Pseudomonas syringae phytotoxins: mode of action, regulation, and biosynthesis by peptide and polyketide synthetases.

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.

Analysis of Sweet cherry (Prunus avium L.) Leaves for Plant Signal Molecules That Activate the syrB Gene Required for Synthesis of the Phytotoxin, Syringomycin, by Pseudomonas syringae pv syringae.

An important aspect of the interaction of Pseudomonas syringae pv syringae with plant hosts is the perception of plant signal molecules that regulate expression of genes, such as syrB, required for synthesis of the phytotoxin, syringomycin. In this study, the leaves of sweet cherry (Prunus avium L.) were analyzed to determine the nature of the syrB-inducing activity associated with tissues of a susceptible host. Crude leaf extracts yielded high amounts of total signal activity of more than 12,000 units g-1 (fresh weight) based on activation of a syrB-lacZ fusion in strain B3AR132. The signal activity was fractionated by C18 reversed-phase high-performance liquid chromatography and found to be composed of phenolic glycosides, which were resolved in three regions of the high-performance liquid chromatography profile, and sugars, which eluted with the void volume. Two flavonol glycosides, quercetin 3-rutinosyl-4[prime]-glucoside and kaempferol 3-rutinosyl-4[prime]-glucoside, and a flavanone glucoside, dihydrowogonin 7-glucoside, were identified. The flavonoid glycosides displayed similar specific signal activities and were comparable in signal activity to arbutin, a phenyl [beta]-glucoside, giving rise to between 120 and 160 units of [beta]-galactosidase activity at 10 [mu]M. Although D-fructose exhibits intrinsic low level syrB-inducing signal activity, D-fructose enhanced by about 10-fold the signal activities of the flavonoid glycosides at low concentrations (e.g. 10 [mu]M). This demonstrates that flavonoid glycosides, which represent a new class of phenolic plant signals sensed by P. s. syringae, are in sufficient quantities in the leaves of P. avium to activate phytotoxin synthesis.

Lipopeptide phytotoxins produced by Pseudomonas syringae pv. syringae: comparison of the biosurfactant and ion channel-forming activities of syringopeptin and syringomycin.

The phytopathogenic bacterium Pseudomonas syringae pv. syringae produces two classes of necrosis-inducing lipodepsipeptide toxins commonly referred to as the syringomycins and syringopeptins. Members of the syringomycins class are pore-forming cytotoxins that act by promoting passive transmembrane ion flux. In this study, we test the hypothesis that syringopeptin forms SP22A and SP22B likewise function as pore-forming cytotoxins and are similar in activity to syringomycin in artificial and plant membranes. Correspondingly, syringopeptin increased the conductance of black-lipid membranes in a manner indicative of ion channel formation. In tobacco protoplast assays, syringopeptin forms SP22A and SP22B were equivalent in activity causing lysis of protoplasts and measurable 45Ca2+ influx at a threshold concentration of 50 ng/ml. A mixture of three forms of syringomycin did not show cytotoxic activity appreciably different from that of SP22A or SP22B in tobacco protoplast assays. Both forms of syringopeptin also displayed potent biosurfactant properties demonstrated by lowering of the interfacial tension of high-pressure liquid chromatography-grade water to 36 and 34.5 nm/m, respectively; the critical micellar concentration was 0.8 mg/ml for both forms of toxin. These results demonstrate that both classes of pore-forming lipodepsipeptides secreted by P. syringae pv. syringae are cytotoxic to plant cells at nanomolar concentrations and cause necrosis by forming ion channels that are freely permeable to divalent cations.

Syringomycin production among strains of Pseudomonas syringae pv. syringae: conservation of the syrB and syrD genes and activation of phytotoxin production by plant signal molecules.

