Ornithine carbamoyltransferase genes and phaseolotoxin immunity in Pseudomonas syringae pv. phaseolicola

Two different DNA fragments encoding ornithine carbamoyltransferase (OCTase) were cloned from Pseudomonas syringae pv. phaseolicola NPS3121. These fragments did not cross-hybridize and encoded OCTases which differed with respect to their sensitivity to purified phaseolotoxin, an OCTase inhibitor produced by this phytopathogenic bacterium. Recombinant plasmids carrying these DNA fragments complemented OCTase-deficient strains of Escherichia coli and Pseudomonas aeruginosa. Extracts of the complemented E. coli strain contained OCTase enzyme activities with similar degrees of sensitivity to purified phaseolotoxin as extracts of P.s.phaseolicola grown at either 20 or 30°C. The OCTase activity detectable in extracts of P.s.phaseolicola grown at 20°C is insensitive to phaseolotoxin while that detectable in extracts of cells grown at 30°C is sensitive to the toxin. E.coli HB101 harboring recombinant plasmids carrying the gene(s) encoding the phaseolotoxin-insensitive enzyme activity exhibited resistance to purified phaseolotoxin. The results of Tn5 mutagenesis and Southern blotting and the pattern of complementation of OCTase-deficient and Tox^- mutant strains suggest that the gene(s) encoding the phaseolotoxin-insensitive OCTase is part of a gene cluster involved in phaseolotoxin production.     

Characterization of plasmids from plant pathogenic pseudomonads

Physical characterization of the resident plasmids from Pseudomonas tabaci, P. angulata, and P. coronafaciens strains indicated that they harbored five different plasmid DNA species. Two ATCC strains of P. tabaci contained indistinguishable plasmids that we have named pJP1 and pJP2. An isolate of one of these strains contained a spontaneous variant of pJP1, pJP1¹, which contains an insertion of 3.9 Mdal. This 3.9-Mdal region did not hybridize to pJP1 indicating that the region was foreign DNA and not a duplication of a segment of DNA already present in pJP1. Another P. tabaci strain, PT27881, contained a third plasmid species, pJP27, which had few similarities to pJP1 or pJP2, but was indistinguishable from the plasmids from all three P. angulata strains. pJP27 and pJP1 had a small region, 8.8 Mdal, of sequence homology. The one strain of P. coronafaciens examined contained a plasmid, pJP50, which was different from the P. tabaci plasmids, but had the 8.8-Mdal region and additional regions of sequence homology with pJP1 and pJP27 as well as homology with a portion of the pJP1¹ insertion. A fourth strain of P. tabaci, PTBR-2, a pathogen on beans, contained plasmid pBW, the only plasmid that lacked detectable regions of homology with the other plasmids.     

Construction of Tn3-Containing Plasmids from Plant-Pathogenic Pseudomonads and an Examination of Their Biological Properties

Indigenous plasmids isolated from Pseudomonas tabaci ATCC 11528(pJP1), Pseudomonas angulata 45(pJP30), and P. tabaci BR2(pBPW1) (M. Obukowicz and P. D. Shaw, J. Bacteriol. 155:438-442, 1983) were labeled with Tn3, and the strains were subsequently cured of their respective plasmids. Plasmid-containing and cured isolates caused plant symptoms that were nearly indistinguishable, and the same amount of tabtoxin was produced by P. tabaci strains ATCC 11528 and BR2.     

Apparent Involvement of a Plasmid in Phaseotoxin Production by Pseudomonas phaseolicola

Three naturally occurring toxigenic strains (HB-36, G-50, and HB-33), one nontoxigenic strain (HB-20), and one ultraviolet light-induced toxinless mutant (G-50 Tox⁻) of Pseudomonas phaseolicola were examined by dye-buoyant density equilibrium centrifugation for the presence of plasmid deoxyribonucleic acid. All strains contained plasmid deoxyribonucleic acid. Comparison of the plasmid deoxyribonucleic acid of different strains by agarose gel electrophoresis showed that strain G-50 harbored three plasmids, whereas the rest of the strains contained two plasmids each. Irrespective of their toxigenicity, all strains shared the large-sized first plasmid band, but differed with respect to other plasmids. Restriction endonuclease analyses of the plasmids indicated that a 22.50-megadalton plasmid was common to two of the toxigenic strains (HB-36 and G-50). However, strain HB-33, which is also toxigenic, contained a much smaller plasmid (4.23 megadaltons). It is hypothesized that this small plasmid may have arisen by a recombination event from a larger plasmid.     

