Characterization of Two Aromatic Amino Acid Aminotransferases and Production of Indoleacetic Acid in Azospirillum Strains

CHARACTERIZATION OF TWO AROMATIC AMINO ACID AMINOTRANSFERASES AND PRODUCTION OF INDOLEACETIC ACID IN AZOSPIRILLUM STRAINS

B. E. BACA, L. SOTO-URZUA, Y. G. XOCHIHUA-CORONAand A. CUERVO-GARCIA Department0 de Investigaciones Microbiologicas, Instituto de Ciencias, Universidad Autonoma de Puebla, Apartado Postal 1622, 72000 Puebla Pue, Mexico

(Accepted 30 May 1993)

Sunnnary-Experiments were made to quantify indole-3-acetic acid (IAA) excreted by different wild type strains of Azospirilhun spp. The microorganisms produce IAA, during the late stationary growth phase and show a significant increase in IAA production when tryptophan is present in the medium. Under these conditions with the A. brusilense strain UAP 14, we observed two aromatic amino acid aminotransferases. Their enzymatic activity and electrophoretic mobility under non-denaturating conditions were character- ized using cell-free extracts. Biochemical studies showed that these two enzymes can be distinguished by their electrophoretic mobilities. These enzymes are constitutively produced and they are not repressed by tyrosine or by tyrosine plus phenlylalanine.

INTRODUCTION described in Pseudomonas savastanoi and Agrobac- terium tumefaciens. In Azospirillum this process is Soil bacteria of the genus Azospirillum live in associ- poorly understood. Hartman et al. (1983) and Zim- ation with the root zones of grasses and other plants, mer et al. (1988) identified indole-3-pyruvic acid among which are the economically-important culti- (IPyA), indole-3-acetaldehyde, and IAA as the sub- vated plants maize, wheat, barley, rye, oat and rice. stances that are excreted by A. brasilense Sp7 strain, Reports indicate that Azospirillum can stimulate the suggesting that the transamination pathway is present growth of the host plants (Elmerich, 1984; Okon, in A. brusilense. Ruckdaschell et al. (1988) described 1985). The bacterium may affect plant growth by four amino acid aminotransferases from A. lipoferum. nitrogen fixation (Dobereiner and Pedrosa, 1987). We have found that Azospirillum spp wild type Inoculation with Azospirillum may also improve the strains produced IAA and we demonstrated the efficiency of applied mineral nutrients by helping the presence of two aromatic amino acid aminotrans- plant scavenge limiting nutrients (Lin et al., 1983). ferases associated with the production of IAA in A. These effects have been proposed to be related to the brasilense strain UAP 14. production of plant growth regulators by Azospiril- lwn spp (Morgenstern and Okon, 1987). The pro- MATERIALSAND METHODS duction of phytohormones by the microorganism seems to play an essential role in the Azospiril- Bacterial strains and growth conditions lum-plant interaction. It has been shown that bac- The A. brasilense H18, UAP 14, and A. lipoferum terial production of the phytohormone IAA is UAP 06 wild type strains used were obtained from involved in interactions between microorganisms and Professor J. Caballero-Mellado (Universidad Au- plants (Morris, 1986). In grasses, legumes and tomato tonoma de Puebla, Mexico) and A. brasilense AZ plants, as tested in Petri dishes or pouch systems, 30 INTA was supplied by Professor E. A. Rodriguez- inoculation with Azospirillum increased root diam- Caceres. To estimate IAA production Azospirillum eter, density, and length of root hairs (Jain and strains were grown in a minimal medium (Jain and Patriquin, 1984; Okon et al., 1988). Experiments with Patriquin, 1985), amended with 0.1% of NH,Cl, or suitable concentrations of IAA produced, changes in 0.1% of KNO, as a nitrogen source. The medium was root tip morphology comparable to those produced supplemented with 100 pg ml-’ sterilized tryptophan, by Azospirillum, suggesting the involvement of auxin tyrosine or tyrosine plus phenylalanine as indicated; produced by Azospirillum on root morphology (Jain 50 ml of medium was inoculated with 100 ~1 ml-’ of and Patriquin, 1985). 109cfu ml-’ of different Azospirillum strains and Several pathways have been reported for the con- grown in a 250 ml flask, for 48, 72 or 96 h at 32”C, version of tryptophan to IAA by bacteria (Van with shaking at 160rev min-r. To determine the Onckelen ef al., 1986). The most studied pathway aromatic amino acid aminotransferase activity, A. involved conversion via indole-3-acetamide (IAM) brasilense strain UAP 14 was grown in a 500 ml flask

57 58 B. E. BACA et al.

