell motility but increase cell aggregation and sensitivity to pH.Overall,This work demonstrates that(i)the activity of Xcc amidase AmiC1 to hydrolyze PG needs the activation of the LytM-family regulator protein NlpD,(ii)PG hydrolysis mediated by the amidase AmiC and its activator NlpD is critical for not only cell separation but also virulence in Xcc,and(iii)AmiC/NlpD is involved in T3SS but not T2SS,suggesting that the cleavage of the peptide side-chain by AmiCl/NlpD may be a crucial action to remodel the PG layer for facilitation of the T3S apparatus assembly in Xcc. |
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