Targeted engineering of Azospirillum brasilense SM with indole ...

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Targeted engineering of Azospirillum brasilense SM with indole acetamide pathway for indoleacetic acid over-expression Mandira Malhotra and Sheela Srivastava

Abstract: Rhizospheric bacterial strains are known to produce indole-3-acetic acid (IAA) through different pathways, and such IAA may be beneficial to plants at low concentrations. IAA biosynthesis by a natural isolate of Azospirillum brasilense SM was studied and observed to be tryptophan-inducible and -dependent in nature. While our work demonstrated the operation of the indole pyruvic acid pathway, the biochemical and molecular evidence for the genes of the indole acetamide (IAM) pathway were lacking in A. brasilense SM. This led us to use the IAM pathway genes as targets for metabolic engineering, with the aim of providing an additional pathway of IAA biosynthesis and improving IAA levels in A. brasilense SM. The introduction of the heterologous IAM pathway, consisting of the iaaM and iaaH genes, not only increased the IAA levels by threefold but also allowed constitutive expression of the same genes along with efficient utilization of IAM as a substrate. Such an engineered strain showed a superior effect on the lateral branching of sorghum roots as well as the dry weight of the plants when compared with the wild-type strain. Such an improved bioinoculant could be demonstrated to enhance root proliferation and biomass productivity of treated plants compared with the parental strain. Key words: indole-3-acetic acid, tryptophan, indole-3-acetamide, iaaM-iaaH, metabolic engineering. Résumé : On sait que les souches bact ériennes de la rhizosph ère ont la capacité de produire de l’acide indole-3 acétique (AIA) par l’intermédiaire de différentes voies métaboliques et que cet AIA à faible concentration est bénéfique pour la plante. La biosynthèse de AIA par un isolat naturel de la souche SM de Azospirillum brasilense a été étudiée et s’est révélée inductible par le tryptophane et dépendant du tryptophane dans la nature. Bien que notre travail ait démontré l’implication de la voie métabolique de l’acide indole pyruvique, les preuves moléculaires et biochimiques de l’existence des gènes impliqués dans la voie de l’indole acétamide (IAM) sont absentes chez la souche A. brasilense SM. Ceci nous a amené à utiliser les gènes impliqués dans la voie de l’IAM comme cibles en ingénierie métabolique afin de générer un sentier de biosynthèse de AIA additionnel et améliorer les niveaux de AIA chez A. brasilense SM. L’introduction d’une voie IAM hétérologue comprenant les gènes iaaM et iaaH a non seulement augmenté les niveaux de IAA de trois fois, mais elle a aussi permis l’expression constitutive de ces gènes parallèlement à une utilisation efficace de IAM comme substrat. Une telle souche modifiée a démontré sa supériorité quant à la formation de ramifications secondaires des racines de sorgho ainsi que dans la production en poids sec de plants, comparativement à la souche sauvage. Cet inoculant biologique amélioré pourrait augmenter la prolifération des racines et la productivité des plants traités, comparativement à la souche parentale. Mots clés : acide indole 3-acétique, tryptophane, indole-3-acétamide, iaaM-iaaH, ingénierie métabolique. [Traduit par la Rédaction]

Malhotra and Srivastava

Introduction The rhizosphere, being a nutrient-rich zone, allows higher microbial growth than bulk soil. Rhizospheric bacteria can stimulate plant growth through the production of phytohormones, which may regulate certain physiological processes of plants, leading to consequent changes in root growth (Dobbelaere et al. 2003). It has long been known that AzoReceived 10 April 2006. Revision received 29 June 2006. Accepted 10 July 2006. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 5 December 2006. M. Malhotra and S. Srivastava.1 Department of Genetics, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India. 1

Corresponding author (e-mail: [email protected]).

