The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity.

Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain.

A role for chloroplast-localized Thylakoid Formation 1 (THF1) in bacterial speck disease development.

Coronatine (COR), a jasmonate mimic produced by Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is required for full virulence of Pst DC3000 in tomato and Arabidopsis. COR is shown to induce a range of physiological processes including chlorosis, root growth inhibition and anthocyanin accumulation in tomato. To elucidate the host/signaling genes involved in COR-responses, we utilized a forward genetics approach using Nicotiana benthamiana and virus-induced gene silencing (VIGS) and identified genes that play a role in COR-mediated chlorosis. We designated these genes as altered COR response (ALC). When silenced, one gene designated ALC1 produced a hypersensitive/necrosis-like phenotype after COR application in a coronatine insensitive 1 (COI1)-dependent manner. In pathogenicity assays performed on Arabidopsis thylakoid formation 1 (thf1) knockout lines and SlALC1-silenced tomato plants, Pst DC3000 induced coalescing necrotic lesions in an accelerated manner. Furthermore, we showed that COR affects ALC1 localization in chloroplast in a COI1-dependent manner. In conclusion, our results show the potential of VIGS-based, forward genetic screens to identify new players in COR-mediated signal transduction.

Transcriptional studies of the hrpM/opgH gene in Pseudomonas syringae during biofilm formation and in response to different environmental challenges.

Pseudomonas syringae pv. syringae strain FF5 is a phytopathogen that causes a rapid dieback on ornamental pear trees. In the present study, the transcriptional expression of hrpM/opgH, algD, hrpR and rpoD was evaluated in P. syringae FF5 and FF5.M2 (hrpM/opgH mutant). The temporal expression of these genes was evaluated during biofilm formation, the hypersensitive reaction (HR) on tobacco plants, and when the bacteria were subjected to different environmental stresses. The results indicate that mutations in hrpM negatively impair several traits including biofilm formation, the ability to cause disease in host plants and the HR in non-host plants, and the expression of hrpR, a regulatory gene modulating the latter two traits. Furthermore, FF5.M2 was decreased in swarming motility and unable to respond to different environmental challenges. Interestingly, FF5.M2 showed an exponential increase in the expression of algD, which is the first gene to be transcribed during the biosynthesis of the alginate, a virulence factor in P. syringae. The expression of both hrpM and algD were required for biofilm formation, and hrpM was expressed earlier than algD during biofilm development. These findings indicate that hrpM expression is required for several traits in P. syringae and plays an important role in how this bacterium responds to environmental challenges.

A virus-induced gene silencing screen identifies a role for Thylakoid Formation1 in Pseudomonas syringae pv tomato symptom development in tomato and Arabidopsis.

Pseudomonas syringae pv tomato DC3000 (Pst DC3000), which causes disease in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana), produces coronatine (COR), a non-host-specific phytotoxin. COR, which functions as a jasmonate mimic, is required for full virulence of Pst DC3000 and for the induction of chlorosis in host plants. Previous genetic screens based on insensitivity to COR and/or methyl jasmonate identified several potential targets for COR and methyl jasmonate. In this study, we utilized Nicotiana benthamiana and virus-induced gene silencing to individually reduce the expression of over 4,000 genes. The silenced lines of N. benthamiana were then screened for altered responses to purified COR. Using this forward genetics approach, several genes were identified with altered responses to COR. These were designated as ALC (for altered COR response) genes. When silenced, one of the identified genes, ALC1, produced a hypersensitive/necrosis-like phenotype upon COR application in a Coronatine-Insensitive1 (COI1)-dependent manner. To understand the involvement of ALC1 during the Pst DC3000-host interaction, we used the nucleotide sequence of ALC1 and identified its ortholog in Arabidopsis (Thylakoid Formation1 [THF1]) and tomato (SlALC1). In pathogenicity assays performed on Arabidopsis thf1 mutant and SlALC1-silenced tomato plants, Pst DC3000 induced accelerated coalescing necrotic lesions. Furthermore, we showed that COR affects ALC1 localization in chloroplasts in a COI1-dependent manner. In conclusion, our results show that the virus-induced gene silencing-based forward genetic screen has the potential to identify new players in COR signaling and disease-associated necrotic cell death.

