The phytopathogen Pseudomonas syringae subsp. savastanoi incites the production of galls on olive and oleander plants. Gall formation is dependent on bacterial production of the phytohormone indoleacetic acid (IAA). The genetic determinants for IAA synthesis are located on a plasmid (pIAA) and are organized in an operon in oleander strains of the bacterium. P. syringae subsp. savastanoi further converts IAA to an amino acid conjugate, 3-inidole-acetyl-r-L-lysine (IAA-lysine). The gene for IAA-lysine synthetase (iaaL) was found on the IAA plasmid by screening pIAA deletion mutants for the ability to convert IAA to IAA-lysine. The iaaL locus was then cloned in the vector pUC8 from a bank of P. syringae subsp. savastanoi EW2009 plasmid DNA to construct recombinant plasmid pLG87. The specific activity of IAA-lysine synthetase in Escherichia coli transformed with pLG87 was 47 times higher than that of the enzyme extract from P. syringae subsp. savastanoi. The direction of transcription of the iaaL gene was determined to be opposite to that of the IAA operon. The location of the iaaL gene on pIAAl was mapped by TnS insertion mutagenesis to a 2.5-kilobasepair fragment 2 kilobase pairs from the IAA operon.Pseudomonas syringae subsp. savastanoi (Smith) Stevens incites a disease of oleander (Nerium oleander L.) and olive (Olea europa L.) that is characterized by tumorous outgrowths on the stems and leaves of infected plants (21,26,27). This development of galls is dependent on bacterial production of the phytohormone indoleacetic acid (IAA) (6, 23). The two enzymes involved in the conversion of tryptophan to IAA are tryptophan monooxygenase, which converts tryptophan to indoleacetamide, and indoleacetamide hydrolase, which catalyzes the conversion of indoleacetamide to IAA (16,17). IAA can be further metabolized to an amino acid conjugate, 3-indole-acetyl-e-L-lysine (IAA-lysine) (12, 13).The genes for tryptophan monooxygenase, iaaM, and indoleacetamide hydrolase, iaaH, are organized in an operon in P. syringae subsp. savastanoi (7,8,28); the iaaM locus is the first gene transcribed. In strains isolated from oleander galls, the IAA operon is located on a plasmid (pIAA) (6, 9). Oleander gall isolates selected for resistance to the tryptophan analog a-methyl tryptophan are often incapable of synthesizing IAA and cannot incite the production of galls when inoculated onto oleander plants (23). These avirulent IAA-mutants fall into two classes when surveyed for their plasmid composition: those that lack the IAA operon, either by the loss of the IAA plasmid or by deletion of the IAA genes, and those that contain insertions in the iaaM gene (6,8,9).Certain isolates of P. syringae subsp. savastanoi convert IAA to 13
Bacteriophages offer interesting alternatives to antibodies for the specific capture and detection of pathogenic bacteria onto biosensing surfaces. Procedures for the optimal chemical immobilization of lytic bacteriophages onto surfaces are presented. More specifically, the removal of lysate contaminants from bacteriophage suspensions by size exclusion chromatography significantly increases the resultant planar surface density of immobilized bacteriophages. E. coli T4 and Salmonella enterica serovar Typhimurium P22 phage systems seem to undergo highly heterogeneous adsorption to the surface, possibly explaining the observed phage clustering at higher surface densities. The T4 phage and its E. coli host were initially employed as a model system where we discovered an optimal planar surface density of phages for best bacterial capture: 18.9 ± 0.8 phages/μm2 capturing 18.0 ± 0.3 bacteria/100 μm2. Phage surface clustering ultimately limits the T4 phage-immobilized surface’s ability to specifically capture its host bacteria. Nevertheless, this is to our knowledge the largest surface capture density of E. coli reported using intact T4 bacteriophages. Two additional purified bacteriophage systems (P22 and Campylobacter jejuni phage NCTC 12673) were then similarly studied for their ability to capture their corresponding host bacteria (Salmonella enterica serovar Typhimurium and Campylobacter jejuni respectively) on a surface.
Cytokinin production by strains of the phytopathogenic bacterium Pseudomonas syningae 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. sawastanoi 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-l"-methylzeatin. The amounts secreted were approximately 1000 times greater than those secreted by Agrobacterium tumefaciens 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.Gall-forming phytopathogenic bacteria have long been known to produce plant hormones (11,28) and there has been speculation, supported by recent evidence, that pathogenesis may be hormone-dependent. It is now known that the bacterial pathogens Pseudomonas syringae pv savastanoi and Agrobacterium tumefaciens contain genes which specify the synthesis of IAA. P. savastanoi, the causative organism of olive and oleander knot, contains two plasmid-born genes which specify IAA biosynthesis (6,7, 17); the first, iaaM, encodes the enzyme tryptophan monooxygenase (12) while the second, iaaH, encodes an indoleacetamide hydrolase. Both genes are required for effective
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-e-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 TnS 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.Pseudomonas syringae subsp. savastanoi (Smith 1908) Stevens incites the formation of tumorous outgrowths on olive (Olea europa L.) and oleander (Nerium oleander L.
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