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Plasmid-mediated production of the phytotoxin coronatine by Pseudomonas syringae pv. tomato
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9th World Congress
Lipoxygenase-dependent secretory effect ofcanatoxin. CHRISTINA BARJA-FIDALGO,1 CI~L1AR. CARLIN12and J. A. GUIMAR.7~.ES2 (aDept. of Pharmacology, I.B., Universidade do Estado do Rio de Janeiro; 2Dept. of Biochemistry, CCS/ICB, Universidade Federal do Rio de Janeiro, CP 68041, Rio de Janeiro, R. J., Brazil). CANATOXIN(CNTX), the neurotoxic protein isolated from Canavalia ensiformis seeds (CARLINI and GUIMAR.~S, 1981), induces convulsions and death in mice and rats (LDs0 = 2 - 3 mg/kg), through a process modulated by the central nervous system (CARLINI et al., 1984). CNTX also induces platelet activation through a lipoxygenase pathway (CARLINIet al., 1985). Based on several physiological similarities relating platelets to synaptosomes, the effect of CNTX was investigated on this in vitro model. Synaptosomes prepared from rat brain and loaded with 3H-serotonine were resuspended in a nutritive medium containing 5 #M imipramine and stimulated with micromolar concentrations of CNTX. The toxic protein induced release of the labelled neurotransmitter in a time-, temperature- and dose-dependent manner, without releasing LDH from the vesicles. The releasing effect reached a plateau after 20 min at 37"C. An effect of the same magnitude was obtained with either 6 #M CNTX or 150mM KC1. Thereafter, labelled synaptosomes were pre-incubated ( 0 - 2 0 m i n , r.t.) with 150#M indomethacin or 500 #M nordihydrogualaretic acid (NDGA), a lipoxygenase inhibitor, and then exposed to 4 #M canatoxin. It could be seen that NDGA pretreatment inhibited the secretory effect of CNTX in a timedependent fashion, but indomethacin had no effect. There was no interference of both drugs on 150 mM KClinduced secretion. These data suggest that the secretion promoting action of canatoxin on synaptosomes is also a lipoxygenase-dependent pathway. Supported by FINEP, CNPq, CEPG-UFRJ and IFS (Sweden). REFERENCES CARLINI and G U I M A ~ (1981) Toxicon 19, 667. CARLINI et al. (1984) Acta Pharmac. Toxicol. 54, 161. CARLINI et al. (1985) Br. J. Pharmac. 84, 551. Effect ofNaja nigrocollis phospholipase A 2 on intact and skinned muscle fibers from the frog. ANITA C. BAXTER,1 JAMES B. LARSEN1 and PmLLn" ROSENBERG2 (~Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, MS 39406-5018; 2School of Pharmacy, University of Connecticut, Storrs, CT 06268, U.S.A.). EXPERIMENTS with isolated preparations of frog semitendinosus muscle were conducted to clarify the relationship between enzymatic and myotoxic activities of purified phospholipase A2 (PLA2) from Naja nigricollis venom. Bundles of intact fibers were stimulated directly (0.1 Hz) in physiological saline solution containing PLA2. Mechanically skinned fibers (HELLAM and PODOLSKY, 1969) were tested in a high-Ca 2+ (contracting) solution, and then immersed in a Ca2÷-free (relaxing) solution containing PLA2. Twitches recorded from intact fibers stimulated in the presence of PLA2 (50 #g/ml) were significantly smaller than those recorded from control fibers: at 30 min, average twitch amplitude had decreased by 38% in treated fibers and by only 16% in controls (p < 0.02); at 60 min, average amplitude had decreased by 80% in treated fibers and by only 32% in controls (p < 0.01). Skinned fibers exposed to PLA2 (50 #g/ml) for 30 min developed a sustained contracture which was not observed in controls. Treatment did not modify the normal contraction resulting from subsequent transfer to the high-Ca 2 + solution. The effects of PLA2 on intact fibers may result from enzymatic disruption of cell membranes. However, in skinned fibers the observed contractures, which develop in the absence of a sarcolemma and without Ca 2 +, suggest a separate pharmacological effect (FLETCHER et al., 1981). REFERENCES FLETCHER, J. E., RAPUANO,B. E., CONDREA, E., YANG, C. and ROSENBERG,P. (1981) Toxicol. appl. Pharmac. 