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Author's personal copy Plant Soil (2012) 356:139–150 DOI 10.1007/s11104-011-1009-2 REGULAR ARTICLE The hisC1 gene, encoding aromatic amino acid aminotransferase-1 in Azospirillum brasilense Sp7, expressed in wheat Julio Castro-Guerrero & Angelica Romero & José J. Aguilar & Ma. Luisa Xiqui & Jesús O. Sandoval & Beatriz E. Baca Received: 9 February 2011 / Accepted: 25 September 2011 / Published online: 30 November 2011 # Springer Science+Business Media B.V. 2011 Abstract Background and aims Production of indole-3-acetic acid (IAA) by Azospirillum brasilense is one of the most important mechanisms underlying the beneficial effects observed in plants after inoculation with this bacterium. This study determined the contribution of the hisC1 gene, which encodes aromatic amino acid aminotransferase-1 (AAT1), to IAA production, and analyzed its expression in the free-living state and in association with the roots of wheat. Methods We determined production of IAA and AAT activity in the mutant hisC::gusA-smR. To study the expression of hisC1, a chromosomal gene fusion was analyzed by following β-glucuronidase (GUS) activity in vitro, in the presence of root exudates, and in association with roots. Results IAA production in the hisC mutant was not reduced significantly compared to the activity of the wild-type strain. AAT1 activity was reduced by 50% when tyrosine was used as the amino acid donor, Responsible Editor: Euan K. James. J. Castro-Guerrero : A. Romero : J. J. Aguilar : M. L. Xiqui : J. O. Sandoval : B. E. Baca (*) Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Av. San Claudio s/n, Ciudad Universitaria, 72570 Puebla, Mexico e-mail: [email protected] whereas there was a 30% reduction when tryptophan was used, compared to the activity of the wild-type strain. Expression of the fusion protein was upregulated in both logarithmic and stationary phases by several compounds, including IAA, tryptophan, tyrosine, and phenyl acetic acid. We observed the expression of hisC1 in bacteria associated with wheat roots. Root exudates of wheat and maize were able to stimulate hisC1 expression. Conclusions The expression data indicate that hisC1 is under a positive feedback control in the presence of root exudates and on plants, suggesting that AAT1 activity plays a role in Azospirillum–plant interactions. Keywords Azospirillum brasilense . Aromatic amino acid aminotransferase-1 . Indole acetic production . Radical exudates regulation Abbreviations IAA Indole-3-acetic acid AAT1 Aromatic amino acid aminotransferase-1 IPyA Indole-3-pyruvic acid IAdhl Indole-3-acetaldehyde Trp Tryptophan Tyr Tyrosine Phe Phenylalanine pHPP p-Hydroxyphenylpyruvate PAA Phenyl acetic acid HPLC High-performance liquid chromatography Author's personal copy 140 Introduction The amino acid aminotransferase-1 (AAT1) protein from Azospirillum brasilense Sp7 is encoded by hisC1. AAT1 belongs to the α subfamily of Family I aminotransferases (Jensen and Gu 1996). These aminotransferases participate in several chemical reactions of cellular metabolism, aromatic amino acid biosynthesis, and function in the catabolism of amino acids to provide nitrogen and carbon sources for growth (Sergeeva et al. 2007). Individual subfamily members may be relatively specific for aromatic amino acids, but exhibit broad specificity. In addition to using aromatic amino acids as substrates, AAT1 from A. brasilense is involved in the catabolism of histidine, and produces indol-3-pyruvic acid (IPyA) from tryptophan (Trp) and phenyl pyruvic acid from phenylalanine. These compounds are precursors of indole acetic acid (IAA) and phenyl acetic acid (PAA), respectively (Baca et al. 1994; Soto-Urzúa et al. 1996; Somers et al. 2005). Azospirillum belongs to a group of soil bacteria often referred to as plant-growth promoting rhizobacteria (PGPR; Bashan et al. 2004). Several studies have described their beneficial effects on several plants: cereals; Compositae, such as sunflower; Cruciferae, such as mustard, vegetable crops; legumes, such as chick pea; clover, and alfalfa (Okon and LabanderaGonzález 1994; Bashan et al. 2004). Several mechanisms of plant–microbe interactions can affect plant growth, including N2 fixation, hormonal effects, production of nitric oxide (NO), and