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Applied and Environmental Microbiology, February 1999, p. 849-852, Vol. 65, No. 2
Institute of Genetic Ecology,
Received 9 July 1998/Accepted 12 November 1998
Rhizobitoxine is synthesized by the legume symbiont
Bradyrhizobium elkanii and the plant pathogen
Burkholderia andropogonis. Rhizobitoxine competitively
inhibited 1-aminocyclopropane-1-carboxylate (ACC) synthase bLE-ACS2
from the tomato, a key enzyme in the pathway of ethylene biosynthesis.
Based on this inhibition of ACC synthase, we have
developed a new assay for rhizobitoxine.
Rhizobitoxine
[2-amino-4-(2-amino-3-hydropropoxy)-trans-but-3-enoic
acid] is synthesized by the legume symbiont Bradyrhizobium elkanii (3, 5, 7, 8, 11, 15) and the plant pathogen Burkholderia andropogonis (9, 10). The bacterial
toxin induces foliar chlorosis of the host plants; however, the role of
the toxin in symbiosis and pathogenicity remains unclear.
Enzymatic studies have revealed that rhizobitoxine inhibits
Owens et al. (13) have demonstrated that rhizobitoxine
inhibits the production of ethylene by apple tissues by measuring the incorporation of 14C from 14C-labeled
methionine into ethylene. 1-Aminocyclopropane-1-carboxylase (ACC)
synthase (S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14)
is the rate-limiting enzyme in the biosynthesis of ethylene from
methionine in higher plants (23).
Aminoethoxyvinylglycine (AVG), a structural analogue of
rhizobitoxine, has been used as an inhibitor in the enzymatic studies
of ACC synthase, because AVG is commercially available.
Therefore, rhizobitoxine is expected to be a potent inhibitor of
ACC synthase. The latter may be a target enzyme for rhizobitoxine
during symbiosis between B. elkanii and the host plant, but
rhizobitoxine inhibition of ACC synthase has not yet been verified.
Indeed, ethylene has been suggested to be a component of the signaling
pathway controlling the rhizobial infection of legumes (1, 16,
17).
Assay methods for rhizobitoxine have included the scoring of chlorosis
symptoms in host plants (11), use of an amino acid analyzer
(5), and the inactivation of The objectives of this work are to examine whether rhizobitoxine is a
potent inhibitor of ACC synthase and to develop a new method for
assaying rhizobitoxine by inhibition of the enzyme.
Two enzymatic assays for rhizobitoxine, the ACC synthase assay
described in this work and the previously reported ACC synthase activity was measured by incubating (30°C,
15 min) a reaction mixture containing ACC synthase, 100 µM
S-adenosylmethionine (SAM), 50 µM pyridoxal
phosphate, 100 µg of bovine serum albumin, 125 mM
HEPES-KOH (pH 8.5), and various concentrations of inhibitors such as
rhizobitoxine in a total volume of 0.4 ml. The amount of ACC formed was
determined by the methods of Lizada and Yang (4).
In practice, 50 µl of 0.8 M HEPES-KOH buffer (pH 8.9), 50 µl of 40 µM pyridoxal phosphate, 50 µl of 0.02% bovine serum albumin, 50 µl of sterile water, 50 µl of ACC synthase preparation (9 U), 100 µl of sample solution, and 50 µl of 0.8 mM SAM in 0.001 N sulfate
solution were sequentially pipetted into a blood sampling vacuum tube
(5 ml; TERUMO, Tokyo, Japan) on ice. After incubation (30°C, 15 min),
the reaction was stopped by adding 100 µl of 20 mM CuCl2
and cooling the tube in an ice water bath. Upon the addition of 100 µl of NaOCl reagent (5% sodium hypochlorite solution-saturated NaOH
solution, 1:1 [vol/vol]), to convert ACC into ethylene, a rubber
stopper was immediately placed on the tube. After 5 min of incubation
on ice, the gas phase (1.0 ml) was sampled and injected into a gas
chromatograph (Shimadzu CG-7A) equipped with a Porapack R column
(internal diameter, 2.2 mm; length, 2 m) and a flame ionization
detector in order to determine ethylene concentration.