59, 375. HELLAM, D. C. and PODOLSKY, R. J. (1969) J. Physiol., Lond. 200, 807. Plasmid-mediated production o f the phytotoxin coronatine by Pseudomonas syringae pv. tomato. CAROL L. BENDER,1 DEAN K. MALVICK1 and ROBIN E. MITCHELL2 (1Dept. of Plant Pathology, Oklahoma State University, Stillwater, OK 74078, U.S.A., and 2Dept. of Scientific and Industrial Research, Auckland, New Zealand). THE PHYTOPATHOGENIC bacterium Pseudomonas syringae pv. tomato PT23.2 produces the chlorosis-inducing phytotoxin, coronatine. Thirty-eight chlorosis-defective mutants of PT23.2 were generated using the transposon Tn5. Five mutants contained Tn5 insertions in the indigenous plasmid pPT23A; the remaining 33 mutants were either missing pPT23A (29 mutants) or contained putative deletion derivatives of this plasmid (4 mutants).
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These results suggested that pPT23A was involved in coronatine production in strain PT23.2. This plasmid was introduced into P. syringae pv. syringae PS61, which does not produce the phytotoxin coronatine. A bioassay for coronatine suggested that PS61 transconjugants containing pPT23A were able to make the phytotoxin. In a more critical analysis, organic acids were isolated from PT23.2, PS61 and the transconjugant PS61(pPT23A); these were derivatized to their methyl esters and analyzed by gas chromatography. The organic acids extracted from PT23.2 and PS61(pPT23A) contained a distinct coronatine peak; coronatine production was absent in the wild type PS61. Therefore, the acquisition of pPT23A by PS61 resulted in coronatine biosynthesis, thus proving that pPT23A is involved in coronatine production in P. syringae pv. tomato. This is the first conclusive evidence for the role of plasmid-borne genes in the biosynthesis of a chlorosis-inducing phytotoxin.
lodinated bothropic venoms are unable to cause tissue damage and are devoid of other enzymatic activities. ROGf~RIO X. BICALHO,10RIVALDO A. ROCHA,2 LUIZ G . D . HENEINE,4 ARINOS M A G A L ~ 3 and IBRAHIM FELIPPE HENE1NE1 (1Laborat6rio de Biofisica, Departamento de Fisiologia e Biofisica; 2Departamento de Patologia Geral; 3Departamento de Bioquimica e Imunologia; Instituto de Ci6ncias Biol6gicas, Universidade Federal de Minas Gerais, 31270; and 4FUNED, 30550 - Belo Horizante, Minas Gerais, Brazil).
MANY CROTALIDvenoms are well known to cause severe damage in biological tissues, and toxoiding these venoms by various procedures has been tried in order to minimize these local lesions. By controlled iodinations of the whole Bothrops jararaca venom, a preparation was obtained without the ability to cause dermo- and myonecrosis, hemorrhage, coagulation of fibrinogen and direct hemolysis. Lethality was also abolished up to 32 LD~0. The immunogenicity is somewhat lowered, putatively caused by a rapid excretion of the iodinated derivatives. Other bothropic venoms studied behaved similarly.
REFERENCE HENEINE, I. F., HENEINE, L. G. D., DANIEL, J. P., NASCIMENTO,M. C. S. and ROCHA, O. A. Symposium on Toxic Proteins. Academia Ci~ncias Estado de Sao Paulo (ACIESP), XII, in press.