R-Plasmid Transfer in Zymomonas mobilis

Conjugal transfer of three IncP1 plasmids and one IncFII plasmid into strains of the ethanol-producing bacterium Zymomonas mobilis was obtained. These plasmids were transferred at high frequencies from Escherichia coli and Pseudomonas aeruginosa into Z. mobilis and also between different Z. mobilis strains, using the membrane filter mating technique. Most of the plasmids were stably maintained in Z. mobilis, although there was some evidence of delayed marker expression. A low level of chromosomal gene transfer, mediated by plasmid R68.45, was detected between Z. mobilis strains. Genetic evidence suggesting that Z. mobilis may be more closely related to E. coli than to Pseudomonas or Rhizobium is discussed.     

Overexpression and in vitro reconstitution of the ethylene-forming enzyme from Pseudomonas syringae

By replacing a native promoter with lac and tac promoters, the gene encoding an ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 was overexpressed in Escherichia coli. The EFE protein expressed by a multicopy plasmid accounted for more than 30% of the total cellular protein, resulting in ethylene-forming activities higher than 10 μl of ethylene (mg cell)⁻¹h⁻¹ in recombinant E. coli cells. However, most of the EFE protein accumulated as inactive inclusion bodies particularly at elevated temperatures (>30°C). We present an efficient procedure for reconstituting an active enzyme from inclusion bodies by solubilization with 8 M urea and dialysis. The reconstituted EFE has specific activity identical to that of the native enzyme from P. syringae, suggesting that the EFE protein has an intrinsic folding capability in vitro.     

Heterologous expression of the gene for the ethylene-forming enzyme fromPseudomonas syringae in the cyanobacteriumSynechococcus

Summary Bioconversion of atmospheric carbon dioxide to ethylene was studied in a recombinant cyanobacterium. The gene for the ethylene-forming enzyme ofPseudomonas syringae pv.phaseolicola PK2 was cloned and expressed in the cyanobacteriumSynechococcus PCC7942 R2-SPc by use of a shuttle vector pUC303. The ethylene-forming activityin vivo ofSynechococcus PCC7942 R2-SPc that carried the gene for the ethylene-forming enzyme ofP. syringae pv.phaseolicola PK2 was one-fifth of that ofE. coli JM109 that harbored the same plasmid. The enzyme accounted for 0.021% by weight of the total soluble protein inSynechococcus PCC7942 R2-SPc.     

Cell Surface Properties of Pseudomonas syringae pv. phaseolicola Wild-type and hrp Mutants

The cell surface hydrophobicity and charge as well as surface polysaccharides of eight independent prototrophic hrp::-Tn5 mutants (Lindgren et al., J. Bacteriol. 168 , 512–522, 1986) were compared to the wild-type parent strain NPS3121 of Pseudomonas syringae pv. phaseolicola. No significant differences were found in cell surface charge, but mutant strain NPS4005 exhibited significantly lower cell surface hydrophobicity than the wild-type and the other mutant strains. The mutant strains all retained the ability to produce the exopolysaccharides (EPS) levan, a neutral fructan, and alginate, an acidic polymer. Relative amounts of EPS produced in vitro was dependent on culture conditions. Lipopolysaccharide (LPS) chemotypes were similar for all nine strains. Chemical as well as ¹³C-NMR analyses of the O-antigens from four wild-type strains of P. s. pv. phaseolicola representing two physiological races as well as the O-antigens of two strains of P. s. pv. syringae which belong to the same serogroup as P. s. pv. phaseolicola indicated that all of the O-antigens were very similar if not identical. LPS of three strains of P. s. pv. phaseolicola produced in vitro or in planta were also compared and no significant differences were detected. The altered phenotype of the Tn5 mutants of P. s. pv. phaseolicola does not appear to be due to changes in the ability to produce exopolysaccharides or to an altered composition of cell surface polysaccharides (LPS and EPS). However, a change in an unidentified cell surface component(s) leading to lowered cell surface hydrophobicity of mutant strain NPS4005 may be important.     

