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Keywords:

  • Indole-3-acetic acid synthesis;
  • IAA mutants;
  • Azospirillum brasilense;
  • Tn5

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

The molecular genetics of indole-3-acetic (IAA) synthesis and regulation in Azospirillum brasilense was investigated in this study. Tn5 mutagenesis was performed and five mutants with decreased IAA production were isolated. Five Tn5-inserted genes from these mutants were cloned and sequenced. Four genes were reported for the first time to be involved in IAA production, namely, atrA, ftsA, omaA and aldA that code for GntR-family transcriptional regulator, iron-binding protein component of ABC-type Fe3+ transport system, outer membrane protein, and aldehyde dehydrogenase, respectively. In addition, two genes atrB and atrC, with predicted proteins that showed high homology to aminotransferases, were cloned from the downstream of atrA in this bacterium. Studies also showed that complementation of atrA, ftsA and omaA were able to restore the IAA production of the corresponding IAA mutants. Comparison of Fe3+ concentrations in culture supernatants of the wild-type strain, the ftsA mutant and the complemented strain revealed that the iron-uptake ability of the ftsA mutant was highly reduced. This result also points to the necessity of iron as a metal ion in IAA synthesis. Statistical analysis showed no significant difference in the IAA accumulated in cells between the omaA mutant and the wild-type strain, suggesting the omaA might not affect IAA secretion but be involved in IAA production in other unknown ways.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

The ability to produce indole-3-acetic acid (IAA) is present in a number of organisms, including plants, bacteria, fungi and algae. Many bacteria isolated from plant rhizospheres can synthesize IAA in vitro with or without physiological precursors, mainly tryptophan (Trp) [1,2]. Several IAA biosynthesis pathways in bacteria have been proposed according to their intermediates, and more than one pathway may exist in certain species [3,4].

Azospirillum brasilense is a nitrogen-fixing bacterium with great potential to be used as inoculant to promote growth in grass and cereals [5]. Previous studies have shown that the growth-promoting effect of Azospirillum on plants is attributed to the secretion of IAA [6,7]. In Azospirillum, attempts to isolate a mutant completely deficient in IAA production have not been successful [8,9], suggesting that more than one pathway might be involved in IAA synthesis. Feeding experiments with radioactive labeled indole-3-acetamide (3H-IAM) and tryptophan (3H-Trp) demonstrated that A. brasilense possesses at least 3 IAA biosynthetic pathways, i.e., the two Trp dependent pathways (the indole-3-acetamide (IAM) pathway and the Indole-3-pyruvate (IPyA) pathway) and the Trp independent pathway [10]. The first step in the IPyA pathway is the conversion of Trp to IPyA catalyzed by multispecific aminotransferases. Indole-3-pyruvate decarboxylase (IPDC) then catalyses the conversion of IPyA to indole-3-acetaldehyde (IAald) and the latter can be oxidized to IAA by non-specific aldehyde dehydrogenase (also called aldehyde oxidase) [3]. As in higher plants and some microorganisms, the IPyA pathway is the main route for IAA production in the presence of exogenous Trp in A. brasilense. The ipdC gene, encoding a key enzyme in the IPyA pathway, has been cloned from A. brasilense strains Sp245 and Sp7 [11,12]. An ipdC mutant of strain Sp245 showed 90% reduction in IAA synthesis compared to the wild-type strain [13]. However, the other genes involved in synthesis and regulation of IAA in the IPyA and other pathways have not been identified. Isolation and characterization of the genes involved in IAA production will enable elucidation of the mechanisms of IAA production in A. brasilense. This will be fundamental for the construction of a genetic engineered strain with high IAA production that can be used as an inoculant to improve agricultural yield. Hence, the aim of this study was to isolate the genes involved in IAA production from A. brasilense Yu62.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

2.1Strains, plasmids and growth conditions

Azospirillum brasilense Yu62 with known ability to produce more than 50 μg ml−1 indole-3-acetic acid (IAA) in medium supplemented with Trp was isolated from maize (Zea mays L. cv. Huang 3–4) rhizosphere in Beijing, China [14]. Escherichia coli DH5α was used as the host for cloning and sequencing and E. coli S17-1 was used as the Tn5 donor in transposon mutagenesis. Plasmid pRL1063a carrying the Kmr-transposon Tn5 was introduced into the recipient A. brasilense Yu62 by conjugation with E. coli S17-1, and the wide-host-range vector pLAFR3 was used for complementation study [15].

