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The hypersensitive response (HR) of higher plants elicited by phytopathogenic bacteria is characterized by rapid cell collapse at infection sites and is associated with active defense (19). The hrp (HR and pathogenicity) genes of phytobacteria which are necessary for the HR are conserved among many gram-negative phytobacteria, including Pseudomonas syringae, Ralstonia (Pseudomonas) solanacearum, Xanthomonas campestris, Erwinia amylovora, Erwinia stewartii, and Erwinia chrysanthemi (1, 16). Based on their putative functions, the hrp gene products can be classified into three categories: (i) a delicate regulatory system, (ii) a type III secretion pathway, and (iii) extracellular or surface-associated proteins (24).

A 25-kb hrp-hrm cluster clone (pHIR11) isolated from P. syringae pv. syringae 61 allows saprophytes to cause the HR in tobacco, indicating that the cluster contains sufficient genes for HR elicitation (13). Nine hrp genes, which have been newly designated as hrc (HR and conserved) genes, are widely conserved in the type III secretion apparatus used by Yersinia, Shigella, Salmonella, Pseudomonas, Xanthomonas, and Erwinia spp. (5, 16). HrcC (= HrpH) is homologous to PulD, pIV, and other members of the outer membrane secretin superfamily of the general secretion pathway (1, 14, 30). The hrcJ (= hrpC) gene in the hrpZ operon encodes a putative lipoprotein with extensive similarity to YscJ and MxiJ (2, 15, 26). Eight of the Hrc proteins have additional homologues involved in flagellar biogenesis (1, 15). HrcJ is one of these and is homologous to FliF (15). At least three proteins, InvG, PrgH, and PrgK in Salmonella typhimurium, are identified from purified needle complexes, and InvG and PrgK are homologous to HrcC and HrcJ, respectively (21). Based on sequence conservation, these Hrc proteins might assemble into a supramolecular structure similar to the needle complex found in the S. typhimurium envelope. However, little is known about how many Hrc-Hrp proteins there are and the mechanism of how they are involved in assembly of the complex. In this report, we provide evidence for cellular locations of HrcC and HrcJ, based on cell fractionation with Triton X-100 extraction, sucrose-gradient isopycnic centrifugation, and cell surface immunogold labeling, to gain insight into the roles played by these two proteins in assembly of the type III secretion machinery. Also, our analysis of HrcJ has provided the first evidence for this class of proteins of an association with both membranes.

Biological functions of HrcJ and HrcC proteins. hrcJ and hrcC are the third genes of hrpZ and hrpC operons, respectively. For characterization of their individual function, nonpolar mutations were made by inserting an nptII gene, which lacks a rho-independent transcription terminator, in the coding region, and the resultant mutants are Pss61-N314 (hrcJ::nptII) and Pss61-N393 (hrcC::nptII) (3, 6). Both mutations were confirmed by DNA gel blot hybridization with nptII as a probe (data not shown). Intact hrcJ and hrcC genes were generated by PCR and cloned individually into pRK415 (18) for complementation. After infiltrating into tobacco leaves at 10⁸ CFU/ml, these two mutants were no longer able to elicit the HR, and their complementation clones can restore the ability for HR elicitation (data not shown). Immunoblot analysis with an anti-HrpZ serum was applied to determine the involvement of hrcJ and hrcC genes in harpin secretion. The HrpZ protein was detected in the cell pellet of Pss61-N314 and Pss61-N393, indicating that these mutants cannot secrete HrpZ, and their corresponding genes can restore the phenotype (data not shown). Those results reveal that HrcJ and HrcC proteins are indeed required for HR elicitation and harpin secretion.

