Identification of a novel Pseudomonas syringae Psy61 effector with virulence and avirulence functions by a HrpL-dependent promoter-trap assay.

The hrp pathogenicity island of Pseudomonas syringae encodes a type III secretion system (TTSS) that translocates effectors into plant cells. Most genes encoding effectors are dispersed in the P. syringae genome. Regardless of location, all are regulated coordinately by the alternative sigma factor HrpL. An HrpL-dependent promoter-trap assay was developed to screen genomic libraries of P. syringae strains for promoters whose activity in Escherichia coli is dependent on an inducible hrpL construct. Twenty-two HrpL-dependent promoter fragments were isolated from P. syringae Psy61 that included promoters for known HrpL-dependent genes. One fragment also was isolated that shared no similarity with known genes but retained a near consensus HrpL-dependent promoter. The sequence of the region revealed a 375-amino acid open reading frame encoding a 40.5-kDa product that was designated HopPsyL. HopPsyL was structurally similar to other secreted effectors and carried a putative chloroplast-targeting signal and two predicted transmembrane domains. HopPsyL':'AvrRpt2 fusions were translocated into host cells via the P. syringae pv. tomato DC3000 hrp TTSS. A hopPsyL::kan mutant of Psy61 exhibited strongly reduced virulence in Phaseolus vulgaris cv. Kentucky Wonder, but did not appear to act as a defense response suppressor. The ectopically expressed gene reduced the virulence of Pseudomonas syringae DC3000 transformants in Arabidopsis thaliana Col-0. The gene was shown to be conserved in 6 of 10 P. syringae pv. syringae strains but was not detected in 35 strains of other pathovars. HopPsyL appears to be a novel TTSS-dependent effector that functions as a host-species-specific virulence factor in Psy61.

Enhancer-binding proteins HrpR and HrpS interact to regulate hrp-encoded type III protein secretion in Pseudomonas syringae strains.

In Pseudomonas syringae strains, the hrp-hrc pathogenicity island consists of an HrpL-dependent regulon that encodes a type III protein translocation complex and translocated effector proteins required for pathogenesis. HrpR and HrpS function as positive regulatory factors for the hrpL promoter, but their mechanism of action has not been established. Both HrpR and HrpS are structurally related to enhancer-binding proteins, but they lack receiver domains and do not appear to require a cognate protein kinase for activity. hrpR and hrpS were shown to be expressed as an operon: a promoter was identified 5' to hrpR, and reverse transcriptase PCR detected the presence of an hrpRS transcript. The hrpR promoter and coding sequence were conserved among P. syringae strains. The coding sequences for hrpR and hrpS were cloned into compatible expression vectors, and their activities were monitored in Escherichia coli transformants carrying an hrpL'-lacZ fusion. HrpS could function as a weak activator of the hrpL promoter, but the activity was only 2.5% of the activity detected when both HrpR and HrpS were expressed in the reporter strain. This finding is consistent with a requirement for both HrpR and HrpS in the activation of the hrpL promoter. By using a yeast two-hybrid assay, an interaction between HrpR and HrpS was detected, suggestive of the formation of a heteromeric complex. Physical interaction of HrpR and HrpS was confirmed by column-binding experiments. The results show that HrpR and HrpS physically interact to regulate the sigma(54)-dependent hrpL promoter in P. syringae strains.

The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli.

Regulation of virulence gene expression in enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) is incompletely understood. In EPEC, the plasmid-encoded regulator Per is required for maximal expression of proteins encoded on the locus of enterocyte effacement (LEE), and a LEE-encoded regulator (Ler) is part of the Per-mediated regulatory cascade upregulating the LEE2, LEE3, and LEE4 promoters. We now report that Ler is essential for the expression of multiple LEE-located genes in both EPEC and EHEC, including those encoding the type III secretion pathway, the secreted Esp proteins, Tir, and intimin. Ler is therefore central to the process of attaching and effacing (AE) lesion formation. Ler also regulates the expression of LEE-located genes not required for AE-lesion formation, including rorf2, orf10, rorf10, orf19, and espF, indicating that Ler regulates additional virulence properties. In addition, Ler regulates the expression of proteins encoded outside the LEE that are not essential for AE lesion formation, including TagA in EHEC and EspC in EPEC. delta ler mutants of both EPEC and EHEC show altered adherence to epithelial cells and express novel fimbriae. Ler is therefore a global regulator of virulence gene expression in EPEC and EHEC.

Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenic Escherichia coli.

The locus of enterocyte effacement of enteropathogenic Escherichia coli encodes a type III secretion system, an outer membrane protein adhesin (intimin, the product of eae ) and Tir, a translocated protein that becomes a host cell receptor for intimin. Many type III secreted proteins require chaperones, which function to stabilize proteins, prevent inappropriate protein-protein interactions and aid in secretion. An open reading frame located between tir and eae, previously named orfU, was predicted to encode a protein with partial similarity to the Yersinia SycH chaperone. We examined the potential of the orfU gene product to serve as a chaperone for Tir. The orfU gene encoded a 15 kDa cytoplasmic protein that specifically interacted with Tir as demonstrated by the yeast two-hybrid assay, column binding and coimmunoprecipitation experiments. An orfU mutant was defective in attaching-effacing lesion formation and Tir secretion, but was unaffected in expression of other virulence factors. OrfU appeared to stabilize Tir levels in the cytoplasm, but was not absolutely necessary for secretion of Tir. Based upon the physical similarities, phenotypic characteristics and the demonstrated interaction with Tir, orfU is redesignated as cesT for the chaperone for E. coli secretion of T ir.

Current concepts of active defense in plants.

A growing body of evidence indicates that elicitation of primary active defense responses results from a recognition event frequently involving protein-protein interactions. Most pathogen avirulence determinants eliciting resistance gene-dependent responses have been shown to be proteins with no apparent enzymic activity. Disruption of the tertiary and quaternary structure of these proteins abolishes their elicitor activity. Critical to their elicitor activity is their display by the pathogen. Resistance genes are proposed to function as receptors for the eliciting proteins. The most consistent feature of resistance gene products is the presence of potential protein binding domains in the form of leucine-rich repeat regions, and there is direct evidence for the physical interaction of elicitor proteins and receptor proteins in several cases. Thus in many but not all cases the primary recognition event eliciting an active defense response during incompatible interactions appears to be a protein-protein interaction occurring between a specific pathogen protein and a strategically placed receptor protein in the host cell. The interaction of elicitor protein with the receptor protein activates a signal transduction pathway leading to programmed cell death and an oxidative burst.

Phenotypic expression of Pseudomonas syringae avr genes in E. coli is linked to the activities of the hrp-encoded secretion system.

The specific recognition of elicitors produced by plant pathogenic bacteria carrying avirulence (avr) genes is postulated to initiate cellular defense responses in plants expressing corresponding resistance genes. The biochemical functions of most avr genes, however, are not known. A heterologous system was developed to phenotypically express Pseudomonas syringae avr genes in Escherichia coli cells that required the P. syringae hrp cluster. E. coli MC4100 transformants carrying the plasmic-borne P. syringae pv. syringae Pss61 hrp cluster and p. syringae pv. glycinea avrB expressed from a triple lacUV5 promoter gained the ability to elicit the hypersensitive response in soybean cultivars expressing Rpg1 and in an Arabidopsis thaliana accession expressing RPM1. Inactivation of energy transducing or outer membrane components of the hrp-encoded secretion system blocked phenotypic expression expression of avrB in E. coli, but deletions abolishing harpinPSS production had little effect on the production of the AvrB phenotype by the E. coli transformants. Phenotypic expression of avrA, AvrPto, avrRpm1, avrRpt2, and avrPph3 in E. coli was also shown to require the hrp cluster. The results indicate that generation of the Avr phenotype in P. syringae strains is specifically dependent on the secretion activities of the hrp cluster.

Characterization of the hrpJ and hrpU operons of Pseudomonas syringae pv. syringae Pss61: similarity with components of enteric bacteria involved in flagellar biogenesis and demonstration of their role in HarpinPss secretion.

