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Phosphate Starvation-Inducible Gene ushA Encodes a 5' Nucleotidase Required for Growth of Corynebacterium glutamicum on Media with Nucleotides as the Phosphorus Source

Institute of Biotechnology 1, Research Center Juelich, D-52428 Juelich, Germany

Received 10 December 2004/ Accepted 28 February 2005

	ABSTRACT

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Phosphorus is an essential component of macromolecules, like DNA, and central metabolic intermediates, such as sugar phosphates, and bacteria possess enzymes and control mechanisms that provide an optimal supply of phosphorus from the environment. UDP-sugar hydrolases and 5' nucleotidases may play roles in signal transduction, as they do in mammals, in nucleotide salvage, as demonstrated for UshA of Escherichia coli, or in phosphorus metabolism. The Corynebacterium glutamicum gene ushA was found to encode a secreted enzyme which is active as a 5' nucleotidase and a UDP-sugar hydrolase. This enzyme was synthesized and secreted into the medium when C. glutamicum was starved for inorganic phosphate. UshA was required for growth of C. glutamicum on AMP and UDP-glucose as sole sources of phosphorus. Thus, in contrast to UshA from E. coli, C. glutamicum UshA is an important component of the phosphate starvation response of this species and is necessary to access nucleotides and related compounds as sources of phosphorus.

	INTRODUCTION

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Phosphorus is one of the major constituents of the cell, accounting for about 1.5 to 2.1% of the cell dry weight. Cells contain inorganic phosphates, like orthophosphate, pyrophosphate, or polyphosphate, as well as organophosphates, such as nucleotides, sugar phosphates, or phospholipids. The major phosphorus-containing components are polymers, like RNA, DNA, or polyphosphate. Typically, cells take up phosphate as their preferred phosphorus source. Many organisms have developed mechanisms that allow them to adapt phosphate utilization to phosphate availability. When phosphate is scarce, many bacteria can use organophosphates as phosphorus sources. Either organophosphates are taken up into the cell directly, as in the case of glycerol 3-phophate or hexose phosphates, or they are degraded extracellularly by enzymes, such as alkaline phosphatases, and the inorganic phosphate formed is taken up into the cell subsequently.

Corynebacterium glutamicum, a gram-positive amino acid-producing bacterium, responds to phosphate starvation by changing expression of a number of genes involved in phosphorus metabolism (14, 32). Among the genes of the phosphate starvation stimulon are genes that putatively code for an uptake system for inorganic phosphate, an uptake system for glycerol 3-phosphate, an extracellular nuclease (nucH), and a 5' nucleotidase or related esterase (ushA). While in Bacillus subtilis phosphate starvation also induced genes for the biosynthesis of teichuronic acids and genes for the general stress response (1, 12), in C. glutamicum genes for the general stress response were not induced by phosphate starvation (14, 32). In addition, the cell wall of C. glutamicum lacks teichoic and teichuronic acids, and its genome lacks genes for the biosynthesis of these compounds (15). The phosphate starvation stimulons of C. glutamicum and Escherichia coli are similar, but unlike E. coli (31), C. glutamicum cannot utilize phosphonates and its genome lacks genes for phosphonate metabolism (14, 15, 32). A further difference between the responses of E. coli and C. glutamicum to phosphate starvation is that the 5' nucleotidase gene ushA is not a known member of the phosphate starvation stimulon of E. coli (3, 31), while its C. glutamicum homolog showed increased RNA levels in phosphate-starved cells (14).

In E. coli, the 5' nucleotidase UshA has an important function in nucleotide salvage (21, 33). In addition, it is required for growth on 5'-AMP as a sole carbon source (21, 33). Animal 5' nucleotidases exhibit homology with E. coli UshA (17), and their physiological role is termination of the action of ATP and other nucleotides that serve as extracellular messengers and neurotransmitters in the brain, as well as in the periphery (34). The aims of this study were to characterize the UshA protein of C. glutamicum and ushA gene expression and to determine the physiological role of UshA in this bacterium.

