Functional Characterization of thed-Tyr-tRNATyr Deacylase from Escherichia coli *

The yihZ gene of Escherichia coli is shown to produce a deacylase activity capable of recycling misaminoacylated d-Tyr-tRNATyr. The reaction is specific and, under optimal in vitroconditions, proceeds at a rate of 6 s−1 with aK m value for the substrate equal to 1 μm. Cell growth is sensitive to interruption of theyihZ gene if d-tyrosine is added to minimal culture medium. Toxicity of exogenous d-tyrosine is exacerbated if, in addition to the disruption of yihZ, the gene of d-amino acid dehydrogenase (dadA) is also inactivated. Orthologs of the yihZ gene occur in many, but not all, bacteria. In support of the idea of a general role of thed-Tyr-tRNATyr deacylase function in the detoxification of cells, similar genes can be recognized inSaccharomyces cerevisiae, Caenorhabditis elegans, Arabidopsis thaliana, mouse, and man.

growth rate of yihZ null mutants could be obtained if minimal medium was supplemented with D-tyrosine. Orthologs of the E. coli deacylase gene occur in the genetic materials of many cells including those of mammals and higher plants. MATERIALS AND METHODS D-[methylene- 3 H]Tyrosine (211 GBq/mmol) was custom-prepared by Amersham Pharmacia Biotech; L-[ 14 C]tyrosine was from NEN Life Science Products; and unlabeled D-tyrosine was from Sigma. Sephadex DEAE-A50 and Q-Sepharose were from Amersham Pharmacia Biotech. Hydroxylapatite was from Bio-Rad and Trisacryl GF05 from IBF.
Preparation of Aminoacylated tRNA Tyr -E. coli tRNA Tyr2 was overexpressed in strain JM101TR from a synthetic gene built according to a published procedure (9). The resulting bulk tRNA accepted 320 pmol of L-tyrosine per A 260 unit.

D-[
3 H]Tyr-tRNA Tyr was directly prepared from the above tRNA extract. The reaction mixture (500 l) contained 20 mM Tris-HCl (pH 7.8), 7 mM MgCl 2 , 2 mM ATP, 3.5 M D-[ 3 H]tyrosine (18.5 GBq/mmol), 0.1 mM EDTA, 7.5 M tRNA Tyr , and 1.2 M purified E. coli tyrosyl-tRNA synthetase. Reactions conditions (10 min, 28°C) ensured a nearly complete esterification of the D-amino acid to the tRNA. Note that because of the occurrence of contaminating L-tyrosine (2%) inside the D-[ 3 H]tyrosine sample, the reaction was performed with a concentration of tRNA Tyr in excess over that of D-tyrosine. Under this condition and provided the D-amino acid is fully incorporated in tRNA Tyr , the proportion of L-Tyr-tRNA Tyr (the favored product of tyrosyl-tRNA synthetase) in the prepared D-Tyr-tRNA Tyr sample cannot exceed 2%. The reaction was quenched by 500 l of 600 mM sodium acetate (pH 5.0) plus 500 l of phenol saturated with a 100 mM sodium acetate solution (pH 5.0). The solution was vigorously shaken and centrifuged for 15 min at 15,000 ϫ g. The interphase and the phenol phase were re-extracted with 250 l of 300 mM sodium acetate (pH 5.0) and centrifuged as above. The two aqueous phases were pooled, precipitated with ethanol, and centrifuged (15 min at 15,000 ϫ g). The pellet was resuspended in 100 l of 20 mM sodium acetate (pH 5.0) containing 100 mM KCl and 0.1 mM EDTA. Finally, the sample was chromatographed on a Trisacryl GF05 column (0.25 ϫ 16 cm) equilibrated in the same solution. Resulting D-Tyr-tRNA Tyr (100 pmol of D-amino acid incorporated per A 260 unit of tRNA) was stored frozen at Ϫ20°C.
N-Acetylation of D-[ 3 H]Tyr-tRNA Tyr or L-[ 14 C]Tyr-tRNA Tyr was performed as described earlier for the preparation of diacetyl-lysyl-tRNA Lys (10).
