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Chemotaxis of Pseudomonas syringae subsp. savastanoi Virulence Mutants.

Several mutants of Pseudomonas syringae subsp. savastanoi were tested for their ability to sense and respond to a chemotactic gradient in low concentrations of yeast extract. The mutants were deficient in one or both of the genes coding for the synthesis of the plant hormones indole-3-acetic acid (IAA) and isopentenyl adenosine. Mutations which resulted in the loss of IAA production were due to the loss of the entire plasmid containing the iaa operon or to an 18-kb deletion of the iaa region. Additional mutants tested were deficient in their ability to produce isopentenyl adenosine as a result of the loss of the ptz-bearing plasmid. In all cases, strains which had lost the ability to produce IAA exhibited enhanced motility of up to 2.5 times that of the wild type (IAA) in medium containing 0.01% yeast extract. No differences in motility were observed on medium containing lower concentrations of yeast extract. The presence or absence of the cytokinin plasmid and the presence or absence of inorganic nitrogen in the medium had no effect on the relative mobility of the strains.

Journal Article↗

Evaluation of Four Aerobiological Sampling Methods for the Retrieval of Aerosolized Pseudomonas syringae.

The Andersen six-stage impactor, the SAS (Surface Air System) impactor, the AGI-30 impinger, and gravity plates were evaluated for the retrieval of aerosol-released Pseudomonas syringae. The upper limits of the impactor samplers were exceeded at a spray concentration of 10 CFU/ml, indicating that these samplers are not appropriate for monitoring high airborne concentrations. Decreased cell concentrations were retrieved with increased sampling time for the Andersen and AGI samplers, indicating that a minimum sampling time is preferable for monitoring aerosolized vegetative cells.

Journal Article↗

Evidence for a correlation between auxin production and host plant species among strains of Pseudomonas syringae subsp. savastanoi.

Auxin production by 131 strains of Pseudomonas syringae subsp. savastanoi was investigated with the aim of looking for correlations among this characteristic and the origin of the strains, the types of symptoms, and the host plant. Most of the P.syringae subsp. savastanoi strains, except those isolated from ash, produced auxin and harbored iaa genes. Among ash strains, which were pathogenic only on ash, only 2 out of 33 were found to produce auxin and to harbor iaa genes.

Indoleacetic Acids↗

Effect of phenotypic plasticity on epiphytic survival and colonization by Pseudomonas syringae.

The bacterial epiphyte Pseudomonas syringae MF714R was cultured on agar or in broth or collected from colonized leaves; it was then inoculated onto greenhouse-grown bean plants incubated in a growth chamber at low relative humidity or in the field or onto field-grown bean plants. Cells cultured in liquid medium survived the least well after inoculation of leaf surfaces under all conditions. Cells cultured in solid medium exhibited the highest percent survival and desiccation tolerance in the growth chamber but generally survived less well in the field than did cells harvested from plants. Cells harvested from plants and inoculated onto plants in the field usually exhibited the highest percent survival, started to increase in population earlier, and reached a higher number than did cells cultured in vitro. Differences in field survival were apparently not attributable to differential UV tolerance. The observed effects of phenotypic plasticity on epiphytic survival and colonization should be considered in risk assessment studies, in studies of bacterial epidemiology, and in the use of microbial antagonists for biological pest control.

Bacteriological Techniques↗

Similarity between Copper Resistance Genes from Xanthomonas campestris and Pseudomonas syringae.

Plasmid-borne copper resistance genes from copper-resistant strains of Xanthomonas campestris pv. vesicatoria from California, Florida, and Oklahoma shared structural similarities. A strain of X. campestris pv. campestris also contained plasmid-borne copper resistance genes similar to the resistance genes from X. campestris pv. vesicatoria. Furthermore, a region of the copper resistance genes from X. campestris pv. vesicatoria 07882 hybridized with copA, the first gene of the copper resistance operon (cop) of Pseudomonas syringae pv. tomato. A copper-inducible protein of similar size to CopA was detected by Western blot (immunoblot) analysis from the wild-type strain 07882 and from the cloned copper resistance genes of 07882 introduced into a copper-sensitive strain of X. campestris pv. vesicatoria. A low level of hybridization was observed with chromosomal DNA from other xanthomonads when the copper resistance genes from strain 07882 were used as probes.

Journal Article↗

Development of a gene reporter system in moderately halophilic bacteria by employing the ice nucleation gene of Pseudomonas syringae.