Studies of the sequence of Mojave toxin: the acidic subunit. ALLAN L. BIEBER,1 ROBERT R. BECKER,2 REGINALD MCPARLAND,2 DONALD F. HUNT,3 JEFFRY SHABANOWITZ,3 JOHN R. YATES IIIa and GreBES R. JOHNSON~ (1Department of Chemistry, Arizona State University, Tempe, AZ 85287; 2Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331; and 3Department of Chemistry, University of Virginia, Charlottesville, VA 22908, U.S.A.).
NEUROTOXIC proteins with intrinsic phospholipase activity are present in venoms of several rattlesnake species. These neurotoxic protein complexes consist of a basic phospholipase associated with an acid protein chain by non-covalent interactions. Crotoxin (C. d. terrificus) and Mojave toxin (C. s. scutulatus) are characteristic examples. The amino acid sequence for the basic subunit and a partial sequence for the acidic subunit of crotoxin have been published, but sequences for the corresponding subunits from Mojave toxin have not been reported. This report provides sequence data for the acidic subunit from Mojave toxin and the results demonstrate that the heterogeneity of the native toxin may arise from the existence of multiple forms of acidic subunit. Mojave toxin, purified by ion-exchange chromatography, was dissociated to constituent subunits. The isolated subunits were further purified by HPLC. The reduced, alkylated acidic subunit was resolved into individual peptide constituents by HPLC. The isolated fragments were subjected to sequence analysis using gas phase sequencing, degradation by proteases and high resolution Fourier transform mass spectrometry. Amino acid sequence data generated for each major peptide fragment obtained by reductive alkylation are shown below. PeptideA PeptideB PeptideC
GCYCGAGGQGWPQDAS DRCCFEHDCCYAKLTGCDPTTDVY pEEDGEI VCQDDPCGTQI CGCDKAAAI CFRDSMDT S PENCQGES QPC ENCQGESQPC (Underscored residues need confirmation.) The sequence for each peptide are very similar to the corresponding peptides from crotoxin, consistent with other data that demonstrate the relatedness of these neurotoxins. Supported in part by NSF BNS-8408667.
9th World Congress
Lipoxygenase-dependent secretory effect ofcanatoxin. CHRISTINA BARJA-FIDALGO,1 CI~L1AR. CARLIN12and J. A. GUIMAR.7~.ES2 (aDept. of Pharmacology, I.B., Universidade do Estado do Rio de Janeiro; 2Dept. of Biochemistry, CCS/ICB, Universidade Federal do Rio de Janeiro, CP 68041, Rio de Janeiro, R. J., Brazil). CANATOXIN(CNTX), the neurotoxic protein isolated from Canavalia ensiformis seeds (CARLINI and GUIMAR.~S, 1981), induces convulsions and death in mice and rats (LDs0 = 2 - 3 mg/kg), through a process modulated by the central nervous system (CARLINI et al., 1984). CNTX also induces platelet activation through a lipoxygenase pathway (CARLINIet al., 1985). Based on several physiological similarities relating platelets to synaptosomes, the effect of CNTX was investigated on this in vitro model. Synaptosomes prepared from rat brain and loaded with 3H-serotonine were resuspended in a nutritive medium containing 5 #M imipramine and stimulated with micromolar concentrations of CNTX. The toxic protein induced release of the labelled neurotransmitter in a time-, temperature- and dose-dependent manner, without releasing LDH from the vesicles. The releasing effect reached a plateau after 20 min at 37"C. An effect of the same magnitude was obtained with either 6 #M CNTX or 150mM KC1. Thereafter, labelled synaptosomes were pre-incubated ( 0 - 2 0 m i n , r.t.) with 150#M indomethacin or 500 #M nordihydrogualaretic acid (NDGA), a lipoxygenase inhibitor, and then exposed to 4 #M canatoxin. It could be seen that NDGA pretreatment inhibited the secretory effect of CNTX in a timedependent fashion, but indomethacin had no effect. There was no interference of both drugs on 150 mM KClinduced secretion. These data suggest that the secretion promoting action of canatoxin on synaptosomes is also a lipoxygenase-dependent pathway. Supported by FINEP, CNPq, CEPG-UFRJ and IFS (Sweden). REFERENCES CARLINI and G U I M A ~ (1981) Toxicon 19, 667. CARLINI et al. (1984) Acta Pharmac. Toxicol. 