JOURNAL OF BACTERIOLOGY, Oct. 1995, p. 5762–5766 Vol. 177, No.

20
0021-9193/95/$04.0010
Copyright q 1995, American Society for Microbiology

Catabolism of Indole-3-Acetic Acid and 4- and 5-Chloroindole-

3-Acetic Acid in Bradyrhizobium japonicum
JOHN BECK JENSEN,1 HELGE EGSGAARD,2 HARRY VAN ONCKELEN,3
1
AND BJARNE U. JOCHIMSEN *

Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C,1 and Department of Combustion
Research, Risø National Laboratory, DK-4000 Roskilde,2 Denmark, and Department of Biology,
University of Antwerp, B-2610 Antwerp, Belgium3
Received 16 March 1995/Accepted 6 August 1995

Some strains of Bradyrhizobium japonicum have the ability to catabolize indole-3-acetic acid. Indoleacetic

Downloaded from http://jb.asm.org/ on March 11, 2021 by guest

acid (IAA), 4-chloro-IAA (4-Cl-IAA), and 5-Cl-IAA were metabolized to different extents by strains 61A24 and
110. Metabolites were isolated and analyzed by high-performance liquid chromatography and conventional
mass spectrometry (MS) methods, including MS-mass spectroscopy, UV spectroscopy, and high-performance
liquid chromatography-MS. The identified products indicate a novel metabolic pathway in which IAA is
metabolized via dioxindole-3-acetic acid, dioxindole, isatin, and 2-aminophenyl glyoxylic acid (isatinic acid) to
anthranilic acid, which is further metabolized. Degradation of 4-Cl-IAA apparently stops at the 4-Cl-dioxindole
step in contrast to 5-Cl-IAA which is metabolized to 5-Cl-anthranilic acid.

Bradyrhizobium japonicum reduces atmospheric nitrogen to
ammonia when the bacterium is located in the root nodules of
soybean (Glycine max) plants. The formation of root nodules
results from a complex series of interactions between the bac-
terium and the plant and requires the growth and differentia-
tion of both partners. Indoleacetic acid (IAA) has long been
assumed to play a role in one or more aspects of nodule growth
and development (11), although no specific role for IAA in
nodule formation or development has been suggested.
Bradyrhizobia have the enzymatic capacity to produce IAA
in culture (7, 8), and it has been demonstrated that nodules
induced by an IAA-overproducing B. japonicum strain contain
much greater amounts of IAA than nodules induced by the
parental strain (5, 6), thus indicating that the bacterium can
affect nodule IAA levels.
In addition to being capable of IAA synthesis, some strains
of B. japonicum are able to catabolize IAA. It was hypothe-
sized that the first enzyme catalyzes an oxygen-consuming
opening of the indole structure. A degradation pathway with
anthranilic acid as the eventual degradation product was pro-
posed (2). In this paper we report the analysis of the degrada-
tion products from IAA, 4-chloro-IAA (4-Cl-IAA), and 5-Cl-
IAA, determined by conventional mass spectrometry (MS) and
by high-performance liquid chromatography (HPLC)-thermo-
spray MS. Using these methods, we have identified the inter-
mediates in IAA catabolism in order to compare the degrada-
tion products with those of the degradation pathway postulated
for B. japonicum (2).
MATERIALS AND METHODS
Strains and media. B. japonicum strain 110 (4), a derivative of USDA110, and
strain 61A24 (14), showing different kinetics of IAA degradation, were used in
this study. The strains were grown in yeast broth medium (1) on a rotary shaker
with gentle shaking (100 rpm) at 288C until turbidity reached an optical density
at 450 nm of 0.35 to 0.45 (3 to 5 days).
Preparing cultures for in vivo experiments. IAA was added to a final concen-
tration of 0.2 mM to a stationary liquid culture and incubated overnight to induce
IAA-catabolizing enzymes. The following day, the culture was centrifuged at
20,000 3 g for 10 min at 48C. The pellet was washed twice in a 20 mM potassium
phosphate buffer, pH 7, and resuspended in the same buffer at 1/10 the initial
volume.
Metabolic studies. When the cultures were prepared for in vivo experiments,
IAA, 5-Cl-IAA, or 4-Cl-IAA (3) was added to the cultures at a final concentra-
tion of 0.2 mM. At given times, samples of the culture supernatants were cen-
trifuged through 0.22-mm-pore-size Durapore filters (Millipore) and analyzed
immediately by the methods described below.
Reverse-phase HPLC. HPLC analysis was performed with a 200-mm Waters
mBondapak C18 column (particle size, 5 mm) and a model SP8800 ternary pump.
Samples (50 ml) of the supernatants were injected onto the column. Elution was
monitored by a model 757 Applied Biosystems detector, set at 260 nm, coupled
to a model SP4290 integrator. The mobile phase consisted of 40% methanol–
0.5% acetic acid, pH 2.9, at a flow rate of 1 ml/min.
Reverse-phase HPLC-thermospray MS. Chromatography was performed with
an Alltech C18 column (100 by 4 mm; particle size, 3 mm) and an HPLC pump
(model 600 MS; Waters). Samples were injected onto the column with a Waters
700 Satellite WISP injector, and elution was monitored by a Waters model 486
variable wavelength absorbance detector, set at 260 nm. The flow rate was 0.8
ml/min. The mobile phases used for the metabolic studies of IAA, 5-Cl-IAA, and
4-Cl-IAA were 5% methanol with 95% 0.1 M ammonium acetate–0.1 M acetic
acid, 10% methanol with 90% 0.1 M ammonium acetate–0.1 M acetic acid, and
35% methanol with 65% 0.1 M ammonium acetate–0.1 M acetic acid, respec-
tively. The eluent was vaporized via a thermospray interface (the vaporizer tip
temperature was between 210 and 2308C and the source temperature was 2508C)
and then was subjected to analysis in a VG TRIO2000 quadrupole mass spec-
trometer.
Metabolite purification. Culture supernatants were acidified by the addition of
H2SO4 to a final concentration of 0.1 M and were partitioned against diethyl
ether. In order to extract the compounds represented by peaks A3 (Fig. 1a) and
B2 (Fig. 1b), the culture supernatants were partitioned against equal volumes of
diethyl ether. To perform differential ether extraction of the compound repre-
sented by peak A1 (Fig. 1a), the supernatant was evaporated to 10 ml and a
sequential series of partitionings was carried out. The partitioning was per-
formed four times against 2 ml of diethyl ether, the products of which were
discarded, and then four times against 20 ml of diethyl ether, the product of which
was collected. The diethyl ether fractions were evaporated to dryness and analyzed
by UV spectroscopy and direct probe (70 eV) electron impact ionization MS.
UV spectroscopy. For UV spectroscopy, the purified metabolites were dis-
solved in methanol and the spectra were recorded on a Shimadzu UV260 or
UV2101PC spectrophotometer.
MS. MS analyses were carried out with a Varian MAT CH 5D double-focusing
spectrometer equipped with a combined EI/FI/FD ion source. Collision activa-
tion was carried out by introducing helium as target gas in the second field-free
region. Samples were introduced via a direct inlet system with Al crucibles.