	MATERIALS AND METHODS

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Bacteria, plasmids, and growth conditions.
Strain ATCC 13032 of C. glutamicum was used as the wild type (WT), and an ushA deletion mutant was generated from it as described below. E. coli DH5 was used for cloning (9). Luria-Bertani (LB) medium was used as the standard medium for E. coli (26), while brain heart infusion medium (Difco) was used as the complex medium for C. glutamicum. CGXII was used as a minimal medium for C. glutamicum (16), but it contained 30 mg/liter protocatechuic acid. Glucose (40 g liter^–1) was used as the carbon source. When appropriate, E. coli strains were cultured with ampicillin (100 µg ml^–1) or kanamycin (50 µg ml^–1), and C. glutamicum strains were cultured with kanamycin (50 µg ml^–1). A reduced concentration of kanamycin (15 µg ml^–1) was used to obtain transformants of C. glutamicum. E. coli was grown at 37°C, and C. glutamicum was grown at 30°C in 60 ml of medium in 500-ml baffled shake flasks at 130 rpm.

The phosphate starvation conditions used for growth of C. glutamicum have been described previously (14). For growth of C. glutamicum on different sources of phosphorus, CgXII medium lacking potassium phosphate was prepared and used for a 24-h preculture to exhaust the intracellular stored phosphorus. In the main cultures, CgXII medium lacking potassium phosphate but containing 1 mM UDP-glucose, 1 mM AMP, or a limiting concentration of phosphate (0.065 mM) as the sole source of phosphorus was used (14).

Construction of plasmids and strains.
Plasmids were constructed in E. coli DH5 from PCR-generated fragments (Expand High Fidelity PCR kit; Roche Diagnostics) by using C. glutamicum WT DNA prepared as described by Eikmanns et al. (7) as a template. E. coli was transformed by the RbCl₂ method (11). C. glutamicum was transformed via electroporation (30). All transformants were analyzed by plasmid analysis and/or PCR with appropriate primers.

For overexpression of C. glutamicum ushA, primers ushA(5'-AAGGAGAATTTAATGAAGAGGCTTTCC-3') and ushArev (5'-ATTACATGAACTGCGCAAACATAGCC-3') (the underlined nucleotides correspond to bp 343625 and 345721, respectively, of the accession no. NC003450 sequence) were used to amplify ushA from genomic DNA. The PCR product was subcloned into pGEM-T (Promega, Mannheim, Germany), and after NcoI/SpeI restriction and blunt ending it was cloned into EcoRI-restricted and blunt-ended pEKEx2 (6).

To amplify flanking regions of ushA, primers ush1 (5'-TGGTCTAGAATCCAACAAGATTGTTGC-3'; the underlined nucleotide corresponds to bp 343379 of the NC003450 sequence, and the XbaI site is indicated by boldface type), ush2rev (5'-CCCATCCACTAAACTTAAACAACGGGAAAGCCTCTTCAT-3'; the underlined nucleotide corresponds to bp 343653 of the NC003450 sequence, and the linker sequence is indicated by boldface type), ush3 (5'-TGTTTAAGTTTAGTGGATGGGCTCAGCTTCAGGCTATG-3'; the underlined nucleotide corresponds to bp 345686 of the NC003450 sequence, and the linker sequence is indicated by boldface type), and ush4rev (5'-CAGTAAGCTTGAGAATCGTGTTGTTC-3'; the underlined nucleotide corresponds to bp 345955 of the NC003450 sequence, and the HindIII site is indicated by boldface type) were used. The crossover PCR product obtained with primers ush1 and ush4rev and both of the PCR fragments generated as templates was cloned into pK19mobsacB (27) via its primer-attached XbaI and HindIII sites. Gene deletion mutagenesis with pK19mobsacBushA was carried out as described previously (24). The correct genotype of the deletion mutant was verified by PCR analysis using primers ushA-k-for (5'-GCAGATCCTCGGTGTGAACCT-3'; the underlined nucleotide corresponds to bp 343166 of the NC003450 sequence) and ushA-k-rev (5'-CTCACTGCGGACATTAAAACAGCC-3'; the underlined nucleotide corresponds to bp 346217of the NC003450 sequence), and the mutant was designated WTushA.

