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PubMed1989

An ice nucleation reporter gene system: identification of inducible pathogenicity genes in Pseudomonas syringae pv. phaseolicola.

We have constructed derivatives of the transposon Tn3 that allow an ice nucleation gene (inaZ) to be used as 'reporter' of the transcriptional activity of genes into which it is inserted. In these derivatives (Tn3-Ice and Tn3-Spice), the lacZYA sequences of transposon Tn3-HoHo1 were replaced with inaZ lacking its native promoter. The ice nucleation activity of virB::inaZ fusions in the correct transcriptional orientation was inducible by acetosyringone, a plant metabolite which activates the vir operon of Agrobacterium tumefaciens Ti plasmids, while fusions in the opposite orientation were unresponsive to the inducer. Tn3-Spice was also used to investigate the expression of a cluster of genes (hrp) which control pathogenicity and hypersensitivity elicited by Pseudomonas syringae pv. phaseolicola. An inducible region was identified which is expressed at low levels in vitro but becomes activated when the bacteria come into contact with the susceptible host, bean. Activation of this region occurred within 2 h post-inoculation and was nearly complete by the time the bacteria began to multiply in the leaf tissue. The inaZ reporter appears to be at least 10(5)-fold more sensitive than lacZ in P.s.phaseolicola. Thus, the inaZ fusion system provides a sensitive, convenient and inexpensive tool for the study of bacterial gene expression, particularly during plant pathogenesis, and should be generally useful as a reporter gene system in Gram-negative bacteria.

Amino Acid Sequence↗
PubMed1985

Detection of two ornithine carbamoyltransferases in a phaseolotoxin-producing strain Pseudomonas syringae pv. phaseolicola.

The phaseolotoxin-producing Pseudomonas syringae pv. phaseolicola strain 1321 contains two ornithine carbamoyltransferases which differ in resistance to phaseolotoxin. Whereas ornithine carbamoyltransferase 1 (OCT 1) is inhibited at low concentrations of phaseolotoxin, ornithine carbamoyltransferase 2 (OCT 2) is insensitive to phaseolotoxin. The activity of the insensitive enzyme is correlated with the amount of toxin formed.

Exotoxins↗
PubMed1987

[Demonstration of an NAD-dependent 6-phosphogluconate dehydrogenase in Pseudomonas syringae pv. phaseolicola].

Crude extracts from cells of Pseudomonas syringae pv. phaseolicola, a fluorescent pseudomonad, when grown on glucose contain a NAD-linked 6-phosphogluconate dehydrogenase. The reaction of the enzyme, which produces 14CO2 from 1-14C-6-phosphogluconate, is not inhibited by NaF, a potent inhibitor of the Enter-Doudoroff (ED) pathway enzyme 6-phosphogluconate dehydratase. In the presence of phosphate or arsenate ions the NAD-linked glyceraldehyde-3-phosphate dehydrogenase reacts with glyceraldehyde-3-phosphate which, in the ED pathway, is produced from 6-phosphogluconate and overlaps the 6-phosphogluconate dehydrogenase reaction. Only a small proportion of glucose is metabolized via the 6-phosphogluconate dehydrogenase/oxidative pentose phosphate pathway.

Kinetics↗
PubMed1988

[Detection and characterization of a levansucrase and a sucrase in Pseudomonas syringae pv. phaseolicola].

Pseudomonas syringae pv. phaseolicola, a plant pathogenic pseudomonad, possesses two sucrose-splitting enzymes, a levansucrase and a sucrase. The levansucrase is found both extracellularly and intracellularly, and enzyme synthesis is independent of the carbon source. In addition to levansucrase, cells grown on sucrose contain a sucrase. The two sucrose-splitting enzymes differ in their optimum pH value and optimum temperature as well as in their substrate specificities.

Hexosyltransferases↗
PubMed1989

Detection of an insensitive ornithine-carbamoyltransferase in strains of Pseudomonas syringae pv. phaseolicola with different phytotoxin-generating capacities.