~n~ining 250 ml of medium, inoculated with 2 ml of perature was 45°C. The reaction was stopped with 1.6 x 109cfuml-‘, then samples were collected at (5 M) KOH and the amount of pHPP produced from different times as indicated, Bacterial growth was tyrosine was estimated by its absorbance at 331 nm. measured photometrically with a Klett Summerson Production of IPyA was measured following the (green filter) or by plating and viable cell counting. method of Truelsen (1972). The reaction temperature was 45°C. The formation of IPyA was measured at IAA determination 328 nm. Bacterial cultures were harvested by centrifugation at 10,OOOgfor 10 min at 4°C. The supernatants were Polyacrylamide gel electrophoresis and aromatic extracted with ethyl-acetate (Tien et al., 1979). The amino acid aminotransferase activity staining ethyl-acetate extracts were evaporated to dryness at Electrophoretic analyses under non-denaturing 39°C under vacuum and residue redissolved in 2 ml of conditions was done in a discontinuous system, the methanol and the IAA ~n~ntration was determined running buffer being T~sglycine (25 rn&&and 192 M, by the Salkowski reaction (Stahl, 1969). pH 8.3). The running gel consisted of 10% acryl- Cell-fee extracts preparation amide and the stacking gel was made from 4% acrylamide. All procedures were done at 4°C. Protein Culture of A. brasilense strain UAP 14 were har- (100 pg) extract was loaded in each well and tetra- vested at 48, 72 or 96 h by centrifugation at 10,OOg zolium dye reduction was used to stain aromatic for 10min at 4°C. Subsequent procedures were also amino acid aminotransferase activity. Gels were made at 4°C. The cells (2 g wet wt) were washed in stained at room temperature in the dark. The staining 10 ml (3 M) NaCl. After being stirred for 1 h the cells solution contained: Tris-HCl buffer (0.1 M, pH 8.6); were centrifuged at 10,000 g for 10 min; the super- tryptophan or another L-amino acid, 5.5 mM; pyri- natant was discarded and the cells were resuspended doxal phosphate, 0.2 mM; phenazine methosulfate, in 20 ml of TES buffer pris-NC1 (SOmM, pH 8.5), 98 F”M; 2-oxoglutaric acid, 12.5 mM; and nitroblue EDTA, 50mna; NaCl, 50m~] and lysed with tetrazolium, 0.6 mh9. lysozyme (5~~grnl-‘) the mixture was stirred for 18 h, then centrifuged at 10,000g for 20 min. The Protein ~terminations supematant which is referred to as crude extract, was brought to 30% saturation with solid ammonium Protein was determined by the method of Lowry sulfate. After stirring for JOmin, the sample was et al. (1951) using bovine serum albumin as a stan- centrifuged at 10,OOOgfor 10min. The supernatant dard. was brought to 70% saturation with solid ammonium sulfate. After stirring and centrifugation as before the RESULTS pellet was resuspended in 2 ml of a buffer [Tris-HCI (50 mhr, pH 8.5); dl-dithiothreitol (DTT), 1 mM, Determination of IAA in the culture medium of MgCl,. 7H,O, IOmM, pyridoxal phosphate, lOmhr, Azospirillum sodium azide, 0.02% and glycerol, lo%]. The enzyme We studied IAA accumulation in the culture solution was stored at 4°C. medium produced by Azospiri~lum wild type strains, to understand the influence of nutrient conditions on Enzyme activities assays the synthesis of ph~oho~one by these microorgan- Tyrosine aminotransferase and phenylalanine isms. IAA production was influenced by the nutrient aminotransferase activities were determined by content of the medium and by the incubation time as measuring the p-hydroxyphenyl pyruvate @HPP) shown in Table 1. IAA produced by Azospirillum was and phenyl pyruvate (PP), produced using the pro- released continuously, especially when the bacteria cedure of Diamondstone (1966). The reaction tem- were grown in medium supplemented by the amino