Can. J. Microbiol. 52: 1078–1084 (2006)

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spirilla produce phytohormones, i.e., auxins, cytokinins, and gibberellins (Tien et al. 1979), and that inoculation of such bacteria to seeds results in an improvement of root growth in terms of root hairs and lateral roots (Dobbelaere et al. 1999). Multiple pathways for indole-3-acetic acid (IAA) biosynthesis have been documented from the rhizospheric bacterium Azospirillum (Patten and Glick 1996). The indole pyruvic acid (IPyA) pathway operates in plants like Arabidopsis (Tam and Normanly 1998) as well as in plant-beneficial bacteria like Azospirillum and Rhizobium in an inducible manner and is subjected to extremely tight regulation (Costacurta and Vanderleyden 1995; Patten and Glick 1996). However, Rhizobium, Bradyrhizobium, and the plant pathogens Pseudomonas syringae and Agrobacterium tumifaciens synthesize IAA via a constitutive pathway involving the production of indole-3acetamide (IAM) (Patten and Glick 1996). The IAM pathway has earlier been reported from Azospirillum brasilense Sp7

doi:10.1139/W06-071

© 2006 NRC Canada


based on partial homology with the iaaM gene from P. syringae (Bar and Okon 1993). However, the genes involved, iaaM and iaaH, coding for tryptophan-2-monoxygenase (TMO) and indole-3-acetamide hydrolase (IAH), respectively, have been neither cloned from any of the Azospirilla so far nor documented further. With the idea of creating a recombinant strain that could produce higher levels of IAA and be used as a superior bioinoculant, we have worked on a natural isolate of A. brasilense SM. This strain has been observed to produce IAA via the IPyA pathway in a tryptophan (Trp)-dependent and -inducible manner. This study comprises up-regulation of IAA levels by the introduction of the constitutive IAM pathway genes from a heterologous background (P. syringae) in A. brasilense SM. The consequent positive effects of such a recombinant strain on the growth of sorghum has also been demonstrated. To the best of our knowledge, this is the only report on the expression of heterologous IAM pathway genes in Azospirillum.

Materials and methods Bacterial strains and media A natural isolate of A. brasilense SM (MTCC 4037, isolated from the rhizosphere of sorghum) has been used in this study. The bacterial cultures were maintained on modified Luria–Bertani (LB*) agar (Albrecht and Okon 1980) containing ampicillin (50 µg/mL). The buffered standard succinate medium (SSM, Meyer and Abdallah 1978) was the nutritionally defined medium used. The cells were grown at 30 °C on a shaker at 200 r/min and were subcultured to an initial OD560 (Genesys 10vis, Thermo Spectronic, Rochester, N.Y.) of 0.1 (~107 colony-forming units (CFU) per millilitre) and grown for 24 h in all experiments. All chemicals used were of analytical grade. Growth conditions and IAA detection For detection of IAA, both wild-type and transformant strains were raised for 24 h in SSM with or without Trp, depending on the experimental requirement. For induction of IAA biosynthesis, cultures raised in the presence of 1 mmol/L Trp for 20 h were subsequently subcultured in 5 mmol/L Trp. To account for the variation caused by the growth, the IAA values were normalized to the cell density in each set. The amount of IAA was quantified by high-performance liquid chromatography (HPLC), as per the method of CarreñoLopez et al. (2000). The culture filtrate samples extracted with ethyl acetate were vacuum dried (Eyela, Tokyo, Japan) and reconstituted in 2 mL of methanol of which 10 µL aliquots were analyzed with a Merck Lichrospher 100 RP18e (250 mm × 4 mm, 5 µm; Darmstadt, Germany) column in a Shimadzu Class10 HPLC system (Class-VP release 6.13SP1; Kyoto, Japan). Samples were analyzed on the basis of standards (Sigma-Aldrich, St. Louis, Mo.), in methanol – 1% acetic acid (40:60 v/v) at a flow rate of 1 mL/min at 280 nm with a UV detector. For IAM feeding experiments, cultures were raised with 5 mmol/L IAM and the control sets were raised with 5 mmol/L Trp. For all other experiments, the controls consisted of cultures raised in SSM only.