The expression of the ice nucleation gene inaZ of Pseudomonas syringae in several moderate halophiles was investigated to establish its utility as a reporter for promoter activity and gene expression studies in these biotechnologically and environmentally important bacteria. A promoterless version of inaZ was introduced in two different restriction sites and at both orientations in a recombinant plasmid able to replicate in moderate halophiles and, in particular, within the sequence of its pHE1 part, a native plasmid of Halomonas elongata. One orientation of both recombinant constructs expressed high levels of ice nucleation activity in H. elongata and Volcaniella eurihalina cells, indicating that inaZ was probably introduced in the correct orientation downstream of putative native promoters. A recombinant construct carrying a tandem duplication of inaZ at the same orientation gave significantly higher ice nucleation activity, showing that inaZ is appropriate for gene dosage studies. The ice nucleation gene was also expressed in H. elongata and V. eurihalina under the control of Pbla (the promoter of the beta-lactamase gene of Escherichia coli) and Ppdc (the promoter of the pyruvate decarboxylase gene of Zymomonas mobilis). One of the inaZ reporter plasmids expressing high levels of ice nucleation activity under the control of a native putative promoter was also transferred in Halomonas subglaciescola, Halomonas meridiana, Halomonas halodurans, and Deleya halophila. In all cases, Ice+ transconjugants were successfully isolated, demonstrating that inaZ is expressed in a wide spectrum of moderately halophilic species.

Bacterial Outer Membrane Proteins↗

Altered Epiphytic Colonization of Mannityl Opine-Producing Transgenic Tobacco Plants by a Mannityl Opine-Catabolizing Strain of Pseudomonas syringae.

The plasmid pYDH208, which confers the ability to catabolize the mannityl opines mannopine and agropine, was mobilized into the nonpathogenic Pseudomonas syringae strain Cit7. The growth of the mannityl opine-catabolizing strain Cit7(pYDH208) was compared with that of the near-isogenic non-opine-catabolizing strain Cit7xylE on leaves of wild-type tobacco (Nicotiana tabacum cv. Xanthi) and transgenic mannityl opine-producing tobacco plants (N. tabacum cv. Xanthi, line 2-26). The population size of Cit7(pYDH208) was significantly greater on the lower leaves of transgenic plants than on middle or upper leaves of those plants. The population size of Cit7(pYDH208) on lower leaves of transgenic plants was also significantly greater than the population size of Cit7xylE on similar leaves of wild-type plants. High-voltage paper electrophoresis demonstrated higher levels of mannityl opines in washings from lower- and mid-level leaves than in washings from upper-level leaves. The ability of Cit7(pYDH208) to catabolize mannityl opines in the carbon-limited phyllosphere increased the carrying capacity of the lower leaves of transgenic plants for Cit7(pYDH208). In coinoculations, the increase in the ratio of population sizes of Cit7(pYDH208) to Cit7xylE on transgenic plants was apparently due to a subtle difference in the growth rates of the two strains and to the difference in final population sizes. An ability to utilize additional carbon sources on the transgenic plants also enabled Cit7(pYDH208) to achieve a higher degree of coexistence with Cit7xylE on transgenic plants than on wild-type plants. This supports the hypothesis that the level of coexistence between epiphytic bacterial populations can be altered through nutritional resource partitioning.

Journal Article↗

Ice nucleation induced by pseudomonas syringae.

Broth cultures of suspensions of Pseudomonas syringae isolated from decaying alder leaves (Alnus tenuifolia) were found to freeze at very warm (-1.8 to -3.8 C) temperatures. The initiation of freezing appears associated with the intact cell and not with extracellular material. Chemical treatments and physical destruction of the cell destroy activity. Bacteria must be in concentrations of approximately 10(6)/ml before freezing at warm temperatures occurs.

Air Microbiology↗

Phaseolotoxin-insensitive ornithine carbamoyltransferase of Pseudomonas syringae pv. phaseolicola: basis for immunity to phaseolotoxin.

Cell-free extracts from phaseolotoxin-producing strains of Pseudomonas syringae pv. phaseolicola grown at 18 degrees C, the optimum temperature for phaseolotoxin production, contain an ornithine carbamoyltransferase activity that is insensitive to phaseolotoxin. Extracts from the same strains grown at 30 degrees C, a temperature at which little or no detectable phaseolotoxin is produced, and from phaseolotoxin-nonproducing strains contain a phaseolotoxin-sensitive ornithine carbamoyltransferase activity. The phaseolotoxin-insensitive ornithine carbamoyltransferase activity is also less senstive to N delta-(phosphonacetyl)-L-ornithine than the phaseolotoxin-sensitive ornithine carbamoyltransferase activity of the corresponding strain.

Bacterial Toxins↗

Integration and partial excision of a cryptic plasmid in Pseudomonas syringae pv. phaseolicola.