Purification of D-Tyr-tRNA Tyr Deacylase-D-Tyr-tRNA Tyr deacylase was purified from E. coli strain K37 (Table I). All buffers contained 0.1 mM EDTA and 10 mM 2-mercaptoethanol. Cells were grown at 37°C in 16 liters of 2ϫ TY medium and harvested by centrifugation for 35 min at 3,000 ϫ g. The cell pellet was suspended in 20 mM Tris-HCl (pH 7.8) at a cell density of 0.1 g of wet weight per ml of buffer. Cells were disrupted by sonication (10 min, 0°C), and debris removed by centrifugation (35 min at 3,000 ϫ g). Nucleic acids were precipitated by addition of streptomycin (30 g/liter) to the supernatant, which was then centrifuged for 35 min at 3,000 ϫ g. The resulting supernatant was brought to 50% ammonium sulfate saturation, left to stand 1 h at 4°C, and centrifuged (35 min, 3,000 ϫ g). The pellet was discarded, and the supernatant was brought to 80% ammonium sulfate saturation. After centrifugation for 35 min at 3,000 ϫ g, the protein sample was dissolved in 250 ml of 20 mM potassium phosphate (pH 8.0) and dialyzed against 3 liters of the same buffer. The resulting solution was applied on a * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. column of Sephadex DEAE-A50 (4 ϫ 12 cm), equilibrated in 20 mM potassium phosphate (pH 8.0). Elution was carried out with a 3-liter linear gradient of 40 -350 mM potassium phosphate (pH 8.0) at a flow rate of 100 ml/h. Fractions containing activity were pooled, dialyzed against a 10 mM potassium phosphate (pH 6.75) buffer, and applied on a hydroxylapatite column (3 ϫ 13 cm) equilibrated in the same buffer. Enzyme activity was recovered by using a 2ϫ 500 ml linear gradient of 10 -400 mM potassium phosphate (pH 6.75), at a flow rate of 50 ml/h. Fractions containing activity were pooled, dialyzed against a 10 mM potassium phosphate buffer (pH 7.0), and applied on a Q-Sepharose Hi-Load column (3.2 ϫ 10 cm, from Amersham Pharmacia Biotech) equilibrated in the same buffer. This column was eluted with a 1.5-liter linear gradient from 10 to 500 mM NaCl in the buffer of the column. One fraction from the Q-Sepharose chromatography, accounting for 1/3 of the total recovered activity, was concentrated batchwise on a Sephadex DEAE-A50 column (0.25 ϫ 4 cm). 100 l of the recovered sample (600 l) were then loaded on a TSK G3000 SW HPLC column (0.75 ϫ 30 cm, from Tosohaas), which was eluted at a flow rate of 0.5 ml/min with 10 mM potassium phosphate (pH 7.0) containing 150 mM KCl.
N-terminal Sequencing of D-Tyr-tRNA Tyr Deacylase-Aliquots (200 l) of each active fraction recovered from the above TSK column were analyzed by SDS-PAGE 1 (11). After migration, proteins were electrophoretically transferred on a ProBlot membrane (from Applied Biosystems) and stained with Amido Black. About 10 protein bands were visible on the membrane. One of them, the intensity of which was correlated with the D-Tyr-tRNA Tyr deacylase activity measured in the fractions, was cut and submitted to 10 cycles of Edman degradation on an Applied Biosystems 473A Sequencer.
Purification of Tyrosyl-tRNA Synthetase-E. coli tyrosyl-tRNA synthetase was overexpressed in strain JM101TR harboring plasmid pBR322 EcoTyrTS (Table I). Cells were grown overnight in 1 liter of 2ϫ TY medium containing 100 g of ampicillin per ml. Crude extract preparation and removal of nucleic acids by streptomycin precipitation were performed as described above for the purification of D-Tyr-tRNA Tyr deacylase. Then the sample was brought to 70% ammonium sulfate saturation, left to stand 1 h at 4°C, and centrifuged for 20 min at 8,000 ϫ g.
The protein sample was dissolved in 3 ml of 20 mM potassium phosphate (pH 6.75), dialyzed overnight against 3 liters of the same buffer, and applied on an 80-ml Q-Sepharose column (2.8 ϫ 13 cm). The column was eluted with a 1200-ml linear gradient of 100 -400 mM KCl in the column buffer (flow rate 120 ml/h). Recovered fractions containing activity were immediately applied on a hydroxylapatite column (3.8 ϫ 18 cm) equilibrated in 20 mM potassium phosphate (pH 6.75). Tyrosyl-tRNA synthetase activity was recovered at a flow rate of 100 ml/h by using a 5-liter linear gradient of potassium phosphate (pH 6.75), from 40 to 200 mM. As judged by SDS-PAGE analysis, tyrosyl-tRNA synthetase was at least 90% pure at this stage. The enzyme solution was concentrated 100-fold by a batchwise chromatography on a Sephadex DEAE column (2 ϫ 3.2 cm) and then dialyzed against 20 mM Tris-HCl (pH 7.8) containing 60% glycerol and stored at Ϫ20°C.