enhanced mineral uptake (Bashan et al. 1990, 2004; Creus et al. 2005), all of which contribute to the beneficial effects observed in plants (Bashan and de-Bashan 2010). Azospirillum can synthesize four classes of phytohormones; auxins, gibberellins, cytokinins, and abscisic acid (Tien et al. 1979; Carreño-López et al. 2000; Cohen et al. 2008; Croizier et al. 1988; MartínezMorales et al. 2003; Piccoli et al. 1996). Production of auxins is considered the most important (Spaepen et al. 2008). The biosynthesis of IAA involves distinct pathways: three pathways requiring Trp have been proposed. The main pathway in Azospirillum involves the intermediary (IPyA), [Trp IPyA IAdhl IAA] (Costacurta et al. 1994; Carreño-López et al. 2000; Spaepen et al. 2008). The first reaction of this pathway is transamination of Trp to IPyA. The Plant Soil (2012) 356:139–150 enzymatic activity of two AATs is found in several A. brasilense strains (Pedraza et al. 2004) and four A. lipoferum strains (Ruckdäschel et al. 1988). The second step is catalysis by the key enzyme phenyl pyruvate decarboxylase (PPDC; Spaepen et al. 2007a, b) to produce IAdlh, which is further oxidized to IAA. The present study identified the hisC1 gene from A. brasilense Sp7 that encodes AAT1, and examined its contribution to biosynthesis of IAA, as well as its expression in both the free-living state and in association with the plant. Materials and methods Bacterial strains and growth conditions Escherichia coli strains were grown overnight at 37°C in Luria broth (LB, Sambrook et al. 1989) containing antibiotics: 25 μg/mL kanamycin, 100 μg/mL ampicillin, and 75 μg/mL streptomycin (Sm). Azospirillum strains were grown in minimal K-lactate medium (Carreño-López et al. 2000), overnight at 30°C. The mating and isolation of the transcriptional fusion was performed as described by Carreño-López et al. (2000). E. coli S17.1 (pAB144-4A) or E. coli S17.1 (pAB144-4B) strains (Table 1), with the cassette smRgusA or A. brasilense Sp7 were used as the donor and acceptor strain, respectively. For selection of transconjugants, plates (minimal K-lactate medium with 150 μg/mL Sm) were incubated at 30°C for 2 days. Construction of plasmid clones and DNA sequencing Plasmid DNA isolation, restriction analysis, transformation and molecular cloning were performed by methods described in Sambrook et al. (1989). For Southern hybridization experiments, genomic, plasmid, and cosmid DNA were digested with BglII, EcoRI, KpnI, EcoRV, or SalI and were blotted on Hybond N+ nylon membranes (Amersham Biosciences, Piscataway, NJ) with a vacuum blotter (Fisher Scientific, Waltham, MA). DNA was fixed by exposure to a 312-nm transilluminator for 4 min. Prehybridization, labeling, and purification of probes, as well as hybridization, were performed as described by Carreño-López et al. (2000). The physical maps of the inserts for this work Author's personal copy Plant Soil (2012) 356:139–150 141 Table 1 Bacterial strains and plasmids used in this study Strains/plasmids Description Reference or source Escherichia coli DH5α E. coli S17.1 endA1 hsdR17 supE44 thi-1 λ- recA1 gyrA96 relA1 ΔlacU169 Ø80 (ΔlacZΔM15) pro, thi hsd, recA, :: RP4-2 Tc:: Mu- Km Tn7 Invitrogen, Carlsbad, CA Azospirillum brasilense Sp7 A. brasilense7445 Wild-type strain ATCC29729 Derivative of Sp7 hisC1:: gusA- smR This study A. brasilense7446 Derivative of Sp7 hisC1::smR-gusA This study Strain Simon et al. 1983 Plasmids pSUP202 Suicide vector ApR, CmR, TcR Simon et al. 1983 SuperCos 1 Cloning vector ApR, KmR, Stratagene, La Jolla, CA pCR 2.1 Cloning vector Invitrogen pBSL197 Vector pBSL carrying the cassette smR-gusA pABaat1 Derivative of pCR2.1 carrying a fragment of 432 bp of hisC1 gene This study pAB144 This study pAB144-1 Cosmid from A. brasilense Sp7 library, carrying a fragment of 20 kb harboring the hisC1 gene Derivative of pAB144 carrying a 13 kb EcoRI fragment pAB144-2 Derivative of pSUP202 carrying a 4.2 kb BglII fragment of pAB1444-2 This study pAB144-3 Derivative of pAB144-1 carrying a 4.2 kb BglII fragment, cloned in pCR2.1 This study pAB144-4A Derivative of pAB144-2 carrying the cassette hisC1:: gusA- smR inserted in the 5’ 3’ direction Derivative of pAB144-2 carrying the cassette hisC1::smR-gusA This study pAB144-4B are shown in Fig. 1. Both DNA strands were sequenced