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.     

The Erwinia chrysanthemi type III secretion system is required for multicellular behavior

Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of extracellular polysaccharide, cellulose, and protein filaments, known as curli. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here, we demonstrate that Erwinia chrysanthemi, which does not carry curli genes, requires the TTSS for pellicle formation. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found that hrpY, which encodes a two-component system response regulator homolog, is required for activity of hrpS, which encodes a sigma54-dependent enhancer-binding protein homolog. In turn, hrpS is required for activity of the sigma factor homolog hrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36 degrees C, even though TTSS genes were expressed at this temperature. We found that cellulose is a component of the E. chrysanthemi pellicle but that pellicle formation still occurs in a strain with an insertion in a cellulose synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors.     

Visualization of secreted Hrp and Avr proteins along the Hrp pilus during type III secretion in Erwinia amylovora and Pseudomonas syringae

Pili are required for protein and/or DNA transfer from bacteria to recipient plant or bacterial cells, based on genetic evidence. However, it has never been shown directly that the effector proteins or DNA are localized along or inside the pili in situ. Failure to visualize an association of effector proteins/DNA with pili is the central issue in the debate regarding the exact function of pili in protein and DNA transfer. In this study, a newly developed in situ immunogold labelling procedure enabled visualization of the specific localization of type III effector proteins of Erwinia amylovora and Pseudomonas syringae pv. tomato along the Hrp pilus, but not along the flagellum or randomly in the intercellular space. In contrast, PelE, a pectate lyase secreted via the type II protein secretion system, was not associated with the Hrp pilus. These results provide direct evidence that type III secretion occurs only at the site of Hrp pilus assembly and that the Hrp pilus guides the transfer of effector proteins outside the bacterial cell, favouring the 'conduit/guiding filament' model.     

Characterization and virulence properties of Erwinia chrysanthemi lipopolysaccharide-defective, phi EC2-resistant mutants.

Outer membrane alterations were characterized in spontaneous mutants of the Erwinia chrysanthemi 3937jRH, which were selected for resistance to bacteriophage phi EC2. All but one of the mutants analyzed were affected in their lipopolysaccharide (LPS) structure, lacking the entire heterogeneous region of apparent high molecular weight present in the wild-type E. chrysanthemi LPS. At least two 3937jRH mutants, one selected as phi EC2 resistant (RH6065) and the other previously selected (D. Expert and A. Toussaint, J. Bacteriol. 163:221-227, 1985) as bacteriocin resistant (R1456), were cross-resistant to bacteriophage Mu and had rough LPSs with an altered core structure. Two phi EC2r mutants (RH6053 and RH6065) were most severely affected in their outer membrane integrity and also lost their virulence on saintpaulia plants, although they still possessed normal extracellular levels of pectinolytic and cellulolytic activities. The two Mur mutants RH6065 and R1456 were also able to induce systemic resistance in the challenged plant. All the other phi EC2r mutants retained the virulence of 393jRH.     

Cloning and characterization of a phospholipase gene from Erwinia chrysanthemi EC16.

A single gene (plcA) was cloned from a cosmid library of Erwinia chrysanthemi EC16 DNA that encoded an extracellular phospholipase. The gene was subcloned and DNA sequence data showed the presence of a single open reading frame encoding a protein with a predicted size of 39kDa. The coding region was G+C-rich and the protein had a predicted basic isoelectric point. The protein showed no significant homology with others in the PIR library, including other phospholipases. When overexpressed in Escherichia coli cells, the plcA gene directed production of a c. 39kDa protein that was largely localized in the periplasm, but its N-terminal amino acid sequence was that of the native protein predicted from DNA sequence data. Unlike the wild-type bacterium, an E. chrysanthemi EC16 marker exchange mutant of the plcA gene did not secrete extracellular phospholipase activity into the medium. However, no detectable change was observed in terms of the virulence of the mutant strain on potato tubers or chrysanthemum stems.     