Independently of their capacity to produce phytotoxins, strains of Pseudomonas syringae pv. phaseolicola contain two ornithine carbamoyltransferases (OCT, EC 2.1.3.3) which differ in resistance to phaseolotoxin and Orn-P(O) (NH2)-NH-SO3 H (PNSOrn). At 18 degrees C, the optimal temperature for product formation, the balance of the two types of OCT was shifted in favour of the insensitive type in phaseolotoxin producing strains, and in favour of the sensitive one in strains with little or no toxin production. The results suggest a causal relationship between the existence of an insensitive enzyme and the synthesis of toxins.

Drug Resistance↗
PubMed1989

Phaseolotoxin production by Pseudomonas syringae pv. phaseolicola: the influence of temperature.

Phaseolotoxin (N-sulphodiaminophosphinyl-ornithyl-alanyl-homoarginine) is a phytotoxic secondary metabolite produced by Pseudomonas syringae pv. phaseolicola. The production of the phytotoxin is strongly regulated by temperature. The optimal temperature for phaseolotoxin production is 18 degrees C. Temperatures in the range between 18 degrees C and 30 degrees C inhibit the production of phaseolotoxin in an increasing manner. By temperature shift experiments and the inhibition of protein synthesis by chloramphenicol it was demonstrated, that the synthesis of enzymes related to toxin synthesis and not the regulation of the activity of the responsible enzyme system is affected by higher temperatures.

Cell Division↗
PubMed1993

Dodecylguanidine monoacetate (dodine) causes severe membrane damage in Pseudomonas syringae above the critical micelle concentration.

The release of K+ from Pseudomonas syringae cells treated with dodecylguanidine monoacetate (dodine) was followed with a K(+)-selective glass electrode. Treatment of the cells with 5-15 mumol/l dodine resulted in low levels of K+ release, but higher surfactant concentrations caused extensive and rapid K+ efflux. Dodine concentrations that caused high K+ release also induced significant leakage of inorganic phosphate. The addition of 5-10 mumol/l dodine also caused an increase in the rate of oxygen consumption in the presence of glycerol or succinate, but an increase in concentration from 10 to 40 mumol/l resulted in a concomitant decrease in O2 consumption. The results from this and previous work suggest that dodine inhibits respiration firstly by causing drainage of coenzymes, and then by a direct interaction with the components of the respiratory chain. Previous work showed that above 25 mumol/l, dodine molecules aggregate to form micelles. The results therefore suggests that, in contrast with other cationic amphiphiles, the micellar form of dodine is more damaging to the cytoplasmic membrane than the free molecules.

Anti-Bacterial Agents↗
PubMed2003

Structure of the Clade 1 catalase, CatF of Pseudomonas syringae, at 1.8 A resolution.

Catalase CatF of Pseudomonas syringae has been identified phylogenetically as a clade 1 catalase, closely related to plant catalases, a group from which no structure has been determined. The structure of CatF has been refined at 1.8 A resolution by using X-ray synchrotron data collected from a crystal flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are, respectively, 18.3% and 24.0%. The asymmetric unit of the crystal contains a whole molecule that shows accurate 222-point group symmetry. The crystallized enzyme is a homotetramer of subunits with 484 residues, some 26 residues shorter than predicted from the DNA sequence. Mass spectrometry analysis confirmed the absence of 26 N-terminal residues, possibly removed by a periplasmic transport system. The core structure of the CatF subunit was closely related to seven other catalases with root-mean-square deviations (RMSDs) of 368 core Calpha atoms of 0.99-1.30 A. The heme component of CatF is heme b in the same orientation that is found in Escherichia coli hydroperoxidase II, an orientation that is flipped 180 degrees with respect the orientation of the heme in bovine liver catalase. NADPH is not found in the structure of CatF because key residues required for nucleotide binding are missing; 2129 water molecules were refined into the model. Water occupancy in the main or perpendicular channel of CatF varied among the four subunits from two to five in the region between the heme and the conserved Asp150. A comparison of the water occupancy in this region with the same region in other catalases reveals significant differences among the catalases.