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Determination of intracellular Trp The intracellular Trp level was determined according to Kupfer and Atkinson (1964), as it allows detection of indole, anthranilic acid, and Trp in the same sample at 550, 420, and 580 nm, respectively. For estimating the intracellular Trp levels, an aqueous extract of the sample was prepared by harvesting and washing the cells repeatedly with saline (0.9% NaCl) to remove exogenous Trp and by subjecting them to lysis in 0.5 mol/L NaOH. To 5 mL of aqueous extract, 1 mL of p-dimethylaminobenzaldehyde solution (60 mg/mL) was added. After thorough mixing, 3 mL of 30 mol/L H2SO4 was added, mixed again, and the samples were left undisturbed at room temperature for 1 h. To this, 100 µL of 0.1% (m/v) NaNO2 was added to allow color development. After 30 min, OD580 was read for Trp, and the concentrations were calculated based on standards prepared. Construction of the IAA-over-expressing strain of A. brasilense SM The plasmid pME3468 (Beyeler et al. 1999) carrying the iaaM-iaaH genes of P. syringae was introduced into A. brasilense SM electro-competent cells by the method of Vande Broek et al. (1989), using the MicroPulser apparatus (Bio-Rad Laboratories, Hercules, Calif.). Briefly, 40 µL of the competent cell suspension (~109 CFU/mL) mixed with 100 ng of plasmid DNA in a 0.2 cm (interelectrode gap) electroporation cuvette was exposed to a single electric pulse (25 µF capacitance and 7.5 kV/cm peak voltage). The transformation mixture was incubated in LB* for 3 h, and transformants were selected on LB* supplemented with tetracycline (10 µg/mL, selectable marker for pME3468). Transformants were characterized for presence of the plasmid by alkaline lysis (Sambrook et al. 1989) and confirmed by restricting with XbaI-NcoI (New England Biolabs Inc., Beverly, Mass.) that yielded the iaaM-iaaH fragment along with the constitutive chloramphenicol resistance promoter driving the expression of the genes on pME3468. In addition, a digoxigenin-labeled iaaH probe (a 506 bp EcoRI-NcoI fragment from pME3468) was prepared to enable detection of this gene by Southern blotting, as per the manufacturer’s instructions (Roche Diagnostics, GmbH, Germany). To determine TMO activity by nondenaturing PAGE (polyacrylamide gel electrophoresis) (Bar and Okon 1993), cell-free extracts of bacterial cultures were prepared by cell sonication (Sonics and Materials Inc., Newtown, Conn.) for 6 min at 250 W followed by centrifugation at 30 000g at 4 °C. Seed bacterization Sorghum (‘Sudex chari’) seeds were surface sterilized with 0.1% (m/v) HgCl2 for 5 min and washed thoroughly with sterile water. They were immersed in sterile water (in the case of the untreated control) or in cultures (cells were washed twice with saline to remove the residual medium and resuspended in saline prior to treatment) containing various levels of colony-forming units per millilitre of A. brasilense SM and A. brasilense SMp30 for bacterization for 30 min with slow shaking. This was done to optimize the level of inoculum to be used for seed bacterization experiments. After thorough rinsing with sterile water, growth of the seeds was monitored for 14 days by transferring them to sterile sand in © 2006 NRC Canada

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Can. J. Microbiol. Vol. 52, 2006 Table 1. Effect of exogenous tryptophan (Trp) addition on indole-3-acetic acid (IAA) production and the intracellular tryptophan levels of wild-type Azospirillum brasilense SM and recombinant A. brasilense SMp30 cultures. IAAb (µg/OD) Exogenous Trpa

A. brasilense SM

No Trp Trp at: 1 mmol/L 5 mmol/L

Trp (µmol/L)

