Involvement of plasmid deoxyribonucleic acid in indoleacetic acid synthesis in Pseudomonas savastanoi.

Olive (or oleander) knot is a plant disease incited by Pseudomonas savastanoi. Disease symptoms consist of tumorous outgrowths induced in the plant by bacterial production of indole-3-acetic acid (IAA). Synthesis of IAA occurs by the following reactions: L-tryptophan leads to indoleacetamide leads to indoleacetic acid, catalyzed by tryptophan 2-monooxygenase and indoleacetamide hydrolase, respectively. Whereas the enzymology of IAA synthesis is well characterized, nothing is known about the genetics of the system. We devised a positive selection for the presence of tryptophan 2-monooxygenase based on its capacity to use as a substrate the toxic tryptophan analogue 5-methyltryptophan. Efficient curing of the bacterium of tryptophan 2-monoxygenase, indoleacetamide hydrolase, and IAA production was obtained by acridine orange treatment. Further, loss of capacity to produce IAA by curing was correlated with loss of a plasmid of 34 X 10(6) molecular weight. This plasmid, here called pIAA1, when reintroduced into Iaa- mutants by transformation, restored tryptophan 2-monooxygenase and indoleacetamide hydrolase activities and production of IAA.     

A mutation in the indole-3-acetic acid biosynthesis pathway of Pseudomonas syringae pv. syringae affects growth in Phaseolus vulgaris and syringomycin production.

Homologs of the genes for indole-3-acetic acid (IAA) biosynthesis from Pseudomonas syringae pv. savastanoi were retrieved from a genomic library of P. syringae pv. syringae, and their nucleotide sequences were determined. Sequence relatedness between the P. syringae pv. syringae and P. syringae pv. savastanoi iaa operons is greater than 90% within the iaaM and iaaH loci but declines dramatically at a position approximately 200 bp 5' of the iaaM translation initiation codon. A third open reading frame was detected downstream of iaaH. Production of IAA was undetectable in mutant strain Y30-53.29, which was generated by transposition of Tn5 into the iaaM gene of P. syringae pv. syringae Y30. The IAA-deficient (IAA-) mutant retained the ability to colonize the bean phylloplane and induced disease symptoms on bean which were similar to those produced by the parental strain. However, the population dynamics of the IAA- strain during the parasitic phase in leaves differed from those of both the parental strain and the mutant genetically restored for IAA biosynthesis. The mutant was capable of inducing disease symptoms when established in bean tissues at a lower initial cell density than either IAA-producing strain. Syringomycin biosynthesis by the IAA- strain was diminished in comparison with the parental strain or the mutant genetically restored for IAA production. The results indicate that bacterially derived IAA, or its biosynthesis, is involved in the regulation of in planta growth and in the expression of other factors that affect the host-pathogen interaction.     

Regulation of 3-indoleacetic acid production in Pseudomonas syringae pv. savastanoi. Purification and properties of tryptophan 2-monooxygenase

The oxidative decarboxylation of L-tryptophan to yield 3-indoleacetamide, catalyzed by tryptophan 2-monooxygenase, represents a controlling reaction in the synthesis of indoleacetic acid by Pseudomonas savastanoi (Pseudomonas syringae pv. savastanoi), a gall-forming pathogen of olive (Olea europea L.) and oleander (Nerium oleander L.). Production of indoleacetic acid is essential for virulence of the bacterium in its hosts. Tryptophan 2-monooxygenase was characterized to determine its role in indoleacetic acid metabolism in the bacterium. The enzyme was purified to apparent homogeneity from Escherichia coli cells containing the genetic locus for this enzyme obtained from P. savastanoi. The preparation contained a single polypeptide with a mass of 62,000 that cross-reacted immunologically with a homologous protein in P. savastanoi. The holoenzyme contained one FAD moiety/subunit with properties consistent with a catalytic function. The enzyme preparation catalyzed an L-tryptophan-dependent O2 uptake and yielded 3-indoleacetamide as a product. Enzyme activity fit simple Michaelis Menten kinetics with a Km for L-tryptophan of 50 microM. 3-Indoleacetamide and 3-indoleacetic acid were identified as regulatory effectors. The apparent Ki for 3-indoleacetamide was 7 microM; that for indoleacetic acid was 225 microM. At Km concentrations of tryptophan, enzyme activity was inhibited 50% by 25 microM 3-indoleacetamide. In contrast, 230 microM indoleacetic acid was required to effect a similar inhibition. Phenylalanine and tyrosine were ineffective as regulatory metabolites. These results indicate that IAA synthesis in P. savastanoi is regulated by limiting tryptophan and by feedback inhibition from indoleacetamide and indoleacetic acid.     