Preparation of cell extracts, membrane fractions, and supernatants.
For determination of enzyme activities, cells and medium supernatant were separated by centrifugation (5,000 x g, 10 min, 4°C). Supernatants were passed through a 0.2-µm sterile filter and concentrated about 50-fold by ultrafiltration using Amicon Ultra 10,000-molecular-weight-cutoff membranes according to the manufacturer's recommendations. The cell pellet was washed with ice-cold 100 mM Tris-HCl, pH 7.6, resuspended in 1 ml of the same buffer, and disrupted by ultrasonic treatment at 4°C with a Branson 250 Sonifier (Branson, Danbury, CT) at an output of 2.5 and a duty cycle of 25% for 10 min. After centrifugation for 30 min at 16,000 x g and for 2 h at 190,000 x g, the supernatant was used as the cleared cell extract, and the pellet was resuspended in 50 mM Tris, pH 7.6, 5 mM MgCl₂, 5 mM cysteine, 30% (vol/vol) glycerol and used as the membrane fraction.

5' Nucleotidase assay.
5' Nucleotidase activity was determined using a modification of the assay described by Edwards et al. (5). The reaction mixtures (1 ml) contained 75 mM sodium acetate, pH 6.0, 4.5 mM CoCl₂, and 14.5 mM CaCl₂, and the reactions were started by addition of 1 mM 5'-AMP. The reaction mixtures were incubated at 37°C for 0, 1, 5, 10, and 15 min before the reactions were stopped by addition of 0.04 N HCl and the phosphate released by the activity of 5' nucleotidase was determined. Then, after incubation of the stopped reaction mixtures on ice for 5 min and centrifugation at 4°C and 16,000 x g for 5 min, 1% (wt/vol) ascorbic acid, 0.25% (wt/vol) ammonium molybdate, and 0.6 NH₂SO₄ were added. After 20 min of incubation at 45°C and twofold dilution in water, the absorbance at 820 nm was determined. Phosphate concentrations were determined with appropriate standards. The 5'-AMP concentration was varied from 0.2 mM to 20 mM to determine the kinetic constants. To determine the pH optimum, the pH was varied from 4.5 to 6.0 with increments of 0.5 U. To determine the substrate spectrum of UshA, instead of 5'-AMP, 1 mM or 5 mM ADP, ATP, dATP, CMP, GMP, UMP, IMP, XMP, 2',3'-cAMP, or 3',5'-cAMP was used.

UDP-sugar hydrolase assay.
UDP-sugar hydrolase activity was determined at 30°C by a coupled spectrophotometric assay as described previously (5). The reaction mixtures (1 ml) contained 35 mM Tris-HCl, pH 8.0, 35 mM MgCl₂, 3.1 µM glucose 1,6-bisphosphate, 0.7 mM NADP⁺, rabbit muscle phosphoglucomutase (1 U/ml), and Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (2.5 U/ml), and the reactions were started by addition of 1.4 mM UDP-glucose. Glucose 1-phosphate formed by the reaction of UDP-sugar hydrolase was converted to glucose 6-phosphate and subsequently to 6-phosphogluconate by coupling to phosphoglucomutase and glucose-6-dehydrogenase, and concomitant formation of NADPH (_{340 nm} = 6.3 mM^–1 cm^–1) was monitored at 340 nm.