RESULTS
* Corresponding author. Mailing address: Department of Molecular
Biology, University of Aarhus, DK-8000 Aarhus C, Denmark. Phone: Identification of intermediates of 5-Cl-IAA degradation.
45-89422659. Fax: 45-86196500. Following the addition of 5-Cl-IAA, the 61A24 culture super-

5762
VOL. 177, 1995
FIG. 1. Elution profiles of culture supernatants. (a) Strain 61A24. The peaks
eluting between 2 and 4 min originate from the phosphate buffer. Peak A4
represents 5-Cl-IAA. Peak A3 has an RT identical to that of 5-Cl-AA. (b) Strain
110. Peak B4 represents 4-Cl-IAA.
natants were analyzed by HPLC. The elution profile of a sam-
ple taken after 180 min is shown in Fig. 1a. Four significant
peaks appeared and were numbered A1 to A4. Peak A4 has a
retention time (RT) and a UV spectrum identical to those of
the added 5-Cl-IAA. Upon prolonged (24 h) incubation, A1
and A2 were metabolized into A3. Compound A3 was identi-
fied as 5-Cl-anthranilic acid (5-Cl-AA) since its UV spectrum,
B. JAPONICUM IAA DEGRADATION 5763
Downloaded from http://jb.asm.org/ on March 11, 2021 by guest
FIG. 2. (a) Electron impact MS of 5-Cl-IAA degradation product A1 puri-
fied from culture supernatant of strain 61A24. (b) MS-mass spectroscopy spec-
trum of compound A1. (c) Spectrum of the 4-Cl-IAA degradation product B2
purified from culture supernatant of strain 110.
RT, and mass spectra were identical to those of authentic
5-Cl-AA (results not shown). After 240 min, the culture super-
natant was extracted by differential ether extraction. The final
ether phase, containing chromatographically 99% pure A1,
was subjected to direct probe MS. The result is shown in Fig.
2a. A compound with an apparent molecular ion m/z 241 is
observed together with m/z 243 in the characteristic 3:1 ratio of
chlorine. The MS-mass spectroscopy spectrum of the molecu-
lar ion m/z 241 (Fig. 2b) revealed that the fragments with m/z
182 and 195 are derived from compound A1. The m/z 241
represents a compound with an increase of 32 atomic mass
units (AMU), equivalent to the atomic mass of two oxygen
atoms, compared with 5-Cl-IAA, and the m/z 182 could rep-
resent the loss of a z CH2COOH radical. The UV spectrum of
compound A1 (not shown) revealed a major A259 and a minor
A298, which resembles the UV spectrum of dioxindole-3-acetic
acid (12).
In order to identify peak A2 (present in much smaller
amounts than A1), HPLC-MS was used. A sample taken from
the culture of strain 110 revealed two chromatographic peaks
with m/z 199 and 242, respectively. The m/z 242 (Fig. 3a) is
obviously the [MH]1 equivalent to the m/z 241 found for peak
A1 analyzed by direct probe MS. The peak equivalent to A2
5764 JENSEN ET AL.
FIG. 3. HPLC-MS of 5-Cl-IAA degradation products. Shown are peaks com-
parable to those of A1 (a) and A2 (b) (Fig. 1a). Also shown are the spectra of
IAA-derived compounds eluting at 3.5 min (c) and at 3.9 min (d).
with a m/z of 199 (Fig. 3b) is more difficult to account for, since
the final metabolite of 5-Cl-IAA was found to be 5-Cl-AA.
Thus, the intermediates are assumed to contain one nitrogen
atom. The [MH]1 of such compounds should always add up to
an even number. An explanation of the m/z 199 may be that the
compound represents an NH41 adduct and hence contains two
nitrogen atoms. Peak A2 may therefore represent Cl-dioxin-
