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The available information, technologies and resources that were developed to ensure plant health and utilized for postintroduction plant pathogen forensics are assessed. Recommendations for prioritization of efforts and resource expenditures required to enhance the plant pathogen forensics capabilities are given.

The gene encoding alginate lyase (algL) in Pseudomonas syringae pv. syringae was cloned, sequenced, and overexpressed in Escherichia coli. Alginate lyase activity was optimal when the pH was 7.0 and when assays were conducted at 42 degrees C in the presence of 0.2 M NaCl. In substrate specificity studies, AlgL from P. syringae showed a preference for deacetylated polymannuronic acid. Sequence alignment with other alginate lyases revealed conserved regions within AlgL likely to be important for the structure and/or function of the enzyme. Site-directed mutagenesis of histidine and tryptophan residues at positions 204 and 207, respectively, indicated that these amino acids are critical for lyase activity.

We cloned the rpoN (ntrA and glnF) gene encoding sigma(54) from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. The P. syringae ES4326 rpoN gene complemented Pseudomonas aeruginosa, Escherichia coli, and Klebsiella aerogenes rpoN mutants for a variety of rpoN mutant phenotypes, including the inability to utilize nitrate as sole nitrogen source. DNA sequence analysis of the P. syringae ES4326 rpoN gene revealed that the deduced amino acid sequence was most similar (86% identity; 95% similarity) to the sigma(54) protein encoded by the Pseudomonas putida rpoN gene. A marker exchange protocol was used to construct an ES4326 rpoN insertional mutation, rpoN::Km(r). In contrast to wild-type ES4326, ES4326 rpoN::Km(r) was nonmotile and could not utilize nitrate, urea, C(4)-dicarboxylic acids, several amino acids, or concentrations of ammonia below 2 mM as nitrogen sources. rpoN was essential for production of the phytotoxin coronatine and for expression of the structural genes encoding coronamic acid. In addition, ES4326 rpoN::Km(r) did not multiply or elicit disease symptoms when infiltrated into Arabidopsis thaliana leaves, did not elicit the accumulation of several Arabidopsis defense-related mRNAs, and did not elicit a hypersensitive response (HR) when infiltrated into tobacco (Nicotiana tabacum) leaves. Furthermore, whereas P. syringae ES4326 carrying the avirulence gene avrRpt2 elicited an HR when infiltrated into Arabidopsis ecotype Columbia leaves, ES4326 rpoN::Km(r) carrying avrRpt2 elicited no response. Constitutive expression of ES4326 hrpL in ES4326 rpoN::Km(r) partially restored defense-related mRNA accumulation, showing a direct role for the hrp cluster in host defense gene induction in a compatible host-pathogen interaction. However, constitutive expression of hrpL in ES4326 rpoN::Km(r) did not restore coronatine production, showing that coronatine biosynthesis requires factors other than hrpL.

Pseudomonas aeruginosa and the phytopathogen P. syringae produce the exopolysaccharide alginate, which is a copolymer of D-mannuronic and L-guluronic acids. One of the key regulatory genes controlling alginate biosynthesis in P. aeruginosa is algT, which encodes the alternate sigma factor, sigma(22). In the present study, the algT gene product from P. syringae pv. syringae showed 90% amino acid identity with its P. aeruginosa counterpart, and sequence analysis of the region flanking algT in P. syringae revealed the presence of nadB, mucA, and mucB in an arrangement virtually identical to that of P. aeruginosa. An algT mutant of P. syringae was defective in alginate production but could be complemented with wild-type algT from P. syringae or P. aeruginosa when expressed in trans. The algT mutant also displayed increased sensitivity to heat, paraquat, and hydrogen peroxide (H(2)O(2)); the latter two compounds are known to generate reactive oxygen intermediates. Signals for activation of algT gene expression in P. syringae were investigated with an algT::uidA transcriptional fusion. Like that in P. aeruginosa, algT transcription in P. syringae was activated by heat shock. However, algT expression in P. syringae was also stimulated by osmotic stress and by exposure to paraquat, H(2)O(2), and copper sulfate. The latter two compounds are frequently encountered during colonization of plant tissue and may be unique signals for algT activation in P. syringae.

