Genetic organization of the Pantoea stewartii subsp. stewartii hrp gene cluster and sequence analysis of the hrpA, hrpC, hrpN, and wtsE operons

The hrp/wts gene cluster of Pantoea stewartii subsp. stewartii is required for pathogenicity on sweet corn and the ability to elicit a hypersensitive response (HR) in tobacco. Site-directed transposon mutagenesis and nucleotide sequencing were used to identify hrp/wts genes within the left 20 kb of this cluster. Seventeen open reading frames (ORFs) comprise seven genetic complementation groups. These ORFs share homology with hrp and dsp genes from Erwinia amylovora, Erwinia chrysanthemi, and Pseudomonas syringae pathovars and have been designated, in map order, wtsF, wtsE, hrpN, hrpV, hrpT, hrcC, hrpG, hrpF, hrpE, hrpD, hrcJ, hrpB, hrpA, hrpS, hrpY, hrpX, and hrpL. Putative hrp consensus promoter sequences were identified upstream of hrpA, hrpF, hrpN, and wtsE. Expression of the hrpA, hrpC, and wtsE operons was regulated by HrpS. Transposon mutations in all of the hrp operons abolished pathogenicity and HR elicitation, except for the hrpN and hrpV mutants, which were still pathogenic. hrpS, hrpXY, and hrpL regulatory mutations abolished HrpN synthesis, whereas secretory mutations in the hrpC, hrpA, and hrpJ operons permitted intracellular HrpN synthesis. wtsEF mutants were not pathogenic but still produced HrpN and elicited the HR. wtsE encodes a 201-kDa protein that is similar to DspE in E. amylovora and AvrE in P. syringae pv. tomato, suggesting that this protein is a major virulence factor involved in the elicitation of water-soaked lesions.     

水稻条斑病菌hrcC、hpa3和hrpE基因表达不依赖hrpG和hrpX基因调控

摘要：【目的】决定水稻条斑病菌（Xanthomonas oryzae pv. oryzicola）在非寄主植物上激发过敏反应（hypersensitive response, HR）和在寄主水稻上致病性（pathogenicity）的hrp基因簇是受hrpG和hrpX基因调控的,但还不清楚hrpG和hrpX基因是否共同决定着所有hrp基因的表达。【方法】本文通过基因敲除方式获得了水稻条斑病菌的hrpG和hrpX基因的双突变体。【结果】烟草和水稻上测定结果显示,双突变体与单突变体一样,均在烟草上失去HR激发能力和丧失在水稻上的致病性;相应地,功能互补后双突变体恢复至野生表型。细菌在水稻悬浮细胞、hrp诱导培养基XOM3和营养丰富的培养基NB中生长后的RT-PCR结果显示,NB中hrp基因低水平表达,XOM3和水稻细胞能够高水平诱导hrp基因表达。无论何种生长条件,hrpG单突变体中hrcC、hrcT、hpa3和hrpE基因表达,而hpa1、hpa2、hpaB、hrcJ和hrpG基因不表达;hrpX单突变体中hpa2、hrcC、hpa3、hrpE和hrpG基因表达,而hpa1、hrcT、hpaB和hrcJ基因不表达;hrpG和hrpX双突变体中hrcC、hpa3和hrpE基因表达,而hpa1、hpa2、hpaB、hrcT、hrcJ和hrpG基因不表达。【结论】这提示,水稻条斑病菌的hrcC、hrpE和hpa3基因不受hrpG和hrpX基因单独或同时调控,而hrcT基因受HrpG调控。由此推测,水稻条斑病菌III型分泌系统关键组份的表达有可能通过另外的信号途径进行调控,这为进一步分析III型分泌途经的形成提供了线索。     

Analysis of Erwinia chrysanthemi EC16 pelE::uidA, pelL::uidA, and hrpN::uidA mutants reveals strain-specific atypical regulation of the Hrp type III secretion system