Purification of the D-Tyr-tRNA Tyr Deacylase from Overproducing E. coli Cells-E. coli strain JM101TR transformed by plasmid pYtH (Table  I) was grown at 37°C in 2 liters of 2ϫ TY medium containing 200 g of ampicillin per ml. When the optical density of the culture reached 0.3 at 650 nm, 1 mM isopropyl-1-thio-␤-D-galactopyranoside was added, and growth was continued overnight. Conditions for crude extract preparation, removal of nucleic acids, and ammonium sulfate precipitation were the same as those used for the purification of the deacylase from the strain K37. Further chromatographies on Q-Sepharose and hydroxylapatite columns were similar to those of tyrosyl-tRNA synthetase purification. However, in the case of the deacylase (i) all buffers were supplemented with 0.1 mM EDTA and 10 mM 2-mercaptoethanol, (ii) NaCl rather than KCl was used in the elution buffer of the Q-Sepharose column, and (iii) the potassium phosphate linear gradient for the elution of the hydroxylapatite column was from 10 to 400 mM. Recovered deacylase was concentrated by an ammonium sulfate precipitation (80% saturation). After centrifugation at 10,000 ϫ g for 30 min, the pellet was dialyzed against a 20 mM Tris-HCl buffer (pH 7.8) containing 60% glycerol, 0.1 mM EDTA and 10 mM 2-mercaptoethanol, and stored at Ϫ20°C.
Assay of D-Tyr-tRNA Tyr Deacylase Activity-Unless otherwise stated, D-Tyr-tRNA Tyr deacylase activity was measured for 5 min at 28°C in 100-l assays containing 50 nM D-[ 3 H]Tyr-tRNA Tyr , 20 mM Tris-HCl (pH 7.8), and 0.1 mM EDTA. The reaction was quenched by the successive addition of 100 l of 10% trichloroacetic acid and 20 l of carrier RNA from yeast (4 mg/ml). The sample was centrifuged (5 min, 15,000 ϫ g), and 200 l of the supernatant were mixed with 6 ml of Picofluor scintillation mixture (from Packard) and counted in a Beckman LS1801 counter. Concentration of D-Tyr-tRNA Tyr deacylase was determined using the light-absorption coefficient calculated from the amino acid sequence of the protein (0.571 A 280 units⅐mg Ϫ1 ⅐ml). K m and k cat values were derived from iterative non-linear fits to the theoretical Michaelis equation to the experimental values, using the Levenberg-Marquardt algorithm. Confidence limits on the fitted values were obtained by 100 Monte Carlo simulations followed by least squares fitting, using the experimental standard deviations on individual measurements (12).
Cloning of the yihZ Gene-The yihZ gene was amplified by PCR using oligonucleotides DTyrFront (CCGAATTCCATGATTGCATTAATTCAAC-GCGTAAC) and DTyrEnd (CCAGCCAAGCTTTCATACCTGCAACCAG-AATGTCACG) and genomic DNA from strain K37. The amplified DNA fragment (460 bp) was purified using the Qiagen Plasmid Mini Kit 100, digested by EcoRI and HindIII, and inserted into the corresponding sites of plasmid pKK223-3, to finally give plasmid pYtH. The sequence of the insert was verified by DNA sequencing.
The EcoRI-HindIII fragment of plasmid pYtH containing the yihZ gene was also subcloned between the corresponding sites of plasmid pBluescript(Ϫ)KS (pBS). The resulting plasmid, designated pBSKSyihZ, harbored the yihZ gene in the opposite orientation to the lacZ promoter.
