Structure of rhizobitoxine, an antimetabolic enol-ether amino-acid from Rhizobium japonicum

Owens, Lowell D.; Thompson, John F.; Pitcher, Ross G.; Williams, Thomas H.

doi:10.1039/c39720000714

Cited by 85 publications

(41 citation statements)

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“…Four of the nine strains in DNA homology group I1 are strains of bradyrhizobia which produce foliar chlorosis ( 5 ) caused by the anti-metabolite rhizobitoxine (19). Of the 10 strains tested which are not characterized by relative DNA homology, 8 are chlorosis producers (6, 7).…”

Section: Discussionmentioning

confidence: 99%

Fatty Acids, Antibiotic Resistance, and Deoxyribonucleic Acid Homology Groups of Bradyrhizobium japonicum

Kuykendall

Roy

O'Neill

et al. 1988

International Journal of Systematic Bacteriology

914

621

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The fatty acid compositions and multiple antibiotic resistance patterns of 32 strains of Bradyrhizobium japonicum correlated with two major deoxyribonucleic acid homology groups. In group I, the fatty acid composition was 1.3% 16:l cis9 acid, 3.6% 16:lC acid, 8.8% 16:O acid, 1.2% 19:0 cyclopropane acid, and 81.2% 18:l acid. Group I1 contained 0.5% 16:lC acid, 11.1% 16:O acid, 0.8% 17:O cyclopropane acid, 24.7% 19:O cyclopropane acid, and 62.3% 18:l acid. Group I strains were susceptible to rifampin (500 pglml), tetracycline (100 pglml), streptomycin (100 pg/ml), chloramphenicol (500 pg/ml), erythromycin (250 pglml), carbenicillin (500 pg/ml), and nalidixic acid (50 pg/ml), whereas group I1 strains were resistant to these antibiotics. Both groups were resistant to trimethoprim (50 pg/ml) and vancomycin (100 pglml).Bradyrhizobium japonicum (Jordan 1982) (formerly Rhizobium japonicum) is the slow-growing symbiont of soybeans. This species is comprised of two highly divergent taxonomic groups which should be phenotypically distinguishable. The deoxyribonucleic acid (DNA)-DNA hybridization studies of Hollis et al. (8) established DNA homology groups I, Ia, and 11. These groups were characterized with respect to acidic extracellular polysaccharide composition and phenotypic ex planta nitrogenase expression. Groups I and Ia were reported to be quite distinct from DNA homology group I1 according to both of these criteria (9). Using some of the same strains, Stanley et al. (21) characterized the DNA nucleotide sequence divergences in and proximal to known symbiotic genes and concluded that strains of B . japonicum comprise two symbiotic genotypes (sTI and sTII) consistent with separate species designations.Whole-cell fatty acid profiles of strains grown under standardized conditions have been used for identification (1, 4,18) and classification of many bacteria (10, 17), but not in the genus Bradyrhizobium. Pankhurst et al. (20) correlated rifampin resistance in some slow-growing rhizobial strains with in vitro nitrogenase activity. This observation, together with that of Huber et al. (9) that only DNA homology group I1 strains express nitrogenase in vitro, suggested to us that patterns of antibiotic resistance might also distinguish the groups. In this paper we report that whole-cell fatty acid profiles correlate with DNA homology groups I and I1 of Hollis et al. (8) and that patterns of high-level resistance to antibiotics distinguish the two DNA homology groups. MATERIALS AND METHODSBacterial strains. All USDA strains were obtained from H. H. Keyser and D.Weber, U. S. Department of Agriculture (USDA), Beltsville, Md.; strain ATCC 10324 was obtained from the American Type Culture Collection, Rockville, Md.; strains 5631, 5633, D193, D209, THA6, and 8' were obtained from G. H. Elkan, North Carolina State University, Raleigh; and strains 61A.50 and 61A76 were obtained from S. Smith, Nitragin Co., Milwaukee, Wis. Strains that exhibit rifampin resistance and tetracycline resistance were purified by streaking onto a mediu...

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Section: Discussionmentioning

confidence: 99%

Fatty Acids, Antibiotic Resistance, and Deoxyribonucleic Acid Homology Groups of Bradyrhizobium japonicum

Kuykendall

Roy

O'Neill

et al. 1988

International Journal of Systematic Bacteriology

914

621

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“…The absorbance of the RT peak at 440 nm was larger than that at 570 nm. This was considered to be due to the yellow reaction product of RT with ninhydrin (1,3). When the purified DRT was analyzed, one peak appeared between the positions of DL-plus-allo-o-hydroxylysine and ornithine (Fig.…”

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Determination of Rhizobitoxine and Dihydrorhizobitoxine in Soybean Plants by Amino Acid Analyzer