Table 1. IAA production by AzospiriNum spp wild type strains pg MA ml-’ of the culture medium* NH&it’ KNO,tb NH&l-Trp’ KNO,-Trpd 48 72 96 48 72 96 48 72 96 48 72 9s strains A. iipofenun UAP 06 0.02 0.07 0.15 ND$ 0.15 1.1 0.27 313 33.1 7 38 54 A. bradense AZ 30 INTA 0.04 0.02 0.04 ND 0.2 0.88 0.04 5.3 29.7 34 39 45 H18 0.03 0.05 0.27 ND 0.15 0.61 4.3 27 33.5 23.5 25.5 30 UAP 14 0.16 0.4 0.8 ND 0.1 0.13 15 48 63.5 5.5 25 64 lIAA concentration as measured with the Salkowski reaction. (Data represent the means of three experimental determinations+ standard errors: ‘It5 x IO-’ to +0.015; b+0.04S to J20.2; ‘f0.56 to +4.3; df 1.2 to 6.7.) t’fhe medium contained either 260 mg N I-’ as NH,CI or 140 mg N I-’ as KNO,. fND is not determined. $Hours of incubation. Aromatic aminotransferase in Azospirillum strains 59

(l.OOOE+ 08) 250 225 (LOOOE + 07)

(l.OOOE+ 06)

(l.OOOE+ 05)

(l.OOOE+ 04) 50

(l.OOOE+ 03) 0 0 5 10 15 20 25 30 35 40 45 50 Hours Fig. 1. Growth curves of Azospiriffum spp strains were grown on Jain and Patriquin medium supplemented with 100pg ml-’ L-tryptophan. Log number of viable cells ml-‘: HI8 (*-*); AZ 30 INTA (O-0); UAP 14 (O-0) A. lipoferum UAP 06 ( + - + ). Dotted lines absorbance as Klett units. acid precursor. IAA excreted in medium containing 50pgml-’ with the addition of tryptophan to the KNO, was about 2-10 fold (0.139-1.15 pgml-‘) as medium. compared to IAA produced in medium supplemented Aminotransferase activities were determined in with NH,Cl, in A. lipoferum UAP 06, A. brasilense crude extracts and in precipitated extracts (30-70% AZ 30 INTA, and in A. brasilense H 18 strains, but of ammonium sulfate saturation) [Fig. 2 (C)l. Trypto- this effect was not observed in A. brasilense strain phan was used as an aromatic amino acid donor, and UAP 14. A significant increase in IAA production 2-oxoglutaric acid as the amino group acceptor in the was observed when the medium was supplemented reaction mixture. Specific activities in extracts from with tryptophan, 5&100 fold higher (30-64 pg ml-‘) cultures without amino acids ranged from 16 to as compared to IAA produced in KNOj or NH,Cl 22 nmol of IPyA mgg’ protein ml-‘. However, in the supplemented medium. With the amino acid precur- presence of amino acids added to the culture medium, sor added at the beginning, it was also observed that the specific activities ranged from 27 to 38 nmoles of IAA liberation was stimulated within 48 h. Azospiril- IPyA mg-’ protein ml-‘. lum wild-type strains grow at similar rates (Fig.1). Therefore, differences in IAA production are prob- Polyacrylamide gel electrophoresis (PAGE) of aro- ably not due to non-normal growth (Table 1; Fig. 1). matic amino acid aminotransferase When protein extracts from cultures grown under Kinetics of ZAA production and spectfk activity of four different nutrient conditions were subjected to aromatic amino acid aminotransferases (AAT)from A. electrophoresis on non-denaturing gels and were brasilense strain UAP 14 stained for AAT activity two bands were detected Previous data have suggested that IAA biosyn- (Fig. 3) both convert tryptophan to IPyA in the thesis in Azospirillum could occur through an IPyA presence of 2-oxoglutaric acid. Omitting either intermediate derived from tryptophan by an amino- tryptophan or 2-oxoglutaric acid from the mixture transferase activity (Hartmann et al., 1983; Zimmer, reaction completely eliminated the two bands. Substi- et al., 1988). To examine this biosynthetic pathway tution for tryptophan by either tyrosine or phenyl- we investigated IAA production from A. brasilense alanine [Fig. 4 (B)] revealed the same two activity strain UAP 14 grown at four different growth con- bands. Only the large band showed aminotransferase ditions for 72 h [Fig. 2 (A)]. The IAA excreted by A. activity when histidine was used as amino donor brasilense UAP 14 varied from 0.05 to 2.6 pg ml-‘, [Fig. 4 (C)l. however, the two AAT isoenzymes did when cultured in a medium supplemented with tyro- not show aminotransferase activity when valine, sine, phenylalanine plus tyrosine or without amino isoleucine or aspartate (Fig. 5) were used as donor acid. Under such growth conditions IAA production substrates. Protein extracts derived from cultures was almost the same, when compared on the basis of grown with tryptophan, tyrosine or tyrosine plus biomass as determined as wet weight [Fig. 2 (A) and phenylalanine tested for AAT activity showed two (C)l. However, IAA production increased from 1.6 to isoenzymes pattern (Figs 3 and 4). This result indi- 60 B. E. BACAef a/. cates that both of these two AATs are not regulated Azospiri~lum wild-type strains excreted IAA in by the aromatic amino acid added to the medium. stationary phase culture. This concentration of IAA (20-88 ng ml-‘) is within a physiologicaIly active