dole which, during analysis, is oxidized into isatin and then
undergoes conversion into an NH41 adduct.
Identification of intermediates of 4-Cl-IAA degradation. Af-
ter the addition of 4-Cl-IAA to a culture of strain 110, super-
natants were analyzed by HPLC. The elution profile at 60 min
is shown in Fig. 1b. Three significant peaks appear and are
numbered B1, B2, and B4. Peak B4 has an RT and a UV
spectrum identical to those of the added 4-Cl-IAA. Upon pro-
longed (12 h) incubation, B1 was metabolized into B2. The
supernatant was extracted with ether. The ether phase contain-
ing chromatographically 99% pure B2 was subjected to MS
J. BACTERIOL.
Downloaded from http://jb.asm.org/ on March 11, 2021 by guest
FIG. 4. Conversion of isatin in a suspension culture of strain 110 induced
with IAA. Supernatants were analyzed by HPLC as described in the text. ç,
isatin; }, peak 1 (RT, 3.6 min); å, peak 2 (RT, 3.9 min).
(Fig. 2c). A compound with m/z 183 is observed. This repre-
sents a decrease of 58 AMU compared with the m/z 241 of the
compound A1 in 5-Cl-IAA degradation. A decrease of 58
AMU correlates with the loss of the acetic acid side chain. The
UV spectrum of the B2 compound revealed a major A247 and
a minor A297 (not shown), with strong resemblance to the
spectrum of the compound represented by peak A1, indicative
of B2 being dioxindole. HPLC-MS of a culture supernatant
from strain 110 revealed two chromatographic peaks, both with
mass spectra (not shown) equivalent to those shown in Fig. 3b,
indicating the formation of chloro-isatin NH41 adducts, as
occurs with 5-Cl-IAA degradation.
Identification of intermediates of IAA degradation. In order
to identify intermediates of IAA degradation, HPLC-MS was
used, since the intermediates due to an extensive metabolism
are present in only small amounts. Culture supernatants of
strain 110 revealed two chromatographic peaks with m/z 165
and 208 as characteristic ions. The mass spectra are shown in
Fig. 3c and d. The apparent [MH]1 (m/z 208) is an increase of
32 AMU compared with that of IAA. The compound with an
m/z 165 is equivalent to the m/z observed in 5-Cl-IAA degra-
dation (without chlorine). Thus, the m/z 165 is thought to be an
NH41 adduct of a compound with a molecular weight of 147.
The oxidation of dioxindole, the assumed compound in the
second peak which eluted from the HPLC column when IAA
degradation products were examined, could give rise to isatin
having a molecular weight of 147. Subjecting authentic isatin to
HPLC-MS revealed a chromatographic peak with the expected
m/z 148 [MH]1 accompanied by a predominant m/z 165, in-
dicating the formation of an ammonium adduct. Addition of
isatin to a culture of strain 110 gave rise to two major com-
pounds (Fig. 4), both having RTs similar to those of the peaks
observed during IAA degradation. The compound with an RT
of 3.6 min has a UV spectrum identical to the recorded spec-
trum of a culture supernatant of strain 110 exposed to multiple
additions of IAA (data not shown) and identical to that of
isatinic acid (2-aminophenyl glyoxylic acid) (13). Repeated
chromatography of the collected compound (RT, 3.6 min)
results in anthranilic acid as seen earlier with a metabolite
derived from IAA degradation (2). The other compound (RT,
3.9 min) has a UV spectrum similar to that of dioxindole-3-
VOL. 177, 1995 B. JAPONICUM IAA DEGRADATION 5765