Escherichia coli was grown at 37°C in Luria broth medium. A. brasilense strains were routinely grown at 30°C in LD medium (per liter: tryptone, 10 g; yeast extract, 5 g; NaCl, 2.5 g) [16]. For the IAA production, A. brasilense strains were grown in MAZ medium (per liter: K2HPO4· 3H2O, 6.0 g; KH2PO4, 4.0 g; MgSO4· 7H2O, 0.2 g; NaCl, 0.1 g; CaCl2, 0.02 g; FeCl3· 6 H2O, 0.01 g; H3BO3, 2.8 mg; MnSO4· H2O, 2.1 mg; NaMoO4· 2H2O, 2 mg; ZnSO4· 7H2O, 0.24 mg; CuSO4· 5H2O, 0.016 mg) supplemented with 34 mM malate (carbon source), 100 μg ml−1 Trp (IAA precursor) and 10 mM NH4Cl (nitrogen source) [17]. Antibiotic concentrations used in the medium were: 25 μg ml−1 ampicillin (Ap) and 5 μg ml−1 nalidic acid (Nx) for the A. brasillense Yu62 wild-type; 30 μg ml−1 kanamycin (Km) for the A. brasillense mutants and complemented strains, and 50 μg ml−1 for E. coli; and 12.5 μg ml−1 tetracycline (Tc) for complemented strains and for E. coli.

2.2Tn5 mutagenesis and selection of IAA mutants

Transposon mutants were generated by conjugating A. brasilense Yu62 with E. coli S17–1 carrying the suicide plasmid pRL1063a, which contains the Kmr-transposon Tn5. Conjugations were performed on LD plates and incubated at 30°C for 24 h. Transconjugants were selected on LD plates containing Ap 25 μg ml−1, Nx 5 μg ml−1 and Km 30 μg ml−1. Colonies of transconjugants were streaked on the selective medium twice to yield single colonies. IAA-secreting mutants were selected from the transconjugants by measuring their IAA production with colorimetric and HPLC assays.

2.3Colorimetric assay for indolic compounds

Indolic compounds were estimated using the colorimetric assay as described [18]. Cultures were centrifuged and the resulting supernatants were diluted to appropriate concentration. One milliliter of reagent PC (12 g l−1 FeCl3 in 7.9 M H2SO4), which is based on the Salkowski reagent, was added to 1 ml of the sample solutions. The mixtures were left in the dark for 30 min at room temperature and then their concentration of indolic compounds were determined at 540 nm on a spectrophotometer (Beckman DU 640, USA).

2.4Verification of IAA levels by HPLC

Since colorimetric assay is a general and rapid method that gives only preliminary quantification of indolic compounds, including IAA, IAM, IPyA, etc. [18], IAA levels were further verified by HPLC. Samples for HPLC were prepared as described [4]. Ten milliliter cultures were centrifuged. Supernatants obtained were adjusted to pH 2.5 by dropwise addition of 3 M HCl. The solutions were then extracted three times with ethyl acetate. The extracts were evaporated to dryness at 39°C under vacuum and the resulting concentrates were dissolved in 2 ml of methanol [4]. HPLC analysis was performed with 20 μl aliquots of methanol extract. Indolic compounds were separated on a 4.6 mm × 15 cm, 5 μm Agilent C-18 reverse column on a Waters Liquid Chromatograph (USA Waters Company). Samples were analyzed under isocratic conditions with the mobile phase (methanol: 1% acetic acid in water 40:60 (vol/vol)) at 1 ml min−1 flow rate [4]. Elutes were detected by UV absorbance at 280 nm. IAA was identified by retention time, UV absorbance profile and co-elution of standard IAA. Quantification was based on a standard curve generated using the IAA standard (Sigma).