Both Pseudomonas aeruginosa and the phytopathogen P. syringae produce the exopolysaccharide alginate. However, the environmental signals that trigger alginate gene expression in P. syringae are different from those in P. aeruginosa with copper being a major signal in P. syringae. In P. aeruginosa, the alternate sigma factor encoded by algT (sigma22) and the response regulator AlgR1 are required for transcription of algD, a gene which encodes a key enzyme in the alginate biosynthetic pathway. In the present study, we cloned and characterized the gene encoding AlgR1 from P. syringae. The deduced amino acid sequence of AlgR1 from P. syringae showed 86% identity to its P. aeruginosa counterpart. Sequence analysis of the region flanking algR1 in P. syringae revealed the presence of argH, algZ, and hemC in an arrangement virtually identical to that reported in P. aeruginosa. An algR1 mutant, P. syringae FF5.32, was defective in alginate production but could be complemented when algR1 was expressed in trans. The algD promoter region in P. syringae (PsalgD) was also characterized and shown to diverge significantly from the algD promoter in P. aeruginosa. Unlike P. aeruginosa, algR1 was not required for the transcription of algD in P. syringae, and PsalgD lacked the consensus sequence recognized by AlgR1. However, both the algD and algR1 upstream regions in P. syringae contained the consensus sequence recognized by sigma22, suggesting that algT is required for transcription of both genes.

Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents.

Coronatine (COR) is a plasmid-encoded phytotoxin synthesized by several pathovars of phytopathogenic Pseudomonas syringae. The COR biosynthetic gene cluster in P. syringae pv. glycinea PG4180 is encoded by a 32-kb region which contains both the structural and regulatory genes needed for COR synthesis. The regulatory region contains three genes: corP, corS, and corR. corS is thought to function as a histidine protein kinase, whereas corP and corR show relatedness to response regulators of the two-component regulatory paradigm. In the present study, we investigated whether CorR is a positive activator of COR gene expression. We also studied whether CorR specifically binds the DNA region located upstream of cfl, a gene located at the 5' end of the gene cluster encoding coronafacic acid, the polyketide portion of COR. Complementation analysis with a corR mutant, PG4180.P2, and transcriptional fusions to a promoterless glucuronidase gene (uidA) indicated that CorR functions as a positive regulator of COR gene expression. Deletion analysis of the 5' end of the cfl upstream region was used to define the minimal region required for COR gene expression. A 360-bp DNA fragment located over 500 bp upstream from the cfl transcriptional start site was used in DNase I protection assays to define the specific bases bound by CorR. An area extending from -704 to -650 with respect to the cfl transcriptional start site was protected by DNase I footprinting, indicating a rather large area of protection. This area was also conserved in the promoter region for cmaA, which encodes a transcript containing genes for coronamic acid synthesis, another intermediate in the COR biosynthetic pathway. The results obtained in the current study suggest that both the coronafacic acid and the coronamic acid structural genes are controlled by CorR, a positive activator of COR gene expression.