Insertion specificity and trans-activation of IS801

The transposable element IS801, isolated from plasmid pMMC7105 of Pseudomonas syringae pv. phaseolicola, transposes in Escherichia coli to plasmid targets, expressing a relatively relaxed target specificity. The target sequences are tetramers with homology with the left terminus (GAAC) of the transposing unit, the alternative targets being GAAC, GGAC, CAAG, and CGAC. In the areas flanking IS801 in 13 different locations, no similarities other than the target tetramer were observed. The transposase is physically and functionally separable from the transposing unit since transposition of constructs carrying marker genes occurs with the transposase expressed in trans. The IS801 transposase shows amino acid sequence homology to the transposases of the E. coli elements IS91 and IS1294. These tranposases contain conserved amino acid motifs found in the replicases of certain plasmids that replicate as rolling circles.     

Sodium Transport and Compartmentation in Spergularia marina: Partial Characterization of a Functional Symplasm

Cytokinin production by strains of the phytopathogenic bacterium Pseudomonas syringae pv savastanoi was measured by immunoaffinity chromatography of the culture medium on immobilized anti-cytokinin antibodies, followed by high performance liquid chromatography, radioimmunoassay and mass spectrometry. P. savastanoi strain PB213-2 secretes zeatin (80 nanograms per milliliter) and ribosylzeatin (80 nanograms per milliliter). Even higher levels of zeatin (400 nanograms per milliliter) are produced by the olive-specific strain EW1006, which also produces 180 nanograms per milliliter of the recently identified cytokinin, ribosyl-1″ -methylzeatin. The amounts secreted were approximately 1000 times greater than those secreted by Agrobacterium tumefaciens (DA Regier, RO Morris 1982 Biochem Biophys Res Commun 104: 1560-1566). Examination of cytokinin production by plasmid deletion mutants of PB213-2 and EW1006 indicated that cytokinin biosynthesis was specified, at least in part, by plasmid-borne genes. A fragment of the 105 kilobase pair plasmid from EW1006 was cloned into Escherichia coli where its expression resulted in dimethylallyl transferase activity and the secretion of zeatin.     

Interactions between Pseudomonas syringae pv. tabaci and Two Rhizosphere Hosts, Medicago sativa and Avena sativa

The interactions between Pseudomonas syringae pv. tabaci and either nodulating alfalfa (Medicago sativa) or oat (Avena sativa) seedlings were examined to further our understanding of this rhizosphere association. P. syringae pv. tabaci produces and releases a toxin, tabtoxinine-β-lactam (TβL), that inactivates glutamine synthetase (GS). Sinorhizobium meliloti grew well in the presence of TβL in culture and on alfalfa roots. The alfalfa symbiont, S. meliloti, and its bacteroids contained TβL-sensitive glutamine synthetases and TβL detoxifying-β-lactamase. The GS of alfalfa leaves is also sensitive to TβL, but GS activity was unaffected in infested plants. Toxin production was apparently suppressed in the alfalfa and nitrate-fed oat rhizospheres since these plants survived and retained significant amounts of leaf GS activity. The water-soluble extracts of these rhizospheres inhibited TPL production in culture and the inhibition was correlated with the amount of reduced nitrogen present. Furthermore, representative mixtures of pure ammonium and amino acids inhibited TβL production in culture in a concentration dependent manner. Thus, a bi-directional interaction occurs between the nitrogen metabolism of alfalfa and oat and TβL production by P. syringae pv. tabaci.     

Metabolic engineering of E. coli for pyocyanin production

Pyocyanin is a secondary metabolite from Pseudomonas aeruginosa that belongs to the class of phenazines, which are aromatic nitrogenous compounds with numerous biological functions. Besides its antifungal and antimicrobial activities, pyocyanin is a remarkable redox-active molecule with potential applications ranging from the pharma industry to the development of microbial fuel cells. Nevertheless, pyocyanin production has been restricted to P. aeruginosa strains, limiting its practical applicability. In this study, the pyocyanin biosynthetic pathway was engineered for the first time for high level production of this compound in a heterologous host. Escherichia coli cells harboring the nine-gene pathway divided into two plasmids were able to produce and secrete pyocyanin at higher levels than some Pseudomonas aeruginosa strains. The influence of culture and induction parameters were evaluated, and the optimized conditions led to an increase of 3.5-fold on pyocyanin accumulation. Pathway balancing was achieved by testing a set of plasmids with different copy numbers to optimize the expression levels of pyocyanin biosynthetic genes, resulting in a fourfold difference in product titer among the engineered strains. Further improvements were achieved by co-expression of Vitreoscilla hemoglobin Vhb, which relieved oxygen limitations and led to a final titer of 18.8 mg/L pyocyanin. These results show promise to use E. coli for phenazines production, and the engineered strain developed here has the potential to be used in electro-fermentation systems where pyocyanin plays a role as electron-shuttle.     