The plant pathogen Erwinia chrysanthemi produces a variety of factors that have been implicated in its ability to cause soft-rot diseases in various hosts. These include HrpN, a harpin secreted by the Hrp type III secretion system; PelE, one of several major pectate lyase isozymes secreted by the type II system; and PelL, one of several secondary Pels secreted by the type II system. We investigated these factors in E. chrysanthemi EC16 with respect to the effects of medium composition and growth phase on gene expression (as determined with uidA fusions and Northern analyses) and effects on virulence. pelE was induced by polygalacturonic acid, but pelL was not, and hrpN was expressed unexpectedly in nutrient-rich King's medium B and in minimal salts medium at neutral pH. In contrast, the effect of medium composition on hrp expression in E. chrysanthemi CUCPB1237 and 3937 was like that of many other phytopathogenic bacteria in being repressed in complex media and induced in acidic pH minimal medium. Northern blot analysis of hrpN and hrpL expression by the wild-type and hrpL::omegaCmr and hrpS::omegaCmr mutants revealed that hrpN expression was dependent on the HrpL alternative sigma factor, whose expression, in turn, was dependent on the HrpS putative sigma54 enhancer binding protein. The expression of pelE and hrpN increased strongly in late logarithmic growth phase. To test the possible role of quorum sensing in this expression pattern, the expI/expR locus was cloned in Escherichia coli on the basis of its ability to direct production of acyl-homoserine lactone and then used to construct expI mutations in pelE::uidA, pelL::uidA, and hrpN::uidA Erwinia chrysanthemi strains. Mutation of expI had no apparent effect on the growth-phase-dependent expression of hrpN and pelE, or on the virulence of E. chrysanthemi in witloof chicory leaves. Overexpression of hrpN in E. chrysanthemi resulted in approximately 50% reduction of lesion size on chicory leaves without an effect on infection initiation.     

Relative effects on virulence of mutations in the sap, pel, and hrp loci of Erwinia chrysanthemi

We constructed strains of Erwinia chrysanthemi EC16 with multiple mutations involving three virulence systems in this bacterium, namely pel (coding for the major pectate lyases pelABCE), hrp (hypersensitive response and pathogenicity), and sap (sensitivity to antimicrobial peptides). The relative effects on virulence of those mutations have been analyzed on potato tubers and chicory leaves. In potato tubers, the sap mutation (BT105) had a greater effect in the reduction of the virulence than the pel (CUCPB5006) and hrp (CUCPB5039) mutations. This reduction was similar to that observed in the pel-hrp double mutant (CUCPB5037). The analysis of the strains affected in Pel-Sap (BT106), Hrp-Sap (BT107), and Pel-Hrp-Sap (BT108) suggested that the effects of these mutations are additive. In chicory leaves, the mutation in the sap locus appeared to have a greater effect than in potato tubers. The competitive indices of strains BT105, UM1005 (Pel-), CUCPB5039, and CUCPB5037 have been estimated in vivo and in vitro. These results indicate that the mutation in the hrp locus can be complemented in vivo by coinfection, whereas the mutations in pel and sap cannot.     

Use of a pooled transposon mutation grid to demonstrate roles in disease development for Erwinia carotovora subsp. atroseptica putative type III secreted effector (DspE/A) and helper (HrpN) proteins

Soft rot Erwinia spp., like other closely related plant pathogens, possess a type III secretion system (TTSS) (encoded by the hrp gene cluster) implicated in disease development. We report the sequence of the entire hrp gene cluster and adjacent dsp genes in Erwinia carotovora subsp. atroseptica SCRI1039. The cluster is similar in content and structural organization to that in E. amylovora. However, eight putative genes of unknown function located within the E. carotovora subsp. atroseptica cluster do not have homologues in the E. amylovora cluster. An arrayed set of Tn5 insertional mutants (mutation grid) was constructed and pooled to allow rapid isolation of mutants for any given gene by polymerase chain reaction screening. This novel approach was used to obtain mutations in two structural genes (hrcC and hrcV), the effector gene dspE/A, and the helper gene hrpN. An improved pathogenicity assay revealed that these mutations led to significantly reduced virulence, showing that both the putative E. carotovora subsp. atroseptica TTSS-delivered effector and helper proteins are required for potato infection.     

The ybiT gene of Erwinia chrysanthemi codes for a putative ABC transporter and is involved in competitiveness against endophytic bacteria during infection

We investigated the role in bacterial infection of a putative ABC transporter, designated ybiT, of Erwinia chrysanthemi AC4150. The deduced sequence of this gene showed amino acid sequence similarity with other putative ABC transporters of gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa, as well as structural similarity with proteins of Streptomyces spp. involved in resistance to macrolide antibiotics. The gene contiguous to ybiT, designated as pab (putative antibiotic biosynthesis) showed sequence similarity with Pseudomonas and Streptomyces genes involved in the biosynthesis of antibiotics. A ybiT mutant (BT117) was constructed by marker exchange. It retained full virulence in potato tubers and chicory leaves, but it showed reduced ability to compete in planta against the wild-type strain or against selected saprophytic bacteria. These results indicate that the ybiT gene plays a role in the in planta fitness of the bacteria.     