Assay for alkaline or acid phosphatase activity.
Acid and alkaline phosphatase activities were determined using p-nitrophenylphosphate as the substrate, and the phosphate released during 15 to 60 min of incubation was quantified as described above after the reaction was stopped by addition of 0.5 ml of 2 N NaOH. In assays for alkaline phosphatase activity, the reaction mixtures contained 2 mM MgCl₂, 1 M Tris-HCl, pH 8.0, and 5 mg/ml p-nitrophenylphosphate. The reaction mixtures for acid phosphatase assays contained 2 mM MgCl₂, 100 mM sodium acetate, pH 5.0, and 5 mg/ml p-nitrophenylphosphate.

Primer extension analysis.
Total RNA was prepared as described by Lange et al. (19). Nonradioactive primer extension analysis was performed using IRD800-labeled oligonucleotides (MWG Biotech) as described previously (8). In short, total RNA (20 µg) was transcribed to cDNA using the IRD800-labeled oligonucleotide 5'-GCGAAAGCTGGAACTGCGAGTGCGC-3' (the underlined nucleotide corresponds to bp 343717 of the NC003450 sequence). The primer extension products were separated by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and the lengths of the reaction products were determined by running the four lanes of a sequencing reaction setup using the same oligonucleotide that was used for reverse transcription alongside the primer extension products. The template for the sequencing reaction was generated by PCR amplification of genomic DNA using primers 5'-TTAACACCCCATAAAGAGGGTGAAGAT-3' and 5'-CCACTGGCACCCATTTCTGAGTAAC-3' (the underlined nucleotides correspond to bp 343450 and 343843, respectively, of the NC003450 sequence).

SDS-PAGE and mass spectrometry.
SDS-PAGE and determination of protein concentrations were performed using standard techniques (26) as described previously (25). To identify proteins separated by SDS-PAGE, the bands of interest were excised and digested with trypsin, and the peptide masses determined by matrix-assisted laser desorption ionization—time of flight (MALDI-TOF) mass spectrometry were compared to the masses in a tryptic digest database of 3,746 predicted C. glutamicum proteins as described previously (28). To determine the length of UshA from supernatants, the bands of interest were excised and electroeleuted using a DiaEx midi kit (Serva, Heidelberg, Germany) according to the manufacturer's recommendations, and the protein mass was determined using MALDI-TOF mass spectrometry as described previously (28).

	RESULTS

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C. glutamicum gene ushA encodes a secreted enzyme with 5' nucleotidase and UDP-sugar hydrolase activities.
The ushA (NCgl0322) gene is located 181 bp downstream of an operon putatively encoding RNase H and a glycosyltransferase (NCgl0320 and NCgl0321) and is located 708 bp upstream of NCgl0323, which codes for a hypothetical protein. The ushA gene is predicted to code for a protein consisting of 694 amino acids and having a molecular mass of 72,288 Da. Upon cleavage of the putative signal peptide consisting of 28 amino acids, a 666-amino-acid protein with a molecular mass of 69,485 Da can be predicted. This protein might be secreted or might be an integral membrane protein, as suggested by a possible transmembrane helix at the C terminus.

Based on the fact that 29% of the amino acids of the encoded protein are identical to amino acids of the periplasmic UshA protein from E. coli, it was suggested that these two proteins have similar enzymatic activities. Thus, we examined whether C. glutamicum UshA, like E. coli UshA, is active as a secreted 5' nucleotidase, a UDP-sugar hydrolase, and an alkaline phosphatase. To overexpress ushA, C. glutamicum strain WT(pEKEx2-ushA) was constructed. This strain and the control strain WT(pEKEx2) were grown on LB complex medium with 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). SDS-PAGE of supernatants of C. glutamicum WT(pEKEx2-ushA) revealed a protein with a molecular mass of about 70 kDa that was not present in WT(pEKEx2) (Fig. 1, lanes 1 and 2). The band was excised and digested with trypsin, and the peptide masses determined by MALDI-TOF mass spectrometry were compared to the masses in a tryptic digest database of 3,746 predicted C. glutamicum proteins as described previously (28). Thus, we verified that supernatants of C. glutamicum WT(pEKEx2-ushA) contained UshA. The specific activities of 5' nucleotidase and UDP-sugar hydrolase were higher in supernatants of WT(pEKEx2-ushA) than in supernatants of control strain WT(pEKEx2) (Table 1). On the other hand, the specific activities of alkaline phosphatase and acid phosphatase were not increased noticeably (Table 1). Thus, C. glutamicum WT(pEKEx2-ushA) secreted UshA, which lacked activity as an acid or alkaline phosphatase but was active as a 5' nucleotidase (EC 3.1.4.1) and a UDP-sugar hydrolase (EC 3.1.3.5).