Disruption of the yihZ Gene-Disruption of the yihZ gene was achieved by the procedure of Hamilton et al. (13) using plasmid pMAK705, which contains a thermosensitive replicon and a chloramphenicol resistance gene. A DNA fragment containing the first 190 bp of the yihZ ORF, the kanamycin resistance cassette from pUC4K (14), and the last 187 bp of the yihZ ORF was inserted in pMAK705. E. coli strain K37 was transformed by the resulting plasmid (pKK4). The integration of pKK4 into the chromosome was selected by plating transformants at 42°C on LB agar medium containing chloramphenicol. After growth at 30°C in liquid medium for ϳ30 generations, cells no longer carrying the plasmid in their chromosome were identified as chloramphenicol-sensitive colonies at 42°C. Approximately 60% of selected cells were also kanamycin-resistant at 42°C, thus suggesting that (i) the ⌬yihZ::kan mutation carried by pKK4 was now located on the chromosome and (ii) the yihZ gene was not essential to the growth of E. coli. After subsequent growth at 42°C, the loss of the thermosensitive plasmid was verified by ensuring that the cells had become chloramphenicol-sensitive at 30°C. One of the resulting clones was named K37⌬TyrH and was used in further studies. Disruption of the yihZ gene in this clone was confirmed by PCR amplification of chromosomal DNA using the above oligonucleotides DTyrFront and DTyrEnd. The ⌬yihZ::kan mutation could be transferred from strain K37⌬TyrH into strains EC989, FB8, and FB8r (Table I) by P1 transduction. Resulting strains were named EC989⌬TyrH, FB8⌬TyrH, and FB8r⌬TyrH, respectively. Strain K37⌬TyrH⌬recA was further obtained by P1 transduction of the ⌬recA938::cat mutation from the strain GW5552 (15). Strains EC989d and EC989d⌬TyrH were constructed by P1 transduction of the dadA ϩ allele, from strain K37 into strain EC989 or EC989⌬TyrH, respectively. In the latter case, selection of transductants was performed on M9 minimal plates containing 20 mM L-alanine as carbon source (16,17).

Cloning of the Gene Encoding E. coli D-Tyr-tRNA Tyr Deacylase-
The presence of D-Tyr-tRNA Tyr hydrolytic activity in E. coli strain K37 was verified by adding cellular extract to D-or L-Tyr-tRNA Tyr (Tris 20 mM (pH 7.8), 28°C, final concentration of total protein in the assay: 20 -200 g/ml). The rate of deacylation of D-Tyr-tRNA Tyr was at least 100-fold faster than that of L-Tyr-tRNA Tyr . The activity of the enzyme responsible for the specific D-Tyr-tRNA Tyr hydrolysis was then followed through chromatographies on Sephadex DEAE-A50, hydroxylapatite, Q-Sepharose, and TSK 3000. At each step, a single peak of activity was always recovered. According to the TSK gel filtration, the molecular mass associated to the native D-Tyr-tRNA-Tyr deacylase activity could be estimated equal to 35 Ϯ 3 kDa. In this experiment, marker proteins of known M r included lysyl-tRNA synthetase (18), truncated methionyl-tRNA synthetase (19), ovalbumin, carbonic anhydrase, peptidyl-tRNA hy-drolase (20), and egg white lysozyme.
At the last step of the purification, the recovered enzyme was less than 10% pure, according to SDS-PAGE analysis. However, along the fractions recovered from the TSK column, the activity varied proportionally to the intensity on the gel of one protein band having an apparent mass of 16 Ϯ 2 kDa. After transfer from the gel to a polyvinylidene difluoride membrane, this protein material was submitted to 10 cycles of Edman degradation. Its N-terminal sequence, MIALIQRVTR, designated an open reading frame (ORF), yihZ, at 87.81 min on the E. coli genetic map. The predicted M r of the yihZ product (15,950) was in agreement with the M r of the selected protein.
To assess whether the yihZ gene actually encoded the D-Tyr-tRNA Tyr deacylase activity in the crude extract, a DNA fragment encompassing this gene was amplified by PCR and inserted into the expression vector pKK223-3. Upon transformation by the resulting plasmid (pYtH), E. coli strain JM101TR overexpressed one protein with the expected mass of ϳ16 kDa. In addition, D-Tyr-tRNA Tyr deacylase activity in crude extract was increased 1500-fold as compared with cells transformed by the control plasmid pKK223-3 (Table II). We therefore concluded that the yihZ gene encodes the E. coli D-Tyr-tRNA Tyr deacylase and that, upon TSK chromatography in non-denaturing conditions, this deacylase shows the mass of a dimer.
The yihZ gene is the third ORF of a four-gene operon also including yihX, rbn (or yihY), and yiiD (21) (Fig. 1). A putative promoter sequence can be recognized 30 -63 bp upstream of the yihX initiator codon. rbn codes for RNase BN, an enzyme involved in the maturation of the 3Ј-end of tRNAs (22). According to sequence comparison using BLAST program (23), the yihX protein resembles various dehalogenases or epoxide hydrolases. The yiiD product has no significant homology with any other protein stored in the data bases.