The syrB and syrD genes of Pseudomonas syringae pv. syringae are predicted to encode proteins that function in the synthesis and export of syringomycin, respectively. Using portions of the syr genes as DNA probes, both genes were shown to be conserved as single copies within a 15-kb or smaller DNA region among a broad spectrum of P. s. pv. syringae strains that produce syringomycin or one of its amino acid analogs, syringotoxin and syringostatin. Strains representative of P. viridiflava and six pathovars of P. syringae failed to hybridize with the gene probes, demonstrating that syr sequences are highly specific to P. s. pv. syringae and related nonpathogenic strains. Maximum parsimony analysis of restriction fragment length polymorphism profiles was used to evaluate relatedness among strains within the syrB and syrD gene region. A tree, conveying the smallest number of evolutionary changes among strains, revealed considerable diversity within the syr gene region; subclusters of strains were identified that appear to share specific qualities relevant to the plant-pathogen interaction. Because both the syrB gene and syringomycin production can be induced in response to plant signal molecules, 42 strains containing homologous syr sequences were tested for signal-mediated induction of toxin production. Over 90% of the toxigenic strains produced larger quantities of toxin when the plant signal molecules, arbutin and D-fructose, were added to syringomycin-minimal medium; 13 of the strains produced > or = 10-fold higher toxin levels. Some strains, such as 5D428, produced toxin only in the presence of these signals.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of biosurfactant and ion channel-forming activities of syringomycin in transmembrane ion flux: a model for the mechanism of action in the plant-pathogen interaction.

Syringomycin is a necrosis-inducing lipopeptide toxin synthesized and secreted by the phytopathogen, Pseudomonas syringae pv. syringae. Although small quantities of syringomycin are known to activate a cascade of physiological events in plasma membranes, the mechanism of action of the phytotoxin has never been fully characterized. The objective of this study was to test the hypothesis that the primary mode of action of syringomycin is to form transmembrane pores that are permeable to cations. Accordingly, direct measurement of ion fluxes were performed using artificial bilayers. The hemolytic properties and surface activity of HPLC-purified syringomycin were quantified by use of an erythrocyte lysis assay and by the drop weight method. Assays were performed using syringomycin form SRE alone or a mixture containing all forms of the phytotoxin. At a threshold concentration of 500 ng/ml, syringomycin induced hemolysis by forming ion channels in membranes. Osmotic protection studies indicated a channel radius of between 0.6 and 1 nm. The ion channel-forming activity was insensitive and permeable to both monovalent and divalent cations, suggesting that syringomycin causes lysis of erythrocytes by colloid osmotic lysis. In addition, syringomycin, like other lipopeptide antibiotics, is a potent biosurfactant capable of lowering the interfacial tension of water to 31 mN/m. The critical micellar concentration of syringomycin was calculated to be 1.25 mg/ml and the gamma CMC was 33 mN/m. A model is presented depicting the mechanism of action of syringomycin in the plant-pathogen interaction. The model integrates known effects of the toxin on ion flux in plasma membranes with formation of ion channels and the consequential cascade of effects associated with cellular signalling.

A physical map of the syringomycin and syringopeptin gene clusters localized to an approximately 145-kb DNA region of Pseudomonas syringae pv. syringae strain B301D.

Genetic and phenotypic mapping of an approximately 145-kb DraI fragment of Pseudomonas syringae pv. syringae strain B301D determined that the syringomycin (syr) and syringopeptin (syp) gene clusters are localized to this fragment. The syr and syp gene clusters encompass approximately 55 kb and approximately 80 kb, respectively. Both phytotoxins are synthesized by a thiotemplate mechanism of biosynthesis, requiring large multienzymatic proteins called peptide synthetases. Genes encoding peptide synthetases were identified within the syr and syp gene clusters, accounting for 90% of the DraI fragment. In addition, genes encoding regulatory and secretion proteins were localized to the DraI fragment. In particular, the salA gene, encoding a regulatory element responsible for syringomycin production and lesion formation in P. syringae pv. syringae strain B728a, was localized to the syr gene cluster. A putative ATP-binding cassette (ABC) transporter homolog was determined to be physically located in the syp gene cluster, but phenotypically affects production of both phytotoxins. Preliminary size estimates of the syr and syp gene clusters indicate that they represent two of the largest nonribosomal peptide synthetase gene clusters. Together, the syr and syp gene clusters encompass approximately 135 kb of DNA and may represent a genomic island in P. syringae pv. syringae that contributes to virulence in plant hosts.

The contribution of syringopeptin and syringomycin to virulence of Pseudomonas syringae pv. syringae strain B301D on the basis of sypA and syrB1 biosynthesis mutant analysis.