2.5Cloning of the Tn5-inserted genes and other DNA manipulation methods

The plasmid pRL1063a used in this study carries a Tn5 derivative, the Tn5-1063. Since Tn5–1063 contains an E. coli replication origin, DNA contiguous with the transposon was recovered from the A. brasilense Yu62 genome by excision, recircularization, and transfer to E. coli[19]. The DNA sequence flanking the transposon was determined directly with the primers 5′-TATCAATGAGCTCGGTACCC-3′ and 5′-GATGAAGAGCAGAAGTTATC-3′ that were designed based on the DNA sequence of pRL1063a.

Other DNA manipulation methods, such as isolation of chromosomal DNA and plasmids, restriction enzymes digestions, ligations, transformation, PCR amplification and Southern blotting were performed as described [20].

2.6Complementation study

To complement the three mutants A3, A14 and A16 by the corresponding genes atrA, ftsA and omaA, respectively, the three genes were amplified as follows. A 1379 bp fragment of atrA was amplified with the primers 5′-AATTGGATCCAGCGGGTGATGATAGAG-3′ and 5′-CGTTAAGCTTACTTCAGCACATAGCCC-3′, a 1345 bp fragment of ftsA was amplified with the primers 5′-AATTGGATCCTCGGGAGTCACGAGGAT-3′ and 5′-CGTTAAGCTTGTCTTCGAGGGATCGGT-3′, and a 1708 bp fragment of omaA was amplified with the primers 5′-AATTGGATCCGAGTGCTTCGCCCAGTA-3′ and 5′-CCGTAAGCTTCCCTTCTTGACGTTGGT-3′. The three PCR fragments were digested with Bam HI and Hin dIII and cloned into pLAFR3 to yield plasmids pLAFR-atrA, pLAFR-ftsA and pLAFR-omaA, respectively. These constructs were then introduced into the mutants A3, A14 and A16, respectively, to make complementation.

2.7Measurement of Fe3+ concentration in culture supernatants

Cultures grown in MAZ medium were harvested at 24, 48 and 72 h by centrifugation. Pellets were discarded, and 20 μl of 16.0 M HNO3 was added into 2 ml culture supernatant. The concentration of Fe3+ in the culture supernatant was measured by atomic absorption on a PE 2100 atomic absorption spectrophotometer (Perkin–Elmer Co., Norwalk, USA) with a UV detector at 248.3 nm and a slit distance 0.2 nm [21].

2.8Measurement of IAA concentration within the A. brasilense cells

Cultures were grown in MAZ medium for 48 h, and the media were then diluted until an OD600 of 2.0 was reached. Cells from 20 ml cultures were pelleted by centrifugation and then washed three times with 50 mM Tris–HCl buffer (pH 8.0) until IAA could not be detected in the culture supernatant. Next, the pellets were dissolved in 3 ml of 50 mM Tris–HCl buffer (pH 8.0), and cells in the solution were ruptured by ultrasonic cell crasher (Sonics & Materials Inc., Danbury, CT) until the solutions were clarified. Concentration of IAA in the solution was measured by HPLC and expressed as the ratio of IAA content-to-volume of the original culture.

2.9Statistical analysis

Data were analyzed by analysis of variance (ANOVA) using the general linear model (Version 8.0; SAS Institute Inc., Cary, NC, USA). Duncan's multiple range test was used to determine differences between samples. Significance levels were within confidence limits of 0.05 or less. All treatments were triplicated.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

3.1Isolation of IAA mutants

Transposon mutagenesis was carried out by conjugative transfer of Tn5 from E. coli S-17 to A. brasilense Yu62. More than 5000 Kmr transconjugants were analyzed for IAA production, and 26 mutants with decreased or increased IAA production were isolated with colorimetric assays during the initial screening. IAA levels of the 26 mutants were further verified by HPLC and finally five mutants showing decreased IAA production were obtained. Mutants that were totally devoid of IAA production were not found in this study. The results are in agreement with the report of Hartmann and Zimmer [8]. Southern blotting using the Tn5 fragment as a probe showed that each mutant contained only one hybridizing band. This suggests that each of the five mutants had only single transposon inserted (data not shown).

