Azospirillum brasilense Produces the Auxin-Like Phenylacetic Acid by ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2005, p. 1803–1810 0099-2240/05/$08.00⫹0 doi:10.1128/AEM.71.4.1803–1810.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 71, No. 4

Azospirillum brasilense Produces the Auxin-Like Phenylacetic Acid by Using the Key Enzyme for Indole-3-Acetic Acid Biosynthesis E. Somers,1 D. Ptacek,1 P. Gysegom,1 M. Srinivasan,2 and J. Vanderleyden1* Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, B-3001 Heverlee, Belgium,1 and Yara Technology Centre, Hydro Research Park, Porsgrunn, Norway2 Received 23 August 2004/Accepted 28 October 2004

An antimicrobial compound was isolated from Azospirillum brasilense culture extracts by high-performance liquid chromatography and further identified by gas chromatography-mass spectrometry as the auxin-like molecule, phenylacetic acid (PAA). PAA synthesis was found to be mediated by the indole-3-pyruvate decarboxylase, previously identified as a key enzyme in indole-3-acetic acid (IAA) production in A. brasilense. In minimal growth medium, PAA biosynthesis by A. brasilense was only observed in the presence of phenylalanine (or precursors thereof). This observation suggests deamination of phenylalanine, decarboxylation of phenylpyruvate, and subsequent oxidation of phenylacetaldehyde as the most likely pathway for PAA synthesis. Expression analysis revealed that transcription of the ipdC gene is upregulated by PAA, as was previously described for IAA and synthetic auxins, indicating a positive feedback regulation. The synthesis of PAA by A. brasilense is discussed in relation to previously reported biocontrol properties of A. brasilense. Azospirillum is a well-studied genus of plant growth-promoting bacteria (PGPB), which colonizes the rhizosphere of numerous crop plants in tropical and subtropical regions (5, 6). Different mechanisms, such as phytohormone production, nitrate reduction, and nitrogen fixation, have been proposed to explain improved plant growth following inoculation with Azospirillum (5, 7, 9, 22, 37, 38, 53). The production of phytohormones, and more specifically the auxin indole-3-acetic acid (IAA), has been recognized as an important factor in direct plant-growth-promoting abilities of A. brasilense (5, 18, 19, 37). Azospirillum sp. are not typical biocontrol agents of soilborne plant pathogens (5). Apart from some reports on bacteriocins and siderophores, no other antibacterial substances in Azospirillum sp. have been identified so far (39, 40, 50, 55, 60). However, there have been reports of moderate biocontrol capabilities of Azospirillum brasilense against crown gall disease, bacterial leaf blight of mulberry, and bacterial leaf and/or vascular diseases of tomato (1, 3, 4, 46, 54). In addition, A. brasilense can restrict the proliferation of other nonpathogenic rhizosphere bacteria (21). Nevertheless, the exact mechanisms involved in Azospirillum acting as a putative biocontrol agent are not yet known. Some reports therefore indicate that the protective mechanism may be indirectly explained by the plant growth promotion effect or by outcompeting other bacteria hosted by the same plant (3, 46). In the present study, we attempted to further screen A. brasilense supernatant (extracts) for the presence of metabolites (besides IAA), which may be involved in the persistence of Azospirillum in the rhizosphere. This screening led to the iden-

tification of phenylacetic acid (PAA), an auxin-like molecule with antimicrobial activity. MATERIALS AND METHODS Strains, plasmids, media, and culture conditions. Strains and plasmids used in this study are listed in Table 1. A. brasilense was grown at 30°C in Luria-Bertani (LB) medium supplemented with 2.5 mM CaCl2 and 2.5 mM MgSO4 (LB*) as a rich bacterial growth medium. Alternatively, A. brasilense was grown at 30°C in MMAB minimal medium with 0.5% malate as a carbon source and 20 mM NH4Cl as as a nitrogen source (59). Agrobacterium tumefaciens NT1 was grown aerobically in ABM medium or in TY medium at 30°C (14). Escherichia coli and Chromobacterium violaceum were grown aerobically in LB medium at 37° and 30°C, respectively. Pseudomonas syringae and Erwinia carotovora were grown aerobically in TY medium at 30°C (8). Solid media contained 15 g of agar per liter. Antibiotics were used at the following concentrations: kanamycin, 25 ␮g/ml; and tetracycline, 10 ␮g/ml. Amino acids (L-phenylalanine, L-tryptophan, and L-tyrosine) or precursors thereof (phenylpyruvate, prephenate, and chorismate) were added from filtersterilized 100⫻ stock solutions to a final concentration of 0.5 mM. Fungal strains were cultured on half-strength potato dextrose broth agar (Difco) at 25°C. Screening of A. brasilense supernatant extracts. Increasing evidence that several PGPB can use quorum-sensing-based signaling such as N-acylhomoserine lactones (AHLs) to communicate, thereby enhancing their efficacy, in the rhizosphere incited us to screen A. brasilense culture supernatant for the presence of population density-dependent cell-cell signaling (or autoinducer) molecules. The detection of autoinducer molecules, which are produced at very low concentrations, has been greatly facilitated by the development of sensitive bioassays that allow fast screening for diffusible signal molecules (11). We assessed culture supernatant extracts from A. brasilense Sp7 and Sp245 for the presence of putative AHL signal molecules through thin-layer chromatography (TLC)-mediated fractionation (C18 reversed phase), coupled to detection by means of the A. tumefaciens AHL bioreporter system as described previously (45, 51). Briefly, Azospirillum strains were grown to stationary phase in 100-ml volumes of LB* or MMAB. Cells were removed by centrifugation. If mentioned in the text, supernatant was acidified to pH 2 by addition of HCl. Culture fluid was extracted twice with equal volumes of ethyl acetate. The organic phases were concentrated to dryness, redissolved in 100-␮l volumes of ethyl acetate, and separated by chromatography on C18 reversed-phase TLC plates with a solvent system of methanol:water (60:40 [vol/vol]). Visualization of active AHL was performed by means of overlays with ABM soft agar (0.7%) supplemented with X-Gal (5bromo-4-chloro-3-indolyl-a¯-D-galactopyranoside) and containing the A. tumefaciens AHL reporter strain NT1(pJM749, pSVB33) (14, 45). The TLC plates were incubated overnight at 30°C.