DISCUSSION range that may affect plants roots (Plazinski and Roife, 985). Also this production is further stimulated We have studied the excretion into culture medium by the addition of tryptophan to the medium. Both of IAA by Azospirilium spp, in order to know the species produce large quantities of IAA in the pres- influence of nutrient conditions on the synthesis of ence of an amino acid precursor. These data agree phytohormones. The data obtained showed that the well with those communicated by others for the A. presence of KNO, has little effect on IAA production brasilense Sp7 strain (Tien et al., 1979; Jain and as compared with NH,CI. Tien et al. (1979), also Patriquin, 1985). In our tests A. brasilense strain IJAP reported that combined N has little effect on the 14 was the best IAA producer; it is interesting to note production of IAA by A. bradense. Moreover that this bacte~um was isolated from rhizosphere soil

IAA production from A. brasilense UAP 14 strain

60 (A) 55 F

0 12 24 36 48 60 72 Hours

Biomass wet weight A. h&lease UAP 14 strain Specific activity AATs A. brasiiense UAP 14 strain lor W

0 12 24 36 48 60 72 0 12 24 36 48 60 72 Hours Hours Fig. 2. (A) IAA production of A. brdense strain.UAP 14 grown in a medium supplemented with tyrosine (*--‘); tryptophan (O-0); tyrosine plus phenylalanine ( + - + ); or without amino acid O-J-_O). (B) Biomass as wet weight strain UAP 14 grown under same nutrient conditions. (C) Total specific activity of aromatic amino acid aminotransferase, one unit catalyzes the formation of I PM of IPyA mg-’ protein ml-‘, media conditions tyrosine (*-*); tryptophan (C-0); tyrosine plus phenylalanine ( + - + ); or without amino acid (O-c]). Aromatic aminotransferase in Azospirillum strains 61

AATt AATZ

Fig. 3. Non-denaturing PAGE of AATs from cell-free extracts of A. brusilense strain UAP 14. Nutrient conditions: cultures grown without amino acids (wells 1 and 2 ); with tryptophan (wells 3 and 4); tyrosine (wells 5 and 6); tyrosine plus phenylalanine (wells 7 and 8). Wells 1, 3, 5 and 7 were crude extracts; wells 2,4,6 and 8 were 30-70% ammonium sulfate fractions. Gel stained with tryptophan as amino acid donor.

from a cactaceous plant (Mascarua-Esparza et al., to the endogenous synthesis of tryptophan. However 1988). the amounts of IAA production were similar to those Strain UAP 14 was compared in its ability to obtained in the absence of either tyrosine or tyrosine produce IAA in four different nutrient conditions. plus phenylalanine. On the other hand, large amounts Our results show that this strain released small of IAA were present in the culture medium when the amounts of IAA in minimal medium, and minimal bacterium UAP 14 was grown in the presence of medium supplemented with tyrosine and phenyl- tryptophan. To know if aromatic amino acid amino- alanine. In E. coli, the aromatic amino acid aminotransferase (AAT) could be involved in biosynthesis transferase is repressed by tyrosine (Gelfand and of IAA we studied and identified aminotransferase Steinberg, 1977; Powell and Morrison, 1978). In view activity that reacted with tryptophan in crude extracts of the results obtained with this bacterium, the amino and the ammonium sulfate fractions. The enzymatic acid tyrosine was chosen because it could presumably activity was little affected by the type of aromatic inhibit tyrosine and phenylalanine biosynthesis as is amino acid added to culture medium. Indeed a slight done by E. cd, and, therefore, diverts chorismic acid increase in the specific activity was observed when

A 1 2 3 4 5 6 7 8 9 10 11 12

AAT, -“, AAT, - m .’ ,.