Simple and rapid capillary zone electrophoresis method for the detection of coronamic acid, a precursor to the Pseudomonas syringae phytotoxin coronatine.

The phytotoxin coronatine (COR) is produced by various pathovars of the plant pathogen Pseudomonas syringae, which infects a wide variety of crops. COR consists of two distinct moieties, coronafacic acid (CFA) and coronamic acid (CMA), which are derived from a modified polyketide pathway and isoleucine, respectively. Mutants defective in the CMA or CFA structural gene clusters have been used to study COR biosynthesis, and these mutants are commonly characterized using high-performance liquid chromatography (HPLC). Although the same extraction and HPLC method can be used for detection and quantification of COR and CFA, the detection of CMA by HPLC requires different fractionation and HPLC separation procedures, which are tedious and labor intensive. In this study, we used capillary zone electrophoresis (CZE) as a fast and accurate detection method for the quantification of CMA present in the culture supernatant of P. syringae pv. glycinea (Psg) PG4180 and P. syringae pv. tomato (Pst) DC3000. Analysis was performed by CZE using 100 mM phosphate buffer (pH 2.5) as a separating buffer, an applied voltage of 12 kV, and UV detection at 214 nm. Selected mutants defective in COR biosynthesis were used to validate CZE as a detection method. CMA production by Psg strain 18a/90, which lacks the COR gene cluster, and derivatives of 18a/90 was also evaluated. Furthermore, a procedure for the extraction and detection of CMA present inside the cells of Psg 18a/90 was developed. In conclusion, CZE was shown to be a rapid and sensitive method for the detection and quantification of CMA in P. syringae.

The phytotoxin coronatine induces light-dependent reactive oxygen species in tomato seedlings.

The phytotoxin coronatine (COR), which is produced by Pseudomonas syringae pv. tomato DC3000 (DC3000), has multiple roles in virulence that lead to chlorosis and a reduction in chlorophyll content. However, the physiological significance of COR-induced chlorosis in disease development is still largely unknown. Global expression analysis demonstrated that DC3000 and COR, but not the COR-defective mutant DB29, caused reduced expression of photosynthesis-related genes and result in a 1.5- to 2-fold reduction in maximum quantum efficiency of photosystem II (F(V)/F(M)). Tomato (Solanum lycopersicum) seedlings inoculated with DC3000 and incubated in a long daily photoperiod showed more necrosis than inoculated seedlings incubated in either dark or a short daily photoperiod. The accumulation of reactive oxygen species (ROS) was detected in cotyledons inoculated with either purified COR or DC3000 but not in tissues inoculated with DB29. Interestingly, COR-induced ROS accumulated only in light and was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and diphenylene iodonium, which function to inhibit electron transport from PSII. Furthermore, COR and DC3000 suppressed expression of the gene encoding the thylakoid Cu/Zn superoxide dismutase but not the cytosolic form of the same enzyme. In conclusion, these results demonstrate a role for COR-induced effects on photosynthetic machinery and ROS in modulating necrotic cell death during bacterial speck disease of tomato.

Pathogenicity of Pseudomonas syringae pv. tomato on tomato seedlings: phenotypic and gene expression analyses of the virulence function of coronatine.

