Journal of Bacteriology, October 2003, p . 5854-5861, Vol . 185, No . 19

Identification of Three Genes Encoding P_II-Like Proteins in Gluconacetobacter diazotrophicus: Studies of Their Role(s) in the Control of Nitrogen Fixation

Olena Perlova,¹ Alejandro Ureta,² Stefan Nordlund,^2* and Dietmar Meletzus¹

Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden,² Department of Gene Technology and Microbiology, University of Bielefeld, DE-33615 Bielefeld, Germany¹

Received 24 March 2003/ Accepted 8 July 2003

Biological nitrogen fixation is a highly energy-demanding process catalyzed by nitrogenase . The most common form of nitrogenase is composed of two different enzymes, the Fe protein (dinitrogenase reductase) and the MoFe protein (dinitrogenase) (33) . The synthesis and activity of nitrogenase require the expression of several specific nif genes . Recently, the major nif gene cluster of G . diazotrophicus was isolated and partially genetically characterized . Among these genes, the nifA gene, encoding the transcriptional activator NifA, and the nifHDK genes, encoding the nitrogenase Fe and MoFe proteins, were identified (21) . Gene expression analysis indicated that the nitrogen-sensing system, i.e., the enzyme GlnD and the regulatory P_II protein(s), regulates the expression of the nifA gene in response to nitrogen availability; nifA transcription is repressed in cultures of G . diazotrophicus exposed to high levels of ammonium but induced under conditions of nitrogen limitation (40) . In some diazotrophs, the activity of NifA is also controlled by the glnB-encoded P_II protein (11, 22) . The glnB gene has been cloned from a number of bacterial species and characterized . In addition to glnB, many organisms have multiple genes encoding P_II proteins (3) . The majority of the -proteobacteria, including the genera Sinorhizobium, Bradyrhizobium, Azorhizobium, Azospirillum, and Rhodobacter, harbor two P_II protein-encoding genes, a glnB-like gene and a glnK-like gene linked to an amtB homolog (except in Azospirillum brasilense) . The photosynthetic bacterium Rhodospirillum rubrum (47) and a member of the ß-proteobacteria, Azoarcus sp . strain BH72 (26), were found to carry three genes encoding P_II-like proteins . Previous reports indicated that in Klebsiella pneumoniae and Azotobacter vinelandii, GlnK is involved in sensing of the nitrogen status by NifL (a regulator of NifA activity), although the mechanisms are not the same in these two organisms (14, 23, 35) . The roles of the different P_II proteins in the physiology of nitrogen fixation and ammonium assimilation are clearly beginning to be revealed in many diazotrophs (9, 20, 47) .

In this study, we describe the identification and mutagenesis of three glnB-homologous genes, glnB, glnK1, and glnK2, as part of the ammonium-sensing cascade in G . diazotrophicus . In addition, we describe the characterization of the phenotypes of strains with interposon mutations with respect to nitrogenase expression and activity .

 
Media and growth conditions. G . diazotrophicus was grown at 30°C on C2 medium (rich medium) (40) or minimal medium (43) with or without ammonium chloride at a final concentration of 1 or 20 mM . Escherichia coli was grown in medium containing 10 g of tryptone, 5 g of NaCl, and 5 g of yeast extract per liter (pH 7.5) .

To investigate the expression of nif genes, wild-type G . diazotrophicus and the P_II mutants were grown overnight at 30°C in C2 medium to a final optical density at 600 nm of 0.8 to 1.0, harvested by centrifugation, washed in 100 mM citrate-phosphate buffer (pH 5.5), and finally resuspended in minimal medium with or without 20 mM NH₄Cl as a sole N source .

G . diazotrophicus mutants were grown in the presence of the relevant antibiotics (kanamycin at 200 µg/ml, chloramphenicol at 200 µg/ml, and tetracycline at 20 µg/ml), but these were omitted once cells were transferred to minimal medium during studies of the control of nif gene expression .

DNA preparations. Chromosomal DNA was prepared from G . diazotrophicus grown to stationary phase in liquid C2 medium as described previously (27) . Plasmid DNA was isolated and purified by using a miniplasmid kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations .

Construction of a G . diazotrophicus genomic library. Chromosomal DNA from wild-type G . diazotrophicus PAL5 was used for the construction of a phage genomic library .

