Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity

Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGH operon of E. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus mdoGH of Escherichia coli. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the opgG or opgH gene abolish OPG synthesis. The opg mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.     

Identification of plant-inducible genes in Erwinia chrysanthemi 3937.

We present a method for identifying plant-inducible genes of Erwinia chrysanthemi 3937. Mutagenesis was done with the Mu dIIPR3 transposon, which carries a promoterless neomycin phosphotransferase gene (nptI), so upon insertion, the truncated gene can fuse to E. chrysanthemi promoters. Mutants containing insertions in plant-inducible genes were selected for their sensitivity to kanamycin on minimal plates and for their acquired resistance to this antibiotic when an S. ionantha plant extract was added to kanamycin minimal plates. The selection allowed the identification of E. chrysanthemi promoters inducible by host factors present in the S. ionantha plant extract. Using this method, we isolated 30 mutants and characterized 10 of them. Two mutants were defective in cation uptake, one was defective in the galacturonate degradation pathway, and another was altered in the production of the acidic pectate lyase. The functions of the other mutated genes are still unknown, but we show that most of them are involved in pathogenicity.     

hrp genes of Erwinia chrysanthemi 3937 are important virulence factors

We developed improved virulence assays for Erwinia chrysanthemi 3937 on African violet varieties and devised a new method for the construction of precise bacterial gene knockouts. These methods were tested by constructing mutations in genes suspected to be involved with plant interactions. The virulence of the hrpG and hrcC mutant strains (both gene products presumed to be involved in protein secretion) was greatly reduced on leaves of semitolerant African violet varieties. An hrpN mutant strain produced delayed symptoms on African violet leaves and an hrpN delta pel (delta pel = five major pectate lyase genes deleted) double mutant was nonpathogenic. The hrcC and hrpG mutants did not produce a rapid hypersensitive response (HR) in tobacco, unlike the wild-type bacterium, and the hrpN mutant gave a reduced HR. The results, therefore, establish the importance of hrp genes in the virulence of E. chrysanthemi and their ability to elicit HR on nonhosts. The data also suggest that other effector proteins secreted by the Hrp system are required for full virulence and HR elicitation.     

Regulation and role in pathogenicity of Erwinia chrysanthemi 3937 pectin methylesterase.

The gene pem, encoding the pectin methylesterase (PME) of Erwinia chrysanthemi 3937, was cloned and mutagenized by mini-Mu transposable elements. A second gene, pecY, which could act as a negative regulator of PME was found 5' to the pem gene. A PME-E. chrysanthemi derivative inoculate onto Saintpaulia plants was shown to be clearly noninvasive, demonstrating the important role of this enzyme in soft rot disease.     

The phytopathogen Dickeya dadantii (Erwinia chrysanthemi 3937) is a pathogen of the pea aphid

Dickeya dadantii (Erwinia chrysanthemi) is a phytopathogenic bacterium causing soft rot diseases on many crops. The sequencing of its genome identified four genes encoding homologues of the Cyt family of insecticidal toxins from Bacillus thuringiensis, which are not present in the close relative Pectobacterium carotovorum subsp. atrosepticum. The pathogenicity of D. dadantii was tested on the pea aphid Acyrthosiphon pisum, and the bacterium was shown to be highly virulent for this insect, either by septic injury or by oral infection. The lethal inoculum dose was calculated to be as low as 10 ingested bacterial cells. A D. dadantii mutant with the four cytotoxin genes deleted showed a reduced per os virulence for A. pisum, highlighting the potential role of at least one of these genes in pathogenicity. Since only one bacterial pathogen of aphids has been previously described (Erwinia aphidicola), other species from the same bacterial group were tested. The pathogenic trait for aphids was shown to be widespread, albeit variable, within the phytopathogens, with no link to phylogenetic positioning in the Enterobacteriaceae. Previously characterized gut symbionts from thrips (Erwinia/Pantoea group) were also highly pathogenic to the aphid, whereas the potent entomopathogen Photorhabdus luminescens was not. D. dadantii is not a generalist insect pathogen, since it has low pathogenicity for three other insect species (Drosophila melanogaster, Sitophilus oryzae, and Spodoptera littoralis). D. dadantii was one of the most virulent aphid pathogens in our screening, and it was active on most aphid instars, except for the first one, probably due to anatomical filtering. The observed difference in virulence toward apterous and winged aphids may have an ecological impact, and this deserves specific attention in future research.     

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.     

The GacA global regulator of Vibrio fischeri is required for normal host tissue responses that limit subsequent bacterial colonization

Harmful and beneficial bacterium-host interactions induce similar host-tissue changes that lead to contrasting outcomes of association. A life-long association between Vibrio fischeri and the light organ of its host Euprymna scolopes begins when the squid collects bacteria from the surrounding seawater using mucus secreted from ciliated epithelial appendages. Following colonization, the bacterium causes changes in host tissue including cessation of mucus shedding, and apoptosis and regression of the appendages that may limit additional bacterial interactions. We evaluated whether delivery of morphogenic signals is influenced by GacA, a virulence regulator in pathogens, which also influences squid-colonization by V. fischeri. Low-level colonization by a GacA mutant led to regression of the ciliated appendages. However, the GacA mutant did not induce cessation of mucus shedding, nor did it trigger apoptosis in the appendages, a phenotype that normally correlates with their regression. Because apoptosis is triggered by lipopolysaccharide, we examined the GacA mutant and determined that it had an altered lipopolysaccharide profile as well as an increased sensitivity to detergents. GacA-mutant-colonized animals were highly susceptible to invasion by secondary colonizers, suggesting that the GacA mutant's inability to signal the full programme of light-organ responses permitted the prolonged recruitment of additional symbionts.