Download Molecular and General Genetics.

Mol Gen Genet (2000) 264: 521±530
DOI 10.1007/s004380000340
O R I GI N A L P A P E R
R. CarrenÄo-Lopez á N. Campos-Reales á C. Elmerich
B. E. Baca
Physiological evidence for differently regulated tryptophan-dependent
pathways for indole-3-acetic acid synthesis in Azospirillum brasilense
Received: 13 January 2000 / Accepted: 20 July 2000 / Published online: 8 September 2000
Ó Springer-Verlag 2000
Abstract Disruption of ipdC , a gene involved in indole3-acetic acid (IAA) production by the indole pyruvate
pathway in Azospirillum brasilense Sp7, resulted in a
mutant strain that was not impaired in IAA production
with lactate or pyruvate as the carbon source. A tryptophan auxotroph that is unable to convert indole to
tryptophan produced IAA if tryptophan was present but
did not synthesise IAA from indole. Similar results were
obtained for a mutant strain with additional mutations
in the genes ipdC and trpD . This suggests the existence
of an alternative Trp-dependent route for IAA synthesis.
On gluconate as a carbon source, IAA production by the
ipdC mutant was inhibited, suggesting that the alternative route is regulated by catabolite repression. Using
permeabilised cells we observed the enzymatic conversion of tryptamine and indole-3-acetonitrile to IAA,
both in the wild-type and in the ipdC mutant. IAA
production from tryptamine was strongly decreased
when gluconate was the carbon source.
Key words Azospirillum brasilense á Indole-3-acetic
acid synthesis á ipdC gene á Tryptamine á Indole-3acetonitrile
Introduction
Azospirillum is a gram-negative, nitrogen-®xing bacterium that lives in soil and in association with the roots of
grasses and cereals. This bacterium increases plant
R. CarrenÄo-Lopez á C. Elmerich (&)
Unite de Physiologie Cellulaire, CNRS URA D2172,
DeÂpartement des Biotechnologies, Institut Pasteur,
25±28, Rue du Dr. Roux, 75724 Paris Cedex, France
E-mail: [email protected]
Tel.: +33-1-45688817
Fax: +33-1-45688790
R. CarrenÄo-Lopez á N. Campos-Reales á B. E. Baca
Centro de Investigaciones MicrobioloÂgicas,
Universidad AutoÂnoma de Puebla, Apdo. Postal 1622,
C. P. 72000, Puebla, Pue. MeÂxico
growth primarily by colonising the root surface, causing
an increase in the proliferation of the root hairs and root
system of the host plant. This e€ect is thought to result
from the production of auxin-like compounds, such as
indole-3-acetic acid (IAA), by the bacterium, because
application of IAA mimics the e€ect of inoculation with
the bacteria (for a review see Costacurta and Vanderleyden 1995). No mutant strain that is totally devoid of
IAA production has yet been described in Azospirillum
brasilense (Hartmann and Zimmer 1994).
There are several biosynthetic pathways for IAA in
prokaryotes (Fig. 1) and a given bacterial species may
use more than one pathway (Hartmann and Zimmer
1994; Patten and Glick 1996; Vande Broek et al. 2000).
The indole pyruvate (IPyA) route, which is believed to
be the main pathway for IAA synthesis in plants, has
recently been reported in bacteria (Koga et al. 1991;
Costacurta et al. 1994; Zimmer et al. 1994, 1998; Brandl
and Lindow 1996). The ®rst step in this pathway is the
conversion of Trp to IPyA by transamination. Indole-3pyruvate decarboxylase (IPDC) then catalyses the conversion of IPyA to indole-3-acetaldehyde (IAald), and a
non-speci®c aldehyde oxidase produces IAA (Fig. 1).
Biochemical and genetic evidence for this pathway has
been provided for A. brasilense . Two aromatic aminotransferases have been puri®ed (Soto-UrzuÂa et al.
1996). The structural gene that encodes indole-3-pyruvate decarboxylase, ipdC , has been characterised in the
A. brasilense strains Sp7 and Sp245 (Costacurta et al.
1994; Zimmer et al. 1998). An ipdC mutant of strain
Sp245 produced only 10% of the amount of IAA produced by the wild type after growth in minimal medium
containing Trp, supporting the idea that there are alternative routes for IAA synthesis (Prinsen et al. 1993;
Vande Broek et al. 2000). Based on the use of radiolabelled IAA precursors, Prinsen et al. (1993) proposed
that the major route for IAA synthesis is independent of
tryptophan when no Trp is supplied to the culture. The
indole-3-acetamide (IAM) pathway, if present, could
account for less than 0.1% of IAA synthesis (Prinsen
et al. 1993). Genes coding for the tryptophan-2-mono-
522
route for IAA synthesis, we have constructed a mutant
strain of Sp7 in which the ipdC gene is disrupted by a
spectinomycin resistance cartridge. The ipdC mutation
was introduced into two di€erent Trp auxotrophs and
we studied IAA production in the presence of Trp, indole (Ind) and indole-3-lactic acid (ILA). Our results are
consistent with the existence of a tryptophan-dependent
route for IAA production, other than the IPyA pathway, that is repressed when gluconate is the carbon
source. Physiological evidence suggests that TAM is an
intermediate in this second pathway. Conversion of IAN
into IAA was also observed and may constitute another
route.