Bacterial speck disease, which is caused by Pseudomonas syringae pv. tomato, is an economically important disease on tomato. In the present study, we show that P. syringae pv. tomato DC3000 is a pathogen of tomato seedlings, an aspect of pathogen biology that has not been previously investigated. This resulted in the development of a virulence assay on tomato seedlings that has several advantages over labor-intensive foliar assays, including a shorter growth and incubation period, ease of inoculation and handling, and rapid generation of larger sample sizes per experiment. The utility of this assay was investigated by exploring the virulence function of coronatine (COR) on tomato seedlings. Using the COR- mutant DB29 and a MAPMAN display of transcript data from TOM1 microarrays, COR-dependent expression of genes involved in secondary metabolism, polyamine biosynthesis, reactive oxygen species homeostasis, and the novel transcription factor SlNAC2 were identified. Furthermore, during pathogenesis, genes involved in photosynthetic light reactions and the Calvin-Benson cycle were strongly repressed by COR. In conclusion, we show that P. syringae pv. tomato infects tomato seedlings and that COR is required for virulence in seedlings. The seedling assay can be used in high-throughput screens for the identification of molecular targets for COR and for the identification of genes involved in pathogenesis.

The phytotoxin coronatine contributes to pathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringae pv. tomato DC3000.

The roles of the phytotoxin coronatine (COR) and salicylic acid (SA)-mediated defenses in the interaction of Pseudomonas syringae pv. tomato DC3000 and tomato (Solanum lycopersicum) were investigated. Unlike findings reported for Arabidopsis thaliana, DC3000 mutants impaired for production of COR or one of its components, coronafacic acid (CFA) or coronamic acid (CMA), induced distinctly different disease lesion phenotypes in tomato. Tomato plants inoculated with the CFA- CMA- mutant DB29 showed elevated transcript levels of SlICS, which encodes isochorismate synthase, an enzyme involved in SA biosynthesis in S. lycopersicum. Furthermore, expression of genes encoding SA-mediated defense proteins were elevated in DB29-inoculated plants compared with plants inoculated with DC3000, suggesting that COR suppresses SlICS-mediated SA responses. Sequence analysis of SlICS revealed that it encodes a protein that is 55 and 59.6% identical to the A. thaliana ICS-encoded proteins AtICS1 and AtICS2, respectively. Tomato plants silenced for SlICS were hypersusceptible to DC3000 and accumulated lower levels of SA after infection with DC3000 compared with inoculated wild-type tomato plants. Unlike what has been shown for A. thaliana, the COR- mutant DB29 was impaired for persistence in SlICS-silenced tomato plants; thus, COR has additional roles in virulence that are SA independent and important in the latter stages of disease development. In summary, the infection assays, metabolic profiling, and gene expression results described in this study indicate that the intact COR molecule is required for both suppression of SA-mediated defense responses and full disease symptom development in tomato.

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

SUMMARY 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 algT gene of Pseudomonas syringae pv. glycinea and new insights into the transcriptional organization of the algT-muc gene cluster.

The phytopathogenic bacterium Pseudomonas syringae pv. glycinea infects soybean plants and causes bacterial blight. In addition to P. syringae, the human pathogen Pseudomonas aeruginosa and the soil bacterium Azotobacter vinelandii produce the exopolysaccharide alginate, a copolymer of d-mannuronic and l-guluronic acids. Alginate production in P. syringae has been associated with increased fitness and virulence in planta. Alginate biosynthesis is tightly controlled by proteins encoded by the algT-muc regulatory gene cluster in P. aeruginosa and A. vinelandii. These genes encode the alternative sigma factor AlgT (sigma(22)), its anti-sigma factors MucA and MucB, MucC, a protein with a controversial function that is absent in P. syringae, and MucD, a periplasmic serine protease and homolog of HtrA in Escherichia coli. We compared an alginate-deficient algT mutant of P. syringae pv. glycinea with an alginate-producing derivative in which algT is intact. The alginate-producing derivative grew significantly slower in vitro growth but showed increased epiphytic fitness and better symptom development in planta. Evaluation of expression levels for algT, mucA, mucB, mucD, and algD, which encodes an alginate biosynthesis gene, showed that mucD transcription is not dependent on AlgT in P. syringae in vitro. Promoter mapping using primer extension experiments confirmed this finding. Results of reverse transcription-PCR demonstrated that algT, mucA, and mucB are cotranscribed as an operon in P. syringae. Northern blot analysis revealed that mucD was expressed as a 1.75-kb monocistronic mRNA in P. syringae.