Sequencing of an approximately 3.9-kb fragment downstream of the syrD gene of Pseudomonas syringae pv. syringae strain B301D revealed that this region, designated sypA, codes for a peptide synthetase, a multifunctional enzyme involved in the thiotemplate mechanism of peptide biosynthesis. The translated protein sequence encompasses a complete amino acid activation module containing the conserved domains characteristic of peptide synthetases. Analysis of the substrate specificity region of this module indicates that it incorporates 2,3-dehydroaminobutyric acid into the syringopeptin peptide structure. Bioassay and high performance liquid chromatography data confirmed that disruption of the sypA gene in strain B301D resulted in the loss of syringopeptin production. The contribution of syringopeptin and syringomycin to the virulence of P. syringae pv. syringae strain B301D was examined in immature sweet cherry with sypA and syrB1 synthetase mutants defective in the production of the two toxins, respectively. Syringopeptin (sypA) and syringomycin (syrB1) mutants were reduced in virulence 59 and 26%, respectively, compared with the parental strain in cherry, whereas the syringopeptin-syringomycin double mutant was reduced 76% in virulence. These data demonstrate that syringopeptin and syringomycin are major virulence determinants of P. syringae pv. syringae.

Acute respiratory failure and pulmonary fibrosis secondary to administration of mycophenolate mofetil.

BACKGROUND: We report the first documented case of pulmonary toxicity to mycophenolate mofetil in this article. METHODS: A 51-year-old woman experienced systemic reactions beginning 10 days after cadaveric renal transplantation. RESULTS: Recurrent respiratory failure and documented progressive pulmonary fibrosis ensued. Cultures were negative and other agents were discontinued. It was not until the mycophenolate was stopped did the patient improve. CONCLUSIONS: Mycophenolate mofetil can cause acute respiratory failure simulating opportunistic infection or pulmonary edema. If not recognized, this may lead to the rapid development of severe pulmonary fibrosis, some of which may not be reversible.

Preoperative chemotherapy is beneficial for geriatric patients with locally advanced and metastatic breast cancer.

The relationship between aging and drug toxicity has been documented. We reviewed patients with Stage IIIB and Stage IV breast cancer with the primary tumor intact at presentation to compare the toxicity secondary to chemotherapy, the disease-free survival, and the survival in both young and elderly women. Fifty-three patients were studied. All received preoperative chemotherapy and mastectomy as part of treatment for breast cancer. Sixty years was used arbitrarily to divide the patients into two groups. Data were analyzed by unpaired T-test. Hematologic, nutritional, infectious, and cardiac toxicities were noted and compared between groups. Disease-free survival was studied in patients with Stage IIIB disease. Survival was examined in patients with Stage IIIB and Stage IV disease. We could not detect any statistically significant differences between young and elderly patients in any of these groups. The elderly patients in this study seemed to derive as much benefit from chemotherapy as did younger patients.

Regulation of syringomycin synthesis in Pseudomonas syringae pv. syringae and defined conditions for its production.

Production of the phytotoxin, syringomycin (SR), by Pseudomonas syringae pv. syringae strain B301D was regulated by both iron and inorganic phosphate similar to that of many bacterial secondary metabolites. Iron concentrations of 2 mumol/l or more in deferrated potato-dextrose broth (PDB) resulted in the production of 1024 SR units/ml, a yield comparable to that produced in non-deferrated PDB. Moreover, production of one SR unit required approximately 0.4 ng of available FeCl3. No SR was produced by strain B301D in deferrated PDB despite growth nearly identical with that of B301D in deferrated PDB supplemented with 10 mumol/l FeCl3. Furthermore, a phosphate concentration of 1 mmol/l or more was suppressive to SR production. Of the amino acids tested, L-histidine at a concentration of ca 20 mmol/l was the most effective nitrogen source for SR synthesis under defined conditions. Based on these observations, a synthetic medium, SR minimal, was formulated for SR or syringotoxin production by representative strains of Ps. syringae pv. syringae. The regulation of phytotoxin production is discussed in relation to pathogen survival and virulence.

Physical and functional analyses of the syrA and syrB genes involved in syringomycin production by Pseudomonas syringae pv. syringae.

