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.     

Biosynthesis of coronatine,a thermoregulated phytotoxin produced by the phytopathogen Pseudomonas syringae

Coronatine (COR) is a non-host-specific phytotoxin that is produced by several different pathovars in the species Pseudomonas syringae. COR consists of two distinct components: coronafacic acid (CFA), which is synthesized via the polyketide pathway, and coronamic acid (CMA), a cyclized derivative of isoleucine. Both CFA and CMA function as intermediates in the pathway to COR and must be joined together by an amide bond to form the phytotoxin. Although the mode of action for COR remains obscure, the CFA moiety is a structural and functional analogue of jasmonic acid, a compound that is produced in a variety of plants in response to stress. The COR biosynthetic gene cluster generally occurs on large plasmids in P. syringae, an observation that helps to explain the production of COR by multiple pathovars. Mutagenesis, feeding studies, and complementation analyses have been used to divide the COR biosynthetic gene cluster into functional regions. Nucleotide sequencing of the regions involved in CFA and CMA biosynthesis has revealed relatedness to genes encoding polyketide and peptide synthetases, respectively. The deduced amino acid sequence of the gene responsible for catalyzing amide bond formation between CMA and CFA shows relatedness to enzymes that activate cyclic carboxylic acids by adenylation. Coronatine biosynthesis has been shown to be temperature-sensitive and regulated by a modified two-component regulatory system. Received: 12 February 1996 / Accepted: 8 May 1996     

Cloning and Expression of Genes Required for Coronamic Acid (2-Ethyl-1-Aminocyclopropane 1-Carboxylic Acid), an Intermediate in the Biosynthesis of the Phytotoxin Coronatine

Coronamic acid (CMA; 2-ethyl-1-aminocyclopropane 1-carboxylic acid) is an intermediate in the biosynthesis of coronatine (COR), a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. Tn5 mutagenesis and substrate feeding studies were previously used to characterize regions of the COR biosynthetic gene cluster required for synthesis of coronafacic acid and CMA, which are the only two characterized intermediates in the COR biosynthetic pathway. In the present study, additional Tn5 insertions were generated to more precisely define the region required for CMA biosynthesis. A new analytical method for CMA detection which involves derivatization with phenylisothiocyanate and detection by high-performance liquid chromatography (HPLC) was developed. This method was used to analyze and quantify the production of CMA by selected derivatives of P. syringae pv. glycinea which contained mutagenized or cloned regions from the CMA biosynthetic region. pMU2, a clone containing a 6.45-kb insert from the CMA region, genetically complemented mutants which required CMA for COR production. When pMU2 was introduced into P. syringae pv. glycinea 18a/90 (a strain which does not synthesize COR or its intermediates), CMA was not produced, indicating that pMU2 does not contain the complete CMA biosynthetic gene cluster. However, when two plasmid constructs designated pMU234 (12.5 kb) and pKTX30 (3.0 kb) were cointroduced into 18a/90, CMA was detected in culture supernatants by thin-layer chromatography and HPLC. The biological activity of the CMA produced by P. syringae pv. glycinea 18a/90 derivatives was demonstrated by the production of COR in cosynthesis experiments in which 18a/90 transconjugants were cocultivated with CMA-requiring mutants of P. syringae pv. glycinea PG4180. CMA production was also obtained when pMU234 and pKTX30 were cointroduced into P. syringae pv. syringae B1; however, these two constructs did not enable Escherichia coli K-12 to synthesize CMA. The production of CMA in P. syringae strains which lack the COR biosynthetic gene cluster indicates that CMA production can occur independently of coronafacic acid biosynthesis and raises interesting questions regarding the evolutionary origin of the COR biosynthetic pathway.     

Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonas syringae pv. tomato DC3000

To identify Pseudomonas syringae pv. tomato genes involved in pathogenesis, we carried out a screen for Tn5 mutants of P. syringae pv. tomato DC3000 with reduced virulence on Arabidopsis thaliana. Several mutants defining both known and novel virulence loci were identified. Six mutants contained insertions in biosynthetic genes for the phytotoxin coronatine (COR). The P. syringae pv. tomato DC3000 COR genes are chromosomally encoded and are arranged in two separate clusters, which encode enzymes responsible for the synthesis of coronafacic acid (CFA) or coronamic acid (CMA), the two defined intermediates in COR biosynthesis. High-performance liquid chromatography fractionation and exogenous feeding studies confirmed that Tn5 insertions in the cfa and cma genes disrupt CFA and CMA biosynthesis, respectively. All six COR biosynthetic mutants were significantly impaired in their ability to multiply to high levels and to elicit disease symptoms on A. thaliana plants. To assess the relative contributions of CFA, CMA, and COR in virulence, we constructed and characterized cfa6 cmaA double mutant strains. These exhibited virulence phenotypes on A. thalliana identical to those observed for the cmaA or cfa6 single mutants, suggesting that reduced virulence of these mutants on A. thaliana is caused by the absence of the intact COR toxin. This is the first study to use biochemically and genetically defined COR mutants to address the role of COR in pathogenesis.     

Physical and functional characterization of the gene cluster encoding the polyketide phytotoxin coronatine in Pseudomonas syringae pv. glycinea.

Pseudomonas syringae pv. glycinea PG4180 produces the polyketide phytotoxin coronatine. The coronatine synthesis genes in PG4180 were previously shown to reside on a 90-kb plasmid designated p4180A. In the present study, clones containing a 34-kb region of p4180A were saturated with Tn5, and 71 unique mutations were recombined into p4180A by marker exchange. The effect of each mutation on coronatine synthesis was determined by analyzing the organic acids produced by the mutants by reverse-phase high-performance liquid chromatography. The organic acids of selected mutants were derivatized to their methyl esters and analyzed by gas chromatography and gas chromatography-mass spectrometry. Mutations in a 20.5-kb region of p4180A completely blocked the synthesis of coronafacic acid and coronatine. Mutations within a 4.4-kb region of p4180A prevented the formation of coronatine but allowed for production of coronafacic acid, coronafacoylvaline, coronafacoylisoleucine, and coronafacoylalloisoleucine. The phenotypes of selected mutants were further confirmed in feeding experiments in which coronafacic acid or coronamic acid was added to the culture media. The results of this study allow us to speculate on the likely sequence of steps in the later stages of coronatine biosynthesis.     

Thermoregulated Expression and Characterization of an NAD(P)H-Dependent 2-Cyclohexen-1-one Reductase in the Plant Pathogenic Bacterium Pseudomonas syringae pv. glycinea

The phytopathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 causes bacterial blight of soybeans and preferably infects its host plant during periods of cold, humid weather conditions. To identify proteins differentially expressed at low temperatures, total cellular protein fractions derived from PG4180.N9 grown at 18 and 28°C were separated by two-dimensional gel electrophoresis. Of several proteins which appeared to be preferentially present at 18°C, a 40-kDa protein with an isoelectric point of approximately 5 revealed significant N-terminal sequence homology to morphinone reductase (MR) of Pseudomonas putida M10. The respective P. syringae gene was isolated from a genomic cosmid library of PG4180, and its nucleotide sequence was determined. It was designated ncr for NAD(P)H-dependent 2-cyclohexen-1-one reductase. Comparison of the 1,083-bp open reading frame with database entries revealed 48% identity and 52% similarity to the MR-encoding morB gene of P. putida M10. The ncr gene was overexpressed in Escherichia coli, and its gene product was used to generate polyclonal antisera. Purified recombinant Ncr protein was enzymatically characterized with NAD(P)H and various morphinone analogs as substrates. So far, only 2-cyclohexen-1-one and 3-penten-2-one were found to be substrates for Ncr. By high-pressure liquid chromatography analysis, flavin mononucleotide could be identified as the noncovalently bound prosthetic group of this enzyme. The distribution of the ncr gene in different Pseudomonas species and various strains of P. syringae was analyzed by PCR and Southern blot hybridization. The results indicated that the ncr gene is widespread among P. syringae pv. glycinea strains but not in other pathovars of P. syringae or in any of the other Pseudomonas strains tested.     