54, 161. CARLINI et al. (1985) Br. J. Pharmac. 84, 551. Effect ofNaja nigrocollis phospholipase A 2 on intact and skinned muscle fibers from the frog. ANITA C. BAXTER,1 JAMES B. LARSEN1 and PmLLn" ROSENBERG2 (~Department of Biological Sciences, University of Southern Mississippi, Hattiesburg, MS 39406-5018; 2School of Pharmacy, University of Connecticut, Storrs, CT 06268, U.S.A.). EXPERIMENTS with isolated preparations of frog semitendinosus muscle were conducted to clarify the relationship between enzymatic and myotoxic activities of purified phospholipase A2 (PLA2) from Naja nigricollis venom. Bundles of intact fibers were stimulated directly (0.1 Hz) in physiological saline solution containing PLA2. Mechanically skinned fibers (HELLAM and PODOLSKY, 1969) were tested in a high-Ca 2+ (contracting) solution, and then immersed in a Ca2÷-free (relaxing) solution containing PLA2. Twitches recorded from intact fibers stimulated in the presence of PLA2 (50 #g/ml) were significantly smaller than those recorded from control fibers: at 30 min, average twitch amplitude had decreased by 38% in treated fibers and by only 16% in controls (p < 0.02); at 60 min, average amplitude had decreased by 80% in treated fibers and by only 32% in controls (p < 0.01). Skinned fibers exposed to PLA2 (50 #g/ml) for 30 min developed a sustained contracture which was not observed in controls. Treatment did not modify the normal contraction resulting from subsequent transfer to the high-Ca 2 + solution. The effects of PLA2 on intact fibers may result from enzymatic disruption of cell membranes. However, in skinned fibers the observed contractures, which develop in the absence of a sarcolemma and without Ca 2 +, suggest a separate pharmacological effect (FLETCHER et al., 1981). REFERENCES FLETCHER, J. E., RAPUANO,B. E., CONDREA, E., YANG, C. and ROSENBERG,P. (1981) Toxicol. appl. Pharmac. 59, 375. HELLAM, D. C. and PODOLSKY, R. J. (1969) J. Physiol., Lond. 200, 807. Plasmid-mediated production o f the phytotoxin coronatine by Pseudomonas syringae pv. tomato. CAROL L. BENDER,1 DEAN K. MALVICK1 and ROBIN E. MITCHELL2 (1Dept. of Plant Pathology, Oklahoma State University, Stillwater, OK 74078, U.S.A., and 2Dept. of Scientific and Industrial Research, Auckland, New Zealand). THE PHYTOPATHOGENIC bacterium Pseudomonas syringae pv. tomato PT23.2 produces the chlorosis-inducing phytotoxin, coronatine. Thirty-eight chlorosis-defective mutants of PT23.2 were generated using the transposon Tn5. Five mutants contained Tn5 insertions in the indigenous plasmid pPT23A; the remaining 33 mutants were either missing pPT23A (29 mutants) or contained putative deletion derivatives of this plasmid (4 mutants).
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These results suggested that pPT23A was involved in coronatine production in strain PT23.2. This plasmid was introduced into P. syringae pv. syringae PS61, which does not produce the phytotoxin coronatine. A bioassay for coronatine suggested that PS61 transconjugants containing pPT23A were able to make the phytotoxin. In a more critical analysis, organic acids were isolated from PT23.2, PS61 and the transconjugant PS61(pPT23A); these were derivatized to their methyl esters and analyzed by gas chromatography. The organic acids extracted from PT23.2 and PS61(pPT23A) contained a distinct coronatine peak; coronatine production was absent in the wild type PS61. Therefore, the acquisition of pPT23A by PS61 resulted in coronatine biosynthesis, thus proving that pPT23A is involved in coronatine production in P. syringae pv. tomato. This is the first conclusive evidence for the role of plasmid-borne genes in the biosynthesis of a chlorosis-inducing phytotoxin.