View larger version (131K): [in this window] [in a new window]   FIG. 1. SDS-PAGE analysis of supernatant fractions from C. glutamicum strains WT(pEKEx2) and WT(pEKEx2-ushA) and of cytoplasmic, membrane, and supernatant fractions from strains WT and WTushA. The SDS-PAGE analysis was performed with 3 µg of protein from supernatants of WT(pEKEx2-ushA) (lane 1) and WT(pEKEx2) (lane 2) after growth on LB medium containing 1 mM IPTG and with 10 µg of protein from supernatants, membrane fractions, and cell extracts from phosphate-starved WTushA (lanes 3, 5, and 7, respectively) and from phosphate-starved WT (lanes 4, 6, and 8, respectively). Lane M contained molecular mass standards (191 kDa, 64 kDa, 51 kDa, 39 kDa, 28 kDa, 19 kDa, and 14 kDa).

The ushA deletion mutant WTushA exhibited no detectable 5' nucleotidase activity before and after the onset of phosphate starvation in the cytoplasmic, membrane, and supernatant fractions. However, UDP-sugar hydrolase activity could be detected in cell extracts of phosphate-starved WTushA cells. This suggests that C. glutamicum possesses a cytoplasmic UDP-sugar hydrolase(s) in addition to UshA. On the other hand, ushA encodes the only 5' nucleotidase of C. glutamicum active under the conditions tested.

Expression of ushA is phosphate starvation inducible.
The phosphate starvation stimulon of C. glutamicum included 25 genes which showed increased mRNA levels after phosphate starvation compared to the levels after growth with sufficient phosphate (14). Among these genes was ushA, whose E. coli homolog is not a known member of the phosphate starvation stimulon (31). To confirm that expression of ushA is phosphate starvation inducible in C. glutamicum, we performed primer extension analysis of C. glutamicum WT before and after the onset of phosphate starvation. Primer extension products could be detected only after the onset of phosphate starvation and not before (Fig. 2). The primer extension analysis also revealed the transcriptional start site of ushA and indicated that transcription starts at the cytosine 60 bp upstream of the translational start codon, which is preceded by a ribosome binding site (Fig. 2). The promoter of ushA contains a conserved –10 region but lacks a conserved –35 region (Fig. 2); this was shown to be typical for C. glutamicum promoters (22, 23).

View larger version (40K): [in this window] [in a new window]   FIG. 2. Expression analysis and determination of the transcriptional start sites of the C. glutamicum ushA gene. (A) For primer extension analysis, 20 µg of total RNA isolated from C. glutamicum WT at zero time (lane 1) and 90 min after the shift to a medium without phosphate (lane 2) was used. The transcriptional start site is indicated by an asterisk. The corresponding sequencing reactions (lanes A, C, G, and T) were performed using the same IRD800-labeled oligonucleotide that was used in the primer extension reactions and PCR products which covered the region of the corresponding transcriptional start site as the template DNA. (B) Sequence of the intergenic region between NCgl0321 and ushA. The stop codon of NCgl0321 and the start codon of ushA are indicated by boldface type, and the transcriptional start site (+1), the –10 promoter region, and the ribosome binding site (RBS) are underlined.