Materials and methods
Fig. 1 Schematic representation of known metabolic pathways
for IAA synthesis from Trp: the-indole-3-acetamide (IAM) route
(1); the indole pyruvate (IPyA) route (2) via indole acetaldehyde
(IAald), indole lactic acid (ILA), and indole ethanol (IEth); the
tryptamine (TAM) route (3); the tryptophan side chain oxidation
(TSO) route (4); the indole-3-acetonitrile (IAN) route (5) via the
nitrilase; the IAN route via the nitrile hydratase and amidase (6).
Ant, anthranilic acid; Ind, indole; Ind glycerol ph, indole glycerol
phosphate. See Koga et al. (1991), OberhaÈnsli et al. (1991),
Costacurta and Vanderleyden (1995), Kobayashi et al. (1995),
Patten and Glick (1996), and Vande Broek et al. (2000) for
details
oxygenase (iaaM ) and indoleacetamide hydrolase
(iaaH ) have not yet been isolated (Zimmer and Elmerich 1991; Bar and Okon 1993). Moreover, con¯icting
results have been obtained concerning the detection of
tryptophan-2-monooxygenase activity (Zimmer and
Elmerich 1991; Bar and Okon 1993).
Conversion of indole-3-acetonitrile (IAN) to IAA by
both tryptophan-dependent and tryptophan-independent routes has been reported in higher plants (Patten
and Glick 1996; Normanly and Bartel 1999, and references therein). Nitrile hydratase and amidase activities
that are responsible for the conversion of IAN to IAM
and IAA have been found in Agrobacterium
and Rhizobium species (Kobayashi et al. 1995). A nitrilase activity that catalyses the conversion of IAN directly to IAA was recently reported in Bradyrhizobium
japonicum (Vega-Hernandez et al. 1999). Synthesis of
IAA by the tryptamine (TAM) pathway is common to
plants and fungi (Patten and Glick 1996). A tryptophan
decarboxylase catalyses the conversion of Trp to TAM.
This enzymatic activity has been described in Bacillus
cereus (Patten and Glick 1996). An amine oxidase
catalyses the conversion of TAM to IAald, and a nonspeci®c aldehyde oxidase produces IAA (Fig. 1). Possible conversion of TAM into IAA was reported in Azospirillum , but was not further documented (Hartmann
et al. 1983).
To evaluate the importance of the IPyA route in
A. brasilense Sp7 and to try to identify an alternative
Bacterial strains, plasmids, media, growth conditions,
and conjugation
The bacterial strains and plasmids used in this work are listed in
Table 1. Strain Sp13Trp is a Trp auxotroph isolated from strain
Sp13, a strain similar to Sp7 (Tarrand et al. 1978; Vieille et al.
1989). The rich culture medium used was Luria-Bertani broth for
Escherichia coli and nutrient broth (Difco) for A. brasilense .
Minimal medium consisted of the salt base (K) containing 34 mM
lactate or malate as the carbon source and 20 mM NH4Cl as previously described (Galimand et al. 1989). To ensure that the Trp
auxotrophs grew, 10 lg/ml Trp was added to the culture medium.
To compare the e€ect of the various carbon sources, the concentration of the organic acid was adjusted to 34 mM and that of
gluconate to 10 mM. For the colorimetric determination of indoles
and HPLC analysis, the bacteria were grown in rich medium,
centrifuged, washed and then used to inoculate, at an OD600 of 0.1,
the minimal medium containing the carbon source indicated, and
supplemented with 100 lg/ml Trp. In some experiments, 50 lg/ml
Ind, 100 lg/ml anthranilate (Ant) or 50 lg/ml ILA, as indicated,
replaced Trp. Cultures were incubated for up to 48 h during the
stationary phase, at 30 °C, prior to analysis, unless otherwise
stated. Control analyses were performed with supernatants of
cultures grown in the absence of Trp (or supplemented with 10 lg/
ml Trp). Plasmids were transferred into Azospirillum recipients by
conjugation using E. coli S17.1 as a donor, as previously described
(Galimand et al. 1989). Tetracycline (Tc. 10 lg/ml), kanamycin
(Km, 20 lg/ml) and spectinomycin (Spc, 150 lg/ml) were added to
the culture medium as required.
Colorimetric assay for indolic compounds
The term indolic compound is used to refer to substituted indoles
and avoid confusion with indole (Ind) itself. Indolic compounds
were estimated using the colorimetric assay described by Glickman
and Dessaux (1995). The reagent used was the PC reagent (12 g/l
FeCl3 in 7.9 M H2SO4), which is based on the Salkowski reagent.
This reagent does not react with Trp, Ind or Ant. Indolic compounds were determined spectrophotometrically at 540 nm. Results
are expressed in micrograms of total indolic compounds per mg of
protein, using IAA as standard. Protein content was estimated
using the Bio-Rad reagent.