For the preparation of chromosomal DNA, cells were grown to stationary phase in C2 medium . Cell lysis and DNA extraction were carried out as described previously (27) . Fragments (9 to 23 kb) generated by Sau3A partial digestion were cloned by using a EMBL3/BamHI vector kit (Stratagene Europe, Amsterdam, The Netherlands) with E . coli MRA(P2) as a host . In vitro packaging kits were also obtained commercially from Stratagene .

DNA sequencing. Sequencing of both DNA strands was performed by the dideoxy chain termination method (36) with a DNA sequencing kit (USB Corporation, Cleveland, Ohio) on an ALF automated DNA sequencer (Amersham Biosciences, Freiburg, Germany) .

For the nucleotide sequence determination, unidirected deletions were generated after thionucleotide end protection followed by the successive actions of exonuclease III of E . coli and nuclease S1 of Aspergillus oryzae with a double-stranded nested deletion kit (Amersham Biosciences, Freiburg, Germany) .

Comparison of DNA sequences with GenBank data and further analyses were performed by using the BLAST programs (1) and the software package DNASTAR (DNASTAR Inc., Madison, Wis.) .

Southern hybridization. For Southern hybridization experiments, probes were labeled by the random priming method with a digoxigenin DNA labeling and detection kit (Boehringer Mannheim, Mannheim, Germany) . DNA was transferred to nylon membranes (Schleicher & Schüll, Dassel, Germany) by vacuum blotting for 1 h . After 2 h of prehybridization of blots in 20 ml of prehybridization solution (standard buffer [The DIG System User Guide for Filter Hybridization; Boehringer Mannheim]), the probe (5 to 20 ng/ml in 5 ml of prehybridization solution) was added and hybridization was carried out for 16 h at 68°C . Detection was performed according to Boehringer Mannheim protocols with 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium as a substrate .

PCR amplification and cloning of genes encoding P_II-like proteins in G . diazotrophicus. The nucleotide sequences of known glnB genes from different bacteria were aligned, and the degenerate primers 5'ATCATYAAGCCSTTCAARCTCGA3' and 5'GAARATCTTSCCGTCRCCRAT3' were synthesized (TIBmol, Berlin, Germany) and used in PCRs with wild-type G . diazotrophicus PAL5 chromosomal DNA as a template . The PCR products were evaluated by 1.5% agarose gel electrophoresis . Samples with amplified fragments of the expected size (258 bp) were cloned by using a Sure Clone ligation kit (Amersham Biosciences, Freiburg, Germany) .

Electroporation of G . diazotrophicus. For the introduction of DNA into G . diazotrophicus, cells were grown in C2 medium . At the early- to mid-logarithmic growth phase, cells were collected, washed in ice-cold 10% (vol/vol) glycerol in water, and resuspended in a 1/20 volume of 10% glycerol . Aliquots of 200 µl were used for electroporation after the addition of up to 1 µg of the appropriate plasmid DNA . Cells were electroporated in a Gene Pulser apparatus (Bio-Rad Laboratories, Munich, Germany) as described previously (40) . Cells were then washed from the cuvette with 1 ml of C2 medium, incubated at 28°C for 2 h, collected, and plated on C2 medium containing the corresponding antibiotic .

Western blot analysis and nitrogenase activity. To prepare samples for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), G . diazotrophicus cell suspensions were mixed with a buffer containing 130 mM Tris (pH 6.8), 5% (wt/vol) SDS, 20% (vol/vol) glycerol, 0.003% (wt/vol) bromophenol blue, and 10% (vol/vol) 2-mercaptoethanol and incubated for 5 min at 90°C . Equal amounts of protein were loaded in 12% polyacrylamide-SDS (glutamine synthetase and Fe protein) or 18% polyacrylamide-SDS (P_II) gels, which were run as described previously (43) . Gels were electroblotted onto Hybond-P membranes (Amersham Biosciences, Uppsala, Sweden) and probed with antibodies raised against R . rubrum GlnB, glutamine synthetase, or nitrogenase Fe protein . The results were visualized by using an enhanced chemiluminescence detection system (Amersham Biosciences, Uppsala, Sweden) . Nitrogenase activity was measured by the acetylene reduction assay (43) .