The hrp/hrmA gene cluster of Pseudomonas syringae pv. syringae Pss61 has been shown to form a minimum genetic unit sufficient to enable nonpathogenic bacteria, such as Escherichia coli, to elicit the hypersensitive response associated with disease resistance. The biochemical functions of most of these genes have not been established. The nucleotide sequence of a 4.3-kb SstI-BglII fragment carrying hrp apparent translational units V, VI, and VII revealed one partial open reading frame (ORF) and five complete ORFs producing 35,126-, 48,866-, 17,308-, 20,482-, and 26,364-Da gene products (hrpJ3, J4, J5, U1, U2, respectively). The production of these proteins was confirmed by using T7 RNA polymerase-directed expression. The partial ORF was found to be identical to the C terminus of HrpJ2. The absence of apparent transcriptional terminators and promoters between hrpI (hrpJ2), hrpJ3, hrpJ4, and hrpJ5 together with the observation that the HrpL-dependent hrpJ promoter directs expression of hrpJ3-J5 indicates that these genes form a single operon controlled by the HrpL-dependent hrpJ promoter. A second HrpL-dependent promoter consensus sequence was also identified upstream of hrpU1 and demonstrated to function as a HrpL-dependent promoter, thus indicating that hrpU1, hrpU2, and additional downstream genes may be part of a second operon. The deduced product of hrpJ3 exhibits similarity to FliG of Salmonella typhimurium, a cytoplasmic protein that regulates flagellar rotation and biogenesis. HrpJ4 shares extensive similarity with the FliI family of ATPase-like proteins and retains the known functional domains conserved among this family of proteins. HrpJ5 has properties similar to the S. typhimurium FliJ. Neither HrpU1 nor HrpU2 exhibit significant similarity to known proteins. Secretion of HarpinPss by E. coli MC4100 transformants carrying pHIR11::TnphoA derivatives was blocked in hrpJ4, J5, and U2 mutants. In view of the previously reported similarity of HrpJ2 to the LcrD super-family that includes FlhA, these results predict that the gene products of the hrpJ and hrpU operons form an inner membrane complex for translocation of proteins similar to that used by the flagellar biogenesis system of S. typhimurium.

A single promoter sequence recognized by a newly identified alternate sigma factor directs expression of pathogenicity and host range determinants in Pseudomonas syringae.

A conserved sequence motif associated with transcription of avr genes was identified in the promoter regions of six Pseudomonas syringae pv. syringae Pss61 hrp operons. A 34-bp fragment carrying this motif was cloned from the HrpZ promoter region and was shown to confer HrpL-dependent promoter activity. Expression of pathogenicity and host range determinants in P. syringae strains is thus directed by the apparent alternate sigma factor HrpL.

Identification of a putative alternate sigma factor and characterization of a multicomponent regulatory cascade controlling the expression of Pseudomonas syringae pv. syringae Pss61 hrp and hrmA genes.

The Pseudomonas syringae hrp and hrmA genes controlling pathogenicity and elicitation of the hypersensitive response and the avr genes controlling host range have been shown previously to be regulated by carbon, nitrogen, pH, osmolarity, and hypothetical plant factors. In P. syringae pv. syringae Pss61, inactivation of hrp complementation groups II and XIII reduced expression of a plasmid-borne hrmA'-lacZ fusion. The hrp regions II and XIII were cloned on separate plasmids and shown to enhance the activity of the hrmA promoter in Escherichia coli MC4100 transformants at least 100-fold. The nucleotide sequence of region XIII revealed two open reading frames (hrpR and hrpS) whose deduced products share homology with P. syringae pv. phaseolicola NPS3121 HrpS and are both related to the NtrC family of two-component signal transduction systems. HrpR and HrpS differ from most members of the protein family by lacking an amino-terminal domain which modulates the regulatory activity. A single open reading frame, hrpL, whose product shares homology with AlgU, a putative alternate sigma factor of P. aeruginosa, as well as with the related alternate sigma factors was identified within region II. Key domains are partially conserved. Inactivation of hrpS in Pss61 repressed expression of a plasmid-borne hrpL'-lacZ fusion carried by pYXPL1R, and transformation of MC4100(pYXPL1R) with a plasmid carrying hrpRS increased hrpL promoter activity at least 200-fold. Neither hrpS nor hrpR, when cloned on separate plasmids, activated the hrpL promoter activity individually. The expression of hrpL when directed by a lac promoter was sufficient to express a set of plasmid-borne hrmA'-, hrpJ'-, and hrpZ'-lacZ fusions independently of other hrp genes. The results indicate that hrpRS and hrpL are part of a regulatory cascade in which HrpR and HrpS activate expression of hrpL and HrpL, a putative sigma factor, induces expression of HrpL-responsive genes.