Minamisawa

Kume

1987

Soil Science and Plant Nutrition

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Rhizobitoxine (RT) (2-amino-4-(2-amino-3-hydropropoxy)-trans-but-3-enoic acid, C7H14N204=190) is a phytotoxin produced by some strains of Bradyrhizobiumjaponicum (1-3). ART-producing B. japonicum often induces chlorosis in new leaves of the host plant as a result of the synthesis of the toxin in the nodules (l, 4). Physiologically, RT is known to inhibit fi-cystathionase in the methionine biosynthetic pathway and ethylene formation (5-7). The ability to produce RT has been reported to be common among Bradyrhizobium strains originating from the U .S.A., Africa, Central America, and South America (8). Dihydrorhizobitoxine (DRT), a structural analogue of RT, is also associated with the occurrence of RT in nodules arid has been identified as O_(2_amino-3-hydroxylpropyl)homoserine (C 7 H 16 N 2 0,=192) (1, 2, 9). RT has been detected semi quantitatively by paper chromatography or bioassay. OWENS and WRIGHT (1, 2) separated RT and DRT (unknown Y) by two-dimensional paper chromatography, detecting them by spraying ninhydrin, and the amounts of RT and DRT were relatively estimated by visual comparison with asparagine standards and alanine (or purified unknown Y) standards, respectively. LA FAVRE and EAGLESHAM (8) separated RT by one-dimensional paper chromatography, and the color density of its yellow reaction product with ninhydrin on paper was classified into 3 levels. The relative amount of RT was also assayed by the degree of chlorosis of soybean plants (1,4,8) and seedlings of various plants (4). We have not been able to find any reference on the accurate determination of RT. This report describes the quantitative measurement of the RT and DRT contents of soybean plants using an amino acid analyzer.RT and DRT were isolated and purified from whole-culture extracts of strain. of B. japonicum USDA 94 using the procedure of GIOVANELLI (6), except for the addition of ion exchange chromatography (pyridine type (10» before the re-chromatography step. Fifteen mg of RT and 22 mg of DRT were purified from 7 liters of culture.

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“…The present data do not permit decisions about the relative contributions of the transsulfuration and the direct sulfhydration pathways to methionine biosynthesis, or whether the pathological effects of rhizobitoxine are due chiefly to inhibition of 8-cystathionase. Rhizobitoxine is an enol-ether amino acid with the structure 2-amino-4-(2-amino-3-hydroxypropoxy)-trans-3-butenoic acid (15). This amino acid is produced by certain strains of the root nodule bacterium Rhizobium japonicum and passes from the bacteria in the nodule to the leaves of the host soybean plant, where it inhibits greening of new leaf growth (17).…”

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β-Cystathionase In Vivo Inactivation by Rhizobitoxine and Role of the Enzyme in Methionine Biosynthesis in Corn Seedlings

Giovanelli

Owens²,

Mudd³

1973

Plant Physiol.

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Rhizobitoxine has previously been shown to inactivate irreversibly ,8-cystathionase isolated from spinach. In the present studies, rhizobitoxine was shown to inhibit partially ,-cystathionase of spinach and corn seedlings in vivo. An activity of 30 to 40% of normal remained in toxin-treated seedlings of both spinach and corn. Possible reasons for the partial inhibition are discussed.Rhizobitoxine-treated and control corn seedlings were allowed to assimilate 35SO42-for 3 or 6 hours, and the radioactivity incorporated into sulfur amino acids at these times was studied. The most striking effect of rhizobitoxine was an increase (up to 22-fold) in radioactive cystathionine. Accumulation of radioactivity in methionine was only slightly inhibited by rhizobitoxine treatment. The results strongly suggest that the transsulfuration pathway contributes to methionine biosynthesis, and that metabolism via this pathway is impaired, but not entirely eliminated, by rhizobitoxine treatment.The present data do not permit decisions about the relative contributions of the transsulfuration and the direct sulfhydration pathways to methionine biosynthesis, or whether the pathological effects of rhizobitoxine are due chiefly to inhibition of 8-cystathionase.Rhizobitoxine is an enol-ether amino acid with the structure 2-amino-4-(2-amino-3-hydroxypropoxy)-trans-3-butenoic acid (15). This amino acid is produced by certain strains of the root nodule bacterium Rhizobium japonicum and passes from the bacteria in the nodule to the leaves of the host soybean plant, where it inhibits greening of new leaf growth (17). Rhizobitoxine has also been isolated from the culture medium of R. japonicum (16). The isolated compound is phytotoxic to soybean and many other higher plant species (11). It also inhibits the growth of the green alga Chlorella (12) and the bacterium Salmonella typhimurium (13). Inhibition of the growth of the latter organism is overcome by addition to the medium of methionine or homocysteine, but not of serine, homoserine, or cystathionine. Available evidence (1) synthesizes methionine by reactions 1 through 5 (Fig. 1). This sequence includes the transsulfuration reactions 3 and 4, whereby the sulfur atom of cysteine is transferred to homocysteine by way of the intermediate thioether, cystathionine. The results of the growth experiments described suggest that rhizobitoxine acts in Salmonella by inhibition of the cleavage of cystathionine (reaction 4), a step catalyzed by the enzyme /3-cystathionase. This hypothesis was supported by demonstration that 83-cystathionase isolated from S. typhimuriutm is inhibited in vitro by rhizobitoxine (13).The mode of action of rhizobitoxine in higher plants remains to be elucidated. We have investigated this problem not only because of the intrinsic interest in defining the pathophysiology of this naturally occurring plant toxin, but also because such investigations might be helpful in answering questions about methionine biosynthesis in higher plants. For example, in vitro enzyme studi...

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Structure of rhizobitoxine, an antimetabolic enol-ether amino-acid from Rhizobium japonicum

Cited by 85 publications

References 0 publications

Fatty Acids, Antibiotic Resistance, and Deoxyribonucleic Acid Homology Groups of Bradyrhizobium japonicum

Fatty Acids, Antibiotic Resistance, and Deoxyribonucleic Acid Homology Groups of Bradyrhizobium japonicum

Determination of Rhizobitoxine and Dihydrorhizobitoxine in Soybean Plants by Amino Acid Analyzer

β-Cystathionase In Vivo Inactivation by Rhizobitoxine and Role of the Enzyme in Methionine Biosynthesis in Corn Seedlings

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