by Macrogen (Rockville, MD). To identify the hisC1 gene, we used primers Faat1 (5′ GCTGACGGTCAATCCGCTGG) and Raat1 (5′ CCGCAGGACCATCTCCGACGG) designed from the sequence of peptides LSLTVNPLGP, and YIPSEMVLR, which were obtained from AAT1 protein E C Sm Bg S K This study This study purified to homogeneity as described by Soto-Urzúa et al. (1996).The amino acid sequences of the two peptides were determined at the Stanford University Protein and Nucleic Acid Facility (Stanford, Palo Alto, CA). PCR amplification was performed according to directions of the manufacturer of Taq polymerase (Invitrogen, Carlsbad, CA), in a final volume of Bg K E pAB144-1 probe hisC1 pABaat1 V EV Bg S K Bg S V pAB144-3 1kb ORF1 ORF2 AAT1 S S ORF3 S pAB144-4A; 7445 S smR S gusA S pAB144-4B; 7446 gusA smR Fig. 1 Schematic map of the sequenced hisC1 region in the plasmid, strains and constructs. The insertion of the smR-gusA cassette is shown, and arrows indicate the direction of transcription. E EcoRI, C ClaI , Sm SmaI, Bg BglI, S SalI, K KpnI, EV EcoRV, V Vector Author's personal copy 142 25 μl, using 50 ng genomic A. brasilense Sp7 DNA. The amplification cycle consisted of 10 min at 96°C, 30 cycles of 1 min at 95°C, 1.5 min at annealing temperature of 67°C, 1 min at 72°C, and a final 10min extension at 72°C. The amplicon of 473 bp was cloned into the pCR2.1 vector (Invitrogen) as described by the manufacturer to obtain the plasmid pABaat1 (Table 1). This amplicon was used as a probe to screen an A. brasilense Sp7 library. The cosmid pAB144 was identified by DNA/DNA hybridization. pAB144-1 was obtained by digestion of pAB144 with EcoRI, hybridization with the probe aat1, and then subcloning of a 13-kb fragment into the Supercos vector (Invitrogen). The cosmid pAB144-1 was digested with enzymes Cla I, Kpn I, Sma I, or BglI after hybridization to probe aat1. A 4.7-kb BglII fragment was subcloned into pCR2.1, which was digested with the enzyme BamHI and transformed into E. coli DH5α. This plasmid was designated pAB144-2. Both strands were sequenced at Macrogen to obtain the full sequence of the hisC1 gene. The GenBank accession number is JF509704.1. Construction of trancriptional fusion and mutation of the hisC1 gene The same 4.7 bp BglII fragment from pAB144-1 was also subcloned into the suicide vector pSUP202 digested with BamHI. The ligated fragments were transformed into E. coli S17.1 to generate the plasmid pAB144-3. This plasmid was digested with KpnI and the gusA-smR cassette was obtained from plasmid pSBL197 by digestion with KpnI (Alexeyev et al. 1995). The fragments were ligated and transformed into E. coli S17.1. Both cloned directions were selected (see Table 1). E. coli strains S17.1 (pAB144-4A) and S17.1 (pAB144-4B) were used as donors and transferred to A. brasilense Sp7 to obtain transcriptional fusions (Fig. 1). The correct orientation (sense strand of gusA) was determined by Southern blot analysis as described by Carreño-López et al. (2000), using aat1 and the gusA-smR cassette as probes. IAA production and AAT1 enzyme assays IAA production was determined from bacteria grown in 10 mL K-malate minimal medium supplemented with 100 μg/mL Trp at 30°C for 48 h with shaking at 120 rpm. Plant Soil (2012) 356:139–150 The cells were then pelleted by centrifugation (10,000 g, 5 min, at 20°C). Aliquots of 20 μl supernatant were taken, and the IAA concentration was determined by HPLC as described by Carreño-López et al. (2000). Three replicates were performed, and the experiment was repeated twice. Total AAT enzyme activity was determined from the cell pellets, which was used to produce a cell-free extract described by Pedraza et al. (2004). Specific activity was defined as micromoles of product [(IPyA and phydroxyphenyl pyruvic acid (pHPP)] per minute per milligram of protein described by Diamondstone (1966). Preparation of wheat and maize root exudates To prepare root exudates, seed from wheat (Triticum aestivum var Zacatecas) and maize (Zea mays var. Criollo) were surface-sterilized by gentle shaking for 1 min in 0.2% HgCl2 with 2% Tween 20 (P1379, Sigma). The sterilized seeds were soaked six times for 30 min in sterile, demineralized water. The seeds were allowed to germinate on Petri plates containing agarwater under dark conditions at 20°C for 2 days. Seedlings were