The syrA and syrB genes involved in syringomycin production in Pseudomonas syringae pv. syringae B301D were identified from an EcoRI-pLAFR3 cosmid library and then physically and functionally analyzed in relation to plant pathogenicity. Homologous recombination of the genes required for syringomycin production from cosmids pGX183 (syrA) and pGX56 (syrB), respectively, introduced into nontoxigenic (Tox-) Tn5 mutants W4S2545 and W4S770 resulted in the concomitant restoration of toxin production and full virulence. The disease indices of the Tox+ strains obtained by recombination of the cloned, homologous DNA into the corresponding Tn5 mutant were essentially equivalent to that of strain B301D-R and significantly higher than those of W4S2545 and W4S770. A 12-kilobase (kb) EcoRI fragment from pGX183 was subcloned (i.e., pGX15) and found to contain the sequences necessary for syringomycin production. A map of pGX15 prepared by a combination of restriction endonuclease digestions and Tn5 mutagenesis showed that the syrA sequence was 2.3 to 2.8 kb. Marker exchange of syrA::Tn5 from pGX15 into B301D-R yielded nonpathogenic phenotypes, indicating that syrA is a regulatory gene since it is necessary for both syringomycin production and pathogenicity. The 4.9-kb EcoRI fragment from pGX56 was subcloned (i.e., pGX4) and shown to carry the syrB sequence which was 2.4 to 3.3 kb. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of protein extracts from B301D-R associated five proteins, ranging from approximately 130,000 to approximately 470,000 in molecular weight, with syringomycin production. The syrA and syrB genes were required for the formation of proteins SR4 (approximately 350,000) and SR5 (approximately 130,000), which are believed to be components of the syringomycin synthetase complex.

Evaluation of the Role of Syringomycin in Plant Pathogenesis by Using Tn5 Mutants of Pseudomonas syringae pv. syringae Defective in Syringomycin Production.

Syringomycin is a necrosis-inducing phytotoxin produced by Pseudomonas syringae pv. syringae. To determine whether syringomycin production is a determinant in virulence or pathogenicity, we isolated nontoxigenic (Tox) Tn5-containing mutants and then quantitatively evaluated them for the ability to multiply and cause disease in immature sweet-cherry fruits. Transposon Tn5 was delivered to Tox strain B301D-R by using the suicide vector, pGS9, and the resultant kanamycin-resistant (Km) colonies were screened for changes in syringomycin production by testing for antibiosis against Geotrichum candidum. Southern blot analysis of KpnI-and EcoRI-digested DNA showed that 15 (0.3%) Tox mutants were isolated which had Tn5 inserted into 1 of 14 distinct loci. Phenotypic characterization of the Tox mutants identified three major groups, which were differentiated by pathogenicity and ability to cause a tobacco hypersensitive reaction (HR). The eight strains in group A were pathogenic (Path) in cherry fruit assays, but the disease index was 17 to 66% lower (significant at P = 0.01) than for the parental Tox strain, B301D-R. The population dynamics of group A strains W4S770 and W4S116 in cherry fruits were, however, indistinguishable from that of strain B301D-R. The remaining seven Tox strains were nonpathogenic; group B strain W4S2545 (Path HR) and group C strain W4S468 (Path HR) developed significantly lower populations (10 to 10 CFU per cherry fruit) 3 days after inoculation than strain B301D-R did (nearly 10 CFU per fruit). The data indicate that syringomycin is not essential for pathogenicity, but contributes significantly to virulence.

Plant signal molecules activate the syrB gene, which is required for syringomycin production by Pseudomonas syringae pv. syringae.

The syrB gene is required for syringomycin production by Pseudomonas syringae pv. syringae and full virulence during plant pathogenesis. Strain B3AR132 containing a syrB::lacZ fusion was used to detect transcriptional activation of the syrB gene in syringomycin minimal medium by plant metabolites with signal activity. Among 34 plant phenolic compounds tested, arbutin, phenyl-beta-D-glucopyranoside, and salicin were shown to be strong inducers of syrB, giving rise to approximately 1,200 U of beta-galactosidase activity at 100 microM; esculin and helicin were moderate inducers, with about 250 to 400 U of beta-galactosidase activity at 100 microM. Acetosyringone and flavonoids that serve as signal molecules in Agrobacterium and Rhizobium species, respectively, did not induce the syrB::lacZ fusion. All syrB inducers were phenolic glucosides and none of the aglucone derivatives were active, suggesting that the beta-glycosidic linkage was necessary for signal activity. Phenyl-beta-D-galactopyranoside containing galactose substituted for glucose in the beta-glycosidic linkage also lacked inducer activity. Phenolic signal activity was enhanced two- to fivefold by specific sugars common to plant tissues, including D-fructose, D-mannose, and sucrose. The effect of sugars on syrB induction was most noticeable at low concentrations of phenolic glucoside (i.e., 1 to 10 microM), indicating that sugars such as D-fructose increase the sensitivity of P. syringae pv. syringae to the phenolic plant signal. Besides induction of syrB, syringomycin biosynthesis by parental strain B3A-R was induced to yield over 250 U of toxin by the additions of arbutin and D-fructose to syringomycin minimal medium. These data indicate that syringomycin production by most strains of P. syringae pv. syringae is modulated by the perception of two classes of plant signal molecules and transduced to the transcriptional apparatus of syringomycin (syr) genes such as syrB.