ND

A. brasilense SMp30 0.96±0.06

A. brasilense SM 5.01±0.27

A. brasilense SMp30 5.49±0.32

4.51±0.40 11.85±0.37

8.06±0.24 36.93±0.10

14.69±0.76 26.61±1.03

16.72±0.61 27.83±0.32

Note: The values represent the averages ± the standard error from their respective mean. ND, not detected. a The triplicate sets were all raised independently in 1 mmol/L Trp and finally grown in the respective concentrations of Trp or without Trp to determine whether IAA biosynthesis in A. brasilense SM and its derivative A. brasilense SMp30 was Trp-dependent and Trp-inducible. b The IAA levels were quantified by HPLC as mentioned before and have been normalized against the respective cell density.

wide-mouthed tubes, in the culture room at 28 °C (each set included 12 seeds). Once the inoculum size was optimized, the same was used for all seed bacterization experiments. Treated seeds were transferred to sterile sand in widemouthed tubes in one set, and growth was monitored for 14 days as mentioned above. In the other set, the seeds were transferred to pots containing a soil–vermiculite mixture and maintained under controlled conditions in a greenhouse for 8 weeks. In both sets, seeds were watered with one-tenth strength nitrogen-free solution (Barbieri et al. 1986). At the end of the monitoring period, when the plants were harvested, the sterile soil collected from the rhizosphere was resuspended in saline, and an aliquot of the suspension was plated on LB* containing ampicillin (for wild-type A. brasilense SM) or tetracycline (for the recombinant A. brasilense SMp30). The data for root and shoot length, lateral root number, tiller length, and number of tillers were recorded at the end of the 14 day period. At the end of 8 weeks, the shoot length and the fresh and dry weights of the plants were determined. Statistical analysis All experiments were carried out in three independent sets and values shown are mean ± standard error from the mean. The data for root and shoot length and number of lateral roots was analyzed by the t test to determine if the observed changes after seed bacterization were significant.

Results and discussion Different IAA biosynthetic pathways have been documented from Azospirilla (Patten and Glick 1996), which have been broadly classified as Trp-dependent and Trp-independent in nature and subsequently subclassified according to the major intermediates produced. Our results have shown that IAA biosynthesis by A. brasilense SM is not only Trp inducible but also dependent on a constant exposure to this amino acid, as is evident from Table 1; a fact also confirmed by HPLC analysis of culture extracts of A. brasilense SM. The IPyA pathway is known to be an inducible pathway in both plants and plant beneficial bacteria, such as Azospirillum (Costacurta and Vanderleyden 1995). We have also observed the operation of this pathway in A. brasilense SM (data not shown). In addition, IAA levels observed in the culture supernatant of A. brasilense SM could be correlated with exogenous Trp concentrations. At 5 mmol/L Trp, an ~2.6-fold

