* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Molecular and General Genetics.
Survey
Document related concepts
Gene expression wikipedia , lookup
Genetic engineering wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Citric acid cycle wikipedia , lookup
Microbial metabolism wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Gene nomenclature wikipedia , lookup
Lactate dehydrogenase wikipedia , lookup
Fatty acid synthesis wikipedia , lookup
Butyric acid wikipedia , lookup
Gene regulatory network wikipedia , lookup
Community fingerprinting wikipedia , lookup
Biochemistry wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Expression vector wikipedia , lookup
Biosynthesis wikipedia , lookup
Point mutation wikipedia , lookup
Transcript
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 eect 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 eect 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 dierent 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 eect 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 dierent 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 dicult, 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 5.60.6 10.31 40.4 8.80.9 13.81.4 13.51.4 11.21 3.00.3 3.60.4 4.60.5 ± ± 5.40.6 6.60.7 ± ± 3.80.4 4.60.2 3.20.1 3.80.3 5.30.2 9.40.3 7.80.3 ± 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 dierent 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 buer 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 buer 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 diers 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: eect 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 aected 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 7.70.7 nmol IAald/mg protein, whereas in the ipdC mutant (2111) the activity detected was 0.490.2 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 40461 and 30294 Miller units in media containing malate and lactate, respectively. The kinetics and rate of ipdClacZ expression were not signi®cantly dierent in medium devoid of Trp: the maximal rate was 41478 Miller units with malate and 388109 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 dierent 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) 1.10.3 0.50.3 1.90.8 ND ND ND 413.8 23.72.6 415.5 39.63 224 28.26 346 314.5 313 ND ND ND 8.84.2 3.21.8 20.9 NDb NDb NDb a Indolic compounds were assayed after 2 days of culture in lactatecontaining media. The results are meansSD 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 dierence 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 ineciently, 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 0.440.2 lg/mg protein for Sp7 and 0.30.2 lg/mg protein for 2111. Data are the means of ®ve determinations SD. ND, not detected; NT, not tested a 6.23.2 3.81.8 ND ND 19.57.5 6.42.3 ND 6.52.3 8.40.2 ND 8.74.6 124.8 NT 5.22.1 NT 21.81.2 2.41.4 NT 3.61.2 NT ND 9.92.4 ND ND ND 499.7 6.41.4 27.212 ND ND ND 13.62.8 ND ND 17.66.7 27.84.1 7.40.7 347.8 193.2 83 ND 14.83.5 ND ND ND 29.34.2 17.53 294.2 ND ND ND 7.12.1 ND 161.8 14.13.7 22.84.6 19.51 23.14.2 184.4 10.40.6 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 eect 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 dierent 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 dier 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 1.40.2 NT 2.50.2 NT 0.30.3 344 3.60.3 5.50.5 3.70.23 NT 0.50.1 NT 3.50.7 NT 0.170.04 21.85.8 1.40.4 6.70.9 2.90.2 Data are meansSD 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 dierent 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 dierently 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 Secretarõ 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). References ArseÁne F, Katupitiya S, Kennedy IR, Elmerich C (1994) Use of the lacZ fusions to study the expression of nif genes of Azospirillum brasilense in association with plants. Mol-Plant Microb Interact 7: 748±757 Baca BE, Soto-UrzuÂa L, Xochihua-Corona YG, Cuervo-Garcõ a A (1994) Characterization of two aromatic amino acid aminotransferases and production of indoleacetic acid in Azospirillum strains. Soil Biol Biochem 26: 57±63 Bar T, Okon Y (1993) Tryptophan conversion to indole-3-acetic acid via indole-3-acetamide in Azospirillum brasilense Sp7. Can J Microbiol 39: 81±86 Brandl M, Lindow SE (1996) Cloning and characterization of a locus encoding an indole pyruvate decarboxylase involved in indole-3-acetic acid in Erwinia herbicola . Appl Environm Microbiol 62: 4121±4128 Brandl M, Lindow SE (1997) Environmental signals modulate the expression of an indole-3-acetic acid biosynthetic gene in Erwinia herbicola . Mol Plant-Microb Interact 10: 499±505 Brandl M, Clark EM, Lindow SE (1996) Characterization of the indole-3-acetic acid (IAA) pathway in an epiphytic strain of Erwinia herbicola and IAA production in vitro. Can J Microbiol 42: 596±592 Costacurta A, Vanderleyden J (1995) Synthesis of phytohormones by plant-associated bacteria. Crit Rev Microbiol 21: 1±18 Costacurta A, Keijers V, Vanderleyden J (1994) Molecular cloning and sequence analysis of an Azospirillum brasilense indole-3pyruvate decarboxylase gene. Mol Gen Genet 243: 463±472 Crozier A, Arruda P, Jasmin JM, Monteiro AM, Sandberg G (1988) Analysis of indole-3-acetic acid and related compounds in culture medium from Azospirillum lipoferum and Azospirillum brasilense . Appl Environ Microbiol 54: 2833±2837 De Lorenzo V, Herrero M, Jakubzik U, Timmis KN (1990) MiniTn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol 172: 6568±6572 Frankenberger WT, Brunner W (1983) Method of detection of auxin-indole-3-acetic acid in soils by high performance liquid chromatography. Soil Sci Soc Amer J 47: 237±241 Galimand M, Perroud B, Delorme F, Paquelin A, Vieille C, Bouzouklian H, Elmerich C (1989) Identi®cation of DNA regions homologous to nitrogen ®xation genes nifE, nifUS, and ®xABC in Azospirillum brasilense Sp7. J Gen Microbiol 135: 1047±1059 Glickman E, Dessaux Y (1995) A critical examination of the speci®city of the Salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Appl Environm Microbiol 61: 793±796 Hartmann A, Zimmer W (1994) Physiology of Azospirillum . In: Okon Y (ed) Azospirillum -plant associations. CRC Press, Boca Raton, pp 15±39 Hartmann A, Singh M, KlingmuÈller W (1983) Isolation and characterization of Azospirillum mutants excreting high amounts of indole acetic acid. Can J Microbiol 29: 916±923 530 Kobayashi M, Suzuki T, Fujita T, Masuda M, Shimizu S (1995) Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant associated bacteria, Agrobacterium and Rhizobium . Proc Natl Acad Sci 92: 714±718 Koga J, Adachi T, Hikeda H (1991) Molecular cloning of the gene for indolepyruvate decarboxylase from Enterobacter cloacae . Mol Gen Genet 226: 10±16 Koga J, Adachi T, Hikeda H (1992) Puri®cation and characterization of indolepyruvate decarboxylase. A novel enzyme for indole-3-acetic acid biosynthesis in Enterobacter. J Biol Chem 267: 15823±15828 Koga J, Syono K, Ichikawa T, Adachi T (1994) Involvement of Ltryptophan aminotransferease in indole-3-acetic acid biosynthesis in Enterobacter cloacae. Biochim Biophys Acta 1209: 241±247 Kokotek W, Lotz W (1989) Construction of a lacZ -kanamycinresistance cassette, useful for site-directed mutagenesis and as a promoter probe. Gene 84: 467±471 Normanly J, Bartel B (1999) Redundancy as a way of life ± IAA metabolism. Curr Opin Plant Biol 2: 207±213 OberhaÈnsli T, DeÂfago G, Haas D (1991) Indole-3-acetic acid (IAA) synthesis in the biocontrol strain CHAO of Pseudomonas ¯uorescens : role of tryptophan side chain oxidase. J Gen Microbiol 137: 2273±2279 Omay SH, Schmidt A, Martin P, Bangerth F (1992) Indoleacetic acid production by the rhizosphere bacterium Azospirillum brasilense Cd under in vitro conditions. Can J Microbiol 39: 187±192 Patten CL, Glick BR (1996) Bacterial biosynthesis of indole-3acetic acid. Can J Microbiol 42: 207±220 Prinsen E, Costacurta A, Michiels K, Vanderleyden J, Van Onckelen H (1993) Azospirillum brasilense indole-3-acetic acid biosynthesis: evidence for a non-tryptophan dependent pathway. Mol Plant-Microb Interact 6: 609±615 Sambrook J, Fritsch EF, Maniatis TA (1989) Molecular cloning : a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY Simon R, Priefer U, PuÈhler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Biotechnology 1: 784±791 Soto-UrzuÂa L, Xochihua-Corona Y, Flores-EncarnacioÂn M, Baca BE (1996) Puri®cation and properties of aromatic amino transferases from Azospirillum brasilense UAP 14 strain. Can J Microbiol 42: 294±298 Tarrand JJ, Krieg NR, DoÈbereiner J (1978) A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov., and two species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Can J Microbiol 24: 967±980 Tien TM, Gaskins MH, Hubbel DH (1979) Plant growth substances produced by Azospirillum brasilense and their eect on the growth of pearl millet (Pennisetum americanum ). Appl Environm Microbiol 37: 1016±1024 Vande Broek A, Lambrecht M, Eggermont K, Vanderleyden J (1999) Auxins upregulate expression of the indole-3-pyruvate decarboxylase gene in Azospirillum . J Bacteriol 181: 1338± 1342 Vande Broek A, Dobbelaere S, Vanderleyden J, Van Dommelen A (2000) Azospirillum -plant root interactions: signaling and metabolic interactions. In: Triplett EW (ed) Prokaryotic nitrogen ®xation: a model system for analysis of a biological process. Horizon Scienti®c Press, Wymondham, UK, pp 761±777 Vega-Hernandez M, LeÂon-Barrios M, Jarabo-Lorenzo A, PerezGaldona R (1999) Indole-3-acetic acid production from indole3-acetonitrile by Bradyrhizobium . In: Abstracts of the 12th International Congress on Nitrogen Fixation, Foz de IguacËu, Brazil. pp 112 Vieille C, Onyeocha I, Galimand M, Elmerich C (1989) Homology between plasmids of Azospirillum brasilense and Azospirillum lipoferum . In: Skinner, FA (ed.) Nitrogen ®xation in nonlegumes. Kluwer Academic, Dordrecht, The Netherlands, pp 165±172 Zimmer W, Elmerich C (1991) Regulation of the synthesis of indole-3-acetic acid in Azospirillum. In: Hennecke H, Verma DPS (eds) Advances in molecular genetic of plant-microbe interactions. Kluwer Academic, Dordrecht, The Netherlands, pp 465±468 Zimmer W, Aparicio C, Elmerich C (1991) Relationship between tryptophan biosynthesis and indole-3-acetic acid production in Azospirillum : identi®cation and sequencing of a trpGDC cluster. Mol Gen Genet 229: 41±51 Zimmer W, Hundeshagen B, Niederau E (1994) Demonstration of indolepyruvate decarboxylase gene in dierent auxin-producing species of Enterobactereaceae . Can J Microbiol 40: 1072±1076 Zimmer W, Wesche M, Timmermans L (1998) Identi®cation and isolation of the indole-3-pyruvate decarboxylase gene from Azospirillum brasilense Sp7: sequencing and functional analysis of the gene locus. Curr Microbiol 36: 327±331 Zakharova EA, Shcherbakov AA, Brudnik VV, Skripko NG, Bulkhin NS, Ignatov VV (1999) Biosynthesis of indole-3-acetic acid in Azospirillum brasilense. Insights from quantum chemistry. Eur J Biochem 259: 572±576