Binding Sites↗
PubMed2001

Persistence and translocation of a benzothiadiazole derivative in tomato plants in relation to systemic acquired resistance against Pseudomonas syringae pv tomato.

A reproducible and accurate procedure, based on HPLC analysis, has been developed to determine simultaneously acibenzolar-S-methyl (CGA 245 704) and its acid derivative (CGA 210 007) in tomato leaves. The limit of detection and quantification of the method are 0.015 and 0.15 mg litre-1 for CGA 245 704 and 0.030 and 0.30 mg litre-1 for CGA 210 007. In tomato plants treated with 250 microM CGA 245 704, it was found that the inducer rapidly translocates from treated leaves (cotyledons, 1st and 2nd) to untreated leaves (3rd to 5th), with the maximum translocation (40% of the total quantity found) occurring 8 h after the treatment. CGA 245 704 residues decreased as time elapsed in both treated and untreated tomato leaves, reaching negligible values 72 h after treatment. The acid derivative, CGA 210 007, was formed in tomato plants as early as 2 h after CGA 245 704 treatment, albeit only in the treated leaves. CGA 210 007 residues decreased in treated tomato leaves with a trend similar to that observed for CGA 245 704. Treatment of tomato plants with CGA 245 704 or CGA 210 007 at 250 microM systemically protected the plants against Pseudomonas syringae pv tomato attacks, the causal agent of bacterial speak disease. Evidence of this were reductions in the degree of infection, the bacterial lesion diameter and the bacterial growth in planta. Since neither CGA 245 704 nor CGA 210 007 inhibited bacterial growth in vitro and the protection against bacterial speak of tomato was observed when the two compounds were completely degraded, the protection must be due to the activation of the plant's defence mechanisms.

Anti-Bacterial Agents↗
PubMed1998

Phospholipid analysis and fractional reconstitution of the ice nucleation protein activity purified from Escherichia coli overexpressing the inaZ gene of Pseudomonas syringae.

Ice nucleation protein was partially purified from the membrane fraction of E. coli carrying inaZ from Pseudomonas syringae. The ice nucleation protein was totally localized in the bacterial envelope and was extracted by either salt (0.25 M NH4Cl) or the nonionic detergent Tween 20. The extracted protein was partially purified by sequential passage through DEAE-52 cellulose and Sephacryl-S400 columns. The activity of the purified protein was lost after treatment with phospholipase C, and its activity was subsequently restored by addition of the naturally occurring lipid phosphatidylethanolamine. These results suggest that ice nucleation proteins have a requirement for lipids that reconstitute a physiological hydrophobic environment similar to the one existing in vivo, to attain and maintain a structure that enables ice catalysis.

Bacterial Outer Membrane Proteins↗
PubMed1993

Characterization of an insertion sequence (IS53) located within IS51 on the iaa-containing plasmid of Pseudomonas syringae pv. savastanoi.

A 2568-base pair (bp) insertion sequence (IS53) was cloned from the iaa-containing plasmid pIAA2 of Pseudomonas syringae subsp. savastanoi. IS53 is located downstream from the iaa operon and is inserted into IS51, a previously described element on pIAA2. IS53 causes an 8-bp target duplication and has nearly perfect terminal inverted repeats of 27 bp. Several degenerate copies of the inverted repeats are located internal, and in direct orientation to the terminal inverted repeats. A putative transposase gene conforms to the transposase ORF/opposite strand overlapping ORF motif described in many gram-negative IS elements. The region 5' to the presumptive transposase gene contains sequences that are homologous to the consensus E. coli heat-shock (sigma 32) promoter.

Amino Acid Sequence↗
PubMed1994

Characterization of high-frequency deletions in the iaa-containing plasmid, pIAA2, of Pseudomonas syringae pv. savastanoi.