The Erwinia chrysanthemi EC16 hrp/hrc gene cluster encodes an active Hrp type III secretion system that is flanked by virulence genes functionally unrelated to the Hrp system

Erwinia chrysanthemi is a host-promiscuous plant pathogen that possesses a type III secretion system (TTSS) similar to that of the host-specific pathogens E. amylovora and Pseudomonas syringae. The regions flanking the TTSS-encoding hrp/hrc gene clusters in the latter pathogens encode various TTSS-secreted proteins. DNA sequencing of the complete E. chrysanthemi hrp/hrc gene cluster and approximately 12 kb of the flanking regions (beyond the previously characterized hecA adhesin gene in the left flank) revealed that the E. chrysanthemi TTSS genes were syntenic and similar (>50% amino-acid identity) with their E. amylovora orthologs. However, the hrp/hrc cluster was interrupted by a cluster of four genes, only one of which, a homolog of lytic transglycosylases, is implicated in TTSS functions. Furthermore, the regions flanking the hrp/hrc cluster lacked genes that were likely to encode TTSS substrates. Instead, some of the genes in these regions predict ABC transporters and methyl-accepting chemotaxis proteins that could have alternative roles in virulence. Mutations affecting all of the genes in the regions flanking or interrupting the hrp/hrc cluster were constructed in E. chrysanthemi CUCPB5047, a mutant whose reduced pectolytic capacity can enhance the phenotype of minor virulence factors. Mutants were screened in witloof chicory leaves and then in potato tubers and Nicotiana clevelandii seedlings. Mu dII1734 insertion in one gene, designated virA, resulted in strongly reduced virulence in all three tests. virA is immediately downstream of hecA, has an unusually low G+C content of 38%, and predicts an unknown protein of 111 amino acids. The E. chrysanthemi TTSS was shown to be active by its ability to translocate AvrPto-Cya (a P. syringae TTSS effector fused to an adenylate cyclase reporter that is active in the presence of eukaryote calmodulin) into N. benthamiana leaf cells. However, VirA(1-61)-Cya was not translocated into plant cells, and virA expression was not affected by mutations in E. chrysanthemi Hrp regulator genes hrpL and hrpS. Thus, the 44-kb region of the E. chrysanthemi EC16 genome that is centered on the hrplhrc cluster encodes a potpourri of virulence factors, but none of these appear to be a TTSS effector.     

Marker-exchange mutagenesis of a pectate lyase isozyme gene in Erwinia chrysanthemi.

The phytopathogenic enterobacterium Erwinia chrysanthemi contains pel genes encoding several different isozymes of the plant-tissue-disintegrating enzyme pectate lyase (PL). The pelC gene, encoding an isozyme with an approximate isoelectric point of 8.0, was mutagenized by a three-step procedure involving (i) insertional inactivation of the cloned gene by ligation of a kan-containing BamHI fragment from pUC4K with a partial Sau3A digest of E. chrysanthemi pelC DNA in pBR322; (ii) mobilization of the pBR322 derivative from Escherichia coli to E. chrysanthemi by the helper plasmids R64drd11 and pLVC9; and (iii) exchange recombination of the pelC::kan mutation into the E. chrysanthemi chromosome by selection for kanamycin resistance in transconjugants cultured in phosphate-limited medium (which renders pBR322 unstable). The resulting E. chrysanthemi mutant was Kanr Amps, lacked pBR322 sequences, and was deficient in only one of the four major PL isozymes, PLc, as determined by activity-stained isoelectric-focusing polyacrylamide gels. The rates of PL induction and cell growth in a medium containing polygalacturonic acid as the sole carbon source were not significantly reduced in the mutant. No difference was detected in the ability of the mutant to macerate potato tuber tissue. The evidence suggests that this isozyme is not necessary for soft-rot pathogenesis.     

An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in gram-negative bacteria by marker exchange-eviction mutagenesis

A technique for marker exchange-eviction mutagenesis that enables the construction of directed, unmarked mutations in Gram-negative bacteria was demonstrated in Erwinia chrysanthemi. The technique employs an nptI-sacB-sacR cartridge that is carried on a 3.8-kb BamHI fragment and confers kanamycin (Km) resistance and sucrose sensitivity (due to the production of levansucrase by sacB) in E. chrysanthemi. The cartridge was inserted into a Sau3A site in a cloned E. chrysanthemi pelC gene (encoding pectate lyase isozyme PLc) and then introduced into the Erwinia genome by gene exchange recombination. The resulting mutant was KmR, sucrose-sensitive, and PLc-deficient. The cartridge was then excised from the plasmid-borne pelC gene by PstI cleavage to leave a 28-bp frame-shifting insertion. The pelC allele containing the 28-bp insertion was exchanged for the chromosomal allele containing the nptI-sacB-sacR cartridge by selection for sucrose tolerance. The resulting E. chrysanthemi mutant was Kms and PLc-deficient. The technique permits the construction of complex strains with many directed mutations without the introduction of a corresponding number of antibiotic resistance markers and should prove useful, for example, in exploring the role of the multiple pel genes in E. chrysanthemi.