Catalytic Constants of D-Tyr-tRNA Tyr Deacylase-D-Tyr-tRNA Tyr deacylase was purified from strain JM101TR transformed by plasmid pYtH. After successive chromatographies on Q-Sepharose and hydroxylapatite columns, the enzyme was recovered homogeneous, as judged by SDS-PAGE analysis. The initial rate of D-Tyr-tRNA Tyr hydrolysis, in a 20 mM Tris-HCl buffer (pH 7.8) containing 0.1 mM EDTA, was increased 3-4fold upon the addition of either 50 mM KCl or 10 mM MgCl 2 (Table III). Higher concentrations of these salts inhibited the enzyme, and the simultaneous addition of both MgCl 2 and KCl showed no effect. These results suggest that ionic strength improves the rate of the reaction through the folding of the tRNA structure. Accordingly, addition of 1 mM spermidine also stimulated 3-fold the hydrolysis of D-Tyr-tRNA Tyr (Table III). However, specific features in the tRNA nucleotide sequence may also be involved in substrate recognition, as indicated by the capacity of the deacylase to hydrolyze D-tyrosyl oligonucleotides (7).
The activity of the deacylase in the presence of 5 mM MgCl 2 was insensitive to the further addition of 0.1 mM various metal ions like CaCl 2 , CoCl 2 , MnCl 2 , or NiCl 2 . Only ZnCl 2 showed a slight stimulatory effect (Table III).
The initial rate of hydrolysis by the deacylase was measured at 5 mM MgCl 2 as a function of D-Tyr-tRNA Tyr concentration. The kinetics were Michaelian, with a K m value of 1.0 Ϯ 0.15 M and a maximal rate of 6.0 Ϯ 0.5 s Ϫ1 . Under the same assay Cells were grown overnight in LB medium. With the JM101TR derivatives, the medium was supplemented with 200 g of ampicillin/ml, and 1 mM isopropyl-1-thio-␤-D-galactopyranoside was added when the optical density of the culture reached 0.3 at 650 nm. Specific deacylase activities were measured in crude extracts obtained by sonication of cells resuspended at an optical density of 100 at 650 nm. Final protein concentration in the extracts was 5-10 mg/ml.  conditions, the spontaneous chemical hydrolysis of D-Tyr-tRNA Tyr occurred at a rate of 2.2 ϫ 10 Ϫ4 s Ϫ1 . Addition of uncharged tRNA (K I Ͼ20 M) or of free D-tyrosine (K I Ͼ2 mM) had no effect on the enzyme activity.
Hydrolysis of D-Tyr-tRNA Tyr by Tyrosyl-tRNA Synthetase-Because several aminoacyl-tRNA synthetases can hydrolyze aminoacyl-tRNAs (24 -26), the AMP-independent deacylation of D-Tyr-tRNA Tyr by tyrosyl-tRNA synthetase deserved attention. Nevertheless, this reaction was so slow that, in order to measure initial rates of hydrolysis, synthetase concentrations (0.2-2.5 M) much higher than the D-Tyr-tRNA Tyr substrate concentration (50 nM) had to be used in the assay. Michaelis constants of the reaction could be estimated, however, by varying the enzyme concentration in the assay. k cat and K m values of 1.5 ϫ 10 Ϫ3 s Ϫ1 and 0.2 M, respectively, were deduced. Therefore, although D-Tyr-tRNA Tyr strongly interacts with tyrosyl-tRNA synthetase, its rate of deacylation by this enzyme remains slow, being only 7-fold faster than that of spontaneous chemical hydrolysis.
Specificity of D-Tyr-tRNA Tyr Deacylase-D-Tyr-tRNA Tyr deacylase and peptidyl-tRNA hydrolase recognize an ester linkage between the 3Ј-terminal adenosine of tRNA and the carboxyl group of an amino acid. Nevertheless, D-Tyr-tRNA Tyr deacylase does not hydrolyze L-aminoacyl-tRNAs, on the one hand (7), and peptidyl-tRNA hydrolase is specific for N-blocked-L-amino acids, on the other hand (8). By using D-Tyr-tRNA Tyr , L-Tyr-tRNA Tyr , N-acetyl-D-Tyr-tRNA Tyr , or N-acetyl-L-Tyr-tRNA Tyr as substrates, these specificities could be confirmed (Table IV). In particular, N-blocked D-or L-aminoacylated tRNAs fully resisted the action of D-Tyr-tRNA Tyr deacylase (Table IV).
Notably, strain JM101TR transformed by pYtH did not grow when LB ampicillin plates contained 1 mM isopropyl-1-thio-␤-D-galactopyranoside, i.e. under conditions of full overexpression of yihZ. One possible interpretation is that an excess of cellular deacylase inhibits the growth of E. coli through hydrolysis of some aminoacylated tRNAs. One possible candidate is Gly-tRNA Gly , which was shown in vitro to be sensitive to the action of the deacylase (7).