The phytopathogenic bacterium Pseudomonas syringae pv. savastanoi causes olive and oleander knot disease. The bacterium induces the formation of tumorous galls by the synthesis and secretion of the plant hormones trans-zeatin riboside and indole-3-acetic acid into host intercellular spaces. An Italian oleander isolate, PB213, has been observed to lose the ability to synthesize IAA at high frequency, thus becoming non-pathogenic. The IAA genes, located on the 72-kb iaa-containing plasmid, pIAA2 were lost mainly due to two classes of deletions: 18 or 22 kb in length. Both classes of deletions had a common endpoint upstream of the IAA genes. The other endpoints were in areas that flanked the insertion sequence element IS51. The endpoints are in regions of repetitive DNA of at least 271 bp that have been designated a/b.

Base Sequence↗
PubMed1994

Characterization of pPT23B, the plasmid involved in syringolide production by Pseudomonas syringae pv. tomato PT23.

Avirulence gene D (avrD) in strain PT23 of Pseudomonas syringae pv. tomato (Pst) specifies the production of syringolides, which are elicitors of plant defense reactions. An 83-kb indigenous plasmid (pPT23B) that carries avrD has been mapped and characterized and a putative par region was identified. pPT23B contains a large amount of DNA that is repeated in other native plasmids in PT23. A putative mobile insertion element that occurs on plasmid pPT23A as well as on the chromosome was also identified in strain PT23. New broad-host-range expression vectors that functioned in Pst were constructed for overexpression of the cloned avrD gene and high-level production of the syringolides. Introduction of an avrD overexpression plasmid into PT23 or plasmid-cured strains led to identical syringolide peaks on HPLC with no new peaks observed. These results suggested that neither pPT23B nor other indigenous plasmids in Pst carry additional genes required for syringolide production or metabolism. Pst strains lacking pPT23B were not impaired in virulence on tomato plants.

Bacterial Toxins↗
PubMed1991

The indoleacetic acid-lysine synthetase gene of Pseudomonas syringae subsp. savastanoi induces developmental alterations in transgenic tobacco and potato plants.

The iaaL gene of Pseudomonas syringae subsp. savastanoi encodes an indoleacetic acid-lysine synthetase that conjugates lysine to indoleacetic acid. A chimaeric gene consisting of the iaaL coding region under the control of the 35S RNA promoter from cauliflower mosaic virus (35SiaaL) has been used to test if iaaL gene expression leads to morphological alterations in tobacco and potato. Transgenic tobacco plantlets bearing this construct have been shown to synthesize IAA-[14C]lysine when fed with [14C]lysine. In late stages of development, their leaves show an increased nastic curvature (epinasty) of the petiole and midvein, a finding suggestive of an abnormal auxin metabolism. The alteration is transmitted to progeny as a dominant Mendelian trait cosegregating with the kanamycin resistance marker. Transgenic potato plants harbouring the construct are also characterized by petiole epinasty. Moreover, 35SiaaL transgenic plants have an increased internode length in potato and decreased root growth in both tobacco and potato. An increased content of IAA-conjugates in leaf blade was found to correlate with the epinastic alterations caused by iaaL gene expression in tobacco leaves. These data provide evidence that IAA conjugation is able to modulate hormone action, suggesting that the widespread endogenous auxin-conjugating activities are of physiological importance.

Chromatography, High Pressure Liquid↗
PubMed1993

Genetic characterization of the Pto locus of tomato: semi-dominance and cosegregation of resistance to Pseudomonas syringae pathovar tomato and sensitivity to the insecticide Fenthion.