Nucleotide sequence accession numbers. The sequences determined in this study have been deposited in GenBank under accession numbers AF365037, AF318040, and AF365041 .

In order to obtain the corresponding chromosomal DNA regions carrying the full-length genes as well as the flanking regions, a genomic phage library of G . diazotrophicus was constructed (see Materials and Methods) . The resulting phage library was subsequently screened with plasmids pOP42 and pOP75 carrying different glnB-like genes as probes . Southern hybridization experiments with isolated phage library DNA along with chromosomal DNA as a control revealed that plasmid pOP75 hybridized to 2.1-kb SalI and 3.5-kb EcoRI fragments, while pOP42 hybridized to 3.4-kb SalI, 10.0-kb BamHI, and 8.0-kb EcoRI fragments (data not shown) . The corresponding restriction fragments generated by the endonucleases EcoRI and BamHI were cloned in E . coli by using vectors pSVB30 and pUC18, resulting in hybrid plasmids pOP7, pOP26, and pOP30 (Table 1) .

In order to localize the open reading frames (ORFs) corresponding to the P_II protein-encoding genes, the nucleotide sequences of a 2.5-kb fragment of plasmid pOP26 and a 2.1-kb fragment of plasmid pOP7 were determined by using subclones derived from unidirectional deletions .

An analysis of the nucleotide sequence obtained from pOP26 showed two ORFs of 339 and 1,461 bp separated by 176 bp, with the deduced sequence of ORF1 showing significant similarities to the sequences of known glnB genes of other proteobacteria, with the highest score of 79% identity for the P_II protein of A . brasilense (12) . ORF2, located 176 bp downstream, encodes a putative protein of 486 amino acids showing high degrees of identity to the glutamine synthetase proteins of A . brasilense (73%) (12), R . rubrum (71%) (18), and Sinorhizobium meliloti (70%) (2) . This linkage resembles the common arrangement of the glnB and glnA genes in the -proteobacteria . These genes in G . diazotrophicus were therefore designated glnB and glnA .

The nucleotide sequence of a 2.1-kb region of plasmid pOP7 contained two ORFs of 339 and 1,386 bp, with the putative start codon (GTG) of ORF2 overlapping the stop codon of ORF1 (TGA) . An additional putative start codon (ATG) was found to be located 83 bp downstream of ORF1 . ORF1 encodes a polypeptide with a molecular mass of 12.4 kDa, the deduced amino acid sequence of which is 67% identical to that of the A . brasilense P_z (GlnZ) protein (11) and 64% identical to those of known glnK gene products from E . coli (45) and K . pneumoniae (14), two members of the -proteobacteria . ORF2 encodes polypeptides with deduced molecular masses of 47.4 kDa for the product of translation from GTG and 44.4 kDa for the translation product starting at the downstream ATG . The deduced amino acid sequence of the product of this ORF is 49% identical to that of the amtB gene product of A . brasilense (44); therefore, the ORF likely encodes a (methyl)ammonium transporter in this organism . The genes localized on pOP7 were named glnK1 and amtB1 .

The physical maps of the corresponding chromosomal DNA regions of G . diazotrophicus were deduced from restriction mapping and hybridization and refined by DNA sequencing data generated from subclones and their deletion derivatives (Table 1 and Fig . 1) .

 
Identification and sequence analysis of a third glnB-like gene. As detailed below, we constructed both single and double mutants affected in glnB and glnK1 in order to investigate whether additional P_II homologs were present in G . diazotrophicus . Western blot analysis of crude protein extracts of the double mutant (UBI 130) with polyclonal anti-R . rubrum P_II antibodies was carried out as described for R . rubrum (19) . Surprisingly, we found that a prominent signal of the size observed for P_II-like proteins was still present in the glnB glnK1 double mutant and that the posttranslational modification of a protein of about 13 kDa was still detectable (data not shown) . These results raised the question of whether G . diazotrophicus carries a third glnB-like gene .

In order to obtain the corresponding chromosomal DNA regions, the genomic phage library of G . diazotrophicus was screened by plaque hybridization followed by Southern hybridization of isolated phage DNA with pOP8 as a probe . Two hybridizing signals of 2.1 and 3.2 kb were detected by using chromosomal DNA digested with restriction endonuclease SalI (data not shown) . One of the hybrid phages isolated carries a SalI fragment of the expected size, 3.2 kb . This fragment was cloned in E . coli by using vector pUC18, with the resulting plasmid being named pOP80, followed by nucleotide sequencing (Fig . 1) .