* Corresponding author. Mailing address: Departement Toegepaste Plantwetenschappen, Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium. Phone: 32 16 321631. Fax: 32 16 321966. E-mail: [email protected]. 1803

1804

APPL. ENVIRON. MICROBIOL.

SOMERS ET AL. TABLE 1. Strains and plasmids Strain or plasmid

Bacterial strains A. brasilense Sp7 A. brasilense Sp245 A. brasilense FAJ009 A. tumefaciens NT1 (pJM749, pSVB33) E. carotovora LMG6663 P. syringae pv. glycinea LMG5066 E. coli DH5␣ C. violaceum CV026

Relevant characteristic(s)

Reference or source

Wild-type strain, isolated from Digitaria decumbens rhizosphere soil, Brazil Wild-type strain, isolated from surface sterilized wheat roots, Brazil ipdC Tn5 mutant of A. brasilense Sp245; Kmr Indicator strain for detecting AHLs; Kmr Cbr Wild-type isolate Wild-type isolate F⫺ ␾80DlacZ⌬M15 endA1 recA1 hsdR17 supE44 thi-1 gyrA96 ⌬(lacZYA-argF) AHL biosensor strain, mini-Tn5 mutant of ATCC31532; Kmr

56 2 15 45 Laboratorium voor Microbiologie, Universiteit Gent, Ghent, Belgium Laboratorium voor Microbiologie, Universiteit Gent, Ghent, Belgium 47 34

Plasmids pFAJ64

pLAFR3 with ipdC-gusA fusion

58

Fungal strains N. crassa MUCL19026

Wild-type strain

Mycothe`que Universite´ Catholique de Louvain, Louvain-la-Neuve, Belgium Mycothe`que Universite´ Catholique de Louvain, Louvain-la-Neuve, Belgium Centraalbureau voor Schimmelcultures, Baarn-Delft, The Netherlands

A. brassicicola MUCL20297 F. oxysporum f. sp. matthiolae 247.61

Wild-type strain Wild-type strain

Determination of in vitro antimicrobial activity spectrum of A. brasilense culture extract. In vitro antimicrobial bioassays were used to perform a quick screening for antibacterial and antifungal activity of stationary-phase-grown A. brasilense supernatant extract. The antibacterial activity of the A. brasilense supernatant extract was measured by a paper disk method with a series of bacteria including A. tumefaciens NT1, E. coli DH5a, and C. violaceum CV026. Sterile Whatmann filters were impregnated with 10 ␮l of A. brasilense supernatant extract (prepared as described previously), dried under sterile air, and layered over the surface of an agar plate inoculated with the strain to be tested. Following overnight incubation at 30 or 37°C (depending on the test strain), the formation of a halo surrounding the disk containing the A. brasilense extract was used as an indication of growth inhibition. The antibacterial effect of A. brasilense supernatant extract was determined by measuring the diameter of the halo of growth inhibition. To assess the potential antifungal activity of A. brasilense supernatant extract, the highest dilution that completely inhibited germination of Neurospora crassa, Alternaria brassicicola, and Fusarium oxysporum f. sp. mathiolae was determined by using a liquid growth inhibition assay. Routinely, tests were performed with 96-well microtiterplates with 20 ␮l of a test solution (1 ␮l of A. brasilense supernatant extract in 19 ␮l of half-strength potato dextrose broth, or serial dilutions thereof) and 80 ␮l of a suspension of fungal spores (at a final concentration of 2 ⫻ 104 spores ml⫺1) in half-strength potato dextrose broth, supplemented with tetracycline (10 ␮g/ml) and cefotaxime (100 ␮g/ml). After 24 h of incubation at 25°C, growth of the fungus was evaluated microscopically with an inverted microscope. The absence of hyphal growth was taken as evidence of antifungal activity, and the highest dilution rate for 100% inhibition of hyphal growth was determined as the maximum inhibitory dilution (MID) for the sample. Positive control microcultures contained 20 ␮l of Ace-AMP1 (final concentration, 100 ␮g/ml), an antimicrobial protein isolated from Allium cepa L., and 80 ␮l of the fungal spore suspension (13). Negative control microcultures each contained 20 ␮l of sterile distilled water and 80 ␮l of the fungal spore suspension. A second negative control was included as well, containing 1 ␮l of blank LB* medium extract in 19 ␮l of half-strength potato dextrose broth (or dilutions thereof) and 80 ␮l of the fungal spore suspension. Preparative HPLC: isolation of a growth inhibitory compound from A. brasilense supernatant extracts for gas chromatography-mass spectrometry (GCMS) analysis. A 50-␮l aliquot of supernatant extract from A. brasilense grown in LB* to stationary phase was injected onto a Bondclone 10-␮m reversed-phase

C18 high-performance liquid chromatography (HPLC) column (300 by 37.8 mm) (Phenomenex). Fractions were eluted with an isocratic mobile phase, consisting of 98% ammonium acetate (NH4Ac) (0.25 mM; pH 6.8) and 2% MeOH or 96% NH4Ac (0.25 mM; pH 6.8) and 4% MeOH over a 30-min period at a flow rate of 4 ml/min, monitored at 254 nm, and collected at 2-min intervals. The activity of the fractions against A. tumefaciens was examined by the paper disk method. The fraction with growth-inhibitory activity towards A. tumefaciens was collected, further concentrated by lyophilization, and resuspended in 100 ␮l of NH4Ac (pH 6.2), and a 10-␮l aliquot was reassessed for antimicrobial activity. GC-MS analysis of A. brasilense supernatant extract. One microliter of the concentrated HPLC fraction containing the antimicrobial compound or, alternatively, 1 ␮l of dilutions of the sample (1/5 or 1/150 dilution) was injected into a gas chromatograph coupled with an MS detector (Thermoquest; Finnigan/ Interscience) with a split ratio of 1/8. GC-MS results were obtained at 350 eV (3.5 scans/s at 200°C). GC separation was carried out by using a Restek RTX-5 column (7 m by 0.32 mm) and He (3 ml/min) as the carrier gas. The following temperature gradient program was used: 2 min at 75°C, followed by an increase from 75 to 175°C at a rate of 5°C/min and finally 7 min at 175°C. The m/z peaks (representing mass-to-charge ratios) characteristic for the antimicrobial fraction, isolated by HPLC, were compared with those in the mass spectrum library (National Institute for Standards and Technology). Analytical HPLC: determination of PAA and IAA in A. brasilense supernatant extracts. Optimization of HPLC-guided identification of PAA and IAA in A. brasilense supernatant extracts revealed a separation with superior resolution (including a reduction of tailing) by means of gradient HPLC, compared to isocratic HPLC. Therefore, analytical HPLC was performed by injection of a 5-␮l aliquot of supernatant extract onto a chromatograph fitted with a Bondclone 10-␮m reversed-phase C18 HPLC column (300 by 3.9 mm) (Phenomenex). The chromatograph was operated at a flow rate of 1 ml/min over a 30-min period, with a linear gradient of NH4Ac (0.25 mM; pH 4.2) and MeOH (98 to 0% [vol/vol] and 2 to 100% [vol/vol], respectively). Absorbance at 254 nm was measured. ␤-Glucuronidase assay. Expression of an ipdC-gusA fusion in A. brasilense was assessed as previously described (58). LB*-grown Sp245(pFAJ64) cultures at exponential-growth phase were supplemented with PAA or IAA and compared for ipdC-gusA induction with cultures to which none of the compounds was added. Briefly, three samples (each, 3 ml) were transferred to sterile test tubes and were supplemented with ethyl alcohol (control samples), IAA, or PAA at a final concentration of 1 mM (or otherwise, as indicated). After 4 h of additional

PRODUCTION OF PHENYLACETIC ACID BY A. BRASILENSE

VOL. 71, 2005

growth at 30°C, quantitative ␤-glucuronidase activity was assayed by using the p-nitrophenyl-␤-D-glucuronide substrate as previously described (24). Reactions were stopped at three different time points to ensure that enzyme activity was increasing linearly with incubation time. For each time point, each sample was measured in triplicate at least. Activity is expressed as Miller units (35). Comparison among the mean values was made by Tukey’s multiple-range test with a 95% confidence limit.

TABLE 2. Antifungal activity of A. brasilense supernatant extract Species

MIDAba

MIDblankb

Ratio MIDAb/ MIDblank

N. crassa A. brassicicola F. oxysporum f. sp. matthiolae

4 16 12

1 2 1

4 8 12

a

RESULTS Detection of growth-inhibitory activity in A. brasilense culture extracts. Screening of A. brasilense supernatant extracts for AHLs (see Materials and Methods) did not show clear-cut results. However, the presence of a rather polar compound in culture extracts, derived from LB*-grown A. brasilense Sp245, was reproducibly detected at a fixed position on a C18 reversedphase TLC plate, where it locally inhibited growth of an A. tumefaciens (reporter) strain seeded in an agar overlay (data not shown). Antimicrobial activity was only observed in extracts from A. brasilense Sp245 cultures at the end of the logarithmic phase and the beginning of the stationary-growth phase. The growth-inhibitory compound was not detected in supernatant extracts originating from MMAB grown A. brasilense Sp245. The same results were obtained for A. brasilense strain Sp7. Data and figures presented in this paper illustrate results for strain Sp245 only. In vitro antimicrobial activity spectrum of A. brasilense culture extract. In vitro antibacterial activity of the A. brasilense supernatant extract was assessed by using a paper disk method. A total of 10 ␮l of A. brasilense supernatant extract had marked growth-inhibitory activity (dhalo ⱖ 20 mm) against A. tumefaciens, E. carotovora, and P. syringae pv. glycinea and moderate growth-inhibitory activity (dhalo ⫽ 15 mm) towards E. coli. In contrast, no activity was found towards C. violaceum. Similarly, no halo was visible around a paper disk containing 10 ␮l of extract from blank LB* medium (negative control) in any of the strains tested. Physicochemical characterization of the antibacterial activity showed that the growth-inhibitory effect towards an A. tumefaciens test strain was stable after heat treatment (110°C for 60 min) and after treatment with proteinase K or pronase E. Furthermore, acidifying the stationary-phase culture supernatant to pH 2 prior to ethyl acetate extraction remarkably increased the growth-inhibitory effect of the A. brasilense extract towards the A. tumefaciens test strain. Antifungal activity of A. brasilense supernatant extract was assessed by using a liquid growth inhibition assay. The MID of the bacterial supernatant extract (MIDAb) was compared to that of the blank medium extract (MIDblank) to preclude possible interference of ethyl acetate (extraction solvent) or (concentrated) growth medium components. The MIDAb/MIDblank ratio was considered a measure of the intrinsic antifungal activity of the bacterial supernatant extract (Table 2). A. brasilense supernatant extract exhibited antifungal activity towards all the fungal strains tested and was found to be most effective against F. oxysporum f. sp. matthiolae, followed by A. brassicicola and N. crassa. In the case of A. brassicola, the delayed spore germination in the presence of A. brasilense supernatant extract caused reduced elongation of hyphae and increased branching (hyperbranching) as well. Analogous to the antibacterial effect, acidifying the stationary-phase culture supernatant to pH 2, prior

1805

b

MID of bacterial supernatant extract. MID of blank medium extract.

to ethyl acetate extraction, increased the antifungal effect of the A. brasilense extract. Structure elucidation of the antimicrobial compound in A. brasilense culture extracts. To obtain information at the molecular level about the antimicrobial activity of A. brasilense culture extracts, a 50-␮l aliquot of supernatant extract from A. brasilense grown in LB* to stationary phase was subjected to bioassay-guided HPLC fractionation. One fraction, obtained from a single large peak with a retention time of 13.8 min, displayed growth-inhibitory activity towards A. tumefaciens (fraction 7). The HPLC chromatograms revealed a second large peak, although without any growth-inhibitory activity towards A. tumefaciens, which eluted at 18.6 min and which was identified as IAA by comparison with authentic samples. A. brasilense is known to produce IAA (15). Neither of the two large peaks could be observed in an ethyl acetate extract derived from blank LB* growth medium. GC-MS analysis revealed a molecule with a retention time and a mass spectrum comparable to the reference spectrum of PAA (Fig. 1). Authentic samples of PAA were subjected to HPLC as well, which revealed that synthetic PAA eluted at a position identical to that of the single peak of antimicrobial activity originating from the A. brasilense supernatant extract. Furthermore, the growth inhibitory activities of synthetic PAA and IAA towards A. tumefaciens were assessed by the paper disk method: only paper disks impregnated with PAA (10 ␮l of a 10 mM solution) but not IAA (10 ␮l of a 10 mM solution) caused significant growth inhibition of the Agrobacterium test strain. These results suggest that the antimicrobial activity in A. brasilense supernatant is caused by PAA. PAA: a secondary metabolite derived from phenylalanine. The fact that the growth inhibitory compound PAA could only be identified in supernatant extracts from A. brasilense cultures grown in LB* medium suggested that PAA is derived from amino acid metabolism. Based on its aromatic structure, it was speculated that PAA might be derived from phenylalanine. To test this hypothesis, supernatant extract from A. brasilense cultures grown in minimal MMAB medium supplemented with 0.5 mM phenylalanine was assayed for growth-inhibitory activity and the presence of PAA by means of a paper disk bioassay and analytical HPLC. While A. brasilense supernatant extract derived from MMAB-grown cultures did not display any growth inhibitory activity or PAA production, addition of phenylalanine to the MMAB growth medium resulted in significant PAA production and concomitant growth-inhibitory activity in A. brasilense culture supernatant extracts (Fig. 2 and Fig. 3). In contrast, the addition of tyrosine or tryptophan to MMAB did not induce PAA production in A. brasilense supernatant. A. brasilense cul-

1806

SOMERS ET AL.


FIG. 1. (A) Electron impact mass spectrum of the A. brasilense Sp245 growth-inhibitory compound found in HPLC fraction 7. (B) Comparison with m/z peaks in the mass spectrum library (National Institute for Standards and Technology) revealed a molecule with a retention time and a mass spectrum comparable with the reference spectrum of PAA. A putative fragmentation scheme for the identified molecules with indication of the ion fragment masses is displayed.

tures supplemented with phenylalanine precursors, including phenylpyruvate, prephenate, and chorismate displayed growthinhibitory activity and PAA production as well, though slightly less than cultures supplemented with phenylalanine (Fig. 3). IpdC is involved in the production of PAA. The key step in the IAA biosynthetic pathway of A. brasilense, namely the decarboxylation of indolepyruvic acid, is catalyzed by in-

dolepyruvate decarboxylase, encoded by the ipdC gene in A. brasilense (15, 29). Decarboxylation of phenylpyruvate to phenylacetaldehyde may implicate the activity of the IpdC protein as well, since the HPLC chromatogram of supernatant extract prepared from an LB*-grown A. brasilense ipdC mutant (FAJ009), which is known to synthesize less than 10% of the wild-type IAA levels

FIG. 2. HPLC chromatograms of A. brasilense Sp245-derived culture supernatant extract. A. brasilense was grown in MMAB (A) or MMAB supplemented with 0.5 mM phenylalanine (B). Fractions were eluted with a linear gradient of NH4Ac (0.25 mM; pH 4.2) and MeOH (98 to 0% [vol/vol] and 2 to 100% [vol/vol], respectively) over a 30-min period at a flow rate of 1 ml/min. The peak originating from PAA in culture supernatant extract, derived from A. brasilense cultures grown in MMAB supplemented with phenylalanine, is indicated.

VOL. 71, 2005


1807

FIG. 3. Determination of the antimicrobial activity of supernatant extracts (10 ␮l) isolated from A. brasilense Sp245 cultures grown in MMAB supplemented with 0.5 mM tyrosine (1), 0.5 mM phenylalanine (2), 0.5 mM tryptophan (3), 0.5 mM phenylpyruvate (4), 0.5 mM prephenate (5), or 0.5 mM chorismate (6) by the paper disk method.

(15), was demonstrated to lack both peaks, derived from PAA and IAA. The loss of growth inhibitory activity of FAJ009derived supernatant extract was also confirmed by the paper disk method (data not shown). PAA induces ipdC expression. It was observed previously that IAA, as well as synthetic auxins, upregulates the expression of the A. brasilense Sp245 IAA biosynthetic gene, ipdC (58). We tested whether this positive feedback regulation also occurs with PAA, the synthesis of which also depends on the activity of the ipdC gene product. ipdC gene expression in the presence of 1 mM PAA was slightly but significantly (P ⬍ 0.05; Tukey’s test) higher than ipdC expression in LB* supplemented with 1 mM IAA (Fig. 4). The same results were found with 0.1 mM PAA and IAA, while no significant differences were found when only 0.01 mM IAA or PAA was used to induce ipdC expression. Since acidic conditions are known to induce ipdC expression, the pH levels of all culture media were monitored. No significant differences in pH were found between cultures supplemented with IAA and those supplemented with PAA, indicating that the slightly

higher expression of the ipdC gene in the presence of PAA can be intrinsically attributed to PAA itself and is not due to a pH effect. DISCUSSION Using an A. tumefaciens bioreporter assay, we were able to demonstrate the presence of antimicrobial acitivity against A. tumefaciens in A. brasilense supernatant extracts. Interestingly, Bakanchikova et al. (1) previously demonstrated the inhibitory effect of A. brasilense on crown gall formation in dicotyledonous plants caused by A. tumefaciens. Preinoculation of wounded tissues of grapevines and carrot disks with live cells of A. brasilense 94-3 or Sp7 inhibited the development of the typical bacterial galls, but their mode of action has not been established (1). In vitro antibiosis studies of A. brasilense supernatant extract also revealed its activity against other gramnegative bacterial test organisms (including E. carotovora, P. syringae pv. glycinea, and to a lesser extent E. coli) as well as its confined antifungal activity against the test fungi A. brassici-

FIG. 4. Effect of exogenously added IAA or PAA on ipdC expression in A. brasilense Sp245. Columns, reading from left to right, show results from the following conditions: LB*, LB* supplemented with 1 mM IAA, LB* supplemented with 0.1 mM IAA, LB* supplemented with 0.01 mM IAA, LB* supplemented with 1 mM PAA, LB* supplemented with 0.1 mM PAA, and LB* supplemented with 0.01 mM PAA. Data are the means of three replicates. Means followed by different letters are significantly different at P ⬍ 0.05 (Tukey test).

1808

SOMERS ET AL.

cola, F. oxysporum f. sp. matthiolae, and to a lesser extent N. crassa. The antimicrobial compound from A. brasilense supernatant extract was isolated and identified as the auxin-like molecule PAA by a combination of bioassay-guided HPLC and GC-MS. Similarly to IAA production in A. brasilense, detectable phenylacetate production was demonstrated to occur only in the stationary-growth phase, correlating with the onset of secondary metabolism (30, 41, 10, 58, 43, 57). Secondary metabolism is often brought on by exhaustion of a nutrient (suboptimal growth conditions), biosynthesis or addition of an inducer, or a growth rate decrease (16). The fact that higher levels of PAAmediated antimicrobial activity from A. brasilense extracts could be obtained by prior acidification of the culture is most likely due to a better extraction of the nondissociated acid in ethyl acetate. PAA is known for its weak auxin activity (36, 64). Numerous data on the plant growth promoting the effect of auxins have been published, but only a few studies focused on the fact that certain auxins, including PAA, show antimicrobial effects as well (12, 23, 26). PAA has previously been demonstrated to display growth-inhibitory activity towards gram-negative bacteria (including P. syringae pv. syringae and E. coli), grampositive bacteria (including Bacillus subtilis and Staphylococcus aureus), and fungi (including Rhizoctonia solani, Pythium ultimum, Phytophtora capsici, Saccharomyces cerevisiae, Colletotrichum orbiculare, Candida albicans, and Gibberella pulicaris) (12, 23, 26). As is the case for many known secondary metabolites, PAA may be involved in defense mechanisms, protecting the producing strain from competing cells and as such providing a way for the strain to survive in its natural environment. However, at this stage we have only worked with concentrated supernatant extracts; as yet, we cannot postulate that the actual (currently unknown) concentration of PAA produced by A. brasilense in vivo is sufficient to be of ecological importance. A common theme in secondary metabolite synthesis is the biotransformation of primary metabolites to form compounds that can then be enzymatically altered to obtain different activities (16). We could demonstrate that PAA production by A. brasilense was dependent on the presence of phenylalanine in the growth medium. Catabolism of amino acids usually initiates with aminotransferase-dependent deamination; likewise, the most plausible PAA biosynthetic route may involve the deamination of phenylalanine (by an aminotransferase), resulting in the formation of phenylpyruvate (32). Aromatic amino acid aminotransferases, which can transfer the ␣-amino group of aromatic amino acids to a ketoacid acceptor, have been studied in various microorganisms including E. coli, Klebsiella aerogenes, A. brasilense, Sinorhizobium meliloti, Bacillus brevis, and Pseudomonas aeruginosa; some of them have already been purified (25, 33, 42, 52, 63). Generally, two or more enzymes with overlapping specificities are found in the same microorganism (28, 44). Aromatic amino acid aminotransferases have already been shown to play a role in IAA and PAA biosynthesis in bacteria and fungi (20, 27, 28, 48). The results of the present study also suggest that ipdC is involved in the PAA biosynthetic pathway. ipdC encodes an indolepyruvate decarboxylase and catalyzes the decarboxylation of indolepyruvic acid in the indole-3-pyruvate-dependent


IAA biosynthetic pathway in A. brasilense (29). Analogous to this pathway, phenylpyruvate (released after deamination of phenylalanine) may be decarboxylated by IpdC to form phenylacetaldehyde, which can be further oxidized to PAA. At the protein level, the sequences of the indolepyruvate decarboxylases from Sp245 and Sp7 (15, 65) display only restricted homology with those identified in other plant-associated bacteria such as Enterobacter cloacae (29% identity, 44% similarity), Pantoea agglomerans (Erwinia herbicola) (22% identity, 38% similarity), and Pseudomonas putida GR12-2 (29% identity, 44% similarity), while these latter proteins display at least 50% mutual identity (30, 10, 43). Interestingly, indolepyruvate decarboxylase from E. cloacae was shown to be a highly specific enzyme with a high affinity for indole-3-pyruvate, while phenylpyruvate was found to function as a competitive inhibitor (31). In contrast, phenylpyruvate decarboxylases (EC 4.1.1.43) are known to act on phenylpyruvate, as well as on indolepyruvate (17, 27, 49, 61, 62). The results of the present study will help to further elucidate the enzymatic properties of the A. brasilense IpdC protein, which may act as a broad-spectrum phenylpyruvate decarboxylase (with substrate affinity for indole-3-pyruvate as well), rather than as a specific indole-3-pyruvate decarboxylase. However, it will be indispensable to purify the A. brasilense IpdC protein and characterize its physical and regulatory properties to further elucidate the ipdC-dependent IAA and PAA biosynthesis in A. brasilense. Interestingly, ipdC expression was demonstrated to be significantly upregulated by PAA, indicating that a similar positive feedback regulation, as was observed with IAA biosynthesis, may be involved in PAA biosynthesis (58). It can be argued that the concentrations of exogenously added IAA or PAA for induction of the ipdC gene are rather high, but it should be kept in mind that IAA and PAA are produced inside the A. brasilense cells. In the assay performed in this study, the uptake of IAA or PAA by A. brasilense cells might be limiting for the effect observed. ACKNOWLEDGMENTS We acknowledge financial support from Norsk Hydro. We acknowledge scientific advice from P. Goddard. We thank R. De Mot, C. Michiels, Y. Okon, and P. Cornelis for their valuable contributions during discussions while writing the paper and E. Verhaeren and C. De Backer (AVER N.V., Wijnegem, Belgium) for their valuable assistance with the identification of the antimicrobial compound by means of GC-MS. REFERENCES 1. Bakanchikova, T. I., E. V. Lobanok, L. K. Pavlova-Ivanova, T. V. Redkina, Z. A. Nagapetyan, and A. N. Majsuryan. 1993. Inhibition of tumor formation process in dicotyledonous plants by Azospirillum brasilense strains. Mikrobiologiya 62:515–523. 2. Baldani, V. L. D., M. A. B. Alvarez, J. I. Baldani, and J. Do¨bereiner. 1986. Establishment of inoculated Azospirillum spp. in the rhizosphere and in roots of field grown wheat and sorghum. Plant Soil 90:37–40. 3. Bashan, Y., and L. E. de-Bashan. 2002. Protection of tomato seedlings against infection by Pseudomonas syringae pv. tomato by using the plant growth-promoting bacterium Azospirillum brasilense. Appl. Environ. Microbiol. 68:2637–2643. 4. Bashan, Y., and L. E. de-Bashan. 2002. Reduction of bacterial speck (Pseudomonas syringae pv. tomato) of tomato by combined treatments of plant growth-promoting bacterium, Azospirillum brasilense, streptomycin sulfate, and chemo-thermal seed treatment. Eur. J. Plant Pathol. 108:821–829. 5. Bashan, Y., and G. Holguin. 1997. Azospirillum-plant relationships: environmental and physiological advances (1990–1996). Can. J. Microbiol. 43:103– 121.