Occurrence of yihZ Orthologs in Various Genomes-Searches on the GenBank TM /EMBL data bases using BLAST program (23) revealed the presence of yihZ orthologs in several proteobacteria (Hemophilus influenzae, Pseudomonas aeruginosa, Vibrio cholerae, etc.), in various Gram-positive bacteria (Streptococcus equisimilis, Staphylococcus aureus, Mycobacterium tuberculosis, etc.), as well as in Deinococcus radiodurans, Thermotoga maritima, Aquifex aeolicus, fungi (Saccharomyces cerevisiae), animals (Caenorhabditis elegans), and higher plants (Arabidopsis thaliana). Partial cDNA sequences from expressed sequence tags also indicate the presence of genes homologous to yihZ in mouse and man.
On the other hand, genes related to yihZ could not be recognized in the following complete genome sequences: Helicobacter pylori, Rickettsia prowazekii, Mycoplasma genitalium, Mycoplasma pneumoniae, Synechocystis sp. PCC6803, Borrelia burgdorferi, Treponema pallidum, Chlamydia trachomatis or in any sequenced archaebacterial genome.
The case of B. subtilis is special. In this organism, a DNA fragment encodes a protein sequence clearly homologous to that of the yihZ product. However, the lack of initiation codon renders the translation of this putative protein unlikely, in agreement with the earlier observation that D-Tyr-tRNA Tyr deacylase activity could not be detected in a B. subtilis crude extract (7). One possible reason is that the homolog of yihZ in B. subtilis is not functional.
Inactivation of the Chromosomal yihZ Gene-To assess the importance of D-Tyr-tRNA Tyr deacylase in cell function, the yihZ gene of E. coli strain K37 was inactivated (Table I). For this purpose, a kanamycin resistance cassette was placed inside the chromosomal yihZ gene of strain K37, whereas a wild-type copy of the gene was retained on a plasmid bearing a thermosensitive replicon. The resulting strain grew at 42°C, a non-permissive temperature for plasmid replication. As a con- FIG. 1. Map of the E. coli chromosomal region encompassing yihZ and structures of plasmids pYtH and pKK4. The organization of the four-gene yihX-rbn-yihZ-yiiD operon is derived from Plunkett et al. (21). The direction of transcription is indicated by an arrow. A putative promoter sequence (P) can be recognized 30 -63 bp upstream of the yihX initiator codon. A 460-bp DNA fragment harboring the yihZ gene was amplified by PCR and inserted between the EcoRI and HindIII sites of pKK223-3, to give pYtH. The plasmid pKK4, which was used to inactivate the chromosomal yihZ gene, was derived from pMAK705. It harbored the first 190 bp of yihZ, the kanamycin resistance cassette from pUC4K (14), and the last 187 bp of yihZ. sequence, the thermosensitive plasmid carrying the wild-type copy of the yihZ gene was spontaneously lost at this temperature. The resulting ⌬yihZ::kan chromosomal mutant strain was named K37⌬TyrH.

TABLE III Activity of D-Tyr-tRNA Tyr deacylase under various ionic conditions
Upon inactivation of the yihZ gene, the D-Tyr-tRNA Tyr deacylase activity in crude extracts of the strain K37⌬TyrH was reduced by at least 10-fold (Table II). The residual activity is likely to be contributed by the tyrosyl-tRNA synthetase. Actually, this enzyme, which hydrolyzes D-Tyr-tRNA Tyr at a maximal rate of 1.5 ϫ 10 Ϫ3 s Ϫ1 , is present at a concentration of ϳ1 M in the cell extract. For comparison, using the catalytic constant of purified deacylase, the concentration of the yihZ product in the extract of K37 can be calculated on the order of 30 nM. We therefore conclude that in the E. coli wild-type context, the yihZ gene product is the only protein capable of efficiently deacylating D-Tyr-tRNA Tyr .
The generation times of the strains K37⌬TyrH (⌬yihZ) and K37 (yihZ ϩ ) were compared. They were identical in either 2ϫ TY or LB-rich medium and in M9 minimal medium. LB medium under anaerobic conditions was also investigated, without result. In contrast, we observed that, if added at 2.4 mM (near from its limit of solubility) to M9 minimal medium, Dtyrosine significantly increased the generation time of the ⌬yihZ mutant (92 min instead of 69) but did not change that of the control yihZ ϩ strain (Table V).