Genes of an influenza A (H5N1) virus from a human in Hong Kong isolated in May 1997 were sequenced and found to be all avian-like (K. Subbarao et al., Science 279:393-395, 1998). Gene sequences of this human isolate were compared to those of a highly pathogenic chicken H5N1 influenza virus isolated from Hong Kong in April 1997. Sequence comparisons of all eight RNA segments from the two viruses show greater than 99% sequence identity between them. However, neither isolate's gene sequence was closely (>95% sequence identity) related to any other gene sequences found in the GenBank database. Phylogenetic analysis demonstrated that the nucleotide sequences of at least four of the eight RNA segments clustered with Eurasian origin avian influenza viruses. The hemagglutinin gene phylogenetic analysis also included the sequences from an additional three human and two chicken H5N1 virus isolates from Hong Kong, and the isolates separated into two closely related groups. However, no single amino acid change separated the chicken origin and human origin isolates, but they all contained multiple basic amino acids at the hemagglutinin cleavage site, which is associated with a highly pathogenic phenotype in poultry. In experimental intravenous inoculation studies with chickens, all seven viruses were highly pathogenic, killing most birds within 24 h. All infected chickens had virtually identical pathologic lesions, including moderate to severe diffuse edema and interstitial pneumonitis. Viral nucleoprotein was most frequently demonstrated in vascular endothelium, macrophages, heterophils, and cardiac myocytes. Asphyxiation from pulmonary edema and generalized cardiovascular collapse were the most likely pathogenic mechanisms responsible for illness and death. In summary, a small number of changes in hemagglutinin gene sequences defined two closely related subgroups, with both subgroups having human and chicken members, among the seven viruses examined from Hong Kong, and all seven viruses were highly pathogenic in chickens and caused similar lesions in experimental inoculations.

Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. The genes involved in CFA biosynthesis are encoded by a single transcript which encompasses 19 kb of the COR gene cluster. In the present study, the nucleotide sequence was determined for a 4-kb region located at the 3' end of the CFA biosynthetic gene cluster. Three open reading frames were identified and designated cfa8, cfa9, and tnp1; the predicted translation products of these genes showed relatedness to oxidoreductases, thioesterases, and transposases, respectively. The translational products of cfa8 and cfa9 were overproduced in Escherichia coli BL21; however, tnp1 was not translated in these experiments. Mutagenesis and complementation analysis indicated that cfa8 is required for the production of CFA and COR. Analysis of a cfa9 mutant indicated that this gene is dispensable for CFA and COR production but may increase the release of enzyme-bound products from the COR pathway; tnp1, however, had no obvious function in CFA or COR biosynthesis. A genetic strategy was used to produce CFA in a P. syringae strain which lacks the COR gene cluster; this approach will be useful in future studies designed to investigate biosynthetic products of the CFA gene cluster.

The plant-pathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 synthesizes high levels of the polyketide phytotoxin coronatine (COR) at 18 degrees C, whereas no detectable toxin is produced at 28 degrees C. Previously, we reported that the temperature-sensitive activation of three promoters within the COR biosynthetic gene cluster might explain thermoregulation of COR biosynthesis. The present study was aimed at furthering our understanding of the transcriptional as well as the posttranslational effects of temperature on expression of cmaB, which encodes an enzyme involved in COR biosynthesis. Transcriptional fusions using a promoterless glucuronidase gene and Northern blot analyses were used to monitor promoter activities and transcript abundance for the cmaABT operon during bacterial growth at 18 and 28 degrees C. Promoter activity and transcription rates were maximal when cells were incubated at 18 degrees C and sampled at mid-logarithmic phase. Transcription declined moderately during the transition to stationary phase but remained higher at 18 C than at 28 degrees C. Western blot analysis indicated that CmaB accumulated in the late stationary phase of P. syringae cultures grown at 18 degrees C but not in cultures incubated at 28 degrees C. Temperature shift experiments indicated that CmaB stability was more pronounced at 18 degrees C than at 28 degrees C. Although temperature has a strong impact on transcription of COR biosynthetic genes, we propose that thermoregulation of protein stability might also control COR synthesis.

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.