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.     

Physical comparison of parathion hydrolase plasmids from Pseudomonas diminuta and Flavobacterium sp

Restriction maps of two plasmids encoding parathion hydrolase have been determined. pPDL2 is a 39-kb plasmid harbored by Flavobacterium sp. (ATCC 27551), while pCMS1 is a 70-kb plasmid found in Pseudomonas diminuta (strain MG). Both plasmids previously have been shown to share homologous parathion hydrolase genes (termed opd for organophosphate degradation) as judged by DNA-DNA hybridization and restriction mapping. In the present study, we conducted DNA hybridization experiments using each of nine PstI restriction fragments from pCMS1 as probes against Flavobacterium plasmid DNA. The opd genes of both plasmids are located within a highly conserved region of approximately 5.1 kb. This region of homology extends approximately 2.6 kb upstream and 1.7 kb downstream from the opd genes. No homology between the two plasmids is evident outside of this region.     

Pathogenicity of Pseudomonas syringae subsp. savastanoi Mutants Defective in Phytohormone Production

The present study compares the pathogenicity on olive and oleander plants of three wild-type strains of Pseudomonas syringae subsp. savastanoi (ITM317 and PBa230 from olive and ITM519 from oleander) and three phytohormone-deficient mutants of ITM519: ITM519-41 (Iaa⁺/cytokinins-), ITM519-7 (Iaa⁻/cytokinins⁺), ITM519-6 (Iaa⁻/cytokinins⁻), Mutants not producing IAA (ITM519-7and ITM519-6) only induced necrosis of the inoculated tissues (ITM519-,6) or swellings on the stems attributed to cytokinin production accompanied by necrosis (ITM519-7). By contrast, the Iaa⁺/cytokinins⁻ mutant (ITM519-41) induced attenuated symptoms on stems and knots similar to those obtained with the parent strain on oleander leaves. Olive strains induced necrosis of oleander leaves and were virulent and avirulent, respectively, on olive and oleander stems.
Strain ITM519 and its three mutants were all able to multiply in oleander leaves at similar rates, reaching the same final populations. By contrast, the two olive strains multiply poorly, reaching populations c. 10²-fold lower.
These results confirm that expression of IAA genes alone is sufficient to initiate the development of knots on oleander while cytokinins are necessary for the full expression of the disease symptoms (determining knot size). The findings also indicate that the plant tissues (stems and leaves) react differently to the various strains of the bacterium and, furthermore, suggest that, besides phytohormones, other pathogenetic factors could be involved in this host-pathogen interaction. The necrotic reaction of oleander leaves heavily inoculated with olive strains was interpreted as a possible form of hypersensitivity reaction.     

Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae.