VOL. 71, 2005


6. Bashan, Y., and G. Holguin. 1998. Proposal for the division of plant growthpromoting rhizobacteria into two classifications: biocontrol-PGPB (plant growth-promoting bacteria) and PGPB. Soil Biol. Biochem. 30:1225–1228. 7. Bashan, Y., and H. Levanony. 1990. Current status of Azospirillum inoculation technology: Azospirillum as a challenge for agriculture. Can. J. Microbiol. 36:591–608. 8. Beringer, J. E. 1974. R factor transfer in Rhizobium leguminosarum. J. Gen. Microbiol. 84:188–198. 9. Bothe, H., H. Ko ¨rsgen, T. Lehmacher, and B. Hundeshagen. 1992. Differential effects of Azospirillum, auxin and combined nitrogen on the growth of the roots of wheat. Symbiosis 13:167–179. 10. Brandl, M. T., and S. E. Lindow. 1996. Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola. Appl. Environ. Microbiol. 62:4121–4128. 11. Brelles-Marino, G., and E. J. Bedmar. 2001. Detection, purification and characterisation of quorum-sensing signal molecules in plant-associated bacteria. J. Biotechnol. 91:197–209. 12. Burkhead, K. D., P. J. Slininger, and D. A. Schisler. 1998. Biological control bacterium Enterobacter cloacae S11:T:07 (NRRL B-21050) produces the antifungal compound phenylacetic acid in Sabouraud maltose broth culture. Soil Biol. Biochem. 30:665–667. 13. Cammue, B. P., K. Thevissen, M. Hendriks, K. Eggermont, I. J. Goderis, P. Proost, J. Van Damme, R. W. Osborn, F. Guerbette, J. C. Kader, and W. F. Broekaert. 1995. A potent antimicrobial protein from onion seeds showing sequence homology to plant lipid transfer proteins. Plant Physiol. 109:445– 455. 14. Chilton, M. D., T. C. Currier, S. K. Farrand, A. J. Bendich, M. P. Gordon, and E. W. Nester. 1974. Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc. Natl. Acad. Sci. USA 71:3672–3676. 15. Costacurta, A., V. Keijers, and J. Vanderleyden. 1994. Molecular cloning and sequence analysis of an Azospirillum brasilense indole-3-pyruvate decarboxylase gene. Mol. Gen. Genet. 243:463–472. 16. Demain, A. L. 1998. Induction of microbial secondary metabolism. Int. Microbiol. 1:259–264. 17. Dickinson, J. R., L. E. Salgado, and M. J. Hewlins. 2003. The catabolism of amino acids to long chain and complex alcohols in Saccharomyces cerevisiae. J. Biol. Chem. 278:8028–8034. 18. Dobbelaere, S., A. Croonenborghs, A. Thys, D. Ptacek, J. Vanderleyden, P. Dutto, C. Labandera-Gonzalez, J. Caballero-Mellado, J. Francisco Aguirre, Y. Kapulnik, S. Brener, S. Burdman, D. Kadouri, S. Sarig, and Y. Okon. 2001. Responses of agronomically important crops to inoculation with Azospirillum. Aust. J. Plant Physiol. 28:871–879. 19. Dobbelaere, S., A. Croonenborghs, A. Thys, and A. Vande Broek. 1999. Phytostimulatory effect of Azospirillum brasilense wild type and mutant strains altered in IAA production on wheat. Plant Soil 212:155–164. 20. Gummalla, S., and J. R. Broadbent. 2001. Tyrosine and phenylalanine catabolism by Lactobacillus cheese flavor adjuncts. J. Dairy Sci. 84:1011–1019. 21. Holguin, G., and Y. Bashan. 1996. Nitrogen-fixation by Azospirillum brasilense CD is promoted when co-cultured with a mangrove rhizosphere bacterium (Staphylococcus sp.). Soil Biol. Biochem. 28:1651–1660. 22. Holguin, G., C. L. Patten, and B. R. Glick. 1999. Genetics and molecular biology of Azospirillum. Biol. Fertil. Soils 29:10–23. 23. Hwang, B. K., S. W. Lim, B. S. Kim, J. Y. Lee, and S. S. Moon. 2001. Isolation and in vivo and in vitro antifungal activity of phenylacetic acid and sodium phenylacetate from Streptomyces humidus. Appl. Environ. Microbiol. 67: 3739–3745. 24. Jefferson, R. A. 1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5:387–405. 25. Kanda, M., K. Hori, T. Kurotsu, S. Miura, and Y. Saito. 1987. Purification and properties of the aromatic amino acid aminotransferase from gramicidin S-producing Bacillus brevis. J. Biochem. (Tokyo) 101:871–878. 26. Kim, Y., J. Y. Cho, J. H. Kuk, J. H. Moon, J. I. Cho, Y. C. Kim, and K. H. Park. 2004. Identification and antimicrobial activity of phenylacetic acid produced by Bacillus licheniformis isolated from fermented soybean, Chungkook-Jang. Curr. Microbiol. 48:312–317. 27. Kishore, G., M. Sugumaran, and C. S. Vaidyanathan. 1976. Metabolism of DL-(⫾)-phenylalanine by Aspergillus niger. J. Bacteriol. 128:182–191. 28. Kittell, B. L., D. R. Helinski, and G. S. Ditta. 1989. Aromatic aminotransferase activity and indoleacetic acid production in Rhizobium meliloti. J. Bacteriol. 171:5458–5466. 29. Koga, J. 1995. Structure and function of indolepyruvate decarboxylase, a key enzyme in indole-3-acetic acid biosynthesis. Biochim. Biophys. Acta 1249:1– 13. 30. Koga, J., T. Adachi, and H. Hidaka. 1991. Molecular cloning of the gene for indolepyruvate decarboxylase from Enterobacter cloacae. Mol. Gen. Genet. 226:10–16. 31. Koga, J., T. Adachi, and H. Hidaka. 1992. Purification and characterization of indolepyruvate decarboxylase. A novel enzyme for indole-3-acetic acid biosynthesis in Enterobacter cloacae. J. Biol. Chem. 267:15823–15828. 32. Krings, U., M. Hinz, and R. G. Berger. 1996. Degradation of [2H]phen-

33. 34.

35. 36.