transferred aseptically to Magenta vessels (Sigma-Aldrich, St. Louis, MO) equipped with a stainless steel perforated tray and filled with 150 mL plant nutrient solution (Hoffland 1992). Germinated wheat seedlings (65) and germinated maize seedlings (25), (corresponding to 30 g) were placed on the tray with the root in the solution, and then placed in a cycle of 16-h light at 24°C and 8-h of dark at 16°C for 7 and 10 days. Root exudates were recovered aseptically and checked for bacterial contamination using LB medium before sterile filtration through a 0.45-μm disposable cellulose-nitrate filter (EMD Millipore, Bedford, MA) and then stored at −20°C until use. Only materials from exudates in which no microbial growth could be detected were used. Reporter gene expression in minimal medium and root exudates in the free living-state The activity of β-glucuronidase (GUS) and OD600 nm of A. brasilense 7445 and A. brasilense 7446 strains were determined in K-lactate minimal medium containing: Trp, Phe, pHPP, phenyl-pyruvic acid (PPA), IAA, or PAA, each at a concentration of 0.5 mM. The Author's personal copy Plant Soil (2012) 356:139–150 pH of all media was maintained at 6.8. The strains were grown in K-lactate medium overnight at 30°C with shaking, then inoculated at 0.01 OD600nm in a 125-mL Erlenmeyer flask containing 15 mL of each test medium in individual flasks and incubated at 30°C with shaking at 200 rpm. At various points during the exponential and stationary growth phases, 2 mL culture per flask were centrifuged and resuspended in K-lactate medium (for OD600nm measurements) and frozen at −20°C for later GUS activity determinations. GUS activity was measured by the method of Miller (1972) using PNPG (para-nitrophenyl-β-Dglucuronide, Sigma) as substrate. Enzyme activities were expressed as Ea =[A420(OD600nm ×t)]×1,000, where Ea is GUS activity in Miller units and t is incubation time (in minutes). When required, the total protein was determined with a protein assay reagent (Bio-Rad, Hercules, CA) using serum bovine albumin as the standard (Sigma). Erlenmeyer flasks containing 15 mL root exudates diluted to 50% with a solution described by Hoffland (1992) were inoculated as described above and incubated at 30°C with shaking until reaching 0.3 or 0.4 OD600nm. Cells were then centrifuged and GUS activity determined as indicated above. Plant cultivation and in situ detection of bacteria colonizing the root surface Wheat seeds were germinated as described above, and grown hydroponically under sterile conditions as described by Zeman et al. (1992). One-week-old wheat plantlets were inoculated with 1 mL A. brasilense Sp7 or A. brasilense strains 7445 or 7446 at approximately 108 bacterial cells mL−1 in hydroponic culture as described by Arsène et al. (1994). Five days after inoculation, root material was weighed and homogenized in a mortar with 2 mL sterile phosphate buffer (20 mM at pH 7). Activity of the reporter gene was measure in cells from 1 mL of the homogenized material. As soon as bacteria were isolated from root wheat, GUS activity was determined as described by Miller (1972). The colonization pattern of the root surfaces was estimated by the number of Azospirillum cells present in 1 mL homogenized material from wheat roots by the mostprobable number (MPN) technique. Cell counts for A. brasilense 7445 and 7446 were made in K lactate medium supplemented with Sm (150 μg/mL). In situ 143 detection of GUS activity in 2-cm root segments inoculated with Azospirillum 7445 and 7446 strains was performed as described by Arsène et al. (1994). Afterwards, the segments were stained (XGlc; 5bromo-4-chloro-indolyl-D-glucopyranoside, Sigma). After staining, the segments were examined under a light microscope (Nikon-2000), and sections were viewed and photographed using the Nikon system. Results Identification and mutagenesis of hisC1 We isolated the hisC1 gene using a PCR-generated probe with oligonucleotides designed from peptides obtained from purified and digested AAT1 protein, as described in Materials and methods. The Sp7strain library was screened and a clone including the hisC1 gene was obtained. Figure 1 describes this region. The deduced protein product of ORF1 shares homology with an acetyl transferase of the GNAT family from Rhospirillum centenum SW (YP_002297377.1) with 55% similarity, Roseobacter SpAzwK-3b (ZP_01903442) with 60% similarity, and Azospirillum B510 (AZL012950), with 88% similarity. ORF2 appears to be a hypothetical membrane protein showing homology to the deduced protein products from Aurantimonas manganoxidans SI85-A1 (ZP_01228200.1), with 61% similarity and Rhizobium etli CIAT with 56% similarity. The hisC1 gene, in a convergent orientation, displayed a deduced protein sequence with high similarity to the histidol-aromatic amino acid aminotransferases of various bacteria, in particular the gene hisC (AZL012940) coding for a histidol-phosphate aminotransferase, putatively identified in the recent genome sequence of Azospirillum spp. IB510 (http://genome.kazusa.or.jp/rhizobase). The predicted product from Azospirillum spp. IB510 shares 85% similarity with hisC1 from A. brasilense Sp7. The sequence of AAT1 indicated a protein of 360 amino acids with a predicted molecular mass of 39,248 Da; the pyridoxal 5′ phosphate binding (FLANPNNPTGTY) was also identified. ORF3 corresponded to a partial sequence of a histidine kinase, which is predicted to be a signaling protein. The conservation of gene arrangement in the genetic region identified in A. brasilense Sp7 matched the corresponding region in Azospirillum spp B510; in Author's personal copy 144 Plant Soil (2012) 356:139–150 the latter strain, this region is located on the bacterial chromosome (Kaneko et al. 2010). To analyze the contribution of AAT1 to IAA biosynthesis, we constructed an AAT1 mutant by inserting a gusA-smrR cassette in the natural KpnI site found in the hisC1 sequence. The correct cassette orientation relative to the promoter was created in strain 7445, and strain 7446 was generated by inserting the cassette in the opposite orientation. Growth of strains Sp7, 7445, and 7446 was similar (data not shown). IAA production in 7445 was lower compared with wild type Sp7, but the difference was not significant, as expected: 11.3±1.2 IAA μg/mg protein−1 for the wild type strain, and 8.9±1.1 IAA μg/mg protein−1 for A. brasilense 7445. However, total AAT activity was lower in strain 7445 than in the Sp7 strain (Fig. 2). When Trp was used as the amino acid donor, the reduction was 30% (0.023 μmol/mg protein−1) compared with the wild type (0.039 μmol/mg protein−1). When Tyr was used as the amino acid donor, the reduction was 50% (0.034 to 0.016 μmol/mg 0.05 0.045 Specific Activity AAT1 0.04 0.035 0.03 0.025 0.02 0.015 0.01 0.005 0 1 2 Fig. 2 Aminotransferase-1 (AAT) specific activity. 1 Tryptophan (Trp) AAT activity was assayed by measuring indole-3pyruvic acid (IPyA) at 330 nm (ε=10,000 cm2/mol, Granner and Tomkins 1970), at pH 8.5 with pyridoxal 5′ phosphate (1 mM), 2-oxoglutarate (1 mM), and Trp (1 mM). 2 Tyrosine (Tyr) ATT was measuring as the concentration of p-hydroxyphenylpyruvate (pHPP) at 331 nm (ε=19,900 cm2/mol, Granner and Tomkins 1970), at pH 8.5 with pyridoxal 5′ phosphate (1 mM), 2-oxoglutarate (1 mM), and Tyr (1 mM). Each bar represents the mean of three independent replicates, error bars represent the standard error of the mean, black bars Sp7 strain, grey bars 7445 strain protein−1). The results indicate a role for AAT1 in IPyA intermediate production. Chromosomal gene fusion expression in the free living state To measure the influence of individual compounds on expression of the hisC1 gene, GUS activity of the reporter gene cassette inserted into A. brasilense strains 7445 and 7446 was tested. The cassette insertion was confirmed by Southern blot analysis on A. brasilense 7445 and 7446 genomic DNA probed with the gusA-smR and aat1 DNA cassettes (data not shown). The resulting chromosomal fusion conferred the ability to produce blue colonies on medium containing the GUS indicator X-Glc; the correct orientation for expression was present only in the case of A. brasilense 7445. To check expression of the reporter fusion, GUS activity was determined in both permeabilized A. brasilense 7445 and 7446 cells. As was expected, only A. brasilense 7445 displayed activity. To further study gene expression, A. brasilense 7445 was grown in minimal medium (K-lactate) and K-lactate medium supplemented with Tyr, Trp, Phe, pHPP, IAA, or PAA. GUS activity was determined as described in Materials and methods. Expression of hisC1 was constitutive