Strains of Pseudomonas syringae pv. syringae resistant to copper, streptomycin, or both compounds were recovered from symptomless and diseased tissue of four woody hosts in three nurseries in Oklahoma. In strains resistant to copper and streptomycin (Cur Smr), resistance to both compounds was cotransferred with a single plasmid which was either 68, 190, or 220 kilobase pairs (kb). All Cus Smr strains contained a 68-kb conjugative plasmid. Cur Sms strains contained one plasmid which varied in size from 60 to 73 kb. All conjugative plasmids which transferred streptomycin resistance contained sequences homologous to the strA and strB Smr genes from the broad-host-range plasmid RSF1010. The Smr determinant was subsequently cloned from a 68-kb Cur Smr plasmid designated pPSR1. A restriction map detailing the organization of the homologous Smr genes from pPSR1 and RSF1010 and cloned Smr genes from P. syringae pv. papulans and Xanthomonas campestris pv. vesicatoria revealed the conservation of all sites studied. The Cur genes cloned from P. syringae pv. tomato PT23 and X. campestris pv. vesicatoria XV10 did not hybridize to the Cur plasmids identified in the present study, indicating that copper resistance in these P. syringae pv. syringae strains may be conferred by a distinct genetic determinant.     

Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta.

Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures.     

62-kb Plasmids Harboring <Emphasis Type="Italic">rulAB</Emphasis> Homologues Confer UV-tolerance and Epiphytic Fitness to <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">syringae</Emphasis> Mango Isolates

The presence of genetic determinants homologous to rulAB genes for ultraviolet (UV) radiation resistance was determined in a collection of Pseudomonas syringae pv. syringae strains isolated from mango. The potential role of these plasmids in UV tolerance and ecological fitness in the mango phyllosphere was also evaluated. Nearly all of the 62-kb plasmids present in the P. syringae pv. syringae strains hybridized with a rulAB probe, but these 62-kb plasmids showed differences in restriction patterns. In vitro assays of tolerance to UV radiation of P. syringae pv. syringae strains showed a higher survival of the strains harboring the 62-kb plasmids compared to strains lacking plasmids when exposed to UVC or UVA+B fractions. Similar results were observed when transconjugants harboring the 62-kb plasmid were tested. Survival assays were carried out under field conditions, and a higher survival of P. syringae pv. syringae strains harboring 62-kb plasmids under direct solar radiation on the adaxial surface of leaves was also observed. When the assays were carried out in shady areas or on the abaxial surface of leaves, survival time was comparable for all the assayed strains, whether or not they contained a 62-kb plasmid hybridizing to rulAB. Our results indicate that P. syringae pv. syringae strains harboring 62-kb plasmids show an increase in ecological fitness when colonizing the mango phyllosphere.     

Phenotypes of Tobacco Plants Expressing Genes for the Synthesis of Growth Regulators

The expression of genes for synthesis of auxin (iaaM and iaaH) and cytokinins (ipt) was studied in tobacco plants transformed by two Agrobacterium tumefaciens strains C 58 and LBA 4404. The strain LBA 4404 carried binary vector plasmid pCB 1334 (ipt gene) and plasmid pCB 1349 (iaaM, iaaH and ila genes). Both plasmids carried reportered gene for npt II. Obtained plants expressed incorporated genes. New proteins with molecular masses of about 74, 40, 26, 25, 21 and 17 kDa for wild plasmid pTi C58; 60, 36, 31.5, 27, 26 and 17 kDa for binary vector plasmid pCB 1334 and 74, 49, 36, 31.5, 26 and 25 kDa for binary vector plasmid pCB 1349 were found in the patterns of soluble proteins. Significant changes in the content of chlorophylls, especially chlorophyll a, were detected in the plants carrying ipt gene and in plants transformed by the wild strain C58 of A. tumefaciens. Tobacco plants expressing ipt gene and genes from T-DNA of pTi C58 plasmid were dwarf, and in comparison to the controls, they had thicker stems, and the surface of the leaf blades was reduced to 20 - 50 %. Adventitious roots, growing from the stem, were typical for transformants overproducing auxins. Regenerants and transformants expressing genes from T-DNA of plasmid pTi C58 differed in the shape of the flowers and their fertility. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

Bacteriocin production by Pseudomonas syringae pv. ciccaronei NCPPB2355. Isolation and partial characterization of the antimicrobial compound