Separation of indolic compounds by HPLC
For HPLC, 10-ml cultures were centrifuged, and the pH of the
supernatant was brought to pH 2.5 by the dropwise addition of
4 N HCl. The solution was then extracted three times with ethyl
acetate. The ethyl acetate extracts were evaporated to dryness at
39 °C under vacuum, and the resulting concentrates were
523
Table 1 Strains and plasmids
Strain
Genotype/phenotype
Source/reference
A. brasilense
Sp7
Sp13
Sp13Trp
7853
2111
2112
7853-11
Sp13Trp-11
Sp13Trp-1153
Wild type
Wild type
Trp auxotroph, derivative of Sp13
Sp7 derivative, trpD- Km
Sp7 derivative, ipdC -Spc
Sp7 derivative, ipdC- Spc/lacZ- Km
7853 derivative, trpD- Km, ipdC- Spc
Sp13Trp derivative, trp, ipdC- Spc
Sp13Trp-11 derivative, trp, ipdC- Spc, trpD- Km
Tarrand et al. (1978)
Tarrand et al. (1978)
D.E. Duggan
Zimmer et al. (1991)
This work
This work
This work
This work
This work
Escherichia coli
S17.1
pro,thi, hsd
Plasmid
pSUP202
pAB1053-37
mini-Tn5 Sm/Spc
PCRII
pKOK5
pAB0540
pAB2107
pAB2110
pAB2111
pAB2112
r
, recA , RP4-2Tc::Mu-Km::Tn7
Simon et al. (1983)
Ampr, Tcr, Cmr, suicide vector
pSUP202 derivative containing a Tn5 insertion in trpD, Kmr,
Spcr, source of Sm/Spc cartridge
Ampr, cloning vector for PCR fragments
Source of lacZ- Km cartridge
pVK100 derivative, with an insert of 24 kb containing the 11.5-kb Eco RI
fragment with ipdC
pSUP202 derivative containing the 11.5-kb Eco RI fragment from pAB0540
carrying ipdC
2107 derivative, Sal I deletion
pAB2110 derivative, containing the ipdC gene inactivated by a Sm/Spc cartridge
pAB2111 derivative, containing the lacZ -Km cartridge
dissolved in 2 ml of methanol, as described (Tien et al. 1979).
HPLC analysis was performed with 20-ll aliquots of methanol
extract. Indolic compounds were separated on a 4.6 mm ´ 14.5 cm,
5 lm C-18 reverse phase column on a Beckman Gold Liquid
Chromatograph. Samples were analysed under isocratic conditions
with two di€erent separation solvents (A and B), at a ¯ow rate of
1 ml/min. Eluates were detected by spectrophotometry at 280 nm.
IAA and indole derivatives were quanti®ed by reference to the
peak area obtained for the respective standards (Sigma). The retention times of the indolic compounds and the composition of
solvents A (Tien et al. 1979) and B (Frankenberger and Brunner
1983) are indicated in Table 2. IAA and IAM can be determined
with both solvents. Ind was determined using the data for solvent
B, whereas quanti®cation of ILA was performed using the data
for solvent A. IAald and IPyA are unstable, and are enzymatically
converted to the corresponding reduced products, ILA and IEth,
respectively. Recovery of IEth and TAM from acidic extracts is
dicult, so these compounds were separated using basic extracts
(pH 8) and solvent A as previously described (Tien et al. 1979).
Table 2 Retention times of
indolic compounds separated
by HPLC using various
solvents
Compound
Indole-3-acetamide (IAM)
Indole-3-acetic acid (IAA)
Indole-3-acetaldehyde (IAald)
Indole-3-lactic acid (ILA)
Indole-3-pyruvic acid (IPyA)
Indole (Ind)
Indole-3-acetonitrile (IAN)
Tryptamine (TAM)
a
Simon et al. (1983)
Zimmer et al. (1991)
de Lorenzo et al. (1990)
Invitrogen
Kokotek and Lotz (1989)
Zimmer et al. (1998)
This work
This work
This work
This work
For experiments with indole-3-acetonitrile (IAN) solvent C was
used (Table 2).
Molecular protocols
Genomic and plasmid DNA isolation, restriction analysis, Southern analysis, DNA transformation, and molecular cloning were
carried out according to standard procedures (Sambrook et al.
1989) or as recommended by the manufacturers of the products
used.
Cloning of the ipdC region and construction of ipdC mutant
strains by gene disruption
The physical maps of the inserts in the plasmids constructed for
this work are shown in Fig. 2. The ipdC region of strain Sp7 was
recently isolated (pAB0540) from a library of Sal I fragments of
Retention time (min)
Solvent Aa
Solvent Bb
Solvent Cc
5.6‹0.6
10.3‹1
4‹0.4
8.8‹0.9
13.8‹1.4
13.5‹1.4
11.2‹1
3.0‹0.3
3.6‹0.4
4.6‹0.5
±
±
5.4‹0.6
6.6‹0.7
±
±
3.8‹0.4
4.6‹0.2
3.2‹0.1
3.8‹0.3
5.3‹0.2
9.4‹0.3
7.8‹0.3
±
Solvent A: methanol/1% acetic acid in water (40:60 v/v)
Solvent B: methanol:water acidi®ed to pH 2.5 with phosphoric acid (60:40 v/v), each phase containing 0.01 M 1-heptanesulfonate
c
Solvent C: acetonitrile:1% acetic acid in water (40:60 v/v)
b
524
Fig. 2 Schematic representation of the physical map of the
ipdC region. The construction
of the relevant plasmids is described in Materials and methods. Restriction sites: Bg,
Bgl II; Bam, Bam HI; Pv,
Pvu II; R, Eco RI; S, Sal I;
Stu, Stu I. The arrow indicates the location and orientation of the ipdC gene. Sm/Spc:
spectinomycin/streptomycin
cartridge, lacZ- Km: b-galactosidase cartridge. Plasmid vectors are not shown
A. brasilense Sp7 DNA in the broad-host-range vector pVK100
(Zimmer et al. 1998). The ipdC region in pAB0540 is included in an
11.5-kb Eco RI fragment (Zimmer et al. 1998), that was inserted
into the pSUP202 suicide vector to yield pAB2107 (Fig. 2). The
ipdC coding sequence contains a single Stu I site and a second
Stu I site is present in the adjacent 3.2-kb Sal I fragment (Fig. 2).
The 3.2-kb Sal I fragment containing the unwanted Stu I site was
deleted, to yield pAB2110. pAB2111 was obtained by inserting into
the unique Stu I site in pAB2110 a Sma I fragment containing the
2-kb Sm/Spc cassette from the Tn5 -Sm/Spc minitransposon (de
Lorenzo et al. 1990). Disruption of the ipdC gene in Azospirillum
recipients by marker exchange was subsequently performed using
pAB2111 as previously described (Galimand et al. 1989). Recombination was checked by DNA±DNA hybridisation using the 2-kb
Sm/Spc cartridge and two di€erent ipdC probes. One was a 1.3-kb
internal fragment of the ipdC gene obtained from total Sp7 DNA
by PCR using the oligonucleotides for the ipdC gene described by
Zimmer et al. (1998). The 1.3-kb fragment ampli®ed was inserted
into the PCRII vector (Invitrogen), and the nucleotide sequence of
the insert was established to ensure that it corresponded to the
ipdC gene. The other probe used was a 5.1-kb Bgl II-Sal I fragment that encompasses the ipdC region puri®ed from pAB2110
(Fig. 2). The trpD- Km mutation was introduced into Sp13Trp-11
by marker exchange using the suicide vector pAB1053-37 (Zimmer
et al. 1991). Recombination was checked by hybridisation with
appropriate probes (data not shown).
Construction of the ipdC-lacZ transcriptional fusion
and b-galactosidase activity assay
pAB2112 was obtained by inserting a 4.7-kb Bam HI fragment
carrying lacZ- Km from pKOK5 (Kokotek and Lotz 1989) into
pAB2111 partially digested with Bam HI, to yield pAB2112
(Fig. 2). pAB2112 was introduced into A. brasilense Sp7 by
conjugation using E. coli S17.1 as a donor. After marker ex-
change, correct recombination was checked by DNA±DNA hybridisation with appropriate probes, as described above (data not
shown).
b-Galactosidase activity was assayed as previously described
(ArseÁne et al. 1994). Results are expressed in Miller units per mg of
protein.
Indole pyruvate decarboxylase (IPDC) activity and other
enzyme assays
Bacteria were grown in 200 ml of lactate-containing medium
supplemented with 100 lg/ml Trp. After 48 h the cultures were
harvested by centrifugation. For the IPDC assay the pellet was
resuspended in 10 mM potassium phosphate bu€er containing
5 mM MgCl2 and 0.1 mM thiamine pyrophosphate, pH 6.5
(Koga et al. 1992), and the cells were lysed as described (SotoUrzuÂa et al. 1996). The assay was performed in 3 ml of the bu€er
containing the cell-free extract. The reaction was started by the
addition of 30 ll of 20 mM IPyA (dissolved in ethanol), followed
by incubation for 20 min at 37 °C. The reaction was stopped by
the addition of 6 ml of 0.1 N HCl. HPLC analysis (solvent A)
for IPyA and IAald was conducted according to Koga et al.
(1992).
The assay for tryptophan side chain oxidation (TSO) activity
was performed on crude extracts according to OberhaÈnsli et al.
(1991). Other enzymatic activities were determined using permeabilised cells according to the methods of Brandl et al. (1996).
Bacteria were grown in lactate- or gluconate-containing medium.
After 30 h, the cultures were centrifuged, washed and resuspended
in the same medium without Trp, at an OD600 of 8 and CTAB
(cetyltrimethylammonium bromide) was added at 0.25 mM. Permeabilised cells were incubated with the desired substrate for 24 or
48 h and the reaction products were identi®ed by HPLC using the
appropriate separation solvent (Table 2). IAN was used at 5 mM
®nal concentration and TAM at 2 mM.
525
Results
A. brasilense Sp7 contains a single copy of the ipdC gene
Southern analysis of A. brasilense Sp7 (wild-type), using as a probe the 1.3-kb fragment of ipdC obtained by
PCR, identi®ed restriction fragments of 17 kb with
Eco RI, 7.3 kb with Pvu II, 20 kb with Pst I, 2.3 kb
with Bgl II+Pvu II (Fig. 3), 22 kb with Bgl II and
11 kb with Sal I (not shown). The size of the hybridising
Eco RI fragment di€ers from that in pAB0540 probably
because this plasmid resulted from the cloning of Sal I
fragments that are not contiguous in the genome (Zimmer et al. 1998). We also checked that the sizes of the
fragments carrying the wild-type ipdC gene in the
strains 7853 and Sp13Trp were similar to that in Sp7.
The ipdC gene in the Sp7 strain was disrupted using
the plasmid pAB2111, which carries an Sm-Spc cartridge
inserted in the Stu I site in the ipdC coding sequence.
Figure 3 shows the results of Southern analysis with the
ipdC PCR probe, and indicates that the cartridge had
recombined at the correct location. Indeed, for a given
restriction digest, a single band was observed for both
the wild type and the ipdC mutant (2111). This band
was larger in the case of the mutant, and the increase in
size corresponded to the size of the cartridge. Use of the
Spc cartridge as a probe for Southern analysis of the
ipdC mutant identi®ed the same fragments as the ipdC
probe (not shown). Data obtained using the Bgl II-
Fig. 3 Southern hybridisation analysis of the genomic DNA of
A. brasilense Sp7 and 2111 (ipdC- Spc mutant) with the 1.3-kb
ipdC probe. Lanes 1, 3, 5, and 7 contained Sp7 DNA; lanes 2, 4, 6
and 8, 2111 DNA. The DNAs were digested with Eco RI (lanes 1
and 2), Pvu II (3 and 4), Pst I (5 and 6), Bgl II+Pvu II (7 and 8)
Sal I fragment containing the ipdC gene as a probe
were consistent with those obtained with the PCR probe
(not shown). These results are consistent with there being a single copy of the ipdC gene in strain Sp7. Similarly, Sp13Trp was found to carry a single copy of
ipdC.
Characterisation of the ipdC-Spc and ipdC-lacZ
mutants: e€ect of the carbon source on the
production of indolic compounds
In the case of the wild-type Sp7, and in agreement with
previous reports (Hartmann et al. 1983; Crozier et al.
1988; Omay et al. 1992; Baca et al. 1994), we observed
the accumulation of indolic compounds in culture supernatants using the colorimetric assay on cultures
grown for 2 days or more in the presence of a suitable
IAA precursor, such as Trp, Ind or Ant (at 50 or 100 lg/
ml). A net reduction in the level of indolic compounds
produced was observed when the ipdC mutant strain
(2111) was grown on certain carbon sources, in media
supplemented with Trp (100 lg/ml) (Fig. 4). Similar data
were obtained with the ipdC-lacZ mutant strain (2112;
not shown). In particular, if malate was used as the
carbon source, the level of total indoles was about 25%
that of the wild type, in the same range as that reported
for an ipdC mutant of strain Sp245 grown in MMAB
medium, which contains malate as the carbon source
(Prinsen et al. 1993; Costacurta et al. 1994). Much lower
levels of indolic compounds were produced when gluconate was used as the carbon source. Levels were not
signi®cantly a€ected on succinate, fumarate, lactate or
Fig. 4 Production of indolic compounds by A. brasilense Sp7
(white bars ), the ipdC mutant 2111 (hatched bars ), and the
ipdC mutant carrying pAB0540 (black bars ) after 48 h of culture
in minimal medium containing various carbon sources and
supplemented with Trp at 100 lg/ml. The following carbon sources
were used: malate (1), gluconate (2), succinate (3), lactate (4),
pyruvate (5) and fumarate (6). Data are the means of three
determinations, the error bars indicate SD
526
pyruvate (Fig. 4). No production of indolic compounds
was detected in the absence of Trp, as observed for the
wild type. The IPDC speci®c activity in the wild type
(Sp7) grown in minimal lactate medium containing Trp,
was 7.7‹0.7 nmol IAald/mg protein, whereas in the
ipdC
mutant (2111) the activity detected was
0.49‹0.2 nmol IAald/mg protein and this probably resulted from the chemical instability of the substrate. We
con®rmed that the introduction of pAB0540 into the
mutants restored indolic compound production, regardless of whether the carbon source was malate or
gluconate (Fig. 4). The high level of residual indolic
compound production observed with the ipdC mutant
strain on most carbon sources is consistent with there
being another route for IAA synthesis. The strong decrease in the synthesis of indolic compounds by 2111
grown on gluconate suggests that the alternative route
be regulated by catabolite repression.
Time course of expression of the ipdC-lacZ fusion gene
Figure 5 shows the kinetics of expression of the ipdClacZ fusion during the exponential and stationary
phases of growth on malate (Fig. 5a) and lactate
(Fig. 5b) as the carbon source, in medium supplemented with 100 lg/ml Trp. The expression of the
ipdC-lacZ fusion followed the same kinetics as the
accumulation of indoles; no ipdC expression or synthesis of indolic compounds was detectable during the
exponential phase of growth, and both b-galactosidase
and indolic compounds began to accumulate during
the stationary phase. The maximal level of expression
of the ipdC-lacZ fusion after 48 h was 404‹61 and
302‹94 Miller units in media containing malate and
lactate, respectively. The kinetics and rate of ipdClacZ expression were not signi®cantly di€erent in
medium devoid of Trp: the maximal rate was 414‹78
Miller units with malate and 388‹109 Miller units
with lactate, even when 1 mM IAA was added as an
inducer (data not shown).