and was seen in both exponential and stationary growth phases (Fig. 3). In medium supplemented with IAA, PPA, Trp or Tyr, expression was significantly up-regulated during both phases. Up-regulation was 1.8-fold for Trp, 2.6-fold for Tyr, 2.5-fold for PAA, and 4.6fold for IAA in early stationary phase compared with expression observed in K-lactate medium. These data indicate that the compound that best functioned as a regulator was IAA, followed by PAA, Tyr, and Trp. In view of this, we tested the expression of strain 7445 grown in radical exudates derived from wheat and maize. We found that both exudates drive the transcription of the hisC1 promoter, with maize root exudates showing greater enhancement than wheat exudates (Fig. 4). Maximal bacteria growth was detected with maize root exudates at 10 days (107 CFU/mL), compared with wheat root exudates (106 CFU/mL). Currently, we do not know the concentrations of carbon and nitrogen sources or the nature of the exogenous inducer(s). Additionally, Hoffland solution was inoculated with strain 7445, and wheat exudates Author's personal copy Plant Soil (2012) 356:139–150 25000 Miller Units/mg protein-1 Fig. 3 β-glucuronidase (GUS) activity of the hisC1:: gusA fusion in the freeliving state. Bacteria were grown to both exponential and stationary phase in Klactate (KL) minimal medium supplemented with 0.5 mM Trp, Tyr, IAA and PPA or in their absence. GUS activity was determined at 12 h (white bars), 24 h (grey bars), and 36 h (black bars). The data shown are averages of three independent replicates; error bars standard error of the mean 145 20000 15000 10000 5000 0 KL were inoculated with strain 7446 strain: no GUS activity was detected in either case (1.7±0.15 MU). Chromosomal gene fusion expression in association with plants Expression of the reporter gene in a hydroponic plant was also determined using the GUS assay and MPN. The results shown in Fig. 5 indicate that activity was more significant in A. brasilense 7445 (155.3±43.5 MU) than in A. brasilense 7446 (3.9±0.15 MU), as expected from the direction of cassette insertion (Fig. 5a). The CFU of both strains was set at 107.8 to 108.01 CFU/g root, suggesting that growth of the introduced bacteria occurred in both strains to the same extent (Fig. 5b). To monitor establishment of bacteria expressing the hisC gene on wheat roots, KL + Tyr KL +PAA KL +IAA GUS expression of bacteria on roots was determined at 5 days after inoculation. When plants were inoculated with A. brasilense 7445, hisC expression was detected on roots, whereas no GUS staining was observed in roots inoculated with A. brasilense 7446 (Fig. 5c). Discussion Amino acids in the rhizosphere are of both plant and microbial origin (González-López et al. 2005). The role of amino acids produced by rhizobacteria during their interaction with plants is practically unknown, although it has been established that some amino acids, such as Met and Trp, act in soil as the main precursors for synthesis of the plant hormones 14000 Miller Units/ mg protein-1 Fig. 4 GUS activity of the hisC1::gusA fusion in the presence of exudates from wheat and maize in the freeliving state. GUS activity was determined in cells at OD600 = 0.3 or 0.4. Grey bars Wheat exudates (harvested at 7 and 10 days), black bars maize exudates (harvested at 7 and 10 days). The data shown are averages of three independent replicates; error bars standard error of the mean KL + Trp 12000 10000 8000 6000 4000 2000 0 0.3 0.4 7 Days OD600nm 0.3 0.4 10 Days Author's personal copy 146 Plant Soil (2012) 356:139–150 A C 180 i Miller units/mg protein-1/g Root 160 ii 140 A. brasilense 7445 120 100 iii iv 80 60 A. brasilense 7446 40 20 0 A.brasilense 7445 A.brasilense 7446 B 9.00 Log MPN/g Root 8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 A.brasilense 7445 A.brasilense 7446 Fig. 5a–c Colonization and expression of A. brasilense strains 7445 and 7446 in association with wheat. After 5 days of plant growth, root tips were isolated; bacteria were isolated from the root tip and then plated for determination of MPN and GUS activity as described in Materials and methods. a hisC1 promoter expression: black bar 7445 strain, grey bar 7446 strain. Values represent the average of 15 plants for each experiment; error bars standard error of the mean. b Most