The putative P_II-like protein encoded within this DNA fragment has 112 amino acid residues and showed 66% identity with GlnZ of A . brasilense as well as high levels of similarity to GlnK and GlnB proteins of other organisms . Directly downstream of this gene, an incomplete ORF was found; the amino acid sequence from this ORF showed a high degree of identity with the amino acid sequences of (methyl)ammonium transporters from different organisms . Based on the proposed nomenclature for glnB-like genes (3, 41), the gene described above was named glnK2, and the incomplete ORF was named amtB2 .

Mutagenesis of glnB-like genes in G . diazotrophicus. The arrangement of all three genes directly upstream of other genes encoding proteins involved in nitrogen metabolism suggests that all three are involved in the regulation of assimilation and interconversion of nitrogen compounds in G . diazotrophicus, as in other proteobacteria . In order to examine the physiological roles of the GlnB and GlnK proteins, mutants were constructed by using the kanamycin interposon of plasmid pDM6 (32) for glnB and the promoterless gusA-chloramphenicol resistance gene cassette of plasmid pWM4 (29) for glnK1, resulting in hybrid plasmids pOP16 and pOP5 (Table 1) . For the mutagenesis of glnK2, the tetracycline resistance gene cassette of plasmid p34s-Tc (10) was used . All interposons were cloned as BamHI DNA fragments into unique BglII restriction sites on plasmids pOP30, pOP7, and pOP80 containing G . diazotrophicus glnB, glnK1, and glnK2, respectively . These constructs, located on suicide plasmids, were then introduced to G . diazotrophicus by electroporation and tested for double crossover events by growth on media containing the appropriate antibiotics . Mutants exhibiting an ampicillin-sensitive and kanamycin-, chloramphenicol-, or tetracycline-resistant phenotype were expected to have a chromosomal allele replacement, which was confirmed by Southern hybridization with the chromosomal DNAs of the mutants and the wild-type strain as targets and the entire plasmids carrying glnK1, glnK2, and glnB regions as probes (data not shown) .

The construction of the glnB glnK1 (UBI 130), glnB glnK2 (UBI 133), and glnK1 glnK2 (UBI 135) double mutants as well as the glnB glnK1 glnK2 (UBI 150) triple mutant was carried out by analogous introduction of the corresponding constructs into the verified single (double) mutants followed by selection with the corresponding antibiotic . Allele replacements were confirmed by using pOP80 (for glnK1 and glnK2) and pOP30 (for glnB) as hybridization probes (data not shown) .

Sequence analysis of G . diazotrophicus GlnB, GlnK1, and GlnK2. As shown in Fig . 2, glnB, glnK1, and glnK2 all encode 112-amino-acid proteins with a calculated molecular mass of approximately 13 kDa and with the following overall identities: GlnB and GlnK1, 59%; GlnB and GlnK2, 63%; and GlnK1 and GlnK2, 71% . Sequence analysis of the G . diazotrophicus GlnB, GlnK1, and GlnK2 proteins revealed that they share the common distinctive features of P_II proteins (3) .

 
As determined for other GlnB proteins, G . diazotrophicus GlnB has lysine and glutamate at positions 3 and 5, respectively . The GlnK proteins are distinguished from the GlnB proteins in carrying a hydrophobic residue at position 3 and isoleucine, threonine, or methionine at position 5 . G . diazotrophicus GlnK1 has leucine and threonine at positions 3 and 5, respectively, while in GlnK2, phenylalanine and isoleucine are located at positions 3 and 5, respectively (3) . As for other P_II-like proteins, the significance of the presence of these amino acids at these particular positions is still unknown . A common feature of GlnB, GlnK1, and GlnK2 is the tyrosine residue located at position 51, which is likely to be the site for the posttranslational uridylylation of these paralogs (7) . Stretches of highly conserved amino acids corresponding to the T loop, B loop, and C loop in the three-dimensional conformations of these proteins (16, 31) are clearly present in all three G . diazotrophicus P_II-like proteins (Fig . 2) . Interestingly, although the loops are highly conserved in all three P_II proteins, there are also distinct differences, e.g., in the T loop, where residue 52 is Ile in GlnB, His in GlnK1, and Gln in GlnK2 . Since the loops have been shown to be involved in the interaction of P_II proteins with different effector molecules or target proteins (3), these differences could be an indication of the different responses of these paralogs to cellular effectors and/or differences in their abilities to interact with target proteins .