ushA is required for growth of C. glutamicum on the phosphorus sources AMP and UDP-glucose.
To determine the physiological role of UshA in C. glutamicum, the abilities of C. glutamicum WT and WTushA to utilize AMP and UDP-glucose as sole sources of phosphorus were examined. To exhaust intracellular sources of phosphorus (14, 18), both strains were starved for phosphate before inoculation into phosphate-free CgXII mineral medium containing either 1 mM AMP, 1 mM UDP-glucose, or 0.065 mM phosphate as a sole source of phosphorus. C. glutamicum WT and WTushA could grow with the limiting concentration of phosphate (0.065 mM) (Fig. 3). However, while C. glutamicum WT could utilize AMP and UDP-glucose as sole sources of phosphorus, C. glutamicum WTushA could not grow on these phosphorus sources (Fig. 3 and data not shown). Thus, ushA is required for growth of C. glutamicum on UDP-glucose and nucleotides such as AMP as phosphorus sources.

View larger version (17K): [in this window] [in a new window]   FIG. 3. Growth of C. glutamicum WT and WTushA on UDP-glucose and phosphate as sole sources of phosphorus. C. glutamicum WT (solid symbols) and WTushA (open symbols) were precultured under phosphate-limiting conditions for 24 h before inoculation into phosphate-free CgXII minimal medium containing 40 g/liter glucose and either 1 mM UDP-glucose (squares) or 0.065 mM phosphate (circles). OD600, optical density at 600 nm.

	DISCUSSION

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C. glutamicum UshA belongs to a family of bacterial and animal 5' nucleotidases that have a common structure (17). UshA from C. glutamicum hydrolyzes ribonucleotides, deoxyribonucleotides, and UDP sugars, as do other 5' nucleotidases. However, the physiological roles of 5' nucleotidases differ considerably. Animal 5' nucleotidases in the brain hydrolyze nucleotides to terminate their action as neurotransmitters and mediate cell-matrix and cell-cell adhesion independent of their catalytic activity (10). In enteric bacteria, the main physiological role of 5' nucleotidase is nucleotide salvage. E. coli, but not C. glutamicum, can grow on 5'-AMP as a sole carbon source, and E. coli requires UshA for utilization of 5'-AMP (21, 33). Moreover, the 5' nucleotidase UshA confers sensitivity of E. coli to 5'-fluorouracil under certain conditions. As 5'-fluorouracil is converted to toxic 5'-fluorouridylate in the purine salvage pathways either by uracil phosphoribosyl trasferase (encoded by upp) or, if ribose-1-phosphate is available, by uridine phosphorylase and uridine kinase, ushA mutations render E. coli upp mutants resistant to 5'-fluorouracil with AMP as a source of ribose 1-phosphate (21, 33). In Salmonella enterica serotype Typhimurium, in which the nonhomologous ushB gene encodes a functional 5' nucleotidase, the ushA gene is cryptic due to a missense mutation (13). Hydrolysis of extracellular nucleotides either by UshA or by UshB in enteric bacteria yields inorganic phosphate and may contribute to the availability of extracellular free phosphate. Expression of neither ushA nor ushB is phosphate starvation inducible, and phenotypic manifestations of ushA or ushB mutations for phosphorus metabolism have not been described (31). Therefore, 5' nucleotidases of enteric bacteria do not function primarily in phosphorus metabolism.

In C. glutamicum, ushA expression is phosphate starvation inducible, and UshA is required for growth on UDP-glucose or 5'-AMP as a sole source of phosphorus. Although the possibility of a role in nucleotide salvage or signal transduction cannot be excluded, the clear phenotype of the ushA deletion mutant and the phosphate-dependent control of ushA expression argue for a primary role of C. glutamicum UshA in phosphorus metabolism. C. glutamicum occurs in soil in which the turnover of microbial biomass phosphorus has been estimated to be about 25 kg per hectare per year (2). This turnover results in the release of large amounts of phosphorus in the form of mobile and labile nucleic acids, phospholipids, and sugar phosphates into the soil (2). The breakdown of nucleic acids to nucleotides is catalyzed by extracellular nucleases, such as NucH from Aeromonas hydrophila (4). Expression of the homologous nucH genes from C. glutamicum and also from Synechocystis sp. increased upon phosphate starvation (14, 29). Thus, the transcriptional program of C. glutamicum to adapt to phosphorus limitation includes induction of the nuclease gene nucH and the 5' nucleotidase gene ushA and allows the organism to access nucleic acids and nucleotides in soil as sources of phosphorus for growth.