Pseudomonas syringae pv. glycinea PG4180 produces the polyketide phytotoxin coronatine. The effects of environmental, nutritional, and host factors on growth and coronatine production by PG4180 were examined by varying the components of a defined basal medium which contained the following nutrients per liter: glucose (10 g), NH(4)Cl (1 g), MgSO(4) . 7H(2)O (0.2 g), KH(2)PO(4) (4.1 g), K(2)HPO(4) . 3H(2)O (3.6 g), and FeCl(3) (2 muM). Bacterial growth was recorded as dry weight, and coronatine production was measured by high-performance liquid chromatography. Both growth and the quantity of coronatine synthesized were significantly affected by carbon source, nutrient levels (glucose, NH(4)Cl, phosphate, Mg, and SO(4)), amino acid supplements, and the presence of complex carbon and nitrogen sources. The yield of coronatine generally declined when conditions were varied from those in the basal medium. Coronatine production and growth were not affected when the pH was adjusted from 6.5 to 7.8. Increases in the osmolarity of the basal medium significantly decreased coronatine production without affecting growth. The addition of plant extracts, plant-derived secondary metabolites, or zinc did not affect growth or coronatine production, while the addition of millimolar levels of KNO(3) or micromolar levels of FeCl(3) significantly enhanced coronatine production. The yield of coronatine was maximized after a 7-day incubation at 18 degrees C and 280 rpm. The results of the present study were used to formulate a medium which allowed for enhanced coronatine production in nearly all strains of P. syringae tested. A rapid method for extracting coronatine from small volumes of culture supernatant was also developed.

In Pseudomonas syringae pv. tomato PT23.2, plasmid pPT23A (101 kb) is involved in synthesis of the phytotoxin coronatine (C. L. Bender, D. K. Malvick, and R. E. Mitchell, J. Bacteriol. 171:807-812, 1989). The physical characterization of mutations that abolished coronatine production indicated that at least 30 kb of pPT23A DNA are required for toxin synthesis. In the present study, P-labeled DNA fragments from the 30-kb region of pPT23A hybridized to plasmid DNAs from several coronatine-producing pathovars of P. syringae under conditions of high stringency. These experiments indicated that this region of pPT23A was strongly conserved in large plasmids (90 to 105 kb) that reside in P. syringae pv. atropurpurea, glycinea, and morsprunorum. The functional significance of the observed homology was demonstrated in marker-exchange experiments in which Tn5-inactivated sequences from the 30-kb region of pPT23A were used to mutate coronatine synthesis genes in the three heterologous pathovars. Physical characterization of the Tn5 insertions generated by marker exchange indicated that genes controlling coronatine synthesis in P. syringae pv. atropurpurea 1304, glycinea 4180, and morsprunorum 567 and 3714 were located on the large indigenous plasmids where homology was originally detected. Therefore, coronatine biosynthesis genes are strongly conserved in the plasmid DNAs of four producing pathovars, despite their disparate origins (California, Japan, New Zealand, Great Britain, and Italy).

The efficacy of copper bactericides for control of Xanthomonas campestris pv. vesicatoria in eastern Oklahoma tomato fields was evaluated. Copper bactericides did not provide adequate control, and copper-resistant (Cu) strains of the pathogen were isolated. The Cu genes in these strains were located on a large indigenous plasmid designated pXV10A. The host range of pXV10A was investigated; this plasmid was efficiently transferred into 8 of 11 X. campestris pathovars. However, the transfer of pXV10A to other phytopathogenic genera was not detected. DNA hybridization experiments were performed to characterize the Cu genes on pXV10A. A probe containing subcloned Cu genes from X. campestris pv. vesicatoria E3C5 hybridized to pXV10A; however, a subclone containing Cu genes from P. syringae pv. tomato PT23 failed to hybridize to pXV10A. Further DNA hybridization experiments were performed to compare pXV10A with pXvCu plasmids, a heterogenous group of Cu plasmids present in strains of X. campestris pv. vesicatoria from Florida. These studies indicated that the Cu genes on pXV10A and pXvCu plasmids share nucleotide sequence homology and may have a common origin. Further experiments showed that these plasmids are distinctly different because pXV10A did not contain sequences homologous to IS476, an insertion sequence present on pXvCu plasmids.