3.2Growth and IAA production

The time courses of growth and IAA production for the wild-type strain and IAA mutants are shown in Fig. 1. All five IAA mutants exhibited normal growth behavior when compared to that of the wild-type strain. However, IAA production from the five mutants was significantly lower than that of the wild-type strain throughout the incubation periods. Moreover, the pattern of IAA secretion among the wild-type strain and the IAA mutants was variable. This showed that IAA biosynthesis of A. brasilense was influenced by many complex factors.

image

Figure 1. Growth (—) and IAA production (—) curves of A. brasilense Yu62 wild-type (□) and mutants A3 (•), A14 (▄), A16 (○), A19 (▵) and A24 (▴).

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3.3Cloning and sequencing of the genes involved in IAA synthesis in A. brasilense Yu62

The DNA sequences of the Tn5-inserted genes from the five IAA mutants were determined. The putative functions, highest identities and GenBank accession numbers of the genes involved in IAA synthesis from the five mutants are listed in Table 1.

Table 1. Homology analysis of the Tn5-insertion sites in the IAA mutants
Mutant strainDisrupted genePutative functionHighest identityAccession No.
A3 atrA GntR-family transcriptional regulator Sinorhizobium meliloti 1021 (55%)AY850387
     
A14 ftsA Iron-binding protein component of ABC-type Fe3+ transport system Trichodesmium erythraeum IMS101 (62%)AY850389
     
A16 omaA Major outer membrane protein A. brasillense Sp7 (37%)AY850390
     
A19 aldA Aldehyde dehydrogenase Xanthobacter autotrophicus GJ10 (80%)AY850388
     
A24 trpE Anthranilate synthase component I Rhodospirillum rubrum (61%)AY850391

A 4370-bp DNA region flanking the Tn5 in mutant A3 was obtained and it included three contiguous open reading frames, namely the ORF1, ORF2 and ORF3. The ORF1, ORF2 and ORF3 are designated as atrA, atrB and atrC, respectively, hereafter.

The predicted amino acid sequence of the atrA gene product showed a high degree of homology to the GntR-family transcriptional regulators of Sinorhizobium meliloti 1021, Erwinia carotovora subsp. atroseptica SCRI1043 and Pseudomonas aeruginosa UCBPP-PA14, with identities ranging from 38% to 55%. The GntR-family of bacterial regulators regulate various biological processes and important bacterial metabolic pathways [22]. The transcriptional factors belonging to this family share a similar N-terminal DNA-binding domain, i.e., HTH motif, but they are highly divergent in the C-terminal effector-binding and oligomerization domains [23]. The deduced protein of the atrA gene product of A. brasilense had the typical conserved HTH motif situated at the N-terminus, and mutation in atrA resulted in decreased IAA production, indicating that the protein encoded by atrA is a necessary positive regulator in IAA synthesis. The atrA is the first regulatory gene identified for IAA production in A. brasilense but its functions have yet to be determined.

The predicted amino acid sequence of atrB had about 35–39% identity to the aminotransferases (class III) of Pseudomonas putida KT2440, Brucella suis 1330 and S. meliloti 1021. On the other hand, the predicted product of atrC showed 53–60% identity to the 4-aminobutyrate aminotransferases and the related aminotransferases of Polaromonas sp. JS666, Rhodobacter sphaeroides 2.4.1, Brucella melitensis 16M and Rhizobium sp. NGR234. Aminotransferases are known to catalyze the conversion of Trp to IPyA in the first step of the IPyA pathway in many organisms. As far as we know, this is the first report on the isolation of aminotransferases genes in A. brasilense. Our results are in agreement with a study which found that A. brasilense strain UAP 14 produced two aromatic amino acid aminotransferases (AAT1 and AAT2) [24,25].