Indolic compounds produced by the wild type
and Trp auxotrophs carrying the ipdC mutation
We identi®ed the IAA precursors required for IAA
synthesis when the ipdC strain was grown on lactate by
assessing the levels of indolic compounds produced by
two di€erent Trp auxotrophs with and without the
ipdC mutation. Strain 7853 is a genetically characterised Trp auxotroph that is de®cient in phosphoribosylanthranilate transferase, because it carries a trpD- Km
mutation (Zimmer et al. 1991). This strain cannot convert anthranilate to Trp, but can use Ind for growth. The
level of indolic compound production from Ant (+
10 lg/ml Trp) was very low, whereas signi®cant production was observed with Ind or Trp (Table 3). Strain
Sp13Trp is a Trp auxotroph that cannot convert Ind to
Fig. 5a, b Time course of expression of the ipdC-lacZ fusion in
minimal media, and in minimal media supplemented with Trp
(100 lg/ml). a Malate-containing medium. b Lactate-containing
medium. Growth curves: open squares , Sp7; ®lled squares , 2112
(ipdC-lacZ mutant strain). b-Galactosidase activity: open circles ,
2112. Total indolic compounds: open triangles , Sp7; ®lled
triangles , 2112
Trp. It may carry a mutation in trpA or trpB . Thus, it
cannot grow on Ind or Ant unless 10 lg/ml Trp is added
to supply its requirements for growth. Under these
conditions, no production of indolic compounds was
observed from these two precursors (Table 3). Similar
data were obtained with strains carrying the trpD and
ipdC mutations (strains Sp13Trp-11 and Sp13Trp1153, Table 3). This strongly suggests that the indolic
compounds produced by the alternative route are derived from Trp rather than from Ind or Ant.
Identi®cation of the indolic compounds produced by
the Trp auxotrophs and the ipdC mutant strain
HPLC analysis of extracts of culture supernatant was
performed to determine the amount of IAA accumulated by the wild type and mutants, and to try to identify
intermediates of IAA synthesis via the IPyA pathway or
the putative alternative route. Data are reported in
Table 4 for IAA and ILA production or consumption
only. In no case was the accumulation of IAM, IAald or
IPyA detected. As IPyA is very unstable, it was assumed
527
Table 3 Production of indolic compounds by wild-type and mutant strains of A. brasilense
lactate-containing medium
Strain
Sp7
2111 (ipdC -Spc)
7853 (trpD- Km)
Sp13Trp (trp)
Sp13Trp-11 (trp, ipdC- Spc)
Sp13Trp-5311 (trp, ipdC-Spc, trpD -Km)
during the stationary phase of growth in
Production of indolic compounds (lg/mg protein) in the presence of the indicated substratesa
Trp (10 lg/ml)
Trp (100 lg/ml)
Ind (50 lg/ml) +
Trp (10 lg/ml)
Ant (100 lg/ml) +
Trp (10 lg/ml)
1.1‹0.3
0.5‹0.3
1.9‹0.8
ND
ND
ND
41‹3.8
23.7‹2.6
41‹5.5
39.6‹3
22‹4
28.2‹6
34‹6
31‹4.5
31‹3
ND
ND
ND
8.8‹4.2
3.2‹1.8
2‹0.9
NDb
NDb
NDb
a
Indolic compounds were assayed after 2 days of culture in lactatecontaining media. The results are means‹SD for six independent
experiments. ND, not detected
These strains produced a red pigment that was excreted into the
supernatant. This compound had a maximal absorption peak at
400 nm, and did not react with the PC reagent
that ILA accumulation in the growth medium is proportional to IPyA production.
The wild type Sp7 produced similar amounts of IAA
after growth on lactate or malate whether the precursor
was Trp, Ind or ILA. With Ind, slightly more IAA was
produced if malate was the carbon source. With Trp or
Ind as the precursor, no ILA production was detected.
Insertional inactivation of the ipdC gene in Sp7 (strain
2111) substantially reduced IAA production in malate
cultures, whereas with lactate IAA was produced at a
rate similar to that in the wild type. This is consistent
with the data from the colorimetric assay with Ind or
Trp as the precursor. The major di€erence between the
wild type and the ipdC mutant (2111) was that substantial amounts of ILA accumulated in the mutant,
when either Ind or Trp was supplied as precursor.
Moreover, if ILA was added to the growth medium as
the precursor the level of IAA accumulation by the
mutant was low. This suggests that ILA may also act,
albeit ineciently, as a precursor of IAA in the alternative pathway.
The data obtained with the two Trp auxotrophs are
also consistent with the data for the colorimetric assay.
In minimal malate medium, Sp13 produced the same
amount of IAA as Sp7 and did not accumulate ILA
(data not shown). Sp13Trp produced both IAA and ILA
in lactate minimal medium, if supplied with Trp,
whereas IAA and ILA did not accumulate in this particular strain if the growth medium contained Ind (Table 4). The trpD -Km mutant (7853) produced a similar
amount of IAA from Ind to Sp7. Inactivation of the
ipdC gene in strain Sp13Trp (Sp13Trp-11) resulted in
residual IAA synthesis with either malate or lactate as
the carbon source, and only if a suitable source of Trp
was available. In particular, no IAA was detected when
Ind was added to the growth medium (Table 4). We
observed that in the absence of added tryptophan, ILA
supported the growth of the Trp auxotrophs on solid
and liquid media. If ILA was added to the growth medium, signi®cantly less IAA accumulated in the mutants
(2111, Sp13Trp and Sp13Trp-1153; Table 4) than in the
wild type. This suggests that ILA can serve both as
source of Trp and IAA.