Observation of surface localization of HrcJ and HrcC by electron microscopy. The question whether HrcJ and HrcC were localized on the outer membrane was firstly addressed by immunogold labeling and electron microscopy observation (7). Bacteria grown in 1 ml of Hrp-derepressing minimal medium (17) were harvested, washed with 1× phosphate-buffered saline (PBS), treated with Tris-EDTA (200 mM Tris-HCl [pH 7.4], 2 mM EDTA) for 1 h on ice (12), and then blocked with PBS-1% bovine serum albumin (BSA) for 1 h at room temperature. For cell surface immunogold labeling, anti-HrcC and anti-HrcJ immunoglobulins G (IgGs) were further purified from prepared antisera (6, 29), according to standard procedures (11). To remove nonspecific antibodies, purified anti-HrcJ and anti-HrcC IgG were preabsorbed with Tris-EDTA-treated corresponding mutants at a ratio of 30 µg of IgG to 0.2 mg of wet cell pellets in 250 µl of PBS-1% BSA solution. After prehybridization, an equal volume of fresh PBS-1% BSA and preabsorbed IgGs were mixed with the bacterial pellet, and the mixture was incubated overnight at 4°C. The protein-IgG complex was detected with protein A-gold conjugate (20-nm gold particles) (Zymed Laboratories, Inc., San Francisco, Calif.) under the conditions recommended by the supplier. The labeled bacteria were fixed for 2 h at 4°C with 50 to 100 µl of 1% osmium tetroxide (Merck, Frankfurt, Germany) and observed with a JEOL 200 CX electron microscope at 80 kV. The electron micrographs of P. syringae pv. syringae 61(pHIR11), Pss61-N314, and Pss61-N393 are shown in Fig. 1. The distribution of gold particles over the cell surface was essentially homogeneous for P. syringae pv. syringae 61(pHIR11), and no significant labeling can be seen in Pss61-N314 and Pss61-N393 mutants.

View larger version (39K): [in this window] [in a new window]   FIG. 1.   Immunoelectron microscopic localization of HrcC and HrcJ proteins at the cell surface of P. syringae pv. syringae 61(pHIR11) and nonpolar hrcC (Pss61-N393) and hrcJ (Pss61-N314) mutants. Bacteria were treated with anti-HrcC IgG (A and B) or anti-HrcJ IgG (C and D) and then detected with protein A-gold (20-nm) conjugate as described in the text. Bars, 1 µm.