In the A14 mutant, the predicted product of its interrupted gene, designated as ftsA, shared 48–62% identity to the iron-binding protein components of the ABC-type Fe3+ transport system from Trichodesmium erythraeum IMS101, Thermosynechococcus elongatus BP-1, and Dechloromonas aromatica RCB. On the other hand, the deduced product of omaA identified in the A16 mutant showed 23–37% identity to the major outer membrane proteins of A. brasilense Sp7 and other bacteria. In the A19 mutant, the predicted polypeptide of the interrupted gene aldA had significant homology to aldehyde dehydrogenases found in various bacteria. For instance, it showed 80% identity to the aldehyde dehydrogenase of Bradyrhizobium japonicum USDA 110 and 78% identity to that of Mesorhizobium loti MAFF303099. This is the first report of the aldA sequence in A. brasilense.

The predicted polypeptide of the interrupted gene trpE in the A24 mutant had significant homology to the amino acid sequences of anthranilate synthases component I (TrpE) from many other bacteria, such as 61% identity to TrpE of Rhodospirillum rubrum and 56% identity to that of Magnetospirillum magnetotacticum MS-1. The A. brasilense Yu62 trpE encoded a 531-residue polypeptide with a calculated molecular weight of 58 kDa and the gene trpL, which encoded the leader peptide TrpL, was not found at the upstream of the trpE gene. Our data are consistent with previous reports of a complete trpE gene in Azospirillum lipoferum and the leader peptide was also absent at the upstream of the trpE gene [26]. However, De Troch et al. reported that trpE and trpG were fused together in A. brasilense Sp7 and the fused gene trpE(G) encoded a 732-residue polypeptide with a calculated molecular weight of 78 kDa. Moreover, a 51-bp-long open reading frame encoding TrpL was located 208 bp upstream of the trpE(G) gene [27]. A cluster trpGDC was also identified in Sp7 by Zimmer et al. [28]. Thus, two copies of trpG genes exist in A. brasilense[27]. Comparison of amino acid sequences showed that both TrpEs of A. brasilense strains Yu62 and Sp7 had 44% identity at their last half-parts. The data suggest that the trpE identified in this study existed separately from trpG and there might be two copies of trpE genes in A. brasilense, just as there are two trpG genes in A. brasilense[27,28].

3.4Complementation experiments of IAA mutants

Five genes, specifically atrA, ftsA, omaA, aldA and trpE were disrupted by Tn5 insertion in the IAA mutants. Two of these genes, namely trpE and aldA encode anthranilate synthase and aldehyde dehydrogenase, respectively. The former catalyzes the conversion of chorismate to anthranilate, the precursor of IAA synthesis, while the latter catalyzes the last step in the IPyA pathway. Hence, mutations in the two genes trpE and aldA would result in reduced IAA production.

To determine whether the other three genes atrA, fts A and omaA are really involved in IAA synthesis, these genes were used to complement the corresponding mutants. As shown in Fig. 2, IAA production of the mutant A3 was nearly completely restored by complementation with pLAFR-atrA carrying the atrA gene. The IAA production in mutants A14 and A16 was partially restored by pLAFR-ftsA and pLAFR-omaA, respectively. The data confirm that these genes were involved in IAA synthesis.

image

Figure 2. IAA production of wild-type (white bars), mutants (gray bars) and complemented strains (black bars) after 48 h of culture. 1: wild-type, A3 mutant, A3 complemented by pLAFR-atrA; 2: wild-type, A14 mutant, A14 complemented by pLAFR-ftsA; 3: wild-type, A16 mutant, A16 complemented by pLAFR-omaA.