CorR regulates multiple components of virulence in Pseudomonas syringae pv. tomato DC3000.

The phytotoxin coronatine (COR) is produced by various pathovars of Pseudomonas syringae, including P. syringae pv. tomato DC3000, which is pathogenic on crucifers and tomato, and P. syringae pv. glycinea PG4180, a soybean pathogen. The COR molecule contains two distinct components, coronafacic acid (CFA) and coronamic acid (CMA), which are intermediates in the COR biosynthetic pathway. In P. syringae pv. tomato DC3000, it is not clear whether corR, which encodes a response regulator, positively regulates CFA and CMA synthesis as it does in P. syringae pv. glycinea PG4180. In this study, a corR mutant of P. syringae pv. tomato DC3000 was constructed and was shown to be defective in the production of COR, CFA, and CMA. Furthermore, disease severity was greatly reduced in tomato plants inoculated with the corR mutant compared with wild-type P. syringae pv. tomato DC3000. We also showed that a mutation in hrpL, which encodes an alternate RNA polymerase sigma factor (sigmaL) required for the expression of genes encoding components of the type III secretion system, abrogated production of COR in P. syringae pv. tomato DC3000. The presence of a potential hrp box, the recognition site for sigmaL, upstream of corR suggested that corR might be regulated by hrpL. This was confirmed in reverse-transcription polymerase chain reaction experiments showing that the upstream effector gene holPtoAA, which was associated with the hrp box, was cotranscribed with corR. Furthermore, studies also were conducted to investigate whether mutations in corR had effects on the expression of hrpL. The corR mutant of P. syringae pv. tomato DC3000 showed both a reduction and delay in the expression of hrpL and was impaired in its ability to elicit a hypersensitive response on Nicotiana benthamiana. A putative CorR-binding site was identified upstream of hrpL, and gel shift studies confirmed the binding of CorR to this region. These results indicate that corR directly impacts the expression of the hrp regulon in P. syringae.

Novel virulence gene of Pseudomonas syringae pv. tomato strain DC3000.

Previously, we conducted a mutant screen of Pseudomonas syringae pv. tomato strain DC3000 to identify genes that contribute to virulence on Arabidopsis thaliana plants. Here we describe the characterization of one mutant strain, DB4H2, which contains a single Tn5 insertion in PSPTO3576, an open reading frame that is predicted to encode a protein belonging to the TetR family of transcriptional regulators. We demonstrate that PSPTO3576 is necessary for virulence in DC3000 and designate the encoded protein TvrR (TetR-like virulence regulator). TvrR, like many other TetR-like transcriptional regulators, negatively regulates its own expression. Despite the presence of a putative HrpL binding site in the tvrR promoter region, tvrR is not regulated by HrpL, an alternative sigma factor that regulates the expression of many known DC3000 virulence genes. tvrR mutant strains grow comparably to wild-type DC3000 in culture and possess an intact type III secretion system. However, tvrR mutants do not cause disease symptoms on inoculated A. thaliana and tomato plants, and their growth within plant tissue is significantly impaired. We demonstrate that tvrR mutant strains are able to synthesize coronatine (COR), a phytotoxin required for virulence of DC3000 on A. thaliana. Given that tvrR mutant strains are not defective for type III secretion or COR production, tvrR appears to be a novel virulence factor required for a previously unexplored process that is necessary for pathogenesis.

The phytotoxin coronatine and methyl jasmonate impact multiple phytohormone pathways in tomato.