The Rhizobitoxine strongly inhibited ACC synthase activity (Fig.
2A). Inhibition was detectable in the
presence of 0.02 µM rhizobitoxine, and 93% of the activity was
inhibited at 2 µM rhizobitoxine. AVG was approximately 1.7 times more
effective than rhizobitoxine as an inhibitor of ACC synthase.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
New Assay for Rhizobitoxine Based on Inhibition of
1-Aminocyclopropane-1-Carboxylate Synthase
ABSTRACT
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-cystathionase in the methionine biosynthetic pathway (2, 12,
22).
-cystathionase partially purified from Salmonella typhimurium (12).
-cystathionase assay (18), are schematically shown in Fig.
1. We used ACC synthase bLE-ACS2 purified from the fusion protein of cDNA from a ripe, wounded
tomato pericarp as described previously (20). Rhizobitoxine and dihydrorhizobitoxine were purified from a culture of B. elkanii USDA 94 as described previously (8).
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FIG. 1.
Rhizobitoxine inhibition of enzymes involved in the
pathway of ethylene biosynthesis and two enzymatic assay systems for
rhizobitoxine. The new ACC synthase assay is based on the inhibition of
ACC synthase (tomato bLE-ACS2 expressed in E. coli) by
rhizobitoxine. The -cystathionase assay was reported previously by
Ruan and Peters (18). DNPH, 2,4-dinitrophenylhydrazine.
-cystathionase assay was carried out according to the method of
Ruan and Peters (18), with the exception that the crude enzyme preparation of -cystathionase was isolated from a 1-liter culture of Escherichia coli K-12 grown in M9 medium at
37°C for 20 h.
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FIG. 2.
Inhibition of ACC synthase (bLE-ACS2) (A) and
-cystathionase from E. coli K-12 (B) by
rhizobitoxine (RT), dihydrorhizobitoxine (DRT), and AVG. Enzymatic
activities are expressed as percentages of activities in the absence of
inhibitors, which were 6.28 nmol of ACC produced/h for the ACC synthase
assay (A) and 50 nmol of pyruvate produced/h for the -cystathionase
assay (B). Each striped box indicates the range of rhizobitoxine
concentrations that can be determined by the respective assay system.
Concentrations of rhizobitoxine, AVG, and dihydrorhizobitoxine are
shown in sample solutions (100 µl for the ACC synthase assay and 50 µl for the -cystathionase assay).
Dihydrorhizobitoxine [O-(2-amino-3-hydroxypropyl) homoserine] was thought to be a possible precursor of rhizobitoxine (14, 19) and was always detected in soybean nodules inoculated with a rhizobitoxine-producing B. elkanii strain (5, 6). However, dihydrorhizobitoxine was approximately 100-fold less potent as an inhibitor of ACC synthase than rhizobitoxine (Fig. 2A), indicating that inhibition of ACC synthase depended mainly on rhizobitoxine even when dihydrorhizobitoxine was mixed with rhizobitoxine in a sample solution.
Rhizobitoxine inhibition of -cystathionase isolated from
E. coli K-12 (Fig. 2B) was observed at concentrations
as low as 0.1 µM, and 95% inhibition occurred at about 100 µM
rhizobitoxine. The E. coli K-12 enzyme appeared to be
three times less sensitive to rhizobitoxine than
-cystathionase from S. typhimurium
(18). Considering the goal of developing an assay for
rhizobitoxine, we note that the rhizobitoxine inhibition of the ACC
synthase showed a sharper response than that of the -cystathionase
from E. coli (Fig. 2).
