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The hypersensitive response elicitor harpin (HrpN) of soft rot pathogen Erwinia chrysanthemi strains 3937 and EC16 is secreted via the type III secretion system and remains cell surface bound. Strain 3937 HrpN is essential for cell aggregation, but the C-terminal one-third of the protein is not required for aggregative activity.

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 [[sigma].sup.54]-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.

The host-specific plant pathogen Pseudomonas syringae causes the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture through the Hrp secretion system. The hrpW gene was observed in a region adjacent to the hrp cluster in P. syringae pv. tomato DC3000. hrpW encodes a 42.9-kDa protein with domains similar to harpins and pectate lyases, respectively. HrpW has the main characteristics of harpins.

The modified distribution of the HrpZ hairpin protein in Pseudomonas syringae pv. syringae 61 hrp mutants was found to allow assignment of these four operons to two experimentally identifiable stages in type III protein secretion. The hrpJ and hrpU operons encode an alternative to the Sec system for translocation across the inner membrane, while the hrpC operon is needed for translocation across the outer membrane.

Three pre-defined DNA-based methods were applied to examine the isolates from diseased stems and tubers and understand most reliable classification of isolates through the multilocus sequence analysis (MLSA) method. Hypersensitive response and DNA hybridization assays revealed that strains of both Pectobacterium carotovorum and Pectobacterium wasabiae lack a type III secretion system (T3SS) and the virulence of most of the T3SS-deficient strains are similar to that of T3SS-encoding strains in stems and tubers.

The construction of null knockout indole-3-acetic acid (iaaM) mutant strain Erwinia chrysanthemi (Ech138) is presented. Compared with wild-type Ech3937, Ech138 exhibited reduced ability to produce local maceration, but its multiplication in Saintpaulia ionantha was unaffected.

A screen is developed to identify transposon mutants that are defective in attachment to alfalfa sprouts in order to understand the physiology of Salmonella enterica serovar Newport on plants. Results reveal that Salmonella enterica genes important for virulence in animal systems are also required for colonization of plants, a secondary host that could serve as a vector of Salmonella enterica from animal to animal.

Genetic and biochemical methods are used to examine the genomic density of the enterobacterial plant pathogen Erwina carotovora subsp. Carotovora. The results obtained with each method showed that E. carotovora subsp. Carotovora strains isolated from one ecological niche, potato plants, are surprisingly diverse compared to related pathogens.

Research has been conducted on plant-pathogenic bacterium Pseudomonas syringae B728a. The virulence factor of P. syringae pv. syringae B728a exchangeable effector locus has been investigated via construction of deletion mutation, and the avirulence activity and secretion of the putative effector protein have been tested and discussed.

We used genetic and biochemical methods to examine the genomic diversity of the enterobacterial plant pathogen Erwinia carotovora subsp, carotovora. The results obtained with each method showed that E. carotovora subsp, carotovora strains isolated from one ecological niche, potato plants, are surprisingly diverse compared to related pathogens. A comparison of 23 partial mdh sequences revealed a maximum pairwise difference of 10.49% and an average pairwise difference of 2.13%, values which are much greater than the maximum variation (1.81%) and average variation (0.75%) previously reported for Escherichia coli. Pulsed-field gel electrophoresis analysis of I-CeuI-digested genomic DNA revealed seven rrn operons in all E. carotovora subsp. carotovora strains examined except strain WPP17, which had only six copies. We identified 26 I-CeuI restriction fragment length polymorphism patterns and observed significant poiymorphism in fragment sizes ranging from 100 to 450 kb for all strains. We detected large plasmids in two strains, including the model strain E. carotovora subsp, carotovora 71. The two least virulent strains had an unusual chromosomal structure, suggesting that a particular pulsotype is correlated with virulence. To compare chromosomal organization of multiple enterobacterial genomes, several genes were mapped onto I-CeuI fragments. We identified portions of the genome that appear to be conserved across enterobacteria and portions that have undergone genome rearrangements. We found that the least virulent strain, WPPI7, failed to oxidize cellobiose and was missing several hrp and hrc genes. The unexpected variability among isolates obtained from clonal hosts in one region and in one season suggests that factors other than the host plant, potato, drive the evolution of this common environmental bacterium and key plant pathogen. [ABSTRACT FROM AUTHOR]

Pseudomonas syringae is a plant pathogen whose pathogenicity and host specificity are thought to be determined by Hop/Avr effector proteins injected into plant cells by a type III secretion system. P. syringae pv. syringae B728a, which causes brown spot of bean, is a particularly well-studied strain. The type llI secretion system in P. syringae is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hrp conserved) genes, which are clustered in a pathogenicity island with a tripartite structure such that the hrp/hrc genes are flanked by a conserved effector locus and an exchangeable effector locus (EEL). The EELs of P. syringae pv. syringae B728a, P. syringae strain 61, and P. syringae pv. tomato DC3000 differ in size and effector gene composition; the EEL of P. syringae pv. syringae B728a is the largest and most complex. The three putative effector proteins encoded by the P. syringae pv. syringae B728a EEL-HopPsyC, HopPsyE, and HopPsyV--were demonstrated to be secreted in an Hrp-dependent manner in culture. Heterologous expression of hopPsyC, hopPsyE, and hopPsyV in P. syringae pv. tabaci induced the hypersensitive response in tobacco leaves, demonstrating avirulence activity in a nonhost plant. Deletion of the P. syringae pv. syringae B728a EEL strongly reduced virulence in host bean leaves. EELs from nine additional strains representing nine P. syringae pathovars were isolated and sequenced. Homologs of avrPphE (e.g., hopPsyE) and hopPsyA were particularly common. Comparative analyses of these effector genes and hrpK (which flanks the EEL) suggest that the EEL effector genes were acquired by horizontal transfer after the acquisition of the hrp/hrc gene cluster but before the divergence of modern pathovars and that some EELs underwent transpositions yielding effector exchanges or point mutations producing effector pseudogenes after their acquisition. [ABSTRACT FROM AUTHOR]

Dickeya dadantii is a plant-pathogenic enterobacterium responsible for the soft rot disease of many plants of economic importance. We present here the sequence of strain 3937, a strain widely used as a model system for research on the molecular biology and pathogenicity of this group of bacteria. [ABSTRACT FROM AUTHOR]