Coronatine (COR) is a phytotoxin produced by several pathovars of Pseudomonas syringae and consists of coronafacic acid (CFA), an analog of methyl jasmonic acid (MeJA), and coronamic acid (CMA), which resembles 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor to ethylene. An understanding of how COR functions, is perceived by different plant tissues, and the extent to which it mimics MeJA remain unclear. In this study, COR and related compounds were examined with respect to structure and function. The results indicate that conjugation of CFA to an amino acid is required for optimal activity in tomato, including chlorosis, changes in chloroplast structure, cell wall thickening, accumulation of proteinase inhibitors, induction of anthocyanins, and root growth inhibition. cDNA microarrays were utilized to understand the molecular processes that are regulated by MeJA, COR, CFA and CMA in tomato leaves. A comparison of COR- and MeJA-regulated transcriptomes revealed that COR regulated 35% of the MeJA-induced genes. There was significant overlap in the number of COR and CFA-regulated genes with CFA impacting the expression of 39.4% of the COR-regulated genes. Taken together, the results of biological assays, ultrastructural studies, and gene expression profiling demonstrate that: (1) the intact COR molecule impacts signaling in tomato via the jasmonic acid, ethylene, and auxin pathways; (2) CMA does not function as a structural analog of ACC; (3) COR has a broader range of functions than either CFA or CMA; and (4) COR and MeJA share similar, but not identical activities and impact multiple phytohormone pathways in tomato.

Pseudomonas syringae manipulates systemic plant defenses against pathogens and herbivores.

Many pathogens are virulent because they specifically interfere with host defense responses and therefore can proliferate. Here, we report that virulent strains of the bacterial phytopathogen Pseudomonas syringae induce systemic susceptibility to secondary P. syringae infection in the host plant Arabidopsis thaliana. This systemic induced susceptibility (SIS) is in direct contrast to the well studied avirulence/R gene-dependent resistance response known as the hypersensitive response that elicits systemic acquired resistance. We show that P. syringae-elicited SIS is caused by the production of coronatine (COR), a pathogen-derived functional and structural mimic of the phytohormone jasmonic acid (JA). These data suggest that SIS may be a consequence of the previously described mutually antagonistic interaction between the salicylic acid and JA signaling pathways. Virulent P. syringae also has the potential to induce net systemic susceptibility to herbivory by an insect (Trichoplusia ni, cabbage looper), but this susceptibility is not caused by COR. Rather, consistent with its role as a JA mimic, COR induces systemic resistance to T. ni. These data highlight the complexity of defense signaling interactions among plants, pathogens, and herbivores.

The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis thaliana.

SUMMARY Successful pathogen infection likely involves the suppression of general antimicrobial host defences. One Pseudomonas syringae virulence factor proposed to act in this manner is coronatine (COR), a phytotoxin believed to function as an analogue of one or more jasmonates, a family of plant growth regulators. COR biosynthetic (COR(-)) mutants of P. syringae pv. tomato strain DC3000 exhibit reduced virulence on Arabidopsis thaliana and tomato. In the present study, three genetically and biochemically defined COR(-) mutants of DC3000 were used to explore potential effects of COR and its precursors, coronafacic acid (CFA) and coronamic acid (CMA), on defence signalling pathways in A. thaliana. Inoculation with wild-type DC3000 resulted in the accumulation of several jasmonate-responsive transcripts, whereas infection with a mutant strain that accumulates CFA, which is structurally similar to methyl jasmonate (MeJA), did not. Thus, COR, but not CFA, stimulates jasmonate signalling during P. syringae infection of A. thaliana. The ability of the COR(-) mutants to grow to high levels in planta was fully restored in A. thaliana lines deficient for salicylic acid (SA) accumulation. Although the COR(-) mutants grew to high levels in SA-deficient plants, disease symptoms were reduced in these plants. Collectively, these results indicate that COR is required both for overcoming or suppressing SA-dependent defences during growth in plant tissue and for normal disease symptom development in A. thaliana.

AlgR functions in algC expression and virulence in Pseudomonas syringae pv. syringae.

