Identification of the biosynthetic gene cluster for 3-methylarginine, a toxin produced by Pseudomonas syringae pv. syringae 22d/93.

The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces the rare amino acid 3-methylarginine (MeArg), which is highly active against the closely related soybean pathogen Pseudomonas syringae pv. glycinea. Since these pathogens compete for the same habitat, Pss22d is a promising candidate for biocontrol of P. syringae pv. glycinea. The MeArg biosynthesis gene cluster codes for the S-adenosylmethionine (SAM)-dependent methyltransferase MrsA, the putative aminotransferase MrsB, and the amino acid exporter MrsC. Transfer of the whole gene cluster into Escherichia coli resulted in heterologous production of MeArg. The methyltransferase MrsA was overexpressed in E. coli as a His-tagged protein and functionally characterized (K(m), 7 mM; k(cat), 85 min(-1)). The highly selective methyltransferase MrsA transfers the methyl group from SAM into 5-guanidino-2-oxo-pentanoic acid to yield 5-guanidino-3-methyl-2-oxo-pentanoic acid, which then only needs to be transaminated to result in the antibiotic MeArg.

Complete nucleotide sequence and analysis of pPSR1 (72,601 bp), a pPT23A-family plasmid from Pseudomonas syringae pv. syringae A2.

Plasmid pPSR1 is a conjugative plasmid originally isolated from Pseudomonas syringae pv. syringae A2, and is a member of the recently described pPT23A plasmid family. We have determined the complete sequence of pPSR1 and found the plasmid to be 72,601 bp in length, encoding 55 ORFs. Putative functions were assigned to 49 ORFs; of these, 24 (49.0%) are involved in plasmid replication, maintenance or conjugation, 17 (34.7%) have roles in virulence or ecological fitness, and eight (16.3%) encode transposase functions as part of mobile elements. pPSR1 carries the effector gene orf34, the mutagenic DNA repair operon rulAB which confers tolerance to ultraviolet radiation, and two genes for methyl-accepting chemotaxis proteins, one of which was located within the novel transposon Tn 5395. The streptomycin resistance transposon Tn 5393a, which carries a strA-strB determinant, was found inserted immediately downstream of the pPSR1 repA gene. Functional analysis of the replication region of pPSR1 indicated that the repA gene and flanking upstream and downstream sequences are required for autonomous replication in P. syringae. Hybridization analyses of the distribution of 11 of the pPSR1 ORFs indicated that many of the ecologically important ORFs were confined to the pathovar P. syringae pv. syringae -either to strains from the local population from which pPSR1 was originally isolated, or strains from a worldwide collection. Conjugative transfer genes and a gene encoding a transcriptional regulator were more widely distributed among several P. syringae pathovars. The sequence analysis of pPSR1 suggests that pPT23A-family plasmids evolve by accumulating genes that are important for host-pathogen interactions or growth on plant hosts, which are incorporated onto a conserved backbone encoding conjugation and stability determinants.

Thermoregulated expression of virulence factors in plant-associated bacteria.

Pathogenic bacteria with habitats inside and outside a given host react to changes in environmental parameters by synthesizing gene products specifically needed during pathogenic or saprophytic growth. Temperature effects have been investigated in detail for pathogens of warm-blooded hosts, and major principles governing the temperature-sensing mechanism have been uncovered. Generally, transcription of virulence genes in these pathogens is induced at higher temperatures (37-41 degrees C), which are typical for body cavities and host tissues. However, effects of temperature on virulence determinants in plant pathogenic bacteria have not been focused on in detail. Interestingly, almost all virulence genes of plant pathogenic bacteria studied with respect to temperature exhibit increased transcription at temperatures well below the respective growth optima. This includes virulence determinants such as those directing bacteria-to-plant gene transfer, plant cell-wall-degrading enzymes, phytotoxins, ice nucleation activity, exopolysaccharide production, and the type III protein secretion machinery. Although many of the studied phytopathogens cause "cold-weather" diseases, the ecological rationale for this phenomenon remains to be studied in detail. This mini-review summarizes our current knowledge on thermoregulation of cellular processes taking place in bacterial phytopathogens in response to temperature changes. Since the temperature range of interest is different from that relevant to pathogens of mammals, one envisions novel principles of thermo-sensing in bacteria interacting with plants.

Characterization and mutational analysis of three allelic lsc genes encoding levansucrase in Pseudomonas syringae.