Fig. 4. Non-denaturing PAGE of AATs from cell-free extracts of A. brasdense strain UAP 14. Nutrient conditions: cultures grown with tyrosine. Gel A, wells 1 and 2; cells harvested at 18 h; wells 3 and 4; 24 h; wells 5 and 6; 36 h; wells 7 and 8; 48 h; wells 9 and 10; 72 h. Gel stained with tryptophan as amino acid donor; wells 11 and 12 same as 9 and 10 but stained without amino acid donor as a negative control. Gel B, cells harvested at 18, 24, 36, 48, or 72 h respectively and the gel stained with tyrosine as amino acid donor. Gel C, the cell-free extracts as same as gel B but stained with histidine as amino acid donor. 62 B. E. BACA etal.

Fig. 5. Non-denaturing PAGE of AATs from extracts of Azospiriilum brasilense strain UAP 14. The nutrient conditions are the same as noted in Fig. 4. Cells harvested at 24,36,48 or 72 h respectively. Gel A, wells: 1, 24h; 2, 36 h; 3, 48 h, and 4, 72 h. Gel stained with valine as amino acid donor. Wells 5 and 6 the amino acid donor was omitted as a negative control. Wells 7 and 8 the amino acid donor was tryptophan. Gel B, stained with aspartic acid as amino acid donor; Gel C, stained with isoleucine as amino donor.

tryptophan, tyrosine or tyrosine plus phenylalanine medium. Indeed, data reported in Figs 3 and 4 clearly were added to the medium as compared with specific demonstrate that AATl and AAT activities are not activity obtained in a minimal medium without repressed by tyrosine. These results suggested that amino acids. The addition of tryptophan to the both enzymes are constitutive. However, in cells medium (which promotes a dramatic increase in IAA grown with tryptophan, tyrosine, or tyrosine plus synthesis of A. brasilense UAP 14 strain) did not phenylalanine, total AAT activity had doubled. The show any significant effect on the aminotransferase inability of the aromatic amino acids (phenylalanine activity. and tyrosine) to function as regulatory effecters for To identify the aminotransferase enzymes that IAA biosynthesis suggests that IAA synthesis in A. react with tryptophan, crude extracts and ammonium brasilense may be not subjected to coordinated regu- sulfate fractions were run on PAGE under non-de- lation of phenylalanine and tyrosine synthesis. These natured conditions. Our data clearly demonstrated results suggest that IAA biosynthesis could be regu- the presence of two aromatic amino acid aminotrans- lated by the tryptophan pool in cells. ferases (AATl, and AAT2). Only one of these two isoenzymes showed activity with histidine as sub- Acknowledgements-We thank Dr Miguel Lara F., Depar- strate [Fig. 4 (C)J and might be involved in the tamento de Biologia Molecular de Plantas, Institute de Biot~olo~a-UNAM, for helpful suggestions and critical catabolism of this amino acid. Both enzymes could be reading of the manuscript. This work was partially sup- involved in IAA production. Neither of these en- ported by Subdireccion de Investigation Cientifica y Supera- zymes show activity with branched chain amino acid cion Academica de la SEP. or as an aspartate aminot~nsferase (Fig. 5). The A. brasilense A‘ATl enzyme unlike the E. coli enzyme (Powell and’Morrison, 1978), showed catalytic ac- REFERENCES tivity when histidine replaced tyrosine, phenylalanine Diamondstone T. I. (1966) Assay of tyrosine transaminase or tryptophan as the amino acid donor. Similar activity by conversion of ~-hydroxyphenyIpyruvate to activity has been detected for some AATs from R. hydroxy~~aldehyde. A~aiyt~cal Eiochem~stry 16, 395-401. leguminosarum (Perez-Galdona et al., 1989). We de- Dobereiner J. and Pedrosa F. 0. (1987) In Nitrogen Fixing- tected two other aminotransferase activities that used Bacteria in Non-Leguminous Crop Piants. Brock Springer aspartate, valine and isoleucine as amino acid donors Series in Contemporary Bioscience. Science Tech., (Fig. 5, top arrows). Madison. Our work showed that unlike E. coli (Gelfand and Elmerich C. (1984) Molecular biology and ecology of dia- zotrophs associated with non-leguminous plants. Steinberg, 1977) and A. tumefaciens (Liu and Kado, BiolTechnology 2, 961-978. 1979), AAT activity in A. brasilense strain UAP 14 is Gelfand D. H. and Steinberg R. A. (1977) Escherichia cob’ not affected by the presence of tyrosine in the growth mutants deiicient in the aspartate and aromatic amino Aromatic aminotransferase in A.zuspirillum strains 63

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