Secretion and regulation Hrp mutants of Erwinia amylovora trigger different responses in apple

The interaction between two Hrp mutants of Erwinia amylovora, the parental virulent strain, and a host plant (Malus sp.) was studied with apple seedlings and apple flowers, using inoculation procedures which reduce the possibility of injury. We showed that one hrp regulatory mutant protected apple tissue against the virulent strain to a greater degree than a secretion mutant. The dynamics of bacterial growth in this interaction indicated that the protection was associated with the inhibition of the multiplication of the virulent strain. The activity of two enzymes involved in plant defence responses, phenylalanine-ammonia lyase and guaiacol peroxidase, increased in leaves treated with the regulatory mutant, but not in the leaves treated with the secretion mutant. The role of regulatory genes of the hrp cluster in this interaction is discussed.     

Subset of hybrid eukaryotic proteins is exported by the type I secretion system of Erwinia chrysanthemi

Erwinia chrysanthemi exports degradative enzymes by using a type I protein secretion system. The proteases secreted by this system lack an N-terminal signal peptide but contain a C-terminal secretion signal. To explore the substrate specificity of this system, we have expressed the E. chrysanthemi transporter system (prtDEF genes) in Escherichia coli and tested the ability of this ABC transporter to export hybrid proteins carrying C-terminal fragments of E. chrysanthemi protease B. The C terminus contains six glycine-rich repeated motifs, followed by two repeats of the sequences DFLV and DIIV. Two types of hybrid proteins were assayed for transport, proteins with the 93-residue-protease-B C terminus containing one glycine-rich repeat and both hydrophobic terminal repeats and proteins with the 181-residue C terminus containing all repeat motifs. Although the shorter C terminus is unable to export the hybrids, the longer C terminus can promote the secretion of hybrid proteins with N termini as large as 424 amino acids, showing that the glycine-rich motifs are required for the efficient secretion of these hybrids. However, the secretion of hybrids occurs only if these proteins do not carry disulfide bonds in their mature structures. These latter results suggest that disulfide bond formation can occur prior to or during the secretion. Disulfide bonds may prevent type I secretion of hybrids. One simple hypothesis to explain these results is that the type I channel is too narrow to permit the export of proteins with secondary structures stabilized by disulfide bonds.     

Biogenesis, regulation, and targeting of the type III secretion system

The type III secretion system (T3SS) is employed by a number of Gram-negative bacterial pathogens to inject toxins into eukaryotic cells. The biogenesis of this complex machinery requires the regulated interaction between over 20 cytosolic, periplasmic, and membrane-imbedded proteins, many of which undergo processes such as polymerization, partner recognition, and partial unfolding. Elements of this intricate macromolecular system have been characterized through electron microscopy, crystallography, and NMR techniques, allowing for an initial understanding of the spatiotemporal regulation of T3SS-related events. Here, we report recent advances in the structural characterization of T3SS proteins from a number of bacteria, and provide an overview of recently identified small molecule T3SS inhibitors that could potentially be explored for novel antibacterial development.     

Erwinia chrysanthemi iron metabolism: the unexpected implication of the inner membrane platform within the type II secretion system

The type II secretion (T2S) system is an essential device for Erwinia chrysanthemi virulence. Previously, we reported the key role of the OutF protein in forming, along with OutELM, an inner membrane platform in the Out T2S system. Here, we report that OutF copurified with five proteins identified by matrix-assisted laser desorption ionization-time of flight analysis as AcsD, TogA, SecA, Tsp, and DegP. The AcsD protein was known to be involved in the biosynthesis of achromobactin, which is a siderophore important for E. chrysanthemi virulence. The yeast two-hybrid system allowed us to gain further evidence for the OutF-AcsD interaction. Moreover, we showed that lack of OutF produced a pleiotropic phenotype: (i) altered production of the two siderophores of E. chrysanthemi, achromobactin and chrysobactin; (ii) hypersensitivity to streptonigrin, an iron-activated antibiotic; (iii) increased sensitivity to oxidative stress; and (iv) absence of the FbpA-like iron-binding protein in the periplasmic fraction. Interestingly, outE and outL mutants also exhibited similar phenotypes, but, outD and outJ mutants did not. Moreover, using the yeast two-hybrid system, several interactions were shown to occur between components of the T2S system inner membrane platform (OutEFL) and proteins involved in achromobactin production (AcsABCDE). The OutL-AcsD interaction was also demonstrated by Ni(2+) affinity chromatography. These results fully confirm our previous view that the T2S machinery is made up of three discrete blocks. The OutEFLM-forming platform is proposed to be instrumental in two different processes essential for virulence, protein secretion and iron homeostasis.     