Bacteriocin-like substances produced by Rhizobium japonicum and other slow-growing rhizobia.

Bacteriocin-like substances were commonly produced by slow-growing Rhizobium japonicum and cowpea rhizobia on an L-arabinose medium. Antagonism between strains of R. japonicum was not detected in vitro; however, such strains were often sensitive to some bacteriocins produced by cowpea rhizobia. Inhibitory zones (2 to 8 mm from colony margins), produced by 58 of 66 R. japonicum test strains, were reproducibly detected with Corynebacterium nebraskense as an indicator. Quantitative production was not related to symbiotic properties of effective strains, since nine noninfective strains and one ineffective strain produced bacteriocin. Eight R. japonicum strains that did not produce bacteriocin nevertheless formed effective nodules on soybeans. R. japonicum strains that produced bacteriocin in vitro had no antagonistic effect on nonproducer strains during soybean nodulation. Under controlled conditions, a nonproducer (3I1b135) predominated over a bacteriocin producer (3I1b6) when inoculated at 1:1 and 1:9 ratios. Depending on the particular ratio, up to 38% of the total nodules formed were infected with mixed combinations. The bacteriocin(s) had a restricted host range and antibiotic-like properties which included the ability to be dialyzed and resistance to heat (75 to 80 degrees C, 30 min), Pronase, proteinase K, trypsin, ribonuclease, and deoxyribonuclease. R. japonicum strains representing genetic, serological, cultural, and geographic diversity were differentiated into three groups on the basis of bacteriocin production.

Transformation of Pseudomonas syringae with nonconjugative R plasmids.

Transformation of Pseudomonas syringae strains with plasmid DNA occurs at a frequency of 1 x 10(-3) to 4 x 10(-9) per recipient cell, depending on the strain, plasmid, and conditions for transformation. R plasmids used successfully in transformation were pR0161 (26 x 10(6) molecular weight) and RSF1010 (5.5 x 10(6) molecular weight). Transformation involved growing the recipient cells to approximately 8 x 10(8) colony-forming units per millilitre in 50 mL of a nutrient broth. After washes with a 150 mM CaCl2 - 10% (v/v) glycerol mixture, cells were concentrated 20-fold and resuspended in this solution. The cells then were incubated with purified plasmid DNA for 1 h prior to heat pulse at 45 degrees C for 2 min. Transformants were selected by antibiotic resistance and plasmid presence was verified by agarose gel electrophoresis. With plasmid pCG131 (34 x 10(6) molecular weight; putatively associated with syringomycin production), transformation of syringomycin-negative P. syringae strains that contained no detectable plasmid or were cured of pCG131 was unsuccessful, whether the plasmid was used alone or in combination with either pR0161 or RSF1010.

Bacteriocins of phytopathogenic Corynebacterium species.

The majority (85% of all strains tested) of 12 phytopathogenic Corynebacterium species produced bacteriocin(s) on nutrient broth--yeast extract (NBY) medium. All C. nebraskense, C. michiganense, C. insidiosum, C. oortii, and C. iranicum strains produced bacteriocin(s). The optimal conditions for production of 23 distinct bacteriocins by eight species of Corynebacterium generally were 20 degrees C and 4 days of incubation on NBY or on modified Burkholder's agar that lacked peptone (MBAL). Production in liquid was marginal and not augmented by adding mitomycin C. Bacteriocins generally had little effect on other strains within a species but were inhibitory to other species. Most bacteriocins appeared to be bactericidal proteins resistant to heat (75 to 80 degrees C, 30 min) but sensitive to proteolytic enzymes. Some strains of C. nebraskense, C. michiganense, C. insidiosum, and C. flaccumfaciens produced two bacteriocins which were clearly differentiated by varying or testing one or more of the following: conditions for production, the indicator, heat stability, and susceptibility to proteolysis. Within certain limitations, a convenient and reproducible typing scheme was devised for strain and species differentiation of most phytopathogenic corynebacteria.

Outer membrane protein mediating iron uptake via pyoverdinpss, the fluorescent siderophore produced by Pseudomonas syringae pv. syringae.