Expression of P_II proteins and control of nif genes by P_II proteins in G . diazotrophicus. To verify the expression of all three P_II paralogs in G . diazotrophicus, Western blot analysis of crude protein extracts was carried out with polyclonal antibodies raised against R . rubrum GlnB . As shown in Fig . 3, P_II proteins were expressed in all double mutants, indicating that the expression of one P_II protein is not dependent on the presence of the others . However, the possibility that the level of expression of one paralog is modulated by those of the other two cannot be excluded, as we have not determined whether the antibodies have different sensitivities to the three P_II proteins . Furthermore, no P_II-related signal was found when cell extracts from G . diazotrophicus glnB glnK1 glnK2 triple-mutant strain UBI 150 were used, probably indicating that no additional P_II protein is present in G . diazotrophicus (Fig . 3, lanes 5 and 6) .

 
In bacteria, the core of the nitrogen-sensing system is composed of the bifunctional enzyme GlnD and the regulatory P_II protein(s) . GlnD controls the uridylylation state of P_II in response to nitrogen levels, monitored as the glutamine concentration; in turn, P_II in different uridylylation states controls the overall cellular response to nitrogen (15) . As it has been shown that P_II proteins play different roles in the control of nitrogen fixation in diazotrophic bacteria, the effect of the knockout of these proteins on the expression of nif genes in G . diazotrophicus was studied . The analysis was based on the evaluation of the expression of the Fe protein and nitrogenase activity .

The wild-type and mutant strains were grown in C2 medium (rich medium), and the expression of the nifH gene (encoding the Fe protein) was studied after transfer of the corresponding strains to nitrogen-free minimal medium . The results of this experiment are shown in Fig . 4 . As expected, the induction of the Fe protein and nitrogenase activity occurred in wild-type G . diazotrophicus, and similar results were obtained for the glnB, glnK1, and glnK2 single mutants . Interestingly, nifH was also induced in the P_II protein triple mutant (Fig . 4, lane 8), indicating that in G . diazotrophicus, none of the P_II proteins is required for nif gene expression .

 
However, in strain UBI 130, where GlnK2 is the only P_II paralog present (Fig . 4, lane 5), the expression of the Fe protein was inhibited; no Fe protein was detected after 6 h in nitrogen-free medium . After 20 h, very low levels of the Fe protein were present compared to the levels in the other strains . Since strains UBI 120 (glnB) and UBI 125 (glnK1) showed wild-type induction of nif genes, it can be concluded that GlnK2 has an inhibitory effect on nif gene expression and that GlnB or GlnK1 is required to relieve this effect . In accordance with this observation, strain UBI 133 or UBI 135, expressing only GlnK1 or only GlnB, respectively, showed wild-type induction of nif gene expression .

In many bacteria, the uridylylation state of the P_II proteins controls the cellular response to nitrogen levels . Uridylylated P_II proteins indicate nitrogen limitation, while nonuridylylated forms are related to a cellular state of nitrogen sufficiency (31) . The observation that in G . diazotrophicus the expression of nif genes also occurs when all three proteins are absent suggests that the uridylylated forms are not required per se as positive signals . It is therefore reasonable to propose that in G . diazotrophicus, the nonuridylylated forms of the P_II proteins inhibit nif gene expression under nitrogen sufficiency, as may be the case in other bacteria (30) .

To evaluate this hypothesis, wild-type G . diazotrophicus and all P_II mutant strains were grown in C2 medium, and the regulation of nif gene expression was studied after transfer of the cells to minimal medium containing 20 mM NH₄⁺ . The results of these studies are shown in Fig . 5 . In response to high ammonium levels, nif gene expression was inhibited in the wild type and the G . diazotrophicus P_II single mutants (Fig . 5, lanes 1 to 5) . However, in strain UBI 150, where no P_II proteins are present, nif genes were expressed in spite of high ammonium levels (Fig . 5, lane 8) . This result indicates that in G . diazotrophicus, P_II proteins are in fact required for the repression of nif gene expression in response to ammonium . In strain UBI 133 or UBI 135, expressing GlnK1 or GlnB, respectively, as the only P_II protein, low levels of the Fe protein were present, a finding which could indicate that GlnK2 is required for complete nif repression .

 
Figures 4 and 5 show that as a result of the interposon insertion within glnB, strains UBI 120, UBI 130, UBI 133, and UBI 150 have significantly decreased levels of glutamine synthetase compared to the wild type and the other G . diazotrophicus P_II mutant strains . This finding probably reflects a polar effect of this mutation on the expression of the glnA gene . However, the lack of normal nif regulation in strains UBI 130 and UBI 150 and, to some extent, in strains UBI 133 and UBI 135 cannot be explained by decreased glutamine synthetase levels per se, since strain UBI 120 showed wild-type control of nif gene expression . Similarly, although the GlnK-AmtB interaction plays a regulatory role in, e.g., A . vinelandii (9), we do not believe that potential polar effects of the glnK1 or glnK2 mutations on the corresponding amtB genes can explain the lack of nif control, since strains UBI 125 and UBI 127 showed apparent wild-type control of nif gene expression, while in strain UBI 135, ammonium control was not completely abolished .

It is interesting that none of the strains showed Fe protein expression when grown in C2 medium (rich medium) prior to their transfer to LGI medium (minimal medium) . This result is somewhat surprising for strain UBI 150, since the inhibition of nif gene expression in response to ammonium was lost (Fig . 5, lane 8) . This phenomenon, independent from ammonium-related P_II protein signaling, certainly leads to new questions about the regulation of nif gene expression in G . diazotrophicus .

Our results for nifH gene expression (Fig . 4 and 5; summarized in Table 2) suggest that in G . diazotrophicus, the nonuridylylated forms of P_II proteins are responsible for the inhibition of nif gene expression . However, it was recently reported that a mutation in the 3' end of the G . diazotrophicus glnD gene allows nif gene expression regardless of the levels of ammonium in the medium (32) . This apparent contradiction, i.e., that nif genes are expressed in spite of mutated GlnD, suggests that as a result of this mutation, partially active GlnD with a significantly impaired ability to sense nitrogen (i.e., glutamine) is synthesized, as proposed for the corresponding glnD mutant in E . coli (38) .

 
In conclusion, we report the presence of three P_II proteins in G . diazotrophicus, a finding previously shown only for two other bacteria, R . rubrum (47) and Azoarcus sp . strain BH72 (26) . In G . diazotrophicus, these proteins are expressed independently of the presence of each other, although we cannot distinguish whether the level of expression of one of the P_II proteins is modulated by that of another . This situation is clearly different from that in Azoarcus, where GlnY is expressed only when the other two proteins are deleted (26) . The GlnK2 protein clearly has a function different from those of GlnB and GlnK1, as it represses nif gene expression under all conditions tested in the glnB glnK1 mutant . It thus can be proposed that GlnB and GlnK1 are required as positive signals to efficiently relieve repression by GlnK2 . When either GlnB or GlnK1 is present alone, the repression of nif gene expression is similar to that of the wild type, but not complete, indicating that GlnK2 has a central role in the regulation of nif gene expression . To our knowledge, this function of GlnK2 is a new feature of P_II proteins as central components in the regulation of nitrogen metabolism .

Our studies do not show at what level the three P_II proteins exert their action, i.e., which target proteins are affected . It was previously shown that the NtrBC proteins are not required for nitrogen fixation in G . diazotrophicus (28), a finding which could suggest that NifA is one of the targets for one or more of the P_II proteins, as has been shown in A . brasilense (5, 6) . An important part of future studies will therefore be to establish the effects on nifA expression and/or NifA activity, as well as to determine the capacity of GlnD to catalyze the uridylylation of the three P_II proteins present in G . diazotrophicus .

This work was supported by grants from the Deutsche Forschungsgemeinschaft (Me1254/3-1) and DAAD to D.M . and the Swedish Institute, Carl Tryggers Foundation, and FORMAS to S.N .

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