To confirm that the inhibition of growth by D-tyrosine resulted from the yihZ gene disruption, strain K37⌬TyrH⌬recA was transformed with plasmid pBSKSyihZ, which carries an intact yihZ copy. As expected, addition in trans of this plasmid abolished the growth inhibition caused by D-tyrosine (Table V). Control plasmid pBS had no effect. During exponential growth in M9 medium, an aliquot of the culture was withdrawn to measure the deacylase activity. In cells transformed by pBSKSyihZ or pBS, the D-Tyr-tRNA Tyr deacylase activity amounted to 440 and 14 units per mg of total protein, respectively. Under the same growth conditions, the deacylase activity in the strain K37 was equal to 130 units/ mg. Transport of D-tyrosine in E. coli by the general aromatic amino acid permease is inhibited by the addition of either L-tryptophan or L-tyrosine (27). In agreement with an involvement of this permease in the uptake of D-tyrosine, we observed that supplementation of the M9 medium with 0.2 mM L-tyrosine or 1 mM L-tryptophan fully suppressed the negative effect exerted by 2.4 mM D-tyrosine on the growth of the strain K37⌬TyrH (Table V).
D-Amino Acid Dehydrogenase Protects Against the D-Tyrosine Toxicity-Neurospora crassa mutants with decreased D-amino acid oxidase activity have been reported to be more sensitive to the toxicity of D-tyrosine than their parental strains (28). Consequently, we also studied the effect of the disruption of the yihZ gene in an E. coli strain with reduced D-amino acid dehydrogenase activity (EC989). The dadA ⌬yihZ::kan double mu-tant (EC989⌬TyrH) was obtained by P1 transduction of the ⌬yihZ::kan mutation from strain K37⌬TyrH into strain EC989. However, the parental strain EC972 (dadA ϩ ) could not be used as a control because we observed that it was an auxotroph for tryptophan while EC989 was a prototroph, and the protection exerted by tryptophan against D-tyrosine transport precluded the comparison of this couple of strains. Therefore, dadAϩ derivatives of EC989 and EC989⌬TyrH (EC989d and EC989d⌬TyrH, respectively) were constructed by P1 transduction. In all the ⌬yihZ::kan strains, deacylase activity in crude extracts was lowered by a factor greater than 10-fold when compared with the corresponding yihZ ϩ strains. This factor is identical to that measured when comparing K37⌬TyrH and K37 (Table II).
In the absence of external D-tyrosine, the generation times of strains EC989 (dadA), EC989d (dadA ϩ ), EC989⌬TyrH (dadA ⌬yihZ), and EC989d⌬TyrH (dadA ϩ ⌬yihZ) were the same (80 Ϯ 2 min). In the presence of 2.4 mM D-tyrosine, the growth of the double dadA ⌬yihZ mutant was markedly slowed (230 min) as compared with strain EC989 (106 min). In the dadA ϩ context, the effect of the disruption of yihZ is much less pronounced (136 versus 90 min). These measurements, summarized in Table V, therefore indicate that the toxicity of D-tyrosine is reinforced in the absence of D-amino acid dehydrogenase activity.
In Table V, the two couples of strains K37/K37⌬TyrH and EC989d/EC989d⌬TyrH show small discrepancies in the amplitudes of the response to the D-amino acid. The consequence of the disruption on the growth appeared more pronounced in the EC989d context (136 versus 90 min) than in the K37 one (92 versus 71 min). Such a variation may reflect the different genotypes of the strains (Table I). To exclude an interference of the relA1 mutation with the behavior of the strain EC989d, the following experiment was designed. The ⌬yihZ::kan mutation was transduced into the relA ϩ strain FB8 and into its isogenic relA1 mutant FB8r. The generation times of the four resulting strains were measured in the presence or absence of added D-tyrosine. Values in Table V do not reveal the occurrence of a relation between the D-amino acid toxicity and the relA character in a dadA ϩ context.  a Cells were grown at 37°C in M9 minimal medium containing or not 2.4 mM D-tyrosine. Where indicated, the growth medium was supplemented with 0.2 mM L-tyrosine or 1 mM L-tryptophan. In the case of cells harboring plasmid pBS or pBSKSyihZ, the medium also contained 100 g of ampicillin per ml. In the case of EC989 derivatives, the medium also contained 40 g of methionine per ml. Cultures were inoculated with cells pre-grown to mid-exponential phase (0.2-0.4 OD 650 ) in the medium under study. Measurements were done at least twice. Differences between results never exceeded 5%.

