Angela V. Smirnova

Component and protein domain exchange analysis of a thermoresponsive, two-component regulatory system of Pseudomonas syringae

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.

A temperature-sensing histidine kinase: function, genetics, and membrane topology.

Two-component systems provide a means for bacteria to sense and adapt to environmental signals in order to survive in a continuously changing environment. Understanding of the mechanism by which these systems function is important in combating bacterial infections because many bacterial two-component systems are associated with virulence. The plant pathogenic bacterium Pseudomonas syringae pv. glycinea PG4180 synthesizes high levels of the phytotoxin coronatine at the virulence-promoting temperature of 18 degrees , but not at 28 degrees , the optimal growth temperature. Temperature-dependent coronatine biosynthesis is regulated by a modified two-component system, consisting of the response regulator, CorR, the histidine protein kinase CorS, and a third component, CorP. To elucidate the mechanism by which CorRSP functions, genetic, transcriptional, and biochemical analyses were applied, including in vitro and in planta reporter gene analysis, mRNA quantification, protein expression, mutagenesis, and membrane topology analysis. A combination of these techniques helped to elucidate, to a considerable extent, the temperature-sensing activity of CorS, which seems to act as a membrane-bound molecular thermometer.

Coronatine Gene Expression In Vitro and In Planta, and Protein Accumulation During Temperature Downshift in Pseudomonas syringae

The plant pathogenic bacterium Pseudomonas syringae PG4180 synthesizes high levels of the phytotoxin coronatine (COR) at the virulence-promoting temperature of 18 °C, but negligible amounts at 28 °C. Temperature-dependent COR gene expression is regulated by a modified two-component system, consisting of a response regulator, CorR, the histidine protein kinase CorS, and a third component, termed CorP. We analyzed at transcriptional and translational levels the expression of corS and the cma operon involved in COR biosynthesis after a temperature downshift from 28 to 18 °C. Expression of cma was induced within 20 min and increased steadily whereas corS expression was only slightly temperature-dependent. Accumulation of CmaB correlated with accumulation of cma mRNA. However, cma transcription was suppressed by inhibition of de novo protein biosynthesis. A transcriptional fusion of the cma promoter to a promoterless egfp gene was used to monitor the cma expression in vitro and in planta. A steady induction of cma::egfp by temperature downshift was observed in both environments. The results indicate that PG4180 responds to a temperature decrease with COR gene expression. However, COR gene expression and protein biosynthesis increased steadily, possibly reflecting adaptation to long-term rather than rapid temperature changes.

Site-directed mutagenesis of the temperature-sensing histidine protein kinase CorS from Pseudomonas syringae

Several plant pathogenic bacteria belonging to the species Pseudomonas syringae produce the phytotoxin coronatine to enhance their virulence. Pseudomonas syringae pv. glycinea PG4180 synthesizes coronatine at the virulence-promoting temperature of 18 degrees C, but not at 28 degrees C, its optimal growth temperature. In contrast, temperature has virtually no effect on coronatine synthesis in P. syringae pv. tomato strain DC3000. A modified two-component system controlling coronatine synthesis and consisting of the histidine protein kinase (HPK), CorS, the response regulator, CorR, and a third essential component, CorP, had been identified previously in both strains. CorS had been identified previously as a potential thermo-sensor. Comparison of the amino acid sequences of the HPKs from the two organisms revealed distinct differences. Site-directed mutagenesis of CorS from PG4180 was used to identify amino acyl residues potentially important for temperature signal perception. Point mutations and combinations of these were introduced into corS of PG4180 to generate corS variants with increased similarities to the respective allele from strain DC3000. These mutations resulted in either loss of activity, increase of thermoresponsiveness, or had no effect on CorS activity. Although none of the introduced mutations resulted in a clear conversion of CorS activity from thermo-responsive to temperature-independent, amino acyl residues important for temperature-dependent CorS activity and coronatine biosynthesis were identified.

Impact of Temperature on the Regulation of Coronatine Biosynthesis in Pseudomonas syringae

The plant pathogenic bacterium Pseudomonas syringae pv. glycinea PG4180 synthesizes high levels of the phytotoxin coronatine (COR) at the virulence- promoting temperature of 18°C, but only low amounts at 28°C, the optimal growth temperature. The temperature-dependent COR gene expression is regulated by a modified two-component system, consisting of the histidine protein kinase CorS, a response regulator, CorR, and a third functionally essential protein, CorP. We ana- lyzed at the transcriptional and translational level the expression of corS and the cma operon involved in COR biosynthesis, after a temperature downshift from 28°C to 18°C. The synthesis of cma mRNA was induced within 20 min and then increased steadily and gradually in the 14 h following the shift to 18°C. The synthe- sis of corS mRNA was induced to a lesser extent by the temperature downshift. The induction of cma expression was a result of accelerated transcription rather than increased stability of the cma transcript at 18°C. Accumulation of the COR bio- synthetic protein CmaB correlated with accumulation of cma mRNA. However, cma transcription was suppressed by inhibition of de novo protein biosynthesis.

Strain-specific Sequence Alterations in the Gene Encoding the Histidine Protein Kinase CorS Might be Responsible for Temperature-dependent Production of the Phytotoxin Coronatine by Pseudomonas syringae

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, which is the causal agent of bacterial blight of soybean. COR is produced at 18°C but not at 28°C. It is assumed that CorS responds to a temperature change and subsequently transducer the signal to its cognate response regulator(s), which in turn facilitates transcriptional activation of COR biosynthetic genes. Based on results of experiments with translational fusions between CorS and either alkaline phosphatase (PhoA) or (3-galactosidase (LacZ), CorS possesses six membrane-spanning domains and the location inside or outside of the membrane of certain regions of CorS is altered in dependence of temperature. The genome sequencing project of P. syringae DC3000 has revealed the presence of the CorRSP system in this strain as well. However, DC3000 produces significantly less COR than does PG4180, and the production is temperature-independent. We analysed COR production of various P. syringae strains at 18°C and 28°C and also deduced amino acid sequences of CorS from COR-producing strains. We suggest that PG4180-specific sequence alterations in the sequence of the temperature sensor kinase-encoding gene corS might be responsible for thermoresponsive production. We performed site-directed mutagenesis of CorS from PG4180 by introducing mutations identified in CorS of DC3000. Each of these mutations resulted in a change of the thermoresponsive phenotype for CorS of PG4180.

Temperature-Mediated Differential Gene Expression in Plant Pathogenic Bacteria

Plant pathogenic bacteria cause numerous devastating diseases in worldwide important crops thereby drawing increasing attention to the improved understanding of their infection processes. Phytopathogens, like our model organism, the gram-negative bacterium Pseudomonas syringae, encounter a number of environmental signals during the course of infection of their respective host plants. Foliar plant pathogens causing leaf spot diseases depend on high humidity and the proper temperature conditions to successfully infect the host plant, to rapidly overcome plant defense mechanisms, and to establish in planta population sizes which allow intense disease development. For many P. syringae pathovars high humidity and low temperatures have been shown to foster disease outcome. In analogy to human and animal pathogens that recognize the interior of their respective host organisms by a number of environmental signals, P. syringae might detect the proper conditions for bacterial infection and survival in its host plants. With respect to temperature it has been well established that human and animal pathogenic bacteria express virulence factors at 37–40°C but not at 25–30°C. It has been proposed that by means of temperature regulation - these pathogens economize gene expression: The bacteria synthesize virulence factors preferably at the time point and location when and where they are actually needed - in the interior of the warm-blooded host.