lodinated bothropic venoms are unable to cause tissue damage and are devoid of other enzymatic activities. ROGf~RIO X. BICALHO,10RIVALDO A. ROCHA,2 LUIZ G . D . HENEINE,4 ARINOS M A G A L ~ 3 and IBRAHIM FELIPPE HENE1NE1 (1Laborat6rio de Biofisica, Departamento de Fisiologia e Biofisica; 2Departamento de Patologia Geral; 3Departamento de Bioquimica e Imunologia; Instituto de Ci6ncias Biol6gicas, Universidade Federal de Minas Gerais, 31270; and 4FUNED, 30550 - Belo Horizante, Minas Gerais, Brazil).
MANY CROTALIDvenoms are well known to cause severe damage in biological tissues, and toxoiding these venoms by various procedures has been tried in order to minimize these local lesions. By controlled iodinations of the whole Bothrops jararaca venom, a preparation was obtained without the ability to cause dermo- and myonecrosis, hemorrhage, coagulation of fibrinogen and direct hemolysis. Lethality was also abolished up to 32 LD~0. The immunogenicity is somewhat lowered, putatively caused by a rapid excretion of the iodinated derivatives. Other bothropic venoms studied behaved similarly.
REFERENCE HENEINE, I. F., HENEINE, L. G. D., DANIEL, J. P., NASCIMENTO,M. C. S. and ROCHA, O. A. Symposium on Toxic Proteins. Academia Ci~ncias Estado de Sao Paulo (ACIESP), XII, in press.
Studies of the sequence of Mojave toxin: the acidic subunit. ALLAN L. BIEBER,1 ROBERT R. BECKER,2 REGINALD MCPARLAND,2 DONALD F. HUNT,3 JEFFRY SHABANOWITZ,3 JOHN R. YATES IIIa and GreBES R. JOHNSON~ (1Department of Chemistry, Arizona State University, Tempe, AZ 85287; 2Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331; and 3Department of Chemistry, University of Virginia, Charlottesville, VA 22908, U.S.A.).
NEUROTOXIC proteins with intrinsic phospholipase activity are present in venoms of several rattlesnake species. These neurotoxic protein complexes consist of a basic phospholipase associated with an acid protein chain by non-covalent interactions. Crotoxin (C. d. terrificus) and Mojave toxin (C. s. scutulatus) are characteristic examples. The amino acid sequence for the basic subunit and a partial sequence for the acidic subunit of crotoxin have been published, but sequences for the corresponding subunits from Mojave toxin have not been reported. This report provides sequence data for the acidic subunit from Mojave toxin and the results demonstrate that the heterogeneity of the native toxin may arise from the existence of multiple forms of acidic subunit. Mojave toxin, purified by ion-exchange chromatography, was dissociated to constituent subunits. The isolated subunits were further purified by HPLC. The reduced, alkylated acidic subunit was resolved into individual peptide constituents by HPLC. The isolated fragments were subjected to sequence analysis using gas phase sequencing, degradation by proteases and high resolution Fourier transform mass spectrometry. Amino acid sequence data generated for each major peptide fragment obtained by reductive alkylation are shown below. PeptideA PeptideB PeptideC
GCYCGAGGQGWPQDAS DRCCFEHDCCYAKLTGCDPTTDVY pEEDGEI VCQDDPCGTQI CGCDKAAAI CFRDSMDT S PENCQGES QPC ENCQGESQPC (Underscored residues need confirmation.) The sequence for each peptide are very similar to the corresponding peptides from crotoxin, consistent with other data that demonstrate the relatedness of these neurotoxins. Supported in part by NSF BNS-8408667.