Figure 3 shows double-reciprocal plots of ACC synthase (bLE-ACS2) activity at various SAM concentrations (5 to 50 µM) in the absence or presence of 0.1 µM rhizobitoxine and 0.1 µM AVG. Inhibition of ACC synthase by rhizobitoxine and AVG was competitive with respect to SAM. The Km for SAM was calculated to be 29 µM, which corresponded to the value in previous reports (20, 21). On the other hand, an inhibition constant (Ki) for rhizobitoxine was very low (0.025 µM), indicating that rhizobitoxine is a strong inhibitor of ACC synthase bLE-ACS2 in terms of an inhibition constant. The Ki for AVG was also significantly lower (Ki = 0.019 µM) than those in previous reports (Ki = 0.25 to 2.9 µM) (21), which might be due to differences between dimeric and monomeric molecular forms of ACC synthase (21).
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) or presence of 0.1 µM AVG (
) or 0.1 µM
rhizobitoxine (
) for 15 min at 30°C. From the Lineweaver-Burk
plot, the Km for SAM was 29 µM, and the
Ki values for AVG and rhizobitoxine were 0.019 and 0.025 µM, respectively.
Bradyrhizobium- and soybean-derived -cystathionases were
reportedly less sensitive to rhizobitoxine than -cystathionase from Enterobacteriaceae, including E. coli
(22). For example, -cystathionase from the
rhizobitoxine-sensitive soybean cultivar Forrest and from the
rhizobitoxine-producing B. elkanii strain USDA 94 had
inhibition constants (Ki = 1,048 and 22 µM,
respectively) significantly higher than that of -cystathionase from
E. coli DH52 (Ki = 3 µM).
However, rhizobitoxine strongly inhibited ACC synthase bLE-ACS2
activity with a low Ki (Ki = 0.025 µM) (Fig. 2 and 3). Therefore, it is possible that ACC
synthase is a target enzyme of rhizobitoxine, although the enzyme
source, bLE-ACS2, was not derived from legumes. One could argue that
rhizobitoxine-induced chlorosis and necrosis in leaves, and necrosis in
roots and nodules, of host plants are due to inhibition of ACC synthase
rather than -cystathionase.
Using the ACC synthase assay, we determined the
rhizobitoxine level in a stationary-phase culture (8 days) of
B. elkanii USDA94 growth in Tris-YMRT medium (8).
When the culture supernatant and 10- and 100-fold dilutions (with the
medium) thereof were assayed, the two dilutions yielded the same values
for rhizobitoxine concentrations in the original culture supernatant
(Table 1). This indicated the utility of
the ACC synthase assay for rhizobitoxine.
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Rhizobitoxine is synthesized not only by the symbiotic bacterium B. elkanii (3, 5, 7, 8, 11, 15) but also by the broad-host-range plant pathogen B. andropogonis (9, 10). Moreover, rhizobitoxine-analogous AVG and methoxyvinylglycine have been known to be produced by a Streptomyces sp. and Pseudomonas aeruginosa (19). These compounds are considered to be potent inhibitors of ACC synthase. Indeed, AVG was comparable to rhizobitoxine in terms of ACC synthase inhibition (Fig. 2 and 3). These facts give rise to the interesting idea that both symbiotic and pathogenic bacteria might produce ACC synthase-inhibiting compounds which are structurally and enzymologically similar to rhizobitoxine so as to control the ethylene-induced plant responses that would prevent a successful infection. This new ACC synthase assay will be also useful for surveying these compounds synthesized by plant-associated bacteria as well as for rhizobitoxine determination.
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ACKNOWLEDGMENTS |
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This work was supported in part by a grant from the Joint Research Program of the Institute of Genetic Ecology, Tohoku University (981002).
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Genetic Ecology, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577, Japan. Phone: 81-22-217-5684. Fax: 81-22-263-9845. E-mail: kiwamu{at}ige.tohoku.ac.jp.
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Applied and Environmental Microbiology, February 1999, p. 849-852, Vol. 65, No. 2
0099-2240/99/$04.00+0
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