The Pto locus governs resistance to bacterial speck disease in tomato caused by race 0 strains of Pseudomonas syringae pathovar tomato (Pst). Large populations segregating for the Pto locus were generated and genetically characterized. Analysis of the locus has revealed that Pto acts in a semi-dominant manner and cosegegrates with sensitivity to an organophosphorous insecticide, Fenthion, suggesting that Pto may be a complex locus responsible for both phenotypes. We have redefined its map position on chromosome five of the classical genetic map and assigned its position on the molecular map, thus facilitating the alignment of the two genetic maps of the short arm of chromosome five of tomato. Furthermore, we have screened random amplified polymorphic (RAPD) markers for their ability to differentiate near-isogenic lines that differ only with respect to Pto and have identified and mapped seven of these markers. Our results suggest that Pto may be located in a euchromatic region on chromosome five which will be advantageous for the cloning of this locus by one of several molecular strategies.

Base Sequence↗
PubMed1984

Conjugal transfer of E. coli F'lac from Erwinia chrysanthemi to Pseudomonas syringae pv. glycinea and the apparent stable incorporation of the plasmid into the pv. glycinea chromosome.

The E. coli F'lac plasmid was transferred from an Erwinia chrysanthemi Hfr8 donor to a multiply-auxotrophic, rifampicin-resistant Pseudomonas syringae pv. glycinea recipient. Transfer occurred at a frequency of approximately 10(-5)/donor. Stable transconjugants which were able to utilize lactose as the sole carbon source after several transfers would not donate the F'lac plasmid in detectable frequency to other pv. glycinea or E. coli recipients. The plasmid DNA was shown to be integrated into the pv. glycinea chromosome (Fig. 1).

Conjugation, Genetic↗
PubMed1988

Deletion mutagenesis of the ice nucleation gene from Pseudomonas syringae S203.

The ice nucleation gene inaZ, from Pseudomonas syringae S203, was manipulated to produce a series of defined rearrangements in its coding sequence without changing the reading frame. The effects of these mutations on the ice nucleation phenotype were determined in a heterologous host, Escherichia coli K12. Deletions which disrupted the periodicity of 16 codons, in a repetitive region of inaZ, caused the frequencies of ice nuclei in the bacterial population to be significantly depressed; the nuclei with thresholds at warmer temperatures were most affected. In contrast, when the periodicity was left intact, deletions and duplications in the same region had only slight effects on nucleation activity. Deletions removing part or all of one of the nonrepetitive regions (that encoding the amino-terminal domain of the InaZ protein) did not abolish nucleation activity, but caused it to be limited to cooler threshold temperatures. In contrast, the non-repetitive carboxy-terminal domain of the InaZ protein was shown to be essential for ice nucleation at all temperatures. The differential requirements (for periodicity, and for the amino-terminus) in forming nuclei with different thresholds may be significant for understanding what determines the threshold temperature of an ice nucleus.

Amino Acid Sequence↗
PubMed1984

Characterization of eight excision plasmids of Pseudomonas syringae pv. phaseolicola.

Pseudomonas syringae pv. phaseolicola strain LR719 contains a 150 kilobase pair (kb) plasmid pMC7105, stably integrated into its chromosome. Occasionally, single colony isolates of this strain contain an excision plasmid. Eight unique excision plasmids were selected and characterized by BamHI restriction endonuclease and blot hybridization analyses. These plasmids ranged in size from 35 to 270 kb; the largest contained approximately 130 kb of chromosomal DNA sequences. Restriction maps of pMC7105 were developed to deduce the site of integration and to identify the fragments in which recombination occurred to produce each excision plasmid. The eight excision plasmids were arranged into five classes based on the sites where excision occurs. A 20 kb region of pMC7105, which includes BamHI fragment 9 and portions of adjacent fragments, is present in all excision plasmids and thought to contain the origin of replication. The site of integration on pMC7105 maps within BamHI fragment 8. This fragment shows homology with seven other BamHI fragments of pMC7105 and with five chromosomal fragments identified among the excision plasmids. The data strongly suggest that the integration of pMC7105 may have occurred at a repetitive sequence present on the chromosome and on the plasmid.

Cloning, Molecular↗