probable number (MPN) determination as log CFU/mL: black bar 7445 strain, grey bar 7446 strain. c Bacterial hisC::gusA expression on wheat root. In situ detection of GUS activity of wheat root segments, inoculated with A. brasilense strains 7445 or 7446 strains. Bars: i, iii 100 μm; ii, iv 5 μm ethylene and IAA, respectively (Frankenberger and Arshad 1995; Murcia et al. 1997; Kamilova et al. 2006). Biosynthesis of phytohormones by Azospirillum is documented widely, as is involvement of these substances in plant responses observed after inoculation of plants with bacteria (Dobbelaere et al. 2001; Spaepen et al. 2008). Here, we identified and studied the regulation of the hisC1 gene from A. brasilense Sp7 strain coding for AAT1, which we had previously purified (Soto-Urzúa et al. 1996). We had also demonstrated its substrate specificity and found that the enzyme can convert Trp to IPyA, pHPP to Tyr, and phenyl pyruvate to Phe as reversible reactions of AATs (Carreño-López et al. 2000; Soto-Urzúa et al. 1996). A knock-out mutant of the hisC1 gene showed a significant decrease in specific AAT activity (∼50%). Author's personal copy Plant Soil (2012) 356:139–150 However, IAA production was not as low as expected, which could happen if other AATs isoforms that contribute to IAA production are present in Azospirillum (Pedraza et al. 2004). The lack of a significant effect on IAA production of loss of hisC1 also suggests that AAT does not catalyze the ratelimiting step in the IAA biosynthesis pathway. Ge et al. (2009) identified and mutated the atrC gene from A. brasilense Y62, demonstrating that atrC was involved in IAA production; moreover, a recombinant AtrC protein expressed and purified in E. coli functioned as an aminotransferase with high substrate specificity for Trp. Trp aminotransferase activity was also found to be associated with IAA production in Enterobacter cloacae (Koga et al. 1994), and in Arabidopsis thaliana (Tao et al. 2008). In Pantoea agglomerans rhizosphere isolates, AAT activity was related to the amount of IAA produced in the presence of Trp, but not when Tyr or Phe was used as a sole nitrogen source; all isolates accumulated Trp internally and released IAA (Sergeeva et al. 2007). GUS activity was compared in the presence of two auxins, IAA and PAA, and amino acids Trp and Tyr, both of which are AAT1 substrates. hisC1, which encodes AAT1, catalyzes the first step in the conversion of Trp to IPyA, Tyr to pHPP and Phe to PPA; it is involved in Tyr biosynthesis (by the reverse reaction, pHPP to Tyr), and is up-regulated by PPA and Tyr in addition to IAA. PPA and Tyr are themselves products of the pathway. The results indicate that IAA was the best inducer, followed by PAA, and suggest a mechanism of feedback control. Another positive feedback mechanism has been described for A. brasilense Sp245 by Vande Broek et al. (1999). The expression of the key gene, ipdC, coding for the enzyme PPDC, showed increased expression in the presence of Trp at its maximum during stationary phase, as this study also demonstrated for hisC1. Also, the ipdC gene from A. brasilense strains Sp7 and SM is upregulated by different auxins such as IAA and indole-3-butyric acid (Rothballer et al. 2005; Malhotra and Srivastava 2008). In addition, the ipdC gene from Enterobacter cloacae strain UW5 showed upregulation by TyR, Trp and Phe (Ryu and Patten 2008). Despite the fact that, in A. brasilense, the addition of Trp to the culture medium strongly increased IAA production, Trp did not upregulate ipdC expression (Vande Broek et al. 1999; Carreño-López et al. 2000). Unlike ipdC 147 expression, hisC1 is upregulated by Trp, albeit to a lesser extent than with the other compounds tested. This upregulation could be due to the binding of these compounds to a transcriptional regulator at the hisC1 promoter. Such transcriptional regulation of IAA biosynthesis has been described in E. cloacae UW5 acting on the ipdC promoter (Ryu and Patten 2008). Our data confirm the additional critical role of IAA in gene expression. IAA has a profound effect on bacterial metabolism, as a signaling molecule in bacteria acting during microorganism–plant interactions (Yang et al. 2007; Spaepen et al. 2007a, b). The plant root is thought to be the major source of nutrients for microorganisms living