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Two closely related phytopathogenic bacterial strains, Pseudomonas syringae pv. glycinea PG4180 and P. syringae pv. tomato DC3000, produce the chlorosis-inducing phytotoxin coronatine (COR) in a remarkably divergent manner. PG4180 produces COR at the virulence-promoting temperature of 18 degrees C, but not at 28 degrees C. In contrast, temperature has no effect on COR synthesis in DC3000. A modified two-component system consisting of the histidine protein kinase (HPK), CorS, the response regulator (RR), CorR, and a third component, CorP, governs COR biosynthesis in both strains. A plasmid-based component and domain swapping approach was used to introduce different combinations of RRs, HPKs and hybrid HPKs into corS mutants of both strains. Subsequently, expression levels of the COR biosynthetic cma operon were determined using RNA dot-blot analysis, suggesting that CorRSP of PG4180 mediates a thermoresponsive phenotype dependent on the genomic background of each strain. The reciprocal experiment demonstrated a loss of temperature dependence in the corS mutant of PG4180. The presence of corR from PG4180 led to more pronounced cma expression in DC3000 and was associated with thermoresponsiveness, while corS of PG4180 did not mediate a temperature-dependent phenotype in the DC3000 mutant containing native corR and corP. These findings were substantiated by RT-PCR experiments. The C-terminal domain of CorS of PG4180 mediated thermosensing, while the N terminus did not respond to temperature changes, suggesting cytosolic perception of the temperature signal.

Exopolysaccharides (EPSs) play important roles in the attachment of bacterial cells to a surface and/or in building and maintaining the three-dimensional, complex structure of bacterial biofilms. To elucidate the spatial distribution and function of the EPSs levan and alginate during biofilm formation, biofilms of Pseudomonas syringae strains with different EPS patterns were compared. The mucoid strain PG4180.muc, which produces levan and alginate, and its levan- and/or alginate-deficient derivatives all formed biofilms in the wells of microtitre plates and in flow chambers. Confocal laser scanning microscopy with fluorescently labelled lectins was applied to investigate the spatial distribution of levan and an additional as yet unknown EPS in flow-chamber biofilms. Concanavalin A (ConA) bound specifically to levan and accumulated in cell-depleted voids in the centres of microcolonies and in blebs. No binding of ConA was observed in biofilms of the levan-deficient mutants or in wild-type biofilms grown in the absence of sucrose as confirmed by an enzyme-linked lectin-sorbent assay using peroxidase-linked ConA. Time-course studies revealed that expression of the levan-forming enzyme, levansucrase, occurred mainly during early exponential growth of both planktonic and sessile cells. Thus, accumulation of levan in biofilm voids hints to a function as a nutrient storage source for later stages of biofilm development. The presence of a third EPS besides levan and alginate was indicated by binding of the lectin from Naja mossambica to a fibrous structure in biofilms of all P. syringae derivatives. Production of the as yet uncharacterized additional EPS might be more important for biofilm formation than the syntheses of levan and alginate.

A modified two-component regulatory system consisting of two response regulators, CorR and CorP, and the histidine protein kinase CorS, regulates the thermoresponsive production of the phytotoxin coronatine (COR) in Pseudomonas syringae PG4180. COR is produced at the virulence-promoting temperature of 18 °C, but not at 28 °C, the optimal growth temperature of PG4180. Assuming that the highly hydrophobic N-terminus of CorS might be involved in temperature-signal perception, the membrane topology of CorS was determined using translational phoA and lacZ fusions, leading to a topological model for CorS with six transmembrane domains (TMDs). Interestingly, three PhoA fusions located downstream of the sixth TMD showed a thermoresponsive phenotype. Enzymic activity, immunoblot, and protease-sensitivity assays were performed to localize the CorS derivatives, to analyse the expression level of hybrid proteins and to examine the model. In-frame deletions of the last four, or all six TMDs gave rise to non-functional CorS. The results indicated that the transmembrane region is important for CorS to function as a temperature sensor, and that the membrane topology of CorS might be involved in signal perception.

Plant-pathogenic bacteria may sense variations in environmental factors, such as temperature, to adapt to plant-associated habitats during pathogenesis or epiphytic growth. The bacterial blight pathogen of soybean, Pseudomonas syringae pv. glycinea PG4180, preferentially produces the phytotoxin coronatine at 18 degrees C and infects the host plant under conditions of low temperature and high humidity. A miniTn5-based promoterless glucuronidase (uidA) reporter gene was used to identify genetic loci of PG4180 preferentially expressed at 18 or 28 degrees C. Out of 7500 transposon mutants, 61 showed thermoregulated uidA expression as determined by a three-step screening procedure. Two-thirds of these mutants showed an increased reporter gene expression at 18 degrees C whilst the remainder exhibited higher uidA expression at 28 degrees C. MiniTn5-uidA insertion loci from these mutants were subcloned and their nucleotide sequences were determined. Several of the mutants induced at 18 degrees C contained the miniTn5-uidA insertion within the 32.8 kb coronatine biosynthetic gene cluster. Among the other mutants with increased uidA expression at 18 degrees C, insertions were found in genes encoding formaldehyde dehydrogenase, short-chain dehydrogenase and mannuronan C-5-epimerase, in a plasmid-borne replication protein, and in the hrpT locus, involved in pathogenicity of P. syringae. Among the mutants induced at 28 degrees C, insertions disrupted loci with similarities to a repressor of conjugal plasmid transfer, UV resistance determinants, an isoflavanoid-degrading enzyme, a HU-like DNA-binding protein, two additional regulatory proteins, a homologue of bacterial adhesins, transport proteins, LPS synthesis enzymes and two proteases. Genetic loci from 13 mutants did not show significant similarities to any database entries. Results of plant inoculations showed that three of the mutants tested were inhibited in symptom development and in planta multiplication rates. Temperature-shift experiments suggested that all of the identified loci showed a rather slow induction of expression upon change of temperature.