Enzymatic production of IAA from IAN or TAM
b
Preliminary experiments to identify intermediates in the
alternative route were performed with the wild type
grown in lactate minimal medium containing Trp. No
TSO activity was detected under any of the conditions
used. TAM is a potent growth inhibitor and cannot be
added directly to growing cultures, while IAN is not
taken up by the cells. Using permeabilised cell suspensions of the wild type or the ipdC (2111) mutant strain,
IAA production was detected with TAM and IAN as the
substrates (Table 5). A net increase in IAA production
was observed between 24 and 48 hours of incubation.
When the strains were grown with gluconate as the
carbon source, IAA production from TAM was strongly
decreased (tenfold; Fig. 5), suggesting that the TAM
route is controlled by catabolite repression. IAA production from IAN was also reduced but to a lesser extent.
Discussion
In this work we report the physiological properties of
A. brasilense Sp7 mutant strains in which the ipdC
gene has been disrupted by insertion of a spectinomycin
resistance or a lacZ cartridge. Our results suggest that
at least another tryptophan-dependent route for IAA
synthesis exists. The ®rst and third steps in the IPyA
pathway may be catalysed by non-speci®c aminotransferases and non-speci®c oxidases, respectively
(Koga et al. 1994; Soto-UrzuÂa et al. 1996). It was
therefore important to check for the presence of single or
multiple copies of the ipdC gene in the Sp7 genome, as
suggested by Zimmer et al. (1998), that might account
for the residual synthesis of IAA. Based on the
hybridisation analysis reported (Fig. 3) the presence of a
second copy of the gene is unlikely. A similar conclusion
was also reached in A. brasilense Sp245 (Costacurta et
al. 1994). Moreover, the ipdC mutant strain (2111) is
devoid of IPDC activity, and thus the residual production of IAA is unlikely to be due to another enzyme that
displays the same activity.
The levels of IAA and ILA were determined by HPLC. Levels of IAA production in lactate medium without added Trp were 0.44‹0.2 lg/mg protein for Sp7 and 0.3‹0.2 lg/mg
protein for 2111. Data are the means of ®ve determinations ‹SD. ND, not detected; NT, not tested
a
6.2‹3.2
3.8‹1.8
ND
ND
19.5‹7.5
6.4‹2.3
ND
6.5‹2.3
8.4‹0.2
ND
8.7‹4.6
12‹4.8
NT
5.2‹2.1
NT
21.8‹1.2
2.4‹1.4
NT
3.6‹1.2
NT
ND
9.9‹2.4
ND
ND
ND
49‹9.7
6.4‹1.4
27.2‹12
ND
ND
ND
13.6‹2.8
ND
ND
17.6‹6.7
27.8‹4.1
7.4‹0.7
34‹7.8
19‹3.2
8‹3
ND
14.8‹3.5
ND
ND
ND
29.3‹4.2
17.5‹3
29‹4.2
ND
ND
ND
7.1‹2.1
ND
16‹1.8
14.1‹3.7
22.8‹4.6
19.5‹1
23.1‹4.2
18‹4.4
10.4‹0.6
Sp7
2111 (ipdC -Spc)
7853 (trpD -Km)
Sp13Trp (trp )
Sp13Trp-11
(trp , ipdC- Spc)
Sp13Trp-1153
(trp, ipdC- Spc, trpD -Km)
ILA
IAA
IAA
ILA
IAA
ILA
IAA
ILA
IAA
ILA
Lactate + ILA
(50 lg/ml)
Malate + Trp
(10 lg/ml) +
Ind (50 lg/ml)
Malate + Trp
(100 lg/ml)
Lactate + Trp
(10 lg/ml) +
Ind (50 lg/ml)
Lactate + Trp
(100 lg/ml)
Mediuma
Strain
Table 4 Levels of indole-3-acetic acid (IAA) and indole-3-lactic acid (ILA) produced by wild-type and mutant strains after growth on lactate or malate as carbon source
528
Both ipdC and ipdC-lacZ mutant strains were impaired in the production of indoles when grown with
malate or gluconate (in the presence of Trp), as shown
by the colorimetric assay, but little e€ect on the production of indolic compounds was observed with other
carbon sources including lactate. The colorimetric reagent used reacts with IPyA and ILA, the absorption
maxima of which are di€erent from, but close to, that
of IAA (Glickman and Dessaux 1995). Thus, a decrease in IAA production may be compensated for by
accumulation of one or both of these compounds following ipdC disruption. HPLC analysis showed that
when lactate was the carbon source, IAA production
was not signi®cantly reduced in the mutant, whereas a
net reduction, to 25% of the wild-type level, was observed with malate (Table 4). There was also a net
accumulation of ILA (IPyA) in the ipdC mutant strain
(2111; Table 4). As no ILA accumulation was detected
with the wild type, this suggests that the ipdC gene is
expressed both in lactate- and malate-containing
medium.
The chromosomal transcriptional ipdC-lacZ fusion
(strain 2112) was expressed during the stationary phase.