in the rhizosphere (Lynch and Whipps 1990). Therefore, bacterial growth on compounds present in the rhizosphere is assumed to be essential for efficient colonization and establishment in the rhizosphere. In addition to measuring its expression in the free-living state, we also determined regulation of hisC using maize and wheat root exudates as growth media. Both were able to support growth, but the highest expression was observed in cells growing in root maize exudates. It is well established that exudates from different plant species provide different compounds acting as carbon and nitrogen sources for bacteria associated with plants. Kamilova et al. (2006) found that the total amount of organic acids is much higher than that of total sugar or Trp in the exudates of several plants. However, the concentration differs with plant species and plant age at exudate collection, as observed here. Reporter gene induction in vitro by maize and wheat exudates for genes involved in proline catabolism has been described in Pseudomonas fluorescens and Pseudomonas putida (van Overbeek and van Elsas 1995). Induction of the put promoter by maize exudates collected at 7 days was approximately 20fold (Vilchez et al. 2000). The extent of hisC1 expression was also determined in association with wheat. Bacteria isolated from plants after 5 days of growth showed a substantial difference between the negative control, A. brasilense 7446, and A. brasilense 7445, which carried the reporter gene fusion in the correct orientation. Additionally we tested hisC::gusA expression in situ in wheat roots. In agreement with data obtained from bacteria isolated from roots, significant expression of hisC was detected in plants after 5 days. The tip and emergence point of lateral roots were Author's personal copy 148 favored sites of bacterial microcolonies expressing the reporter gene, as also described by others (Arsène et al. 1994; Vande Broek et al. 1993). Using global analysis of P. putida genes expressed during their interaction with the maize rhizosphere, Matilla et al. (2007) found 90 rhizosphereupregulated genes: genes coding for an aminotransferase (PP3786), an aldhehyde dehydrogenase family protein (PP3281), and phenyl acetic degradation protein (PP2694), as well as genes coding for amino acid transport and metabolism of aromatic compounds. Some of the bacteria were analyzed in competitive colonization, and mutants were founds that were hampered for survival in the rhizosphere in competition with wild type. In our work, both hisC1 strains (7445 and 7446) tested were indistinguishable from each other and from the wild type strain for colonization of wheat under laboratory conditions (data not shown). Furthermore, these mutants also behaved like their parent in this study; A. brasilense 7445 and 7446 were not auxotrophic, as shown by growth in K-lactate minimal medium with NH4Cl as the sole source of nitrogen. On the other hand, both strains were able to use Tyr as sole source of nitrogen. Uncharacterized genes coding for AATs with roles in determining the competitive ability of A. brasilense in the rhizosphere may explain why no difference was observed in colonization, although no such gene conferring a host-specific effect has yet been identified. Knowledge of bacterial growth conditions in the rhizosphere is important for understanding rhizosphere colonization. The composition of root exudates was not analyzed for the presence of carbon sources and amino acids. However, the presence of compounds such as dicarboxylic acids and Trp, Tyr and Phe have been described in the exudates of wheat and maize, suggesting the availability of these particular nutrients (Phillips et al. 2004). Future work should aim to identify the regulatory protein that controls the hisC gene at the transcriptional level, as well as determine the amino acid, IAA, and PAA composition of axenically collected wheat (var Zacatecas) and maize (var. Criollo) root exudates. Acknowledgments This work was supported by a Consejo Nacional de Ciencia y Tecnología (CONACyT) Grant 49227-Z, together with Vicerrectoría de investigación y estudios de posgrado-Secretaría de educación pública (VIEP-SEP), Grant NAT-IC-1. J.G.C. was the recipient of a scholarship from CONACyT. 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