A virulent strain of Pseudomonas syringae pv. phaseolicola, a pathogen of the common bean Phaseolus vulgaris (L.), was shown to harbor a 98-megadalton cryptic plasmid, pMC7105. After exposure of this strain to the plasmid-curing agent mitomycin C, a colony was isolated which had no detectable extrachromosomal DNA. Hybridization of labeled pMC7105 probe to nitrocellulose filters containing Southern-blotted BamHI cleavage products of cellular DNA revealed that pMC7105 was integrated into the chromosome rather than cured from this strain. Imprecise excision of pMC7105 resulted in the formation of three smaller plasmids of 34, 50, and 58 megadaltons. BamHI and EcoRI fingerprint analyses revealed that these plasmids were excised from a common region of pMC7105. The BamHI fragments of pMC7105 which were not present in the excision plasmids remained integrated and could be detected by hybridization of pMC7105 probe to Southern-blotted cellular DNA from these strains. Certain chromosomal fragments also had homology with the pMC7105 probe. The excision plasmids were stably maintained and neither integration nor excision altered the pathogenicity of these strains.

Chromosomes, Bacterial↗

Integration and excision of pMC7105 in Pseudomonas syringae pv. phaseolicola: involvement of repetitive sequences.

The site for integration of pMC7105 into the chromosome of Pseudomonas syringae pv. phaseolicola has been mapped to a 2.6-kilobase-pair (kb) Bg/II-EcoRI fragment on this 150-kb indigenous plasmid. Selected excision plasmids resulting from imprecise excision of pMC7105 were used to identify one of the plasmid-chromosome juncture fragments and to characterize the mechanism of recombination from the chromosome. A 14.2-kb BamHI plasmid-chromosome juncture fragment has been identified in pEX8060 (234 kb), an excision plasmid which carries approximately 90 kb of chromosomal sequences to the left of the site of integration. This fragment contains a portion of the 2.6-kb Bg/II-EcoRI fragment as well as chromosomal sequences. Blot hybridization with a probe made from selected fragments of pMC7105 revealed three distinct repetitive sequences, RS-I, RS-II, and RS-III, on this plasmid. The 2.6-kb fragment, to which the site of integration maps, also contains RS-II. Five copies of RS-II are present in pMC7105, and more than 20 copies are present in the chromosome. Eight small excision plasmids were shown to result from recombination among fragments of pMC7105 that contain common repetitive sequences. The results indicate that integration and excision of pMC7105 occur through general recombination at homologous repetitive sequences.

Chromosomes, Bacterial↗

Identification and cloning of genes involved in phaseolotoxin production by Pseudomonas syringae pv. "phaseolicola".

Genes involved in the production of phaseolotoxin by Pseudomonas syringae pv. "phaseolicola" NPS3121 were identified by Tn5 mutagenesis and cosmid cloning. A total of 5,180 kanamycin-resistant colonies were screened for the loss of phaseolotoxin production by a microbiological assay. Six independent, prototrophic, Tox- mutants were isolated that had Tn5 insertions in five different EcoRI fragments. All six mutants had Tn5 inserted in the same KpnI fragment, which had a length of ca. 28 kilobases including Tn5. The mutants produced residual toxin in vitro. An EcoRI fragment containing Tn5 and flanking sequences from mutant NPS4336 was cloned and used to probe a wild-type genomic library by colony hybridization. Seven recombinant plasmids showing homology to this probe were identified. Each Tox- mutant was restored in OCTase-specific toxin production by two or more of the recombinant plasmids. The data suggest that at least some of the genes involved in phaseolotoxin production were clustered in a large KpnI fragment. No homology was detected between the Tn5 target fragment cloned from mutant NPS4336 and the total genomic DNA from closely or distantly related bacteria that do not produce phaseolotoxin.

Cloning, Molecular↗

Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea.

A wide-host-range cosmid cloning vector, pLAFR3, was constructed and used to make cosmid libraries of partially digested Sau3A DNA from race 0 and race 1 of Pseudomonas syringae pv. glycinea. Two avirulence genes, avrB0 and avrC, cloned from race 0, elicited the hypersensitivity reaction (HR) on specific cultivars of soybean. Race 4 transconjugants containing avrB0 induced a dark brown necrotic HR within 24 h on the soybean cultivars Harosoy and Norchief, whereas race 4 transconjugants containing avrC induced a light brown necrotic HR within 48 h on the soybean cultivars Acme, Peking, Norchief, and Flambeau. An additional avirulence gene, avrB1, cloned from race 1, appeared to be identical to avrB0 from race 0. The avrB0 and avrC genes from race 0 were characterized by restriction enzyme mapping, Southern blot analysis, Tn5 transposon mutagenesis, and site-directed gene replacements. The effects of these three genes on the in planta bacterial growth of race 4 transconjugants have also been examined. The identification and cloning of avrB1 provides genetic evidence for a gene-for-gene interaction in the bacterial blight disease of soybean, as avrB1 from race 1 interacts with the soybean disease resistance locus, Rpg1.

Cloning, Molecular↗

Molecular characterization and nucleic acid sequence of an avirulence gene from race 6 of Pseudomonas syringae pv. glycinea.