The phytotoxin coronatine from Pseudomonas syringae pv. tomato DC3000 functions as a virulence factor and influences defence pathways in edible brassicas

The phytotoxin coronatine (COR) contributes to the virulence of Pseudomonas syringae pv. tomato ( Pst ) strain DC3000 on Arabidopsis thaliana and tomato. However, little is known regarding the role of COR in the virulence of DC3000 on cultivated Brassica spp. In this study, the role of COR and its precursors, coronafacic acid (CFA) and coronamic acid (CMA), were examined in the virulence of Pst DC3000 on collard and turnip, two important edible brassicas. Pst DC3000 and three well-defined COR⁻ biosynthetic mutants of DC3000 exhibited substantial differences in the timing and phenotype of disease lesions on collard and turnip. When examined 3 days post-inoculation (dpi), collard inoculated with DC3000 exhibited visible anthocyanin production and lesions were chlorotic and water-soaked. On turnip, chlorotic and necrotic lesions were evident on DC3000-inoculated leaves 5 dpi. The bacterial population dynamics on plants inoculated with DC3000 and the COR⁻ mutants indicated that COR was essential for DC3000 to maintain high populations in turnip, but not collard. Real-time quantitative PCR revealed that the jasmonic acid pathway responsive genes, LOX2 and CORI1 , were expressed in both hosts inoculated with Pst DC3000. PR1 , a marker associated with the salicylic acid pathway, was expressed in collard and turnip inoculated with the CFA⁻ CMA⁻ mutant DB29, but not DC3000. Further comparison of PR1 and LOX2 expression indicated that CFA plays a subtle role in modulating defence in turnip. This is the first study to investigate the role of COR in the interaction of Pst DC3000 and cultivated brassicas using genetically and biochemically defined COR⁻ mutants.     

The Effect of Light Intensity, Day Length, and Temperature on Fatty Acid Synthesis and Desaturation in Vicia faba L.

Some effects of light intensity, day length, and temperatureon the fatty acid composition of the major glycerolipids ofleaves of Vicia faba L. (cv. Giant Windsor) were observed. Increasinglight intensity caused an increase in the relative concentrationsof 16 : 1 in PG and 18 : 3 in MGDG and DGDG. Increasing daylength during growth (and continuous illumination of leaf tissue)had no effect on 16 : 1 in PG but caused a decrease in the 18: 3 content of PG, PC, MGDG, and DGDG. Since the quantitiesof these lipids increased under these conditions, the decreasewas not due to photodestruction but to the differences in therelative rates of biosynthesis and desaturation of fatty acids.Incubation of leaf tissue in the dark for 4 d had little effecton the fatty acid composition of MGDG, DGDG, and PG. Temperaturealso controls fatty acid synthesis and desaturation. Above theoptimum growth temperature (20 °C), the 18 : 3 content ofMGDG, DGDG, PG, and PC decreased. In mature leaf tissue, thedegree of unsaturation of MGDG may be modified upward in responseto temperature changes. When plants were grown at 30 °Cand transferred to 20 °C the level of 18 : 3 in MGDG ofthe leaf tissue increased to levels found in plants grown onlyat 20 °C. The level of 18 : 3 in MGDG does not decreaseas rapidly when plants grown at 20 °C were transferred to30 °C. This suggests that the lower temperature induceddesaturation of 18 : 2 to 18 : 3.     

Synthetic biology approaches to establish a heterologous production system for coronatines

Coronatine (COR) represents a phytotoxin produced by several pathovars of Pseudomonas syringae. It mediates multiple virulence activities by mimicking the plant stress hormone jasmonoyl-l-isoleucine. Structurally, COR consists of a bicyclic polyketide moiety, coronafacic acid (CFA), which is linked via an amide bond to an unusual ethylcyclopropyl amino acid moiety, coronamic acid (CMA). In our studies, we aimed at establishing and engineering of heterologous COR and CFA production platforms using P. putida KT2440 as host. Based on genetic information of the native producer P. syringae pv. tomato DC3000 a COR biosynthetic gene cluster was designed and reconstituted from synthetic DNA fragments. The applied constructional design facilitated versatile pathway modifications and the generation of various expression constructs, which were evaluated for the production of CFA, COR and its derivatives. By modifications of the gene cluster composition production profiles were directed towards target compounds and valuable information about the function and impact of selected pathway proteins on COR biosynthesis were obtained. Additional engineering of expression vector features, including the use of the constitutive PrpsH promoter and a p15Aori-based transposon backbone, led to the development of an expression strain with promising CFA production yields of > 90 mg/l.