acetic acid and might thus represent a reduction of isatin to

dioxindole.

DISCUSSION
Our experiments have revealed that B. japonicum 110 and
61A24 are able to degrade 4- and 5-Cl-IAA to different ex-
tents. The degradation products are all more polar than the
chloro-IAA. During the degradation process, the characteristic
UV spectrum of the indole structure disappears. When IAA is
degraded by B. japonicum, by a process that is known to re-
quire oxygen, the absorption from the indole structure disap-
pears. An oxygen-consuming opening of the indole structure
giving rise to 2-formaminobenzoylacetic acid was proposed by
Egebo et al. (2). Another possibility may be an oxidation of IAA
leading to dioxindoleacetic acid, which also fulfills the observed

Downloaded from http://jb.asm.org/ on March 11, 2021 by guest

increase in mass of 32 AMU (Fig. 2a and 3d) compared with
the mass of 5-Cl-IAA and IAA. A methyl ester of dioxin-
doleacetic acid has been extracted from rice bran, and hence
dioxindoleacetic acid has been suggested as an intermediate in
the degradation of IAA to 2,6-dihydroxyquinolone-4-acid (9).
The direct probe MS experiments with peak A1 (Fig. 2a) did
not support the presence of 2-formaminobenzoylacetic acid,
since a m/z 212 (241-formyl group) peak of aldehydes was not
seen. The MS-mass spectroscopy experiment (Fig. 2b) revealed
[M2H2O]1 z , [M2H2O2CO]1 z , and [M2CH2COOH]1 z
ions, supporting the hypothesis that the first degradation prod-
uct is (chloro-)dioxindoleactic acid.
The degradation product represented by peak B2, purified
from 4-Cl-IAA metabolism and giving an m/z 183, corresponds
to the loss of the acetic acid side chain from dioxindole-3-acetic
acid, thus giving dioxindole. The UV spectra of the purified
degradation products A1 from 5-Cl-IAA catabolism and B2
from 4-Cl-IAA catabolism are nearly identical, indicating a
structural relationship. Dioxindole-3-acetic acid shows two ab-
sorption bands, a primary band at 250 to 260 nm and a sec-
ondary band between 290 and 300 nm (12), resembling those of
the chloro compounds. Upon analyzing the 5-Cl-IAA degra-
dation by HPLC-MS, no peak corresponding to 5-Cl-dioxin-
dole was found, which may be because of a spontaneous oxi-
dation of the compound to Cl isatin when subjected to HPLC-
MS. Instead, a significant peak corresponding to an NH41 FIG. 5. IAA degradation in B. japonicum, based on the identified interme-
adduct of Cl-isatin is seen. HPLC-MS of authentic isatin gave diates.
two significant signals, the expected m/z 148 [MH]1 and an
m/z 165 [M1NH4]1. Also, when analyzing 4-Cl-IAA degrada-
tion by HPLC-MS the NH41 adduct of Cl-isatin was found hydrolyzed via 2-aminophenyl glyoxylic acid (isatinic acid) to
(data not shown). form anthranilic acid. Anthranilic acid is then subject to fur-
Dissolving isatin in phosphate buffer results in hydrolysis to ther metabolism (not dealt with here), while 5-Cl-AA seems to
2-aminophenyl glyoxylic acid (isatinic acid) (data not shown). be resistant to further degradation. The degradation of 4-Cl-
This conversion occurred much more quickly in the presence IAA seems to stop at 4-Cl-dioxindole. Future work will be
of bacteria. When collected from the HPLC mobile phase, the aimed at establishing the individual enzymatic steps in vitro.
isatinic acid spontaneously disintegrates into anthranilic acid. ACKNOWLEDGMENTS
In a study on IAA catabolism by a Klebsiella strain, isatinic acid We thank K. Engvild for providing the chloro-IAAs, E. L. Esmans