Pseudomonas syringae pv. ciccaronei strain NCPPB2355 was found to produce a bacteriocin inhibitory against strains of Ps. syringae subsp. savastanoi , the causal agent of olive knot disease. Treatments with mitomycin C did not substantially increase the bacteriocin titre in culture. The purification of the bacteriocin obtained by ammonium sulphate precipitation of culture supernatant fluid, membrane ultrafiltration, gel filtration and preparative PAGE, led to the isolation of a high molecular weight proteinaceous substance. The bacteriocin analysed by SDS-PAGE revealed three protein bands with molecular weights of 76, 63 and 45 kDa, respectively. The bacteriocin was sensitive to heat and proteolytic enzymes, was resistant to non-polar organic solvents and was active between pH 5·0–7·0. Plasmid-DNA analysis of Ps. syringae ciccaronei revealed the presence of 18 plasmids; bacteriocin-negative variants could not be obtained by cure experiments.     

Isolation and Characterization of Pseudomonas syringae subsp. savastanoi Mutants Defective in Hypersensitive Response Elicitation and Pathogenicity

Olive strain ITM317 of Pseudomonas syringae subsp. savastanoi , the causal agent of 'Olive and Oleander knot disease' was mutagenized by random transposon (Tn5) insertion. Of the 1 400 transconjugants, four were altered in their ability to induce a hypersensitive reaction (HR) on tobacco; Southern blot analysis showed that a single copy of the Tn5 element was present in their chromosomes. In particular, mutants ITM317–69, ITM317–1010 and ITM317–1194 did not elicit HR whereas mutant ITM317–916 induced a variable response. When assayed for pathogenicity on olive, mutants ITM317–916 and ITM317–1010 induced knots comparable both in size and morphology to those caused by the parental strain. Prototrophic mutant ITM317–1194, still able to produce indole-3-acetic acid and cytokinins, did not cause any knot formation on olive; furthermore, it was unable to multiply in host tissue. Auxotrophic mutant ITM317–69 caused the formation of smaller-sized knots and its prototrophic revertant fully regained the parental phenotypes, suggesting that a single Tn5 insertion had a pleiotropic effect on the mutated phenotypes. Tn5-containing Eco RI fragments from mutants ITM317–69, ITM317–916, ITM317–1010 and ITM317–1194 were cloned into the plasmid vector pBR322. Hybridization of these clones with the hrp gene cluster of P. s. pv. syringae strain 61 was not detected. These results suggest that genes different from those of the above gene cluster might be involved in the interaction of P. s. subsp. savastanoi with olive and with the non-host plant tobacco.     

Auxin polar transport and flower formation in Arabidopsis thaliana transformed with indoleacetamide hydrolase (iaaH) gene

Involvement of auxin polar transport in flower formation of Arabidopsis thaliana was studied using a pinformed (pin) mutant (Rpin) transformed with the indoleacetamide hydrolase (iaaH) gene and the phenocopy of the pin mutant, which was induced by 9-hydroxyfluorene-9-carboxylic acid (HFCA). The application of indoleacetamide (IAM) did not change aberrant structure of the aerial part of Rpin (pin/pin), but extremely inhibited its root growth. Treatment with IAM increased the endogenous concentrations of free and conjugated IAA in Rpin normal (pin/+ or +/+) due to the expression of the iaaH gene, to 140% and 428% of those in non-treated plants, respectively, and those in Rpin to 378% and 120%, respectively. The activity of IAA polar transport in the inflorescence axis of Rpin remained low even in the presence of IAM, the activity being almost similar, to that in the pin mutant. The activity of IAA polar transport in the HFCA-induced phenocopy of the pin mutant was also extremely low, and it was not restored by the simultaneous application of IAA. Arabidopsis thaliana responded to HFCA applied from 7 to 11 d and from 25 to 29 d after germination in the wild-type plant (Enkheim ecotype) and the late flowering mutant (fb mutant), respectively. These results suggest that the construction of the system of auxin polar transport and its normal activities are essential for the differentiation and the formation of floral meristem in the early growth stage of Arabidopsis thaliana.