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3.5Relationship between iron-uptake and IAA synthesis

To determine how the Tn5-insertion impaired the function of the ftsA gene, we also compared the Fe3+ concentrations of the wild-type strain, ftsA mutant and complemented strain (A14 complemented by pLAFR-ftsA) in the culture supernatants. The Fe3+ concentration in the culture supernatant of the ftsA mutant was significantly higher than in the wild-type strain and the complemented strain of cultures grown for 24 and 48 h. In the earlier experiment, the predicted product of ftsA gene was found to possess significant identity to the iron-binding protein component of the ABC-type Fe3+ transporter, which is localized at the cytoplasmic membrane responsible for iron transportation. Hence, mutation in ftsA weakened the iron-uptake ability of A. brasilense (Fig. 3). The IAA phenotype of ftsA mutant was probably due to the lower concentration of iron absorbed into the A. brasilense cells. The results also revealed that iron is a necessary metal ion for IAA production. This is consistent with the report that stated FeSO4· 7H2O was the only metal ion that increased IAA production [29]. However, differences in Fe3+ concentration between cultures of the wild-type strain, the ftsA mutant and the complemented strain in the culture supernatants was not obvious after 72 h of incubation. One possible explanation for this is that Fe3+ was reduced to Fe2+ by acidic compounds secreted by the bacterial cells. The Fe2+ could then be absorbed into the bacterial cells directly. It was previously observed that Fe3+ was gradually reduced by IAA along with the formation of a soluble Fe2+ complex [30]. Another possibility is that the ftsA mutant may uptake Fe3+ by other adapted methods that are independent from the ABC-type iron transporter. It is known that ferric citrate is transported into E. coli via the FecA outer membrane receptor protein and the ABC-type transport system as well as some siderophore-mediated iron-uptake systems independent of ABC-type transporter [31,32]. In A. brasilense, several outer membrane proteins responsible for iron-uptake were specifically induced and a catechol-type siderophore, called the spirilobacin, was secreted under iron deficiency [33,34]. Our previous study also revealed that six outer membrane proteins were apparently induced by iron deprivation in A. brasilense Sp7, and one of them was FhuE, which is a receptor responsible for ferric coprogen transport [35]. Our data, combined the reports of others, suggest that iron transport systems independent of an ABC-type iron transporter may exist in A. brasilense.

image

Figure 3. Measurement of the Fe3+ concentration in the culture supernatants of A. brasilense Yu62 wild-type strain (white bars), A14 mutant (gray bars) and complemented strain (A14 complemented by pLAFR-ftsA, black bars).

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3.6Effect of mutation in omaA on the secretion of IAA

The predicted product of the omaA gene identified in this study had identity to the major outer membrane proteins of A. brasilense Sp7 and other bacteria. The endocellular IAA concentrations of the wild-type strain and the omaA mutant were measured to determine whether the IAA phenotype of omaA mutant was caused by the decreased ability to secret IAA. Since the levels of endocellular IAA for both wild-type strain and omaA mutant were very low, the comparison was performed using statistical analysis. As shown in Table 2, the concentration of IAA within the omaA mutant cells was not significantly different from that within the wild-type cells on cultures grown for 24 and 48 h. Since the amount of IAA accumulated within the omaA mutant cells was only slightly higher than that of the wild-type strain, the reduced IAA production may not have resulted from the inhibition of IAA secretion. This suggests that omaA may be involved in IAA production in another manner besides affecting the secretion of IAA. It was reported that the major outer membrane protein (MOMP) of A. brasilense Sp7 was involved in adhesion when this bacterium interacted with plant roots [36]. Lee et al. [2] identified the function of cytochrome c biogenesis genes in IAA synthesis. However, there are no reports describing the relationship between IAA production and the major outer membrane proteins. Our study points to the complexity of IAA production in A. brasilense Yu62 and the results suggest that bacterial IAA biosynthesis is influenced by many factors.

Table 2. IAA concentrations within cells of the wild-type strain and omaA mutant
StrainsIAA concentration (μg ml−1)
24 h48 h 
  1. aMeans with the same letter in the same column are not significantly different (p < 0.05, n= 3).

Wild-type0.0149 ± 0.0007a0.0158 ± 0.0012a
omaA mutant0.0184 ± 0.0011a0.0190 ± 0.0010a

Acknowledgments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

This work is supported by the National High Technology Research of “863” Program of China (Grant No. 2003AA241170) and China Key Base Research Developing Project Program (Grant No. 001CB108904). This manuscript was edited by Dr. Tan Sze Sze.

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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References
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