In the plant pathogen Pseudomonas syringae pv. glycinea PG4180 and other bacterial species, synthesis of the exopolysaccharide levan is catalyzed by the extracellular enzyme levansucrase. The results of Southern blotting and PCR analysis indicated the presence of three levansucrase-encoding genes in strain PG4180: lscA, lscB, and lscC. In this study, lscB and lscC were cloned from a genomic library of strain PG4180. Sequence analysis of the two lsc genes showed that they were virtually identical to each other and highly similar to the previously characterized lscA gene. lscA and lscC had a chromosomal location, whereas lscB resided on an indigenous plasmid of PG4180. Mutants with impaired expression of individual lsc genes and double mutants were generated by marker exchange mutagenesis. Determination of levansucrase activities in these mutants revealed that the lscB gene product was secreted but not that of lscA or lscC. Our results indicated that lscB and lscC but not lscA contributed to periplasmic levan synthesis of PG4180. The lscB lscC double mutant was completely defective in levan formation and could be complemented by either lscB or lscC. Our data suggested a compartment-specific localization of two lsc gene products, with LscB being the secreted, extracellular enzyme and LscC being the predominantly periplasmic levansucrase. Results of Western blot analyses indicated that lscA was not expressed and that lscA was not associated with levansucrase activities in any particular protein fraction. LscA could be detected in PG4180 only when transcribed from the vector-borne P(lac) promoter. PCR screening in various P. syringae strains with primers derived from the three characterized lsc genes demonstrated the presence of multiple Lsc isoenzymes in other P. syringae pathovars.

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.

The transcriptional activator CorR is involved in biosynthesis of the phytotoxin coronatine and binds to the cmaABT promoter region in a temperature-dependent manner.

A modified two-component regulatory system consisting of the histidine protein kinase CorS and two highly homologous response regulators, CorR and CorP, controls biosynthesis of the polyketide phytotoxin coronatine (COR) by Pseudomonas syringae pv. glycinea PG4180 in a temperature-dependent manner. COR synthesis is maximal at 18 degrees C but does not occur at 28 degrees C. Fusions of CorR and CorP to the maltose-binding protein (MBP) were overproduced in Escherichia coli and P. syringae PG4180, and tested for functionality by complementation of corR and corP mutants of PG4180, respectively. The cmaABT promoter region was defined by deletion mapping, and the DNA-binding capability of CorR and CorP was examined by gel retardation assays. When overproduced in P. syringae at 18 degrees C and purified, MBP-CorR was shown to bind specifically to a 218-bp DNA fragment corresponding to positions -841 to -623 bp upstream of the transcriptional start site of the cmaABT operon. In contrast, MBP-CorP and MBP itself, when overproduced in P. syringae and E. coli at 18 degrees C and 28 degrees C, respectively, did not bind to the 218-bp fragment or to any other DNA fragment analyzed. The CorP protein lacks typical DNA-binding motifs, suggesting that it might modulate the function of CorR. However, addition of purified MBP-CorP did not alter the DNA-binding activity of MBP-CorR. On the other hand, this activity was completely abolished when MBPCorR was overproduced at 28 degrees C or in a corS mutant, indicating that the binding of CorR depended on the growth temperature at which it was produced and was controlled by CorS. In addition, overproduction of MBP-CorR in a corP mutant of PG4180 also yielded inactive protein, underlining the importance of CorP for CorR activation. We propose that CorR is activated by CorS at low temperature and that CorP is required for this activation before CorR can bind to DNA.

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 degreesC were separated by two-dimensional gel electrophoresis. Of several proteins which appeared to be preferentially present at 18 degreesC, 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.

Comparison of Ethylene Production by Pseudomonas syringae and Ralstonia solanacearum.

ABSTRACT Strains of Pseudomonas syringae pv. pisi and Ralstonia solanacearum produced ethylene at rates 20- and 200-fold lower, respectively, than strains of P. syringae pvs. cannabina, glycinea, phaseolicola, and sesami. In the current study, we investigated which ethylene biosynthetic pathways were used by P. syringae pv. pisi and R. solanacearum. Neither the activity of an ethylene-forming enzyme nor a corresponding efe gene homolog could be detected in R. solanacearum, suggesting synthesis of ethylene via 2-keto-4-methyl-thiobutyric acid. In contrast, 2-oxoglutarate-dependent ethylene formation was observed with P. syringae pv. pisi, and Southern blot hybridization revealed the presence of an efe homolog in this pathovar. The efe genes from P. syringae pvs. cannabina, glycinea, phaseolicola, pisi, and sesami were sequenced. Nucleotide sequence comparisons indicated that the efe gene in pv. pisi was not as highly conserved as it was in other P. syringae pathovars. The pv. pisi efe homolog showed numerous nucleotide substitutions and a deletion of 13 amino acids at the C-terminus of the predicted gene product. These sequence alterations might account for the lower rate of ethylene production by this pathovar. All ethylene-producing P. syringae pathovars were virulent on bush bean plants. The overlapping host range of these pathovars suggests that horizontal transfer of the efe gene may have occurred among bacteria inhabiting the same host.