Expanding the results of a high throughput screen against an isochorismate-pyruvate lyase to enzymes of a similar scaffold or mechanism

Antibiotic resistance is a growing health concern, and new avenues of antimicrobial drug design are being actively sought. One suggested pathway to be targeted for inhibitor design is that of iron scavenging through siderophores. Here we present a high throughput screen to the isochorismate-pyruvate lyase of Pseudomonas aeruginosa, an enzyme required for the production of the siderophore pyochelin. Compounds identified in the screen are high nanomolar to low micromolar inhibitors of the enzyme and produce growth inhibition in PAO1 P. aeruginosa in the millimolar range under iron-limiting conditions. The identified compounds were also tested for enzymatic inhibition of Escherichia coli chorismate mutase, a protein of similar fold and similar chemistry, and of Yersinia enterocolitica salicylate synthase, a protein of differing fold but catalyzing the same lyase reaction. In both cases, subsets of the inhibitors from the screen were found to be inhibitory to enzymatic activity (mutase or synthase) in the micromolar range and capable of growth inhibition in their respective organisms (E. coli or Y. enterocolitica).     

Genes required for pathogenicity and hypersensitivity are conserved and interchangeable among pathovars of Pseudomonas syringae

Summary A group of pathogenicity genes was previously identified in Pseudomonas syringae pv. phaseolicola which controls the ability of the pathogen to cause disease on bean and to elicit the hypersensitive response on non-host plants. These genes, designated hrp, are located in a ca. 20 kb region which was referred to as the hrp cluster. Homologous sequences to DNA segments derived from this region were detected in several pathovars of P. syringae but not in symbiotic, saprophytic or other phytopathogenic bacteria. A Tn5-induced Hrp^- mutation was transferred from P. syringae pv. phaseolicola to P. syringae pv. tabaci and to three races of P. syringae pv. glycinea by marker exchange mutagenesis. The resulting progeny were phenotypically Hrp^-, i.e. no longer pathogenic on their respective hosts and unable to elicit the hypersensitive response on non-host plants. These mutants were restored to wild-type phenotype upon introduction of a recombinant plasmid carrying the corresponding wild-type locus from P. syringae pv. phaseolicola. The marker exchange mutants of P. syringae pv. glycinea psg0 and Psg5 which carry different avr genes for race specific avirulence did not elicit a hypersensitive response on incompatible soybean cultivars. It appears, therefore, that P. syringae pathovars possess common genes for pathogenicity which also control their interaction with non-host plants. Furthermore, the expression of race/cultivar specific incompatibility of P. syringae pv. glycinea requires a fully functional hrp region in addition to the avr genes which determine avirulence on single-gene differential cultivars of soybean.     

PsasM2I,a Type II Restriction–Modification System in Pseudomonas savastanoi pv. savastanoi: Differential Distribution of Carrier Strains in the Environment and the Evolutionary History of Homologous RM Systems in the Pseudomonas syringae Complex

A type II restriction–modification system was found in a native plasmid of Pseudomonas savastanoi pv. savastanoi MLLI2. Functional analysis of the methyltransferase showed that the enzyme acts by protecting the DNA sequence CTGCAG from cleavage. Restriction endonuclease expression in recombinant Escherichia coli cells resulted in mutations in the REase sequence or transposition of insertion sequence 1A in the coding sequence, preventing lethal gene expression. Population screening detected homologous RM systems in other P. savastanoi strains and in the Pseudomonas syringae complex. An epidemiological survey carried out by sampling olive and oleander knots in two Italian regions showed an uneven diffusion of carrier strains, whose presence could be related to a selective advantage in maintaining the RM system in particular environments or subpopulations. Moreover, carrier strains can coexist in the same orchards, plants, and knot tissues with non-carriers, revealing unexpected genetic variability on a very small spatial scale. Phylogenetic analysis of the RM system and housekeeping gene sequences in the P. syringae complex demonstrated the ancient acquisition of the RM systems. However, the evolutionary history of the gene complex also showed the involvement of horizontal gene transfer between related strains and recombination events.     