Molecular cloning, nucleotide sequence, and marker exchange mutagenesis of the exo-poly-alpha-D-galacturonosidase-encoding pehX gene of Erwinia chrysanthemi EC16.

The pehX gene encoding extracellular exo-poly-alpha-D-galacturonosidase (exoPG; EC 3.2.1.82) was isolated from a genomic library of the pectate lyase-deficient Erwinia chrysanthemi mutant UM1005 (a Nalr Kanr delta pelABCE derivative of EC16) by immunoscreening 2,800 Escherichia coli HB101 transformants with an antibody against exoPG protein. The cloned pehX gene was expressed highly from its own promoter in E. coli, and most of the enzyme was localized in the periplasm. The nucleotide sequence of pehX revealed the presence of an amino-terminal signal peptide and an open reading frame encoding a preprotein of 64,608 daltons. The cloned pehX gene was insertionally inactivated with TnphoA and used to mutate the chromosomal pehX gene of E. chrysanthemi AC4150 (Nalr) and CUCPB5006 (Nalr Kans delta pelABCE) by marker exchange mutagenesis. Analysis of the resulting mutants, CUCPB5008 (Pel+ Peh-) and CUCPB5009 (Pel- Peh-), indicated that exoPG can contribute significantly to bacterial utilization of polygalacturonate and the induction of pectate lyase in the presence of extracellular pectic polymers. CUCPB5009 retained a slight ability to pit polygalacturonate semisolid agar and macerated chrysanthemum pith tissues when large numbers of bacteria were inoculated.     

Self-regulation of pir, a regulatory protein responsible for hyperinduction of pectate lyase in Erwinia chrysanthemi EC16

Previously, we have cloned and characterized the pir (plant inducible regulator) gene, which is responsible for hyperinduction of the synthesis of an isozyme of pectate lyase (PLe) in Erwinia chrysanthemi EC16 in the presence of potato extract and sodium polypectate (NaPP). The Pir protein purified from Escherichia coli overexpressing pir is able to bind to the promoter region of pir as a dimer. Self-regulation of pir by its own translational product (Pir) was suggested from the findings that Pir binds at the promoter region of pir and that the hyperinduction of the pirlux construct in response to plant extract was observed only in pir+ but not in pir mutant EC16. Thus, hyperinduction of PLe was thought to be mainly due to overproduction of Pir. On the other hand, KdgR and PecS, which have been reported to be the major regulatory proteins for the synthesis of pectic enzymes, did not bind to the promoter region of pir. Thus, the regulation of Pir synthesis seems to be independent of KdgR and PecS. Also, its expression was insensitive to catabolite repression as predicted from failure of cyclic AMP (cAMP)-CRP (cAMP recognizing protein) to bind at the pir promoter region.     

Single-site chromosomal Tn5 insertions affect the export of pectolytic and cellulolytic enzymes in Erwinia chrysanthemi EC16

Exponentially growing cells of Erwinia chrysanthemi EC16 usually export about 98% of their pectate lyase (PL) and protease, about 40% of their polygalacturonase (PG), and about 60% of their cellulase (endoglucanase or carboxymethyl cellulase; CL). By using the R plasmid, pJB4JI (pPH1JI::Mu::Tn5), three independent Tn5 insertion mutants were obtained that exported normal levels of protease but 10% or less of PL, PG, and CL. Physical analysis revealed that single copies of Tn5 had inserted into the E. chrysanthemi chromosome, producing a similar export-defective (Out-) phenotype. The synthesis of PL, PG, and CL was not affected by the Tn5 insertions. These enzymes were released from the mutants on spheroplast formation, indicating that they were located in the periplasmic space. Tn5 insertions caused the loss of a 35-kilodalton periplasmic protein, but did not alter the outer membrane protein composition. The findings are discussed with respect to the current knowledge on protein export in gram-negative bacteria.