Cloning, nucleotide sequence, and expression in Escherichia coli of levansucrase genes from the plant pathogens Pseudomonas syringae pv. glycinea and P. syringae pv. phaseolicola.

Plant-pathogenic bacteria produce various extracellular polysaccharides (EPSs) which may function as virulence factors in diseases caused by these bacteria. The EPS levan is synthesized by the extracellular enzyme levansucrase in Pseudomonas syringae, Erwinia amylovora, and other bacterial species. The lsc genes encoding levansucrase from P. syringae pv. glycinea PG4180 and P. syringae pv. phaseolicola NCPPB 1321 were cloned, and their nucleotide sequences were determined. Heterologous expression of the lsc gene in Escherichia coli was found in four and two genomic library clones of strains PG4180 and NCPPB 1321, respectively. A 3. 0-kb PstI fragment common to all six clones conferred levan synthesis on E. coli when further subcloned. Nucleotide sequence analysis revealed a 1,248-bp open reading frame (ORF) derived from PG4180 and a 1,296-bp ORF derived from NCPPB 1321, which were both designated lsc. Both ORFs showed high homology to the E. amylovora and Zymomonas mobilis lsc genes at the nucleic acid and deduced amino acid sequence levels. Levansucrase was not secreted into the supernatant but was located in the periplasmic fraction of E. coli harboring the lsc gene. Expression of lsc was found to be dependent on the vector-based Plac promoter, indicating that the native promoter of lsc was not functional in E. coli. Insertion of an antibiotic resistance cassette in the lsc gene abolished levan synthesis in E. coli. A PCR screening with primers derived from lsc of P. syringae pv. glycinea PG4180 allowed the detection of this gene in a number of related bacteria.

Occurrence of thermoregulation of genes involved in coronatine biosynthesis among various Pseudomonas syringae strains.

Several pathovars of Pseudomonas syringae produce the polyketide phytotoxin coronatine (COR). In the bacterial blight pathogen of soybean, P. syringae pv. glycinea PG4180, COR is produced at high levels at 18 degrees C whereas no toxin is synthesized at 28 degrees C. Previously, activation of three promoters inside the COR biosynthetic gene cluster by a modified two-component regulatory system was shown to influence thermoregulation of COR biosynthesis. Using phenotypic determination of COR synthesis, a transcriptional reporter gene fusion, and Western blot analysis, we screened a representative number of natural isolates of P. syringae for effects of temperature on expression of cmaA, cmaB, and cmaT, which encode enzymes involved in the biosynthesis of COR. Thermoregulation of cmaABT expression was frequent among the tested strains. However, intensities of the temperature effects varied widely. Coronatine synsthesis was found to differ at up to six-fold among COR producing strains. There was no strain which synthesized COR at 28 degrees C although some of them showed increased basal cmaABT promoter activities at this temperature. Transcriptional fusions between the cmaABT promoter and a promoterless reporter gene were found to be down regulated at 28 degrees C only in COR producing strains but not in the non-producing strains tested. The geographic origin of the bacterial strains did not influence the occurrence of temperature-dependent gene expression.

Growth phase and temperature influence promoter activity, transcript abundance, and protein stability during biosynthesis of the Pseudomonas syringae phytotoxin coronatine.

The plant-pathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 synthesizes high levels of the polyketide phytotoxin coronatine (COR) at 18 degrees C, whereas no detectable toxin is produced at 28 degrees C. Previously, we reported that the temperature-sensitive activation of three promoters within the COR biosynthetic gene cluster might explain thermoregulation of COR biosynthesis. The present study was aimed at furthering our understanding of the transcriptional as well as the posttranslational effects of temperature on expression of cmaB, which encodes an enzyme involved in COR biosynthesis. Transcriptional fusions using a promoterless glucuronidase gene and Northern blot analyses were used to monitor promoter activities and transcript abundance for the cmaABT operon during bacterial growth at 18 and 28 degrees C. Promoter activity and transcription rates were maximal when cells were incubated at 18 degrees C and sampled at mid-logarithmic phase. Transcription declined moderately during the transition to stationary phase but remained higher at 18 C than at 28 degrees C. Western blot analysis indicated that CmaB accumulated in the late stationary phase of P. syringae cultures grown at 18 degrees C but not in cultures incubated at 28 degrees C. Temperature shift experiments indicated that CmaB stability was more pronounced at 18 degrees C than at 28 degrees C. Although temperature has a strong impact on transcription of COR biosynthetic genes, we propose that thermoregulation of protein stability might also control COR synthesis.