Nucleotide sequence and properties of the hrmA locus associated with the Pseudomonas syringae pv. syringae 61 hrp gene cluster.

The hrmA locus, isolated from Pseudomonas syringae pv. syringae 61, is essential for phenotypic expression of the P. s. pv. syringae 61 hrp cluster in Escherichia coli strains and enables bacteria carrying the hrp/hrm gene cluster to elicit the hypersensitive response (HR) associated with plant disease resistance. The phenotype of P. s. pv syringae 61 hrmA mutants (pathogenicity+, delayed HR) was distinct from that of hrp mutants. The locus was localized to a 3.6-kb BamH1-EcoR1 fragment whose nucleotide sequence was determined. A single open reading frame was identified that encodes for a 41,457-Da protein of unknown biochemical function. Production of the deduced protein product was confirmed by using T7 RNA polymerase-directed expression of the locus and N-terminal sequence analysis of the isolated HrmA. The deduced protein product did not exhibit homology with any of the characterized avr genes or the hrpN product of Erwinia amylovora. Transcription was shown to initiate 37 nucleotides upstream of the translational start from an apparent sigma 70 promoter. Two hrp genes were shown to act as positive transcriptional factors for hrmA expression. Expression of hrmA in P. syringae pv. glycinea race 4 did not exhibit the phenotypic properties of an avr gene or HrpN, but suggested that this locus may serve a regulatory function. A homolog to hrmA was present in strains of only three of the 23 P. syringae pathovars tested.

Characterization of the Pseudomonas syringae pv. syringae 61 hrpJ and hrpI genes: homology of HrpI to a superfamily of proteins associated with protein translocation.

The Pseudomonas syringae pv. syringae 61 hrpJ and hrpI genes were sequenced and found to encode predicted proteins of 37,710 Da and 76,490 Da, respectively. The products of these genes were confirmed by T7 polymerase-dependent expression and sodium dodecyl sulfate-polyacrylamide gel analysis. HrpI belongs to a superfamily of proteins represented by Yersinia pestis LcrD.

Multiple periplasmic catalases in phytopathogenic strains of Pseudomonas syringae.

Phytopathogenic strains of Pseudomonas syringae are exposed to plant-produced, detrimental levels of hydrogen peroxide during invasion and colonization of host plant tissue. When P. syringae strains were investigated for their capacity to resist H2O2, they were found to contain 10- to 100-fold-higher levels of total catalase activity than selected strains belonging to nonpathogenic related taxa (Pseudomonas fluorescens and Pseudomonas putida) or Escherichia coli. Multiple catalase activities were identified in both periplasmic and cytoplasmic fluids of exponential- and stationary-phase P. syringae cells. Two of these activities were unique to the periplasm of P. syringae pv. glycinea. During the stationary growth phase, the specific activity of cytoplasmic catalases increased four- to eightfold. The specific activities of catalases in both fluids from exponential-phase cells increased in response to treatment with 0.25 to 10 mM H2O2 but decreased when higher H2O2 concentrations were used. In stationary-growth phase cultures, the specific activities of cytoplasmic catalases increased remarkably after treatment with 0.25 to 50 mM H2O2. The growth of P. syringae into stationary phase and H2O2 treatment did not induce synthesis of additional catalase isozymes. Only the stationary-phase cultures of all of the P. syringae strains which we tested were capable of surviving high H2O2 stress at concentrations up to 50 mM. Our results are consistent with the involvement of multiple catalase isozymes in the reduction of oxidative stress during plant pathogenesis by these bacteria.

Phenotypic expression of the Pseudomonas syringae pv. syringae 61 hrp/hrm gene cluster in Escherichia coli MC4100 requires a functional porin.

Plants, in general, appear to be able to detect the presence of incompatible Pseudomonas syringae strains by a hypothetical cell-cell recognition process to initiate inducible defense mechanisms that contribute to disease resistance. A 25-kb hrp/hrm gene cluster isolated from P. syringae pv. syringae 61(pHIR11) enables Escherichia coli to elicit a hypersensitive response (HR), a plant response generally considered to be a manifestation of recognition and resistance. To identify the nature of the HR-eliciting signal produced by E. coli cells carrying pHIR11, bacterial surface features were surveyed by immunological and biochemical procedures. No immunoreactive epitopes or outer membrane proteins were detected that were associated with expression of the P. syringae pv. syringae 61 hrp/hrm cluster in E. coli MC4100. Phenotypic expression of the P. syringae pv. syringae 61 hrp/hrm cluster in E. coli MC4100, however, was found to be dependent upon ompC and ompF, which control outer membrane permeability to hydrophilic solutes. The results suggest that deployment of the HR-eliciting signal occurs via outer membrane porins and imply that a low-molecular-weight, hydrophilic factor mediates signal exchange between the bacterium and the responding plant cell.

Organization and environmental regulation of the Pseudomonas syringae pv. syringae 61 hrp cluster.

The ability of Pseudomonas syringae pv. syringae 61 to elicit the hypersensitive response in nonhost plant species has been linked to a cluster of hrp/hrm genes whose expression appears to be environmentally regulated. To understand the genetic organization of this hrp/hrm gene cluster and its expression during the interaction with nonhost plant species better, we constructed a set of chromosomal hrp-uidA fusions in P. syringae pv. syringae 61 by Tn5-gusA1 mutagenesis of the cloned hrp/hrm gene cluster and transferred them into the genome by marker exchange mutagenesis. Complementation analysis employing plasmid-borne Tn5-gusA1 insertions and previously characterized chromosomal TnphoA mutations defined at least eight apparent transcriptional units within the hrp/hrm cluster, several of which were multicistronic. The expression of hrp-uidA fusions in seven of these apparent hrp transcriptional units increased following inoculation into tobacco leaves. Enhanced expression from a representative fusion was detected 1 h after inoculation of tobacco leaves. The induction observed in planta was similar to the levels detected following culture of the bacteria in minimal-salts medium: irrespective of the carbon source. Complex amino acid sources, such as peptone, repressed the expression of P. syringae pv. syringae 61 hrp genes at levels exceeding 0.028%. The results indicate that enhanced expression of hrp genes occurs early in the interaction with nonhost plant species in an apparent response to altered nutritional conditions.

Molecular cloning of the structural gene for exopolygalacturonate lyase from Erwinia chrysanthemi EC16 and characterization of the enzyme product.

The ability of Erwinia chrysanthemi to cause soft-rot diseases involving tissue maceration in many plants has been linked to the production of endo-pectate lyase E. chrysanthemi EC16 mutant UM1005, however, contains deletions in the pel genes that encode the known endopectate lyases, yet still macerates plant tissues. In an attempt to identify the remaining macerating factor(s), a gene library of UM1005 was constructed in Escherichia coli and screened for pectolytic activity. A clone (pPNL5) was identified in this library that contained the structural gene for an exopolygalacturonate lyase (ExoPL). The gene for ExoPL was localized on a 3.3-kb EcoRV fragment which contained an open reading frame for a 79,500-Da polypeptide. ExoPL was purified to apparent homogeneity from Escherichia coli DH5 alpha (pPNL5) and found to have an apparent molecular weight of 76,000 with an isoelectric point of 8.6. Purified ExoPL had optimal activity between pH 7.5 and 8.0 and could utilize pectate, citrus pectin, and highly methyl-esterified Link pectin as substrates. A PL- ExoPL- mutant of EC16 was constructed that exhibited reduced growth on pectate, but retained pathogenicity on chrysanthemum equivalent to that of UM1005. The results indicate that ExoPL does not contribute to the residual macerating activity of UM1005.

Influence of Pseudomonas syringae culture conditions on initiation of the hypersensitive response of culture tobacco cells.