increase in IAA along with a 1.8-fold increase in intracellular Trp concentration were observed in comparison to 1 mmol/L Trp. A further increase in Trp concentration did not increase IAA biosynthesis (data not shown). In the absence of exogenous Trp, no IAA was detected, and consequently, the intracellular Trp level dropped to 19% of that observed at 5 mmol/L Trp (Table 1). This suggested that IAA synthesized by A. brasilense SM is upregulated by the substrate, Trp. This is in contrast to the results reported by Prinsen et al. (1993) who suggested that only 10% of IAA is synthesized from Trp, while the remaining 90% is derived in a Trp-independent manner in A. brasilense SpF94. Our results also suggested that growth in the presence of Trp led to an increase in the intracellular Trp pool, which perhaps is being utilized by the bacterium to synthesize IAA. Also, the exposure to a high concentration of Trp would likely keep the Trp pool saturated, and surplus Trp could be channelized towards the production of the secondary metabolite, IAA. This property may be useful in the natural environment, as Trp has been shown to be a component of the root exudates exuded by the plants (Dakora and Philips 2002). HPLC analysis of culture extracts of A. brasilense SM showed no IAM (Fig. 1a), indicating that TMO activity is lacking. This enzyme could also not be detected in cell-free extracts by nondenaturing PAGE (data not shown). The Southern analysis of genomic DNA with a probe from the iaaH gene also suggested the absence of IAM pathway (Fig. 2b). Thus, the lack of conclusive molecular and biochemical evidence for either of the genes, iaaM and iaaH, led us to use these genes as targets for metabolic engineering of A. brasilense SM. For metabolic engineering of A. brasilense SM with IAM pathway genes, the broad host-range pBBR1MCS-based plasmid, pME3468 was electroporated into the cells. This plasmid carried both the genetic determinants of the pathway– iaaM and iaaH, coding for TMO and IAH, respectively, under the constitutive chloramphenicol resistance promoter. A tetracycline resistant transformant of A. brasilense SM, A. brasilense SMp30, was confirmed to carry pME3468 by restriction digestion of the isolated plasmid DNA with XbaINcoI, which yielded a ~4.0 kb fragment carrying the desired genes, not seen in the case of the wild-type A. brasilense SM (Fig. 2a). Moreover, the recombinant strain, A. brasilense SMp30, gave appropriate signals with the digoxigeninlabeled iaaH probe of P. syringae on Southern analysis of its genomic DNA (Fig. 2b) unlike the wild type, which showed © 2006 NRC Canada


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Fig. 1. High-performance liquid chromatography analysis of ethyl acetate extracts of culture filtrates of Azospirillum brasilense SM (a, c, e) and its iaaM-iaaH carrying derivative A. brasilense SMp30 (b, d, f) grown without tryptophan (Trp) (a, b), with 5 mmol/L indole acetamide (IAM) (c, d), or with 5 mmol/L Trp (e, f). Ten microlitre aliquots were analyzed, and the peaks observed were correlated with that obtained for the indole-3-acetic acid (IAA) standard on the basis of retention time (data not shown). Solid arrows represent IAA and broken arrows show IAM. IAA was observed in Figs. 1b and 1d compared with Figs. 1a and 1c, wherein no IAA was observed (values in the figures represent actual concentration of IAA). IAA levels observed in Fig. 1f were almost threefold higher than those observed in case of Fig. 1e. Note: The scale of the chromatograms is different in each case, so the concentrations of the products were calculated prior to confirming the difference between extracts of A. brasilense SM and A. brasilense SMp30. In addition, the concentration of IAA in the samples is represented in micrograms per optical density (µg/OD) to account for the variation in growth.

no signal. This suggested that A. brasilense SMp30 stably maintained the plasmid carrying the IAM pathway genes. pBBR1MCS-based vectors have been shown to be stable in A. brasilense (Rothballer et al. 2005). Azospirillum brasilense SMp30 was screened for its IAA production and intracellular Trp levels. It was observed that

while IAA produced by this strain was higher than that of the wild type, Trp levels were not significantly different between the two strains at any concentration of exogenous Trp tested (Table 1). The IAA produced by A. brasilense SMp30 was threefold higher than that produced by A. brasilense SM in the presence of Trp. Interestingly, A. brasilense SMp30 © 2006 NRC Canada

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Fig. 2. Recombinant nature of Azospirillum brasilense SMp30. (a) XbaI-NcoI restriction profile of the plasmid extracted from transformant A. brasilense SMp30 compared with A. brasilense SM. Lanes: 1, 1 kb ladder; 2, undigested A. brasilense SMp30; 3, A. brasilense SMp30 digested with XbaI/NcoI yielding ~4 kb fragment carrying iaaM-iaaH; 4, undigested A. brasilense SM; 5, A. brasilense SM digested with XbaI/NcoI. (b) Autoradiogram showing the undigested genomic DNA of A. brasilense SM (lane 1) and A. brasilense SMp30 (lane 2) probed with a digoxigenin-labeled region of iaaH. Signals were obtained only with A. brasilense SMp30.

