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.     

Characterization of Plasmids Encoding the Phytotoxin Coronatine in Pseudomonas syringae

Coronatine (COR) is a nonhost-specific phytotoxin that substantially contributes to the virulence of several pathovars (pvs.) of Pseudomonas syringae. The COR gene cluster in P. syringae is generally plasmid-encoded in pvs. atropurpurea, glycinea, morsprunorum, and tomato but chromosomally encoded in pv. maculicola. In the present study, we investigated whether the COR plasmids in four pathovars shared other traits including self-transmissibility, conserved oriV/par loci, and insertion sequences (ISs) known to reside on other plasmids in P. syringae. Three COR plasmids were shown to be self-transmissible, and all COR plasmids shared a related oriV/par region. Two COR plasmids hybridized to IS801, an IS element widely distributed in P. syringae. Further analysis of p4180A, a 90-kb COR plasmid in P. syringae pv. glycinea, indicated that multiple copies of IS801 were present on this plasmid, and all copies mapped outside the COR gene cluster. Sequence analysis of the region adjacent to the COR gene cluster in p4180A indicated the presence of additional IS elements including IS870, IS51, and IS1240. The IS elements borne on p4180A may have contributed to horizontal transfer of the COR gene cluster and the evolution of the COR biosynthetic pathway.     

Interactions of Pseudomonas syringae pv. glycinea with host and nonhost plants in relation to temperature and phytotoxin synthesis

Pseudomonas syringae pv. glycinea PG4180 causes bacterial blight of soybean and produces the phytotoxin coronatine (COR) in a temperature-dependent manner. COR consists of a polyketide, coronafacic acid (CFA), and an amino acid derivative, coronamic acid, and is produced optimally at 18 degrees C whereas no detectable synthesis occurs at 28 degrees C. We investigated the impact of temperature on PG4180 during compatible and incompatible interactions with soybean and tobacco plants, respectively. After spray inoculation, PG4180 caused typical bacterial blight symptoms on soybean plants when the bacteria were grown at 18 degrees C prior to inoculation but not when derived from cultures grown at 28 degrees C. The disease outcome was quantified by determination of bacterial populations in planta. The temperature effect was not observed when PG4180 was artificially infiltrated into soybean leaves, indicating that the pre-inoculation temperature and phytotoxin synthesis were important for bacterial invasion via natural plant openings. In the incompatible interaction, PG4180 elicited the hypersensitive response (HR) on tobacco plants regardless of the bacterial pre-inoculation temperature. However, the HR was significantly delayed when tobacco plants were treated with cells of the CFA-overproducing derivative, PG4180.N9, which were derived from cultures grown at 18 degrees C, compared with parallels incubated at 28 degrees C. CFA biosynthesis by PG4180.N9 was optimal at 18 degrees C and negligible at 28 degrees C. The impact of CFA synthesis on the HR was studied with different growth media, mutants, and transconjugants of PG4180, indicating that the amount of synthesized CFA but not that of COR influenced the outcome of the HR. Feeding experiments with purified coronafacoyl compounds suggested that the observed delay of the HR was mediated by CFA, shedding further light on CFA's putative role as a molecular mimic of the plant signaling molecule, jasmonic acid.     

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.     

The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae

Many plant pathogens suppress antimicrobial defenses using virulence factors that modulate endogenous host defenses. The Pseudomonas syringae phytotoxin coronatine (COR) is believed to promote virulence by acting as a jasmonate analog, because COR-insensitive 1 (coil) Arabidopsis thaliana and tomato mutants are impaired in jasmonate signaling and exhibit reduced susceptibility to P. syringae. To further investigate the role of jasmonate signaling in disease development, we analyzed several jasmonate-insensitive A. thaliana mutants for susceptibility to P. syringae pv. tomato strain DC3000 and sensitivity to COR. Jasmonate-insensitive 1 (jin1) mutants exhibit both reduced susceptibility to P. syringae pv. tomato DC3000 and reduced sensitivity to COR, whereas jasmonate-resistant 1 (jar1) plants exhibit wild-type responses to both COR and P. syringae pv. tomato DC3000. A jin1 jar1 double mutant does not exhibit enhanced jasmonate insensitivity, suggesting that JIN1 functions downstream of jasmonic acid-amino acid conjugates synthesized by JAR1. Reduced disease susceptibility in jin1 mutants is correlated with elevated expression of pathogenesis-related 1 (PR-1) and is dependent on accumulation of salicylic acid (SA). We also show that JIN1 is required for normal P. syringae pv. tomato DC3000 symptom development through an SA-independent mechanism. Thus, P. syringae pv. tomato DC3000 appears to utilize COR to manipulate JIN1-dependent jasmonate signaling both to suppress SA-mediated defenses and to promote symptom development.     

In Pseudomonas syringae pv. phaseolicola, expression of the argK gene, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase, is regulated indirectly by temperature and directly by a precursor resembling carbamoylphosphate

Pseudomonas syringae pv. phaseolicola synthesizes a non-host-specific toxin, phaseolotoxin, and also synthesizes a phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) to protect itself from its own toxin. ROCT is encoded by argK, which is expressed coordinately with phaseolotoxin synthesis at 18 degrees C. To investigate the regulatory mechanisms of this system, null mutants were constructed for argK, argF (encoding the phaseolotoxin-sensitive OCTase [SOCT]), and amtA (encoding an amidinotransferase involved in phaseolotoxin synthesis). The argF mutant did not exhibit arginine auxotrophy when grown in M9 medium at 28 degrees C, because under this condition SOCT was replaced by ROCT. This loss of thermoregulation of argK was apparently caused by accumulation of carbamoylphosphate, one of the substrates of SOCT. Carbamoylphosphate, which has a structure similar to that of the inorganic moiety of phaseolotoxin, was used in induction assays with wild-type P. syringae pv. phaseolicola and was shown to be able to induce argK expression in M9 medium at 28 degrees C. These results indicate that argK expression is independent of temperature and is regulated directly by a compound resembling the inorganic moiety of phaseolotoxin.     

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.     

Characterization of CmaA, an adenylation-thiolation didomain enzyme involved in the biosynthesis of coronatine

Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond. The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited cmaA sequences were corrected. These efforts allowed overproduction of P. syringae pv. glycinea PG4180 CmaA in P. syringae pv. syringae FF5 as a FLAG-tagged protein and overproduction of P. syringae pv. tomato CmaA in Escherichia coli as a His-tagged protein; both proteins were in an enzymatically active form. Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain). ATP-(32)PP(i) exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain L-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for L-allo-isoleucine. Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4'-phosphopantetheinyl group, reacts with the AMP derivative of L-allo-isoleucine to produce an aminoacyl thiolester intermediate. This covalent species was detected by incubating CmaA with ATP and L-[G-(3)H]allo-isoleucine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. It is postulated that the L-allo-isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA. The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid.