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Soil Biol.Biochem.Vol. 26, No. 1, pp. 51-63, 1994
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CHARACTERIZATION
OF TWO AROMATIC AMINO ACID
AMINOTRANSFERASES
AND PRODUCTION OF
INDOLEACETIC
ACID IN AZOSPIRILLUM
STRAINS
B. E. BACA, L. SOTO-URZUA, Y. G. XOCHIHUA-CORONAand A. CUERVO-GARCIA
Department0
de Investigaciones Microbiologicas, Instituto de Ciencias, Universidad
Puebla, Apartado Postal 1622, 72000 Puebla Pue, Mexico
Autonoma
de
(Accepted 30 May 1993)
Sunnnary-Experiments
were made to quantify indole-3-acetic acid (IAA) excreted by different wild type
strains of Azospirilhun spp. The microorganisms produce IAA, during the late stationary growth phase
and show a significant increase in IAA production when tryptophan is present in the medium. Under these
conditions with the A. brusilense strain UAP 14, we observed two aromatic amino acid aminotransferases.
Their enzymatic activity and electrophoretic mobility under non-denaturating conditions were characterized using cell-free extracts. Biochemical studies showed that these two enzymes can be distinguished by
their electrophoretic mobilities. These enzymes are constitutively produced and they are not repressed by
tyrosine or by tyrosine plus phenlylalanine.
described in Pseudomonas savastanoi and Agrobacterium tumefaciens. In Azospirillum this process is
poorly understood. Hartman et al. (1983) and Zimmer et al. (1988) identified indole-3-pyruvic acid
(IPyA), indole-3-acetaldehyde, and IAA as the substances that are excreted by A. brasilense Sp7 strain,
suggesting that the transamination pathway is present
in A. brusilense. Ruckdaschell et al. (1988) described
four amino acid aminotransferases from A. lipoferum.
We have found that Azospirillum spp wild type
strains produced IAA and we demonstrated the
presence of two aromatic amino acid aminotransferases associated with the production of IAA in A.
brasilense strain UAP 14.
INTRODUCTION
Soil bacteria of the genus Azospirillum live in association with the root zones of grasses and other plants,
among which are the economically-important
cultivated plants maize, wheat, barley, rye, oat and rice.
Reports indicate that Azospirillum can stimulate the
growth of the host plants (Elmerich, 1984; Okon,
1985). The bacterium may affect plant growth by
nitrogen fixation (Dobereiner and Pedrosa, 1987).
Inoculation with Azospirillum may also improve the
efficiency of applied mineral nutrients by helping the
plant scavenge limiting nutrients (Lin et al., 1983).
These effects have been proposed to be related to the
production of plant growth regulators by Azospirillwn spp (Morgenstern and Okon, 1987). The production of phytohormones by the microorganism
seems to play an essential role in the Azospirillum-plant interaction. It has been shown that bacterial production of the phytohormone
IAA is
involved in interactions between microorganisms and
plants (Morris, 1986). In grasses, legumes and tomato
plants, as tested in Petri dishes or pouch systems,
inoculation with Azospirillum increased root diameter, density, and length of root hairs (Jain and
Patriquin, 1984; Okon et al., 1988). Experiments with
suitable concentrations of IAA produced, changes in
root tip morphology comparable to those produced
by Azospirillum, suggesting the involvement of auxin
produced by Azospirillum on root morphology (Jain
and Patriquin, 1985).
Several pathways have been reported for the conversion of tryptophan to IAA by bacteria (Van
Onckelen ef al., 1986). The most studied pathway
involved conversion via indole-3-acetamide (IAM)
MATERIALSAND METHODS
Bacterial strains and growth conditions
The A. brasilense H18, UAP 14, and A. lipoferum
UAP 06 wild type strains used were obtained from
Professor J. Caballero-Mellado
(Universidad Autonoma de Puebla, Mexico) and A. brasilense AZ
30 INTA was supplied by Professor E. A. RodriguezCaceres. To estimate IAA production Azospirillum
strains were grown in a minimal medium (Jain and
Patriquin, 1985), amended with 0.1% of NH,Cl, or
0.1% of KNO, as a nitrogen source. The medium was
supplemented with 100 pg ml-’ sterilized tryptophan,
tyrosine or tyrosine plus phenylalanine as indicated;
50 ml of medium was inoculated with 100 ~1 ml-’ of
109cfu ml-’ of different Azospirillum strains and
grown in a 250 ml flask, for 48, 72 or 96 h at 32”C,
with shaking at 160rev min-r. To determine the
aromatic amino acid aminotransferase activity, A.
brasilense strain UAP 14 was grown in a 500 ml flask
57
58
B.
E. BACA
~n~ining 250 ml of medium, inoculated with 2 ml of
1.6 x 109cfuml-‘, then samples were collected at
different times as indicated, Bacterial growth was
measured photometrically with a Klett Summerson
(green filter) or by plating and viable cell counting.
IAA determination
Bacterial cultures were harvested by centrifugation
at 10,OOOgfor 10 min at 4°C. The supernatants were
extracted with ethyl-acetate (Tien et al., 1979). The
ethyl-acetate extracts were evaporated to dryness at
39°C under vacuum and residue redissolved in 2 ml of
methanol and the IAA ~n~ntration
was determined
by the Salkowski reaction (Stahl, 1969).
Cell-fee
extracts preparation
Culture of A. brasilense strain UAP 14 were harvested at 48, 72 or 96 h by centrifugation at 10,OOg
for 10min at 4°C. Subsequent procedures were also
made at 4°C. The cells (2 g wet wt) were washed in
10 ml (3 M) NaCl. After being stirred for 1 h the cells
were centrifuged at 10,000 g for 10 min; the supernatant was discarded and the cells were resuspended
in 20 ml of TES buffer pris-NC1 (SOmM, pH 8.5),
EDTA, 50mna; NaCl, 50m~] and lysed with
lysozyme (5~~grnl-‘)
the mixture was stirred for
18 h, then centrifuged at 10,000g for 20 min. The
supematant which is referred to as crude extract, was
brought to 30% saturation with solid ammonium
sulfate. After stirring for JOmin, the sample was
centrifuged at 10,OOOgfor 10min. The supernatant
was brought to 70% saturation with solid ammonium
sulfate. After stirring and centrifugation as before the
pellet was resuspended in 2 ml of a buffer [Tris-HCI
(50 mhr, pH 8.5); dl-dithiothreitol
(DTT), 1 mM,
MgCl,. 7H,O, IOmM, pyridoxal phosphate, lOmhr,
sodium azide, 0.02% and glycerol, lo%]. The enzyme
solution was stored at 4°C.
Enzyme activities assays
Tyrosine aminotransferase
and phenylalanine
aminotransferase
activities were determined
by
measuring the p-hydroxyphenyl
pyruvate @HPP)
and phenyl pyruvate (PP), produced using the procedure of Diamondstone (1966). The reaction temTable 1. IAA production
et al.
perature was 45°C. The reaction was stopped with
(5 M) KOH and the amount of pHPP produced from
tyrosine was estimated by its absorbance at 331 nm.
Production of IPyA was measured following the
method of Truelsen (1972). The reaction temperature
was 45°C. The formation of IPyA was measured at
328 nm.
Polyacrylamide gel electrophoresis and aromatic
amino acid aminotransferase activity staining
Electrophoretic analyses under non-denaturing
conditions was done in a discontinuous system, the
running buffer being T~sglycine (25 rn&&and 192 M,
pH 8.3). The running gel consisted of 10% acrylamide and the stacking gel was made from 4%
acrylamide. All procedures were done at 4°C. Protein
(100 pg) extract was loaded in each well and tetrazolium dye reduction was used to stain aromatic
amino acid aminotransferase
activity. Gels were
stained at room temperature in the dark. The staining
solution contained: Tris-HCl buffer (0.1 M, pH 8.6);
tryptophan or another L-amino acid, 5.5 mM; pyridoxal phosphate, 0.2 mM; phenazine methosulfate,
98 F”M; 2-oxoglutaric acid, 12.5 mM; and nitroblue
tetrazolium, 0.6 mh9.
Protein ~terminations
Protein was determined by the method of Lowry
et al. (1951) using bovine serum albumin as a standard.