Table 2. Effect of different inoculum size of wild-type Azospirillum brasilense SM and the recombinant strain A. brasilense SMp30 during seed bacterization of sorghum seeds. Root length (cm)

Shoot length (cm)

No. of lateral roots

Inoculum size (CFU/mL)

A. brasilense SM

A. brasilense SMp30

A. brasilense SM

A. brasilense SMp30

A. brasilense SM

A. brasilense SMp30

2×106 2×107 2×108 2×109

6.01±0.09 7.24±0.076 8.10±0.06 7.91±0.05

6.62±0.13* 7.86±0.06* 9.23±0.12* 8.79±0.13*

14.66±0.15 16.13±0.18 17.44±0.19 17.14±0.25

14.86±0.08 16.33±0.08 17.46±0.16 17.21±0.15

13.13±0.67 17.12±0.61 23.13±0.74 20.63±0.60

18.13±0.44* 24.12±0.30* 31.75±0.37* 32.63±0.57*

Note: The values represent the average of 12 plants per set with the standard error from their respective mean (SEM). *Values differ significantly from that of plants treated with wild-type A. brasilense SM.

was also observed to produce IAA independently of exogenous Trp, albeit in extremely low (1.18 µg/OD) amounts (Fig. 1b). As A. brasilense SM lacks the IAM pathway, the observed change in the level of IAA produced by strain A. brasilense SMp30 is likely attributable to the expression of the IAA biosynthetic genes from a constitutive promoter on a multicopy plasmid. In addition to this, higher levels of IAA produced by A. brasilense SMp30 still remained Trpdependent, like A. brasilense SM, as is clear from Table 1. As the introduced genes were derived from P. syringae, their expression needed to be verified in Azospirillum. To confirm that the introduced pathway is operational, the wildtype and recombinant strains were screened for IAH activity by feeding the cultures with IAM and by monitoring the consequent IAA production. With A. brasilense SM raised on IAM, while no IAA was detected, IAM could be observed in the culture filtrate, indicating that the same is not being utilized by the bacterium (Fig. 1c). However, in A. brasilense SMp30 culture extracts raised similarly large amounts of IAA could be detected (114.09 µg/OD, Fig. 1d) along with left-over and unused IAM. This was ~threefold higher than similar cultures raised with Trp (36.19 µg/OD IAA, Fig. 1f), which could also be translated to a ~96-fold increase in comparison with cultures raised in SSM only (1.18 µg/OD IAA, Fig. 1b). This confirmed that the heterologous genes introduced into A. brasilense SM were indeed functional in A. brasilense SMp30 and upregulated IAA

biosynthesis when provided with the substrate. Strongly elevated IAA levels in medium containing Trp have been observed in modified strains of Pseudomonas fluorescens CHA0 carrying pME3468, and such strains were found to stimulate the root growth of cucumber in comparison with the parental strain (Beyeler et al. 1999). We have also observed the stimulatory effect of exogenous Trp on IAA production not only by the wild-type A. brasilense SM (Fig. 1e) but also its genetically engineered derivative, A. brasilense SMp30 (Fig. 1f). Thus, the HPLC analysis proves conclusively that in contrast to A. brasilense SM, the enzymes TMO (Figs. 1b and 1f) and IAH (Fig. 1d) are indeed functional in A. brasilense SMp30. The wild-type strain not only showed an absence of IAM from culture extracts even when supplemented with Trp (Fig. 1e) but also of IAA when exposed to the specific substrate IAM (Fig. 1c). Bacterization of sorghum seeds with increasing inoculum concentrations of A. brasilense strains SM and SMp30 (106–109 CFU/mL) had a pronounced beneficial effect of up to 108 CFU/mL, beyond which no further improvement in the plant response was seen. Thus, 108 CFU/mL was subsequently used for all bacterization experiments. It is worth noting that the plant response to A. brasilense SMp30 treatment was better than that seen for A. brasilense SM at all the inoculum concentrations tested (Table 2). These results are in contrast to those shown by Dobbelaere et al. (1999), wherein they show decreased root development with increasing inoculum © 2006 NRC Canada