was identified as a metabolite. Under acid conditions this me- for critical reading of the manuscript, and W. V. Dongen and H.
tabolite turned into anthranilic acid (13). As seen in Fig. 4, two Hjorth for excellent technical assistance.
compounds arise after isatin addition. One peak identified as This work was supported by grants from the Danish Agricultural and
2-aminophenyl glyoxylic acid (isatinic acid) is visible together Veterinary Research Council and the NOVO Foundation.
with a compound having the same UV spectrum as dioxindole,
REFERENCES
indicating a reduction of isatin to dioxindole, similar to that
1. Dalton, H. 1980. The cultivation of diazotropic organisms, p. 31. In F. J.
catalyzed by a carbonyl reductase (10) and found in the fungus Bergersen (ed.), Methods for evaluating biological nitrogen fixation. Wiley
Mucor ambiguus. Interscience, New York.
On the basis of the above-mentioned results, we propose the 2. Egebo, L. A., S. V. S. Nielsen, and B. Jochimsen. 1991. Oxygen-dependent
degradation pathway for IAA shown in Fig. 5. The degradation catabolism of indole-3-acetic acid in Bradyrhizobium japonicum. J. Bacteriol.
173:4897–4901.
begins with an oxidation of IAA to dioxindole-3-acetic acid. 3. Engvild, K. C. 1977. Preparation of chlorinated 3-indolylacetic acids. Acta
The acetic acid side chain is eliminated, giving rise to dioxin- Chem. Scand. Ser. B Org. Chem. 31:338–339.
dole. Dioxindole is further oxidized to isatin, which is then 4. Hahn, M., and H. Hennecke. 1984. Localized mutagenesis in Rhizobium
5766 JENSEN ET AL.
japonicum. Mol. Gen. Genet. 193:46–53.
5. Hunter, W. J. 1987. Influence of 5-methyltryptophan-resistant Bradyrhizo-
bium japonicum on soybean root nodule indole-3-acetic acid content. Appl.
Environ. Microbiol. 53:1051–1055.
6. Hunter, W. J. 1989. Indole-3-acetic acid production by bacteroids from
soybean root nodules. Physiol. Plant. 76:31–36.
7. Hutzinger, O., and T. Kosuge. 1968. Microbial synthesis and degradation of
indole-3-acetic acid. III. The isolation and characterization of indole-3-
acetyl-e-L-lysine. Biochemistry 7:601–605.
8. Kaneshiro, T., M. E. Slodki, and R. D. Plattner. 1983. Tryptophan catabo-
lism to indolepyruvic and indoleacetic acid by Rhizobium japonicum L-259
mutants. Curr. Microbiol. 8:301–306.
9. Kinashi, H., Y. Suzuki, S. Takeuchi, and A. Kawarada. 1976. Possible met-
abolic intermediates from IAA to b-acid in rice bran. Agric. Biol. Chem.
40:2465–2470.
J. BACTERIOL.
10. Shimizu, S., S. Hattori, H. Hata, and H. Yamada. 1988. A novel fungal
enzyme, NADPH-dependent carbonyl reductase, showing high specificity to
conjugated polyketones. Eur. J. Biochem. 174:37–44.
11. Thimann, K. V. 1936. On the physiology of the formation of nodules on
legume roots. Proc. Natl. Acad. Sci. USA 22:511–514.
12. Tsurumi, S., and S. Wada. 1985. Identification of 3-(O-b-glucosyl)-2-in-
dolone-3-acetylaspartic acid as a new indole-3-acetic acid metabolite in Vicia
seedlings. Plant Physiol. (Bethesda) 79:667–671.
13. Uchino, H. 1969. Studies on the metabolism of indoleacetic acid. I. Isolation
and characterization of isatinic acid as an intermediate in the catabolism by
a Klebsiella strain. Kumamoto Medical J. 22:160–167.
14. Werner, D., E. Mörshel, R. Stripf, and B. Winchenbach. 1980. Development
of nodules of Glycine max infected with an ineffective strain of Rhizobium
japonicum. Planta 147:320–329.
Downloaded from http://jb.asm.org/ on March 11, 2021 by guest