DISCUSSION
The D-Tyr-tRNA Tyr deacylase characterized in this study displays the specificity and the rate parameters expected for an efficient recycling of D-Tyr-tRNA Tyr into free tRNA Tyr and, therefore, for defending the cell against misincorporation of D-tyrosine into proteins. Accordingly, in the absence of functional yihZ gene, D-tyrosine becomes toxic for the bacterium even though all steps of the elongation pathway in translation, from tRNA aminoacylation to peptide bond synthesis, are believed to favor the L-isomers of amino acids (29). This toxicity of D-tyrosine is improved by the concomitant inactivation of the gene of D-amino acid dehydrogenase. Deamination by the latter enzyme enables D-tyrosine to be recycled into 4-hydroxyphenylpyruvate, a precursor of L-tyrosine. Consequently, the absence of this enzyme activity stabilizes intracellular D-tyrosine and exerts a synergistic effect on the yihZ phenotype.
To transport D-tyrosine inside E. coli cells, the general aromatic amino acid permease encoded by the aroP gene is necessary (27). In S. cerevisiae, the uptake of D-tyrosine is sustained by the general amino acid permease encoded by the gap gene. L-Amino acid starvation and, depending on the yeast strain, deprivation of ammonium ions are necessary to promote Dtyrosine transport and, as a consequence, toxicity of this amino acid (30).
In the case of B. subtilis, toxicity of D-tyrosine is exacerbated by the lack of functional D-Tyr-tRNA Tyr deacylase gene. Such a toxicity was indeed observed in strains which efficiently transport this D-amino acid (31). Incorporation of D-tyrosine into proteins could be established (32). In addition, D-tyrosine impairs L-tyrosine biosynthesis through the inhibition of prephenate dehydrogenase (32).
Several D-amino acids like D-methionine, D-phenylalanine, and D-tryptophan are described to be toxic for E. coli. In this case, the toxicity is believed to ensue from incorporation of the D-amino acids in cell walls (33,34) rather than from formation of D-aminoacyl-tRNAs. However, if such misaminoacylated tRNAs occur in vivo, they are possibly recycled by specific hydrolases. Indeed, earlier in vitro studies indicated that the specificity of D-Tyr-tRNA Tyr deacylase was broad enough to ensure the recognition of D-Phe-tRNA Phe or Gly-tRNA Gly (7). In this context, our preliminary results (not shown) suggest that the growth of strain K37⌬TyrH (⌬yihZ::kan) exhibits enhanced sensitivity to the presence of D-tryptophan when compared with K37 (yihZ ϩ ). At this stage, we may hypothesize that D-tryptophan is esterified to tRNA Trp and that it is further released from D-Trp-tRNA Trp by the D-Tyr-tRNA Tyr hydrolase. We also noticed that the toxicity of D-methionine or D-phenylalanine was not improved upon disruption of yihZ. We conclude that either these D-amino acids are not or are weak substrates of the corresponding aminoacyl-tRNA synthetases, or that, if D-aminoacylated tRNAs occur, they are processed by hydrolases distinct from D-Tyr-tRNA Tyr deacylase. In cases where such deacylases would exist, they will not present any sequence homology with the E. coli yihZ gene product.
Gene organization in E. coli suggests that the yihZ gene can be expressed together with the yihX cistron. The product of the latter resembles a detoxification protein. Association of other detoxifying functions with that of the deacylase makes sense for the homeostasis of the bacterium. One other cistron, close to yihZ, is yihY or rbn. It produces an RNase involved in the maturation of functional tRNA molecules. Therefore, one can imagine that, in response to some starvation conditions involving the sequestration of tRNAs under the form of esters of D-amino acids, for instance, the cell must respond by both accelerating the onset of new tRNA molecules and recycling the D-Tyr-misesterified tRNAs. As a benefit of this adaptation, the cell would escape the risk of incorporation of D-amino acids in nascent proteins.
In the H. influenzae genome, the yihZ cistron is adjacent to mioC, a gene which in the E. coli genome context is close to oriC. In A. aeolicus, yihZ appears to be an isolated gene. However, the idea of a physiological link between tRNA starvation and the regulation of the expression of D-Tyr-tRNA Tyr deacylase is again suggested by the genome organizations of B. subtilis, S. equisimilis, and S. aureus. In these bacteria, the yihZ ortholog is systematically located immediately downstream from the rel gene on the chromosome. As already noted above, however, the functionality of the deacylase gene in the B. subtilis genome is questionable. D-Tyr-tRNA Tyr deacylase appears to be widespread in the living world. Possibly, the description of more genomes from more organisms will bring additional indications on the physiological responses involving the mobilization and function of this deacylase activity.