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Fig. 3. Effect of seed bacterization on sorghum seeds. (a) Plant response parameters of root length (thatched box), shoot length (white box), and number of lateral roots (gray box). The average of 12 seeds is represented. The seeds were treated with cultures containing ~108 CFU/mL, and after termination of the experiment at 14 days, (2.55±0.161) × 106 CFU/mL and (2.62±0.183) × 106 CFU/mL were obtained on plating rhizospheric soil suspensions of Azospirillum brasilense SM and A. brasilense SMp30-treated seeds, respectively. (The untreated seeds did not show the presence of any bacteria.) The data were analyzed statistically by t test. * and † represent values significantly different from the untreated set and wild-type A. brasilense SM, respectively, at P ≤ 0.05. (b) Response of treated plants after 8 weeks. 1, A. brasilense SM; 2, untreated control; 3 and 4, A. brasilense SMp30. The data for average shoot length and average dry mass of 12 plants per set, respectively, are given. Note: As the trend of the data observed remained the same between all the sets, one representative treatment set has been shown. In addition, each experiment has been repeated thrice and the trend remained the same in all three cases.

concentrations of A. brasilense Sp245 on wheat. The over-expression of IAA by the recombinant strain can thus be correlated with its plant response. While treatment of seeds with the wild-type strain showed remarkable improvement in overall root response compared with the response in untreated seeds, those treated with the transformant A. brasilense

SMp30 resulted in a significant improvement of lateral branching of roots compared with the wild type (Fig. 3a). Thus, the increased IAA produced by A. brasilense SMp30 could be reflected in a superior root response. The presence of the A. brasilense cells (from strains SM and SMp30) was confirmed at the end of the monitoring period © 2006 NRC Canada

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by screening the respective rhizospheric soil suspensions. When we tested the efficacy of A. brasilense SMp30 treatment over an extended duration of 8 weeks, it was observed that growth of the treated plants was better than that of the untreated plants throughout. The average shoot length and dry weight of the plants increased in the sets treated with A. brasilense SM compared with that in untreated plants, while the same parameters when observed following treatment with A. brasilense SMp30 were still higher than in plants treated with A. brasilense SM (Fig. 3b). IAA at low concentrations is known to beneficially affect the associated plants by promoting the root length as well as the development of lateral roots and root hairs (Dobbelaere et al. 2003). Root proliferation along with the dry weight of the plants have thus been used as the important parameters to study the effect of bioinoculant strains. Thus, as the IPyA pathway is functional in A. brasilense SM (data not shown), the recombinant strain SMp30 is able to utilize the Trp pool more efficiently because of the operation of both the IPyA and IAM pathways. The improvement in lateral branching of sorghum roots and the increased dry weight of the plants after treatment with A. brasilense SMp30 could be attributed to the increased IAA production. That the IAA production and its positive effect on the host plant by A. brasilense SMp30 is over and above the effect shown by the wild-type strain SM suggests that such metabolic engineering can lead to highly improved bioinoculant strains.

Acknowledgements We thank Dr. Anil K. Tripathi (Banaras Hindu University, India) and Dr. Dieter Haas (Universite de Lausanne, Switzerland) for giving us the Azospirillum brasilense strain and plasmid pME3468, respectively. MM acknowledges the fellowship provided by Council of Scientific and Industrial Research, India. The authors also acknowledge financial assistance by the Department of Biotechnology, Government of India to SS, and the facilities supported by University Grants Commission (SAP) and Department of Science and Technology (FIST) in Department of Genetics, UDSC.

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