The inhibitor sensitivity and timing of the ionic response of suspension-cultured tobacco cells were used as a bioassay for the Pseudomonas syringae signal that elicits the hypersensitive response in resistant plants. The ionic response of tobacco cell suspensions inoculated with P. syringae pv. syringae 61 and P. syringae pv. pisi grown in rich media was inhibited by rifampin, tetracycline, and streptomycin during a 2- to 2.5-h induction stage. Coculturing the bacteria with tobacco cells for 3 h or more before inoculating fresh tobacco cells specifically abolished the sensitivity of the ionic response to these inhibitors and reduced the response time of the tobacco cells from 3 to 1 h. The apparent activation of the bacteria during coculture was not dependent on the plant cells and could be achieved by incubating the bacteria in a nitrogen-deficient medium containing a metabolizable carbon source. Addition of proteose peptone and Casamino Acids to this medium suppressed activation of the bacteria. The results suggest that the hypersensitive response-eliciting signal forms late in the induction stage, perhaps as a result of the derepression of some of the P. syringae genes functional in elicitation of the hypersensitive response. The nature of the activated state remains elusive but is consistent with the accumulation of protein(s) whose activity indirectly elicits the ionic response.

Molecular cloning of a Pseudomonas syringae pv. syringae gene cluster that enables Pseudomonas fluorescens to elicit the hypersensitive response in tobacco plants.

A cosmid clone isolated from a genomic library of Pseudomonas syringae pv. syringae 61 restored to all Tn5 mutants of this strain studied the ability to elicit the hypersensitive response (HR) in tobacco. Cosmid pHIR11 also enabled Escherichia coli TB1 to elicit an HR-like reaction when high levels of inoculum (10(9) cells per ml) were infiltrated into tobacco leaves. The cosmid, which contains a 31-kilobase DNA insert, was mobilized by triparental matings into Pseudomonas fluorescens 55 (a nonpathogen that normally causes no plant reactions), P. syringae pv. syringae 226 (a tomato pathogen that causes the HR in tobacco), and P. syringae pv. tabaci (a tobacco pathogen that causes the HR in tomato). The plant reaction phenotypes of all of the transconjugants were altered. P. fluorescens(pHIR11) caused the HR in tobacco and tomato leaves and stimulated an apparent proton influx in suspension-cultured tobacco cells that was indistinguishable from the proton influx caused by incompatible pathogenic pseudomonads. P. syringae pv. tabaci(pHIR11) and P. syringae pv. syringae 226(pHIR11) elicited the HR rather than disease symptoms on their respective hosts and were no longer pathogenic. pHIR11 was mutagenized with TnphoA (Tn5 IS50L::phoA). One randomly chosen mutant, pHIR11-18, no longer conferred the HR phenotype to P. fluorescens. The mutation was marker-exchanged into the genomes of P. syringae pv. syringae strains 61 and 226. The TnphoA insertions in the two pseudomonads abolished their ability to elicit any plant reactions in all plants tested. The results indicate that a relatively small portion of the P. syringae genome is sufficient for the elicitation of plant reactions.

Enzyme regulation in C4 photosynthesis: purification, properties, and activities of thioredoxins from C4 and C3 plants.

Procedures are described for the purification to homogeneity of chloroplast thioredoxins f and m from leaves of corn (Zea mays, a C4 plant) and spinach (Spinacea oleracea, a C3 plant). The C3 and C4f thioredoxins were similar immunologically and biochemically, but differed in certain of their physiochemical properties. The f thioredoxins from the two species were capable of activating both NADP-malate dehydrogenase (EC 1.1.1.37) and fructose-1,6-bisphosphatase (EC 3.1.3.11) when tested in standard thioredoxin assays. Relative to its spinach counterpart, corn thioredoxin f showed a greater molecular mass (15.0-16.0 kDa vs 10.5 kDa), lower isoelectric point (ca. 5.2 vs 6.0), and lower ability to form a stable noncovalent complex with its target fructose bisphosphatase enzyme. The C3 and C4 m thioredoxins were similar in their specificity (ability to activate NADP-malate dehydrogenase, and not fructose-1,6-bisphosphatase) and isoelectric points (ca. 4.8), but differed slightly in molecular mass (13.0 kDa for spinach vs 13.5 kDa for corn) and substantially in their immunological properties. Results obtained in conjunction with these studies demonstrated that the thioredoxin m-linked activation of NADP-malate dehydrogenase in selectively enhanced by the presence of halide ions (e.g., chloride) and by an organic solvent (e.g., 2-propanol). The results suggest that in vivo NADP-malate dehydrogenase interacts with thylakoid membranes and is regulated to a greater extent by thioredoxin m than thioredoxin f.

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.