Role of the parCBA Operon of the Broad-Host-Range Plasmid RK2 in Stable Plasmid Maintenance

The par region of the stably maintained broad-host-range plasmid RK2 is organized as two divergent operons, parCBA and parDE, and a cis-acting site. parDE encodes a postsegregational killing system, and parCBA encodes a resolvase (ParA), a nuclease (ParB), and a protein of unknown function (ParC). The present study was undertaken to further delineate the role of the parCBA region in the stable maintenance of RK2 by first introducing precise deletions in the three genes and then assessing the abilities of the different constructs to stabilize RK2 in three strains of Escherichia coli and two strains of Pseudomonas aeruginosa. The intact parCBA operon was effective in stabilizing a conjugation-defective RK2 derivative in E. coli MC1061K and RR1 but was relatively ineffective in E. coli MV10Δlac. In the two strains in which the parCBA operon was effective, deletions in parB, parC, or both parB and parC caused an approximately twofold reduction in the stabilizing ability of the operon, while a deletion in the parA gene resulted in a much greater loss of parCBA activity. For P. aeruginosa PAO1161Rif^r, the parCBA operon provided little if any plasmid stability, but for P. aeruginosa PAC452Rif^r, the RK2 plasmid was stabilized to a substantial extent by parCBA. With this latter strain, parA and res alone were sufficient for stabilization. The cer resolvase system of plasmid ColE1 and the loxP/Cre system of plasmid P1 were tested in comparison with the parCBA operon. We found that, not unlike what was previously observed with MC1061K, cer failed to stabilize the RK2 plasmid with par deletions in strain MV10Δlac, but this multimer resolution system was effective in stabilizing the plasmid in strain RR1. The loxP/Cre system, on the other hand, was very effective in stabilizing the plasmid in all three E. coli strains. These observations indicate that the parA gene, along with its res site, exhibits a significant level of plasmid stabilization in the absence of the parC and parB genes but that in at least one E. coli strain, all three genes are required for maximum stabilization. It cannot be determined from these results whether or not the stabilization effects seen with parCBA or the cer and loxP/Cre systems are strictly due to a reduction in the level of RK2 dimers and an increase in the number of plasmid monomer units or if these systems play a role in a more complex process of plasmid stabilization that requires as an essential step the resolution of plasmid dimers.     

Homologous Streptomycin Resistance Gene Present among Diverse Gram-Negative Bacteria in New York State Apple Orchards

The streptomycin resistance gene of Pseudomonas syringae pv. papulans Psp36 was cloned into Escherichia coli and used to develop a 500-bp DNA probe that is specific for streptomycin resistance in P. syringae pv. papulans. The probe is a portion of a 1-kb region shared by three different DNA clones of the resistance gene. In Southern hybridizations, the probe hybridized only with DNA isolated from streptomycin-resistant strains of P. syringae pv. papulans and not with the DNA of streptomycin-sensitive strains. Transposon insertions within the region of DNA shared by the three clones resulted in loss of resistance to streptomycin. Colony hybridization of bacteria isolated from apple leaves and orchard soil indicated that 39% of 398 streptomycin-resistant bacteria contained DNA that hybridized to the probe. These included all strains of P. syringae pv. papulans and some other fluorescent pseudomonads and nonfluorescent gram-negative bacteria, but none of the gram-positive bacteria. The same-size restriction fragments hybridized to the probe in P. syringae pv. papulans. Restriction fragment length polymorphism of this region was occasionally observed in strains of other taxonomic groups of bacteria. In bacteria other than P. syringae pv. papulans, the streptomycin resistance probe hybridized to different-sized plasmids and no relationship between plasmid size and taxonomic group or between plasmid size and orchard type, soil association, or leaf association could be detected.     

Mode of Action of the Toxin from Pseudomonas phaseolicola: I. Toxin Specificity, Chlorosis, and Ornithine Accumulation

The specificity of the Pseudomonas phaseolicola toxin for enzyme inhibition and its relationship to toxin-induced chlorosis in bean leaves (Phaseolus vulgaris L.) was examined. The toxin showed no significant inhibitory activity against glutamine synthetase, glutamine transferase, carbamyl phosphate synthetase, aspartate carbamoyltransferase, or arginase at concentrations 100-fold higher than that needed to inhibit ornithine carbamoyltransferase by 50%.