RESULTS
Determination of IAA in the culture medium of
Azospirillum
We studied IAA accumulation in the culture
medium produced by Azospiri~lum wild type strains,
to understand the influence of nutrient conditions on
the synthesis of ph~oho~one
by these microorganisms. IAA production was influenced by the nutrient
content of the medium and by the incubation time as
shown in Table 1. IAA produced by Azospirillum was
released continuously, especially when the bacteria
were grown in medium supplemented by the amino
by AzospiriNum spp wild type strains
pg MA ml-’
of the culture medium*
KNO,tb
NH&it’
KNO,-Trpd
NH&l-Trp’
48
72
96
48
72
96
48
72
96
48
72
9s
38
54
39
25.5
25
45
30
64
strains
A. iipofenun
UAP 06
A. bradense
AZ 30 INTA
0.02
0.07
0.15
ND$
0.15
1.1
0.27
313
33.1
7
0.04
0.02
0.04
ND
0.2
0.88
0.04
5.3
H18
UAP 14
0.03
0.16
0.05
0.4
0.27
0.8
ND
ND
0.15
0.1
0.61
0.13
4.3
15
27
48
29.7
33.5
63.5
34
23.5
5.5
lIAA concentration as measured with the Salkowski reaction. (Data represent the means of three experimental
determinations+ standard errors: ‘It5 x IO-’ to +0.015; b+0.04S to J20.2; ‘f0.56 to +4.3; df 1.2 to 6.7.)
t’fhe medium contained either 260 mg N I-’ as NH,CI or 140 mg N I-’ as KNO,.
fND is not determined.
$Hours of incubation.
Aromatic aminotransferase
in Azospirillum strains
59
(l.OOOE+ 08)
250
225
(LOOOE + 07)
(l.OOOE+ 06)
(l.OOOE+ 05)
15
(l.OOOE+ 04)
50
25
(l.OOOE+ 03)
0
5
10
15
20
25
30
35
40
0
50
45
Hours
Fig. 1. Growth curves of Azospiriffum spp strains were grown on Jain and Patriquin medium supplemented
with 100pg ml-’ L-tryptophan. Log number of viable cells ml-‘: HI8 (*-*); AZ 30 INTA (O-0);
14 (O-0) A. lipoferum UAP 06 ( + - + ). Dotted lines absorbance as Klett units.
IAA excreted in medium containing
KNO, was about 2-10 fold (0.139-1.15 pgml-‘) as
compared to IAA produced in medium supplemented
with NH,Cl, in A. lipoferum UAP 06, A. brasilense
AZ 30 INTA, and in A. brasilense H 18 strains, but
this effect was not observed in A. brasilense strain
UAP 14. A significant increase in IAA production
was observed when the medium was supplemented
with tryptophan, 5&100 fold higher (30-64 pg ml-‘)
as compared to IAA produced in KNOj or NH,Cl
supplemented medium. With the amino acid precursor added at the beginning, it was also observed that
IAA liberation was stimulated within 48 h. Azospirillum wild-type strains grow at similar rates (Fig.1).
Therefore, differences in IAA production are probably not due to non-normal growth (Table 1; Fig. 1).
acid precursor.
Kinetics of ZAA production and spectfk activity of
aromatic amino acid aminotransferases (AAT)from A.
brasilense strain UAP 14
Previous data have suggested that IAA biosynthesis in Azospirillum could occur through an IPyA
intermediate derived from tryptophan by an aminotransferase activity (Hartmann et al., 1983; Zimmer,
et al., 1988). To examine this biosynthetic pathway
we investigated IAA production from A. brasilense
strain UAP 14 grown at four different growth conditions for 72 h [Fig. 2 (A)]. The IAA excreted by A.
brasilense UAP 14 varied from 0.05 to 2.6 pg ml-‘,
when cultured in a medium supplemented with tyrosine, phenylalanine plus tyrosine or without amino
acid. Under such growth conditions IAA production
was almost the same, when compared on the basis of
biomass as determined as wet weight [Fig. 2 (A) and
(C)l. However, IAA production increased from 1.6 to
UAP
50pgml-’
with the addition of tryptophan to the
medium.
Aminotransferase
activities were determined in
crude extracts and in precipitated extracts (30-70%
of ammonium sulfate saturation) [Fig. 2 (C)l. Tryptophan was used as an aromatic amino acid donor, and
2-oxoglutaric acid as the amino group acceptor in the
reaction mixture. Specific activities in extracts from
cultures without amino acids ranged from 16 to
22 nmol of IPyA mgg’ protein ml-‘. However, in the
presence of amino acids added to the culture medium,
the specific activities ranged from 27 to 38 nmoles of
IPyA mg-’ protein ml-‘.
Polyacrylamide gel electrophoresis
matic amino acid aminotransferase
(PAGE)
of aro-
When protein extracts from cultures grown under
four different nutrient conditions were subjected to
electrophoresis on non-denaturing
gels and were
stained for AAT activity two bands were detected
(Fig. 3) both convert tryptophan to IPyA in the
presence of 2-oxoglutaric acid. Omitting either
tryptophan or 2-oxoglutaric acid from the mixture
reaction completely eliminated the two bands. Substitution for tryptophan by either tyrosine or phenylalanine [Fig. 4 (B)] revealed the same two activity
bands. Only the large band showed aminotransferase
activity when histidine was used as amino donor
[Fig. 4 (C)l. however, the two AAT isoenzymes did
not show aminotransferase
activity when valine,
isoleucine or aspartate (Fig. 5) were used as donor
substrates. Protein extracts derived from cultures
grown with tryptophan, tyrosine or tyrosine plus
phenylalanine tested for AAT activity showed two
isoenzymes pattern (Figs 3 and 4). This result indi-
B.
60
E. BACAef a/.
cates that both of these two AATs are not regulated
by the aromatic amino acid added to the medium.
DISCUSSION
We have studied the excretion into culture medium
of IAA by Azospirilium spp, in order to know the
influence of nutrient conditions on the synthesis of
phytohormones. The data obtained showed that the
presence of KNO, has little effect on IAA production
as compared with NH,CI. Tien et al. (1979), also
reported that combined N has little effect on the
production of IAA by A. bradense.
Moreover
wild-type strains excreted IAA in
stationary phase culture. This concentration of IAA
(20-88 ng ml-‘) is within a physiologicaIly active
range that may affect plants roots (Plazinski and
Roife, 985). Also this production is further stimulated
by the addition of tryptophan to the medium. Both
species produce large quantities of IAA in the presence of an amino acid precursor. These data agree
well with those communicated by others for the A.
brasilense Sp7 strain (Tien et al., 1979; Jain and
Patriquin, 1985). In our tests A. brasilense strain IJAP
14 was the best IAA producer; it is interesting to note
that this bacte~um was isolated from rhizosphere soil
Azospiri~lum
IAA production from A. brasilense UAP 14 strain
60
55
(A)
F
0
12
24
36
48
60
72
Hours
Biomass wet weight A. h&lease
lor
0
UAP 14 strain
Specific activity AATs A. brasiiense UAP 14 strain
W
12
24
36
Hours
48
60
72
0
12
24
36
48
60
Hours
Fig. 2. (A) IAA production of A. brdense strain.UAP 14grown in a medium supplemented with tyrosine
(*--‘); tryptophan (O-0); tyrosine plus phenylalanine ( + - + ); or without amino acid O-J-_O). (B)
Biomass as wet weight strain UAP 14 grown under same nutrient conditions. (C) Total specific activity
of aromatic amino acid aminotransferase, one unit catalyzes the formation of I PMof IPyA mg-’ protein
ml-‘, media conditions tyrosine (*-*); tryptophan (C-0);
tyrosine plus phenylalanine ( + - + ); or
without amino acid (O-c]).
72
Aromatic aminotransferase
in Azospirillum strains
61
AATt
AATZ
Fig. 3. Non-denaturing PAGE of AATs from cell-free extracts of A. brusilense strain UAP 14. Nutrient
conditions: cultures grown without amino acids (wells 1 and 2 ); with tryptophan (wells 3 and 4); tyrosine
(wells 5 and 6); tyrosine plus phenylalanine (wells 7 and 8). Wells 1, 3, 5 and 7 were crude extracts; wells
2,4,6 and 8 were 30-70% ammonium sulfate fractions. Gel stained with tryptophan as amino acid donor.
from a cactaceous plant (Mascarua-Esparza
et al.,
1988).
Strain UAP 14 was compared
in its ability to
produce IAA in four different nutrient conditions.
Our results show that this strain released small
amounts of IAA in minimal medium, and minimal
medium supplemented
with tyrosine and phenylalanine. In E. coli, the aromatic amino acid aminotransferase
is repressed by tyrosine (Gelfand and
Steinberg, 1977; Powell and Morrison, 1978). In view
of the results obtained with this bacterium, the amino
acid tyrosine was chosen because it could presumably
inhibit tyrosine and phenylalanine
biosynthesis as is
done by E. cd, and, therefore, diverts chorismic acid
A
AAT,
-“,
AAT,
-
m
1
2
3
4
to the endogenous synthesis of tryptophan.
However
the amounts of IAA production were similar to those
obtained in the absence of either tyrosine or tyrosine
plus phenylalanine.
On the other hand, large amounts
of IAA were present in the culture medium when the
bacterium
UAP 14 was grown in the presence of
tryptophan.
To know if aromatic amino acid aminotransferase (AAT) could be involved in biosynthesis
of IAA we studied and identified aminotransferase
activity that reacted with tryptophan in crude extracts
and the ammonium sulfate fractions. The enzymatic
activity was little affected by the type of aromatic
amino acid added to culture medium. Indeed a slight
increase in the specific activity was observed when
5
6
7
8
9
10
11
12
.’ ,.
Fig. 4. Non-denaturing PAGE of AATs from cell-free extracts of A. brasdense strain UAP 14. Nutrient
conditions: cultures grown with tyrosine. Gel A, wells 1 and 2; cells harvested at 18 h; wells 3 and 4; 24 h;
wells 5 and 6; 36 h; wells 7 and 8; 48 h; wells 9 and 10; 72 h. Gel stained with tryptophan as amino acid
donor; wells 11 and 12 same as 9 and 10 but stained without amino acid donor as a negative control.
Gel B, cells harvested at 18, 24, 36, 48, or 72 h respectively and the gel stained with tyrosine as amino
acid donor. Gel C, the cell-free extracts as same as gel B but stained with histidine as amino acid donor.
62
B. E. BACA etal.
Fig. 5. Non-denaturing PAGE of AATs from extracts of Azospiriilum brasilense strain UAP 14. The
nutrient conditions are the same as noted in Fig. 4. Cells harvested at 24,36,48 or 72 h respectively. Gel
A, wells: 1, 24h; 2, 36 h; 3, 48 h, and 4, 72 h. Gel stained with valine as amino acid donor. Wells 5 and
6 the amino acid donor was omitted as a negative control. Wells 7 and 8 the amino acid donor was
tryptophan. Gel B, stained with aspartic acid as amino acid donor; Gel C, stained with isoleucine as
amino donor.
tryptophan, tyrosine or tyrosine plus phenylalanine
were added to the medium as compared with specific
activity obtained in a minimal medium without
amino acids. The addition of tryptophan to the
medium (which promotes a dramatic increase in IAA
synthesis of A. brasilense UAP 14 strain) did not
show any significant effect on the aminotransferase
activity.
To identify the aminotransferase
enzymes that
react with tryptophan, crude extracts and ammonium
sulfate fractions were run on PAGE under non-denatured conditions. Our data clearly demonstrated
the presence of two aromatic amino acid aminotransferases (AATl, and AAT2). Only one of these two
isoenzymes showed activity with histidine as substrate [Fig. 4 (C)J and might be involved in the
catabolism of this amino acid. Both enzymes could be
involved in IAA production. Neither of these enzymes show activity with branched chain amino acid
or as an aspartate aminot~nsferase (Fig. 5). The A.
brasilense A‘ATl enzyme unlike the E. coli enzyme
(Powell and’Morrison,
1978), showed catalytic activity when histidine replaced tyrosine, phenylalanine
or tryptophan as the amino acid donor. Similar
activity has been detected for some AATs from R.
leguminosarum
(Perez-Galdona et al., 1989). We detected two other aminotransferase activities that used
aspartate, valine and isoleucine as amino acid donors
(Fig. 5, top arrows).
Our work showed that unlike E. coli (Gelfand and
Steinberg, 1977) and A. tumefaciens (Liu and Kado,
1979), AAT activity in A. brasilense strain UAP 14 is
not affected by the presence of tyrosine in the growth
medium. Indeed, data reported in Figs 3 and 4 clearly
demonstrate that AATl and AAT activities are not
repressed by tyrosine. These results suggested that
both enzymes are constitutive. However, in cells
grown with tryptophan, tyrosine, or tyrosine plus
phenylalanine, total AAT activity had doubled. The
inability of the aromatic amino acids (phenylalanine
and tyrosine) to function as regulatory effecters for
IAA biosynthesis suggests that IAA synthesis in A.
brasilense may be not subjected to coordinated regulation of phenylalanine and tyrosine synthesis. These
results suggest that IAA biosynthesis could be regulated by the tryptophan pool in cells.
Acknowledgements-We
thank Dr Miguel Lara F., Departamento de Biologia Molecular de Plantas, Institute de
Biot~olo~a-UNAM,
for helpful suggestions and critical
reading of the manuscript. This work was partially supported by Subdireccion de Investigation Cientifica y Superacion Academica de la SEP.
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