Pseudomonas syringae pv. syringae strain FF5 is a phytopathogen associated with a rapid dieback on ornamental pear trees. P. syringae and the human pathogen Pseudomonas aeruginosa produce the exopolysaccharide alginate, a copolymer of mannuronic and guluronic acid. In P. aeruginosa, the response regulator AlgR (AlgR1) is required for transcription of algC and algD, which encode key enzymes in the alginate biosynthetic pathway. In P. syringae FF5, however, algR is not required for the activation of algD. Interestingly, algR mutants of P. syringae remain nonmucoid, indicating an undefined role for this response regulator in alginate biosynthesis. In the current study, the algC promoter region was cloned from P. syringae pv. syringae strain FF5, and sequence analysis of the algC promoter indicated the presence of potential binding sites for AlgR and sigma(54), the alternative sigma factor encoded by rpoN. The algC promoter from P. syringae FF5 (PsalgC) was cloned upstream of a promoterless glucuronidase gene (uidA), and the PsalgC-uidA transcriptional fusion was used to monitor algC expression in strains FF5.32 (algR mutant of P. syringae FF5) and PG4180.K2 (rpoN mutant of P. syringae pv. glycinea PG4180). Expression of the PsalgC-uidA fusion was fourfold lower in both the algR and rpoN mutants as compared to respective wild-type strains, indicating that both AlgR and sigma(54) are required for full activation of algC transcription in P. syringae pv. syringae. AlgR from P. syringae was successfully overproduced in Escherichia coli as a C-terminal translational fusion to the maltose-binding protein (MBP). Gel shift experiments indicated that MBP-AlgR binds strongly to the algC promoter region. Biological assays demonstrated that the algR mutant was significantly impaired in both pathogenicity and epiphytic fitness as compared to the wild-type strain. These results, along with the gene expression studies, indicate that AlgR has a positive role in the activation of algC in P. syringae and contributes to both virulence and epiphytic fitness. Furthermore, the symptoms observed with wild-type P. syringae FF5 suggest that this strain can move systemically in leaf tissue, and that a functional copy of algR is required for systemic movement.

The Pseudomonas syringae genome encodes a combined mannuronan C-5-epimerase and O-acetylhydrolase, which strongly enhances the predicted gel-forming properties of alginates.

Alginates are industrially important, linear copolymers of beta-d-mannuronic acid (M) and its C-5-epimer alpha-l-guluronic acid (G). The G residues originate from a postpolymerization reaction catalyzed by mannuronan C-5-epimerases (MEs), leading to extensive variability in M/G ratios and distribution patterns. Alginates containing long continuous stretches of G residues (G blocks) can form strong gels, a polymer type not found in alginate-producing bacteria belonging to the genus Pseudomonas. Here we show that the Pseudomonas syringae genome encodes a Ca(2+)-dependent ME (PsmE) that efficiently forms such G blocks in vitro. The deduced PsmE protein consists of 1610 amino acids and is a modular enzyme related to the previously characterized family of Azotobacter vinelandii ME (AlgE1-7). A- and R-like modules with sequence similarity to those in the AlgE enzymes are found in PsmE, and the A module of PsmE (PsmEA) was found to be sufficient for epimerization. Interestingly, an R module from AlgE4 stimulated Ps-mEA activity. PsmE contains two regions designated M and RTX, both presumably involved in the binding of Ca(2+). Bacterial alginates are partly acetylated, and such modified residues cannot be epimerized. Based on a detailed computer-assisted analysis and experimental studies another PsmE region, designated N, was found to encode an acetylhydrolase. By the combined action of N and A PsmE was capable of redesigning an extensively acetylated alginate low in G from a non gel-forming to a gel-forming state. Such a property has to our knowledge not been previously reported for an enzyme acting on a polysaccharide.

Characterization of Cfa1, a monofunctional acyl carrier protein involved in the biosynthesis of the phytotoxin coronatine.

Cfa1 was overproduced in Escherichia coli and Pseudomonas syringae, and the degree of 4'-phosphopantetheinylation was determined. The malonyl-coenzyme A:acyl carrier protein transacylase (FabD) of P. syringae was overproduced and shown to catalyze malonylation of Cfa1, suggesting that FabD plays a role in coronatine biosynthesis. Highly purified Cfa1 did not exhibit self-malonylation activity.

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.