HrcC is an outer membrane protein, and HrcJ is present in outer and inner membranes. To further investigate the cellular location of HrcJ and HrcC, we took a biochemical approach to examining their distribution by cellular fractionation and analyzed the fractions by using immunoblots visualized with HrcC or HrcJ antiserum. The procedure of sucrose-gradient isopycnic centrifugation was modified slightly from a previous protocol (28), and all steps were carried out at 4°C except where specifically stated otherwise. In brief, P. syringae pv. syringae 61(pHIR11) harvested from Hrp-derepressing minimal medium was resuspended in a solution containing 20% (wt/wt) sucrose, 10 µg of DNase per ml, 10 µg of RNase per ml, 10 mM HEPES (pH 7.4), and 1 mM phenylmethylsulfonyl fluoride (PMSF). These cells were disrupted by passage through a prechilled French pressure cell three times at 18,000 lb/in² and centrifuged at 1,000 to 2,000 × g for 20 min to remove unbroken cells. The pellet containing membrane proteins was obtained from ultracentrifugation (1 h at 100,000 × g), resuspended in 20% (wt/wt) sucrose-10 mM HEPES (pH 7.4)-5 mM EDTA, overlaid on top of 30 to 60% sucrose-gradient solutions, and ultracentrifuged at 274,000 × g for 40 h. After centrifugation and fractionation, each fraction was subjected to assays of refractive index (Abbe-3L Refractometer; Milton Roy Co., Rochester, N.Y.), NADH oxidase activity (27), and protein concentration (Pierce Coomassie protein assay reagent) and then precipitated with 5% trichloroacetic acid (TCA) for 1 h. The precipitated proteins were dissolved in 2× loading buffer (0.625 M Tris [pH 6.8], 2% sodium dodecyl sulfate [SDS], 10% glycerol, and 2% -mercaptoethanol) to a final concentration of 1 µg/µl and boiled for 5 min before SDS-polyacrylamide gel electrophoresis. A 10-µg sample of each fraction was applied to the gel, except that 20 µg was used for the detection of OprFan OmpA homologue in Pseudomonas sp.by an OmpA antibody (kindly provided by U. Henning of Max-Planck-Institut für Biologie, Tubingen, Germany). Each fraction was separated by SDS-8% (for HrcC) or SDS-10% (for HrcJ and OprF) polyacrylamide gel electrophoresis, transferred to an Immobilon-P membrane (Millipore Inc., Bedford, Mass.) in a TE70 semidry transfer unit (Hoefer Scientific Instruments, San Francisco, Calif.), and probed individually with anti-HrcC, anti-HrcJ, or anti-OmpA antibodies. Immunodetection was done by an alkaline phosphatase-based chemiluminescent assay with 0.25 mM disodium 2-chloro-5 (4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)-tricyclo(3.3.1.1) decan}-4-yl)-1-phenyl phosphate (CDP-Star; Boehringer Mannheim GmbH, Mannheim, Germany), and the results were quantified by a densitometer (Intelligent Quantifier; Bio Image). NADH oxidase activity and the presence of OprF were used as markers of inner and outer membranes, respectively. In Fig. 2, the majority of HrcC was detected within buoyant densities of 1.27 to 1.19 (fractions 2 to 18), whereas HrcJ was 1.2 to 1.17 (fractions 16 to 23). The distribution of HrcC and HrcJ in different fraction numbers reveals that these two proteins have different cellular localization. Moreover, HrcJ was found in both outer and inner membrane fractions at a ratio of 1:2, indicating that most HrcJ molecules were on the inner membrane.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Isopycnic centrifugation analysis of the total membrane preparation from P. syringae pv. syringae 61(pHIR11). The left vertical axis gives the units of NADH oxidase activity and the density of protein bands from immunoblots (determined by a laser Intelligent Quantifier densitometer). The right axis shows the sucrose buoyant density measured by a refractometer. The symbols are presented at the top.

View larger version (40K): [in this window] [in a new window]   FIG. 3.   Triton X-100 fractionation of P. syringae pv. syringae 61(pHIR11). S, soluble proteins (periplasmic plus cytoplasmic); I, inner membrane proteins; O, outer membrane proteins; NE, nonextracted proteins; P, phenol-denatured proteins. The phenol extraction procedure was described previously by Hancock and Nikaido (9). After each treatment, phenol-denatured proteins were recovered by high-speed centrifugation and finally resuspended in 2× loading buffer for SDS-polyacrylamide gel electrophoresis and immunoblot analyses.

Conclusion. In accordance with the association of HrcJ with both membranes and its similarity to the N terminus of FliF (15), HrcJ may be the major part of a core structure and may be involved in the primary assembly of the Hrp translocation system. In addition to HrcJ, several other Hrc proteins have homology with flagellar components (1). The needle structure isolated from the S. typhimurium type III secretion system (21) shows a structure similar to that of the flagellar export machinery, which further strengthens the intriguing possibility that HrcC (an InvG homologue) and HrcJ (a PrgK homologue) are both involved in forming a supramolecular structure in the bacterial envelope. This structural similarity between the type III and flagellar export systems also suggests that they might have similar mechanisms to assemble their components into a complex. Moreover, the type III systems in plant and animal pathogens are capable of delivering varioushomologous and heterologouseffector proteins to the interior of host cells, indicating that they share a conserved secretion mechanism (for reviews, see references 1 and 16 and the references therein). Given the use of similar secretion systems to secrete different effector proteins, we speculate that these systems may be functionally interchangeable among animal and plant pathogens. However, the fundamental difference between plant and animal cells brings up the key questions of (i) how bacteria use similar structures to translocate effector proteins into host cells with such different surfaces (cell wall versus no cell wall), (ii) how bacteria regulate the translocation process, and (iii) how effector proteins are involved in diseases caused by different pathogens.