A gene was previously cloned from Pseudomonas syringae pv. glycinea race 6, designated avirulence gene A (avrA), that controls the expression of virulence by the pathogen on specific cultivars of soybean. A 3.2-kilobase (kb) AccI subclone from the cosmid clone pPg6L3 was shown to be active when cloned into the broad-host-range vector pRK404. Transposon Tn5 mutagenesis and deletion analysis delineated a span of approximately 2.5 kb of DNA that was necessary for gene activity. The nucleotide sequence of a 3.409-kb segment of DNA which contained the avrA gene has been determined. An open reading frame of 2.721 kb of DNA, which correlates with the region of DNA defined by transposon mutagenesis and deletion analysis, was identified. The open reading frame would encode a protein of 100.866 kilodaltons, which is in good agreement with the 100-kilodalton protein expressed by Escherichia coli maxicells.

Amino Acid Sequence↗

Role of indoleacetic acid-lysine synthetase in regulation of indoleacetic acid pool size and virulence of Pseudomonas syringae subsp. savastanoi.

The phytopathogen Pseudomonas syringae subsp. savastanoi incites the production of galls on olive and oleander plants. Gall formation is dependent upon the bacterial synthesis of the phytohormone indole-3-acetic acid (IAA). Strains isolated from oleander galls are capable of further metabolizing IAA to an amino acid conjugate, 3-indoleacetyl-epsilon-L-lysine (IAA-lysine); bacterial olive gall isolates lack this activity. In this study, the cloned gene for IAA-lysine synthetase (iaaL+) was introduced into strains isolated from olive and oleander galls to determine its effect on the regulation of IAA pool size and virulence. IAA-lysine was synthesized by isolates from olive galls when iaaL+ was introduced by conjugation, but the amount of IAA which accumulated in culture by the transconjugant was reduced by one-third. When the iaaL+ locus of an oleander gall isolate was inactivated by Tn5 mutagenesis, the resulting mutant did not convert IAA to IAA-lysine; however, it accumulated fivefold more IAA in culture than the wild type did. When inoculated into oleander plants, the iaaL mutant did not cause typical gall symptoms, nor did it replicate within host tissue similarly to the wild type.

Conjugation, Genetic↗

Nucleotide sequence and organization of copper resistance genes from Pseudomonas syringae pv. tomato.

The nucleotide sequence of a 4.5-kilobase copper resistance determinant from Pseudomonas syringae pv. tomato revealed four open reading frames (ORFs) in the same orientation. Deletion and site-specific mutational analyses indicated that the first two ORFs were essential for copper resistance; the last two ORFs were required for full resistance, but low-level resistance could be conferred in their absence. Five highly conserved, direct 24-base repeats were found near the beginning of the second ORF, and a similar, but less conserved, repeated region was found in the middle of the first ORF.

Amino Acid Sequence↗

Induction of the copper resistance operon from Pseudomonas syringae.

Cupric sulfate induced mRNA specific to the copper resistance gene cluster previously cloned from Pseudomonas syringae pv. tomato PT23. mRNA from each of the four genes of this cluster responded in a similar manner to induction over time and with different concentrations of cupric sulfate. Promoter fusion constructs indicated the presence of a single copper-inducible promoter upstream from the first open reading frame.

Copper↗

The predicted protein product of a pathogenicity locus from Pseudomonas syringae pv. phaseolicola is homologous to a highly conserved domain of several procaryotic regulatory proteins.

A ca. 20-kilobase (kb) region (hrp) that controls the interaction of Pseudomonas syringae pv. phaseolicola with its host (pathogenicity) and nonhost plants (hypersensitive reaction) was previously cloned and partially characterized. In this study we defined the limits and determined the nucleotide sequence of a hrp locus (hrpS), located near the right end of the hrp cluster. The largest open reading frame (ORF302) in hrpS has a coding capacity for a 302-amino-acid polypeptide. The predicted amino acid sequence of the translation product of ORF302 (HrpS) shows significant similarity to several procaryotic regulatory proteins, including the NtrC, NifA, and DctD proteins of Rhizobium spp., the NtrC and NifA proteins of Klebsiella pneumoniae, and the TyrR protein of Escherichia coli. These proteins regulate diverse operons involved in nitrogen fixation, transport and metabolism of amino acids, and transport of C-4 dicarboxylic acids. The HrpS protein appears to be the shortest naturally occurring member of this family of proteins, corresponding for the most part to the highly conserved central domain of these proteins, which contains a putative ATP-binding site. A C-terminal segment analogous to the less-well-conserved domain, involved in DNA binding of NtrC and NifA, is also present in HrpS. These similarities suggest that HrpS is a regulatory protein. In line with this prediction is the finding that a functional hrpS gene is necessary for the activation of another hrp locus during the plant-bacterium interaction.

Amino Acid Sequence↗