C. glutamicum lacks a gene homologous to the ushB gene in S. enterica serotype Typhimurium (15). However, two C. glutamicum genes, NCgl0021 and NCgl0022, encode proteins which have amino acid sequences similar to those of the larger C. glutamicum UshA protein (694 amino acids). It is likely that an ushA paralogous gene was mutated and now carries a stop codon in its open reading frame, giving rise to NCgl0021 and NCgl0022. Similarly, in S. enterica serotype Typhimurium the ushA gene has been inactivated by a single missense mutation (13). The UshA protein contains two conserved domains: the PF00149 domain conserved in 5' nucleotidases and other calcineurin-type phosphoesterases at its N terminus (amino acids 36 to 252) and the PF02872 domain found in C termini of 5' nucleotidases (amino acids 334 to 501). NCgl0021 encodes a 176-amino-acid polypeptide which contains the PF00149 domain and is 55% identical to amino acids 73 to 250 of UshA. On the other hand, the NCgl0022-encoded polypeptide, which contains the PF02872 domain, is 412 amino acids long and 37% of its amino acids are identical to amino acids of the C terminus of UshA (amino acids 269 to 633). The absence of detectable 5' nucleotidase and UDP-sugar hydrolase activities from supernatants of the C. glutamicum ushA deletion mutant showed that the proteins encoded by NCgl0021 and NCgl0021 cannot functionally replace UshA.

The genome of the related organism Corynebacterium efficiens lacks homologs of NCgl0021 and NCgl0022, but it carries a homolog of ushA (CE0337). Corynebacterium ammoniagenes is another member of the Corynebacteriaceae and is used for biotechnological production of flavor enhancers, such as 5'-IMP (20). In such a process, inosine is synthesized de novo by intracellular dephosphorylation of 5'-IMP. Inosine secreted into the culture medium can be converted to 5'-IMP chemically or enzymatically, and efficient producing strains need to be devoid of extracellular 5' nucleotidase activity (20). It remains to be determined whether C. ammoniagenes possesses genes homologous to ushA and, if so, whether defined ushA deletion mutants perform better in the 5'-IMP production process. In addition to C. glutamicum and C. efficiens, UshA is found in Streptomyces coelicolor (SCO4152), while the genomes of Corynebacterium diphtheriae, Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium leprae do not carry homologs of UshA. This suggests that mycobacteria and C. diphtheriae either cannot utilize nucleotides as sources of phosphorus or do so by a pathway(s) that is an alternative to hydrolysis by 5' nucleotidase.

	ACKNOWLEDGMENTS

 
We thank Hermann Sahm for continuous support, Michael Bott for fruitful discussions, and Christina Mack, Steffen Schaffer, and Axel Niebisch for mass spectrometry analyses.

The support of the Bundesministerium für Bildung und Forschung (BMBF) through grant 031U113D/031U213D is gratefully acknowledged. Ulrike Sorger-Herrmann was supported by a fellowship from the Friedrich-Ebert-Stiftung.

Some of the work described here is from the planned Ph.D. dissertation of Ulrike Sorger-Herrmann at the faculty of Mathematics and Natural Sciences of Heinrich-Heine-Universität Düsseldorf.

	FOOTNOTES

 
* Corresponding author. Mailing address: Institute of Biotechnology 1, Research Center Juelich, D-52425 Juelich, Germany. Phone: 49-2461-615169. Fax: 49-2461-612710. E-mail: v.wendisch{at}fz-juelich.de.

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