37. 38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

1809

ylalanine by the basidiomycete Ischnoderma benzoinum. J. Biotechnol. 51: 123–129. Mavrides, C., and W. Orr. 1975. Multispecific aspartate and aromatic amino acid aminotransferases in Escherichia coli. J. Biol. Chem. 250:4128–4133. McClean, K. H., M. K. Winson, L. Fish, A. Taylor, S. R. Chhabra, M. Camara, M. Daykin, J. H. Lamb, S. Swift, B. W. Bycroft, G. S. Stewart, and P. Williams. 1997. Quorum sensing and Chromobacterium violaceum: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 143:3703–3711. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Muir, R. M., T. Fujita, and C. Hansch. 1967. Structure-activity relationships in the auxin activity of mono-substituted phenylacetic acids. Plant Physiol. 42:1519–1526. Okon, Y. 1985. Azospirillum as a potential inoculant for agriculture. Trends Biotechnol. 3:223–228. Okon, Y., and C. A. Labandera-Gonzalez. 1994. Agronomic applications of Azospirillum—an evaluation of 20 years of worldwide field inoculation. Soil Biol. Biochem. 26:1591–1601. Oliveira, R. G. B., and A. Drozdowicz. 1987. Inhibition of bacteriocin producing strains of Azospirillum lipoferum by their own bacteriocin. Zentbl. Mikrobiol. 142:387–391. Oliveira, R. G. B., and A. Drozdowicz. 1988. Are Azospirillum bacteriocins produced and active in soil?, p. 101–108. In W. Klingmu ¨ller (ed.), Azospirillum IV: genetics, physiology, ecology. Springer Verlag, Berlin, Germany. Omay, S. A., W. A. Schmidt, P. Martin, and F. Bangerth. 1993. Indoleacetic acid production by the rhizosphere bacterium Azospirillum brasilense Cd under in vitro conditions. Can. J. Microbiol. 39:187–192. Paris, C. G., and B. Magasanik. 1981. Tryptophan metabolism in Klebsiella aerogenes: regulation of the utilization of aromatic amino acids as sources of nitrogen. J. Bacteriol. 145:257–265. Patten, C. L., and B. R. Glick. 2002. Regulation of indoleacetic acid production in Pseudomonas putida GR12-2 by tryptophan and the stationaryphase sigma factor RpoS. Can. J. Microbiol. 48:635–642. Pedraza, R. O., A. Ramirez-Mata, M. L. Xiqui, and B. E. Baca. 2004. Aromatic amino acid aminotransferase activity and indole-3-acetic acid production by associative nitrogen-fixing bacteria. FEMS Microbiol. Lett. 233: 15–21. Piper, K. R., S. Beck von Bodman, and S. K. Farrand. 1993. Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction. Nature 362:448–450. Romero, A. M., O. Correa, S. Moccia, and J. G. Rivas. 2003. Effect of Azospirillum-mediated plant growth promotion on the development of bacterial diseases on fresh-market and cherry tomato. J. Appl. Microbiol. 95: 832–838. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Schneider, S., M. E. Mohamed, and G. Fuchs. 1997. Anaerobic metabolism of L-phenylalanine via benzoyl-CoA in the denitrifying bacterium Thauera aromatica. Arch. Microbiol. 168:310–320. Schutz, A., T. Sandalova, S. Ricagno, G. Hubner, S. Konig, and G. Schneider. 2003. Crystal structure of thiamindiphosphate-dependent indolepyruvate decarboxylase from Enterobacter cloacae, an enzyme involved in the biosynthesis of the plant hormone indole-3-acetic acid. Eur. J. Biochem. 270:2312–2321. Shah, S., V. Karkhanis, and A. Desai. 1992. Isolation and characterization of siderophore, with antimicrobial activity, from Azospirillum lipoferum M. Curr. Microbiol. 25:34–35. Shaw, P. D., G. Ping, S. L. Daly, C. Cha, J. E. Cronan, Jr., K. L. Rinehart, and S. K. Farrand. 1997. Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc. Natl. Acad. Sci. USA 94:6036–6041. Soto-Urzua, L., Y. G. Xochinua-Corona, M. Flores-Encarnacion, and B. E. Baca. 1996. Purification and properties of aromatic amino acid aminotransferases from Azospirillum brasilense UAP 14 strain. Can. J. Microbiol. 42: 294–298. Steenhoudt, O., and J. Vanderleyden. 2000. Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiol. Rev. 24:487–506. Sudhakar, P., S. K. Gangwar, B. Satpathy, P. K. Sahu, J. K. Ghosh, and B. Saratchandra. 2000. Evaluation of some nitrogen fixing bacteria for control of foliar diseases of mulberry (Morus alba). Indian J. Sericult. 39:9–11. Tapia-Hernandez, A., M. A. Mascarua-Esparza, and J. Caballero-Mellado. 1990. Production of bacteriocins and siderophore-like activity in Azospirillum brasilense. Microbios 64:73–83. Tarrand, J. J., J. Do ¨bereiner, and J. Do¨bereiner. 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. Thuler, D. S., E. I. Floh, W. Handro, and H. R. Barbosa. 2003. Plant growth

1810

58. 59.

60.

61.

SOMERS ET AL.

regulators and amino acids released by Azospirillum sp. in chemically defined media. Lett. Appl. Microbiol. 37:174–178. Vande Broek, A., M. Lambrecht, K. Eggermont, and J. Vanderleyden. 1999. Auxins upregulate expression of the indole-3-pyruvate decarboxylase gene in Azospirillum brasilense. J. Bacteriol. 181:1338–1342. Vanstockem, M., K. Michiels, J. Vanderleyden, and Van Gool, A. 1987. Transposon mutagenesis of Azospirillum brasilense and Azospirillum lipoferum: physical analysis of Tn5 and Tn5-Mob insertion mutants. Appl. Environ. Microbiol. 53:410–415. Vazquez-Cruz, C., A. Tapia-Hernandez, M. A. Mascarua-Esparza, and J. Caballero-Mellado. 1992. Plasmid profile modification after elimination of bacteriocin activity in some Azospirillum brasilense strains. Microbios 69:195– 204. Vuralhan, Z., M. A. Morais, S. L. Tai, M. D. Piper, and J. T. Pronk. 2003.


62. 63.

64. 65.

Identification and characterization of phenylpyruvate decarboxylase genes in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 69:4534–4541. Ward, O. P., and A. Singh. 2000. Enzymatic asymmetric synthesis by decarboxylases. Curr. Opin. Biotechnol. 11:520–526. Whitaker, R. J., C. G. Gaines, and R. A. Jensen. 1982. A multispecific quintet of aromatic aminotransferases that overlap different biochemical pathways in Pseudomonas aeruginosa. J. Biol. Chem. 257:13550–13556. Wightman, F., and D. L. Lighty. 1982. Identification of phenylacetic acid as a natural auxin in the shoots of higher plants. Physiol. Plantarum 55:17–24. Zimmer, W., M. Wesche, and L. Timmermans. 1998. Identification 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.