In an iron-limited environment Pseudomonas syringae pv. syringae B301D produces a yellow-green fluorescent siderophore called pyoverdinpss which functions in high-affinity iron transport. Two-dimensional electrophoretic comparisons of the outer membrane proteins of strain B301D identified nine proteins which were expressed at low (50 nM) but not at high (10 microM) iron concentrations. Except for the minor protein 8e, the iron-regulated proteins exhibited high molecular weights ranging from approximately 74,000 to 80,000. A mutant of strain B301D incapable of iron uptake (Iu-) from ferric pyoverdinpss lacked the 74,000-molecular-weight protein 4a, which was the major iron-regulated outer membrane protein. In contrast, a nonfluorescent mutant (Flu-) unable to synthesize pyoverdinpss showed no quantitative or qualitative difference in its outer membrane profile from that of the wild-type strain. In plant pathogenicity tests the Iu- and Flu- strains caused typical brown necrotic and sunken lesions in immature sweet cherry fruit which were indistinguishable from those of the wild-type strain. Thus, excretion of pyoverdinpss and subsequent Fe(III) uptake do not have a determinative role in the pathogenicity or virulence of P. syringae pv. syringae.

Characterization of Pyoverdin(pss), the Fluorescent Siderophore Produced by Pseudomonas syringae pv. syringae.

Pseudomonas syringae pv. syringae B301D produces a yellow-green, fluorescent siderophore, pyoverdin(pss), in large quantities under iron-limited growth conditions. Maximum yields of pyoverdin(pss) of approximately 50 mug/ml occurred after 24 h of incubation in a deferrated synthetic medium. Increasing increments of Fe(III) coordinately repressed siderophore production until repression was complete at concentrations of >/= 10 muM. Pyoverdin(pss) was isolated, chemically characterized, and found to resemble previously characterized pyoverdins in spectral traits (absorbance maxima of 365 and 410 nm for pyoverdin(pss) and its ferric chelate, respectively), size (1,175 molecular weight), and amino acid composition. Nevertheless, pyoverdin(pss) was structurally unique since amino acid analysis of reductive hydrolysates yielded beta-hydroxyaspartic acid, serine, threonine, and lysine in a 2:2:2:1 ratio. Pyoverdin(pss) exhibited a relatively high affinity constant for Fe(III), with values of 10 at pH 7.0 and 10 at pH 10.0. Iron uptake assays with [Fe]pyoverdin(pss) demonstrated rapid active uptake of Fe(III) by P. syringae pv. syringae B301D, while no uptake was observed for a mutant strain unable to acquire Fe(III) from ferric pyoverdin(pss). The chemical and biological properties of pyoverdin(pss) are discussed in relation to virulence and iron uptake during plant pathogenesis.

Intrabullous carcinoma: a diagnostic dilemma.

Two cases of bronchogenic carcinoma arising within the walls of bullae are described. They manifested as air-fluid levels within the bullae. Such air-fluid levels are generally considered to be due to infection and conservative management is recommended. This approach delays the recognition of potentially resectable neoplasms, as was the case in our patients. We discuss the clinical and radiographic clues which raise the specter of neoplasm in such cases.

Development, distribution, and characteristics of intrinsic, nonbacterial ice nuclei in prunus wood.

Ice nuclei active at approximately -2 degrees C and intrinsic to woody tissues of Prunus spp. were shown to have properties distinct from bacterial ice nuclei. Soaking 5-centimeter peach stem sections in water for 4 hours lowered the mean ice nucleation temperature to below -4 degrees C, nearly 2 degrees C lower than stems inoculated with ice nucleation-active Pseudomonas syringae strain B301D. Ice nucleation activity in peach was fully restored by air-drying woody stem sections for a few hours. The ice nuclei in woody tissue were inactivated between 40 and 50 degrees C, but unaffected by treatment with bacterial ice nucleation inhibitors (i.e. NaOCl, tartaric acid, Triton XQS-20), sulfhydryl reagents (i.e. p-hydroxymercuribenzoate and iodine) and Pronase. Ice nuclei could not be dislodged from stems by sonication and were shown to be equally distributed in peach bud and internodal stem tissue on a per unit mass basis; outer and inner stem tissues were also indistinguishable in ice nucleation activity. Development of ice nuclei in immature peach and sweet cherry stems did not occur until midsummer and their formation was essentially complete by late August. Once formed the ice nuclei intrinsic to woody stems were stable and unaffected by seasonal changes in growth. The apparent physiological function of the ice nuclei is discussed in relation to supercooling and mechanisms of cold hardiness in Prunus spp.