Maximum speci®c activities on lactate and malate did
not di€er signi®cantly, and were independent of Trp.
Thus, this IAA precursor does not seem to be involved
in the regulation of ipdC transcription. This was also
reported to be the case in the epiphytic bacterium
Erwinia herbicola 299R (Brandl and Lindow 1997). In
A. brasilense Sp245, the expression of a plasmid-borne
ipdC-gus fusion was increased by addition of IAA,
suggesting that the end product acted as an inducer
(Vande Broek et al. 1999). In our work, with a chromosomal ipdC-lacZ fusion, no increase of expression was
observed on addition of IAA. As almost no IAA was
detected in media devoid of Trp, in which the fusion is
fully induced during the stationary phase, it is likely that
other unidenti®ed factors are involved in the expression
of the ipdC gene in strain Sp7.
A recent report based on quantum chemical methods
suggested that Ant and Ind could not act as precursors
of IAA in a Trp-independent pathway (Zakharova et al.
1999). The data reported here, based on the use of Trp
auxotrophs, are consistent with this, showing that no
IAA was produced from Ind by the Sp13Trp strain. As
Sp13Trp cannot use Ind (or Ind + serine) to synthesise
Trp, the lack of synthesis of IAA from Ind suggests that
Trp is probably the IAA precursor. The use of Sp13Trp
derivatives carrying the ipdC mutation showed that no
IAA was produced from Ind, con®rming the tryptophan-dependency of IAA production by the alternative
route.
A particularly interesting result was obtained with
ILA as the precursor, in that Sp13Trp produced small
amounts of IAA. Indeed, the conversion of IPyA to
ILA is known to be a reversible reaction (Crozier et
al. 1988; Koga et al. 1994). In addition, aromatic
amino acid transferases that catalyse the conversion of
Trp to IPyA are not speci®c for amino acid substrates,
529
Table 5 Production of IAA by permeabilised cells supplied with indole-3-acetonitrile (IAN) or tryptamine (TAM) as a substrate
Carbon source
Lactate
Lactate
Gluconate
Lactate
Gluconate
a
Substrate
None
TAM
TAM
IAN
IAN
IAA production (lg/mg protein)a
No bacteria
Sp7
7211
48 h
24 h
48 h
24 h
48 h
NT
ND
NT
ND
NT
NT
1.4‹0.2
NT
2.5‹0.2
NT
0.3‹0.3
34‹4
3.6‹0.3
5.5‹0.5
3.7‹0.23
NT
0.5‹0.1
NT
3.5‹0.7
NT
0.17‹0.04
21.8‹5.8
1.4‹0.4
6.7‹0.9
2.9‹0.2
Data are means‹SD for three independent experiments. ND, not detected; NT, not tested
have a very high K m value for Trp, and the reactions
are also reversible (Koga et al. 1994; Soto-UrzuÂa et al.
1996). For the Trp auxotroph that carries the ipdC
mutation (Sp13Trp-1153), ILA is also a source of IAA
(Table 4). Because IAA synthesis cannot be catalysed
by the ipdC gene product in this latter strain, it is
assumed ILA is a precursor of Trp, which in turn is a
precursor of IAA, through the second Trp-dependent
pathway.
The indole pyruvate pathway is probably the major
pathway for IAA synthesis in cultures grown with malate,
as previously reported (Prinsen et al. 1993; Costacurta et
al. 1994), and with gluconate (Fig. 4). Our data suggest
that the Trp-dependent alternative route is controlled by
catabolite repression. Using permeabilised cells, it was
possible to show the enzymatic conversion of both IAN
and TAM to IAA by both the wild type and the ipdC
mutant. Both activities were decreased when gluconate
was the carbon source. IAN and TAM belong to two
di€erent routes (Fig. 1). TAM can result from the decarboxylation of Trp. This argues in favour of a Trpdependent route for IAA synthesis that uses TAM as an
intermediate in A. brasilense Sp7. The synthesis of IAN
from Trp has been described in plants (Normanly and
Bartel 1999, and references therein). It is tempting to
speculate that a Trp-dependent route for IAA synthesis
that uses IAN as an intermediate is also present in Azospirillum , but this route is not fully repressed by gluconate. This does not rule out the possible existence of Trpindependent routes for synthesis of IAA from either
IPyA, TAM or IAN as intermediates under certain
physiological conditions. To conclude, based on the
physiological evidence reported here, A. brasilense Sp7
appears to possess two di€erently regulated Trp-dependent routes for IAA synthesis (the IPDC and the TAM
routes), as well as an alternative route that uses IAN as an
intermediate.
Acknowledgements We would like to thank Dr. W. Zimmer for the
gift of plasmid pAB0540 and for the communication of data prior to
publication, Dr. Perez-Galdona for helpful discussion on the nitrilase activity, Dr. D.E. Duggan for the Sp13Trp strain and Ms. G.L.
AlaÂvez-Junco for assistance with HPLC. R. CarrenÄo-LoÂpez was a
Pre-doctoral Fellow of the SecretarõÂ a de EducacioÂn PuÂblica (SEP)
and was supported by the Institut Pasteur; N. Campos-Reales holds
a CONACyT fellowship. This work was partially supported by the
ECOS-ANUIES-SEP CONACyT program (France-MeÂxico).
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