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Properties of a D-Glutamic Acid-Requiring Mutant of Escherichia Coli E

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Properties of a D-Glutamic Acid-Requiring Mutant of Escherichia Coli E JouRNAL oF BAcFERIOLoGY, May 1973, p. 499-506 Vol. 114, No. 2 Copyright 0 1973 American Society for Microbiology Printed in U.SA. Properties of a D-Glutamic Acid-Requiring Mutant of Escherichia coli E. J. J. LUGTENBERG,1 H. J. W. WIJSMAN, AND D. vAN ZAANE Laboratory for Microbiology, State University, Catharijnesingel 59, Utrecht, The Netherlands; and Institute of Genetics, University of Amsterdam, Amsterdam, The Netherlands Received for publication 22 January 1973 Some properties of a n-glutamic acid auxotroph of Escherichia coli B were studied. The mutant cells lysed in the absence of n-glutamic acid. Murein synthesis was impaired, accompanied by accumulation of uridine-5'-diphos- phate-N-acetyl-muramyl-L-alanine (UDP-MurNAc-L-Ala), as was shown by incubation of the mutant cells in a cell wall medium containing L- [14C ]alanine. After incubation of the parental strain in a cell wall medium containing L- [14C Iglutamic acid, the acid-precipitable radioactivity was lysozyme degrada- ble to a large extent. Radioactive UDP-MurNAc-pentapeptide was isolated from the L- [4C Iglutamic acid-labeled parental cells. After hydrolysis, the label was exclusively present in glutamic acid, the majority of which had the stereo- isomeric n-configuration. Compared to the parent the mutant incorporated less L- ["4C Iglutamic acid from the wall medium into acid-precipitable material. Lysozyme degraded a smaller percentage of the acid-precipitable material of the mutant than of that of the parent. No radioactive uridine nucleotide pre- cursors could be isolated from the mutant under these conditions. Attempts to identify the enzymatic defect in this mutant were not successful. The activity of UDP-MurNAc-L-Ala: -glutamic acid ligase (ADP; EC 6.3.2.9) (n-gluta- mic acid adding enzyme) is not affected by the mutation. Possible pathways for n-glutamic acid biosynthesis in E. coli B are discussed. In recent years many mutants impaired in in CGPY medium (4) containing salts, 0.5% glucose, the biosynthesis of uridine-5'-diphosphate-N- peptone, and yeast extract. D-Glutamic acid (50 acetyl-muramyl-L-Ala-D-Glu-meso-diaminopi- gg/ml) was supplemented if necessary. The optical melic acid-D-Ala-D-Ala (UDP-MurNAc-penta- density of bacterial cultures, determined at various times, was measured in a Unicam-SP600 spectropho- peptide) have been identified (8). In the collec- tometer at 660-nm wave length. Yeast agar (4) was tion of temperature-sensitive lysis mutants un- used as the solid medium. der study in our laboratories, no mutants were Murein synthesis. Exponentially growing cells found that required D-glutamic acid for growth were harvested and then washed with and suspended (5). Recently W. Messer isolated a D-glutamic in the cell wall synthesis medium CWSM-I, which acid auxotroph of Escherichia coli B (2). Some contains glucose, minimal medium salts, chloram- properties of this mutant are described in the phenicol, uracil, and cell wall amino acids. After present paper. Unfortunately we did not suc- incubation for 15 min at 37 C, L-[U-'4CJalanine was ceed in identifying the enzymatic defect in added to a final concentration of 6.25 x 10-I M (1 or 2 MCi/ml). The suspension was incubated at 37 C. this mutant. Murein synthesis was followed by measuring the acid-precipitable radioactivity of 0.1- or 0.2-ml sam- MATERIALS AND METHODS ples. Under these conditions L-alanine is nearly Bacterial strains. E. coli B/r, F- (relevant mark- completely incorporated into murein (4). Protein ers including leu, pro, trp, his, arg, thy, met, lac, gal, synthesis does not occur under these conditions strA, hsrK, and hsmK) and its n-glutamate-requiring because chloramphenicol is present and a number of mutant were obtained from W. Messer, Max-Planck- amino acids, for which the strains are auxotrophic, Institut fur Molekulare Genetik, Berlin, Germany are not present in CWSM-I medium. Details of the (2). method have been described previously (4). This Growth conditions. The cells were grown at 37 C method was also used to test whether L- [U- 'Present address: University of Connecticut Health Cent- "4Ciglutamic acid (1 or 2 j&Ci/ml) can be incorporated er, Department of Microbiology, Farmington, Conn. 06032. into murein. To obtain a reasonable incorporation, it 499 500 LUGTENBERG, WIJSMAN, AND VAN ZAANE J. BACTERIOL. was necessary to omit unlabeled L-glutamic acid C as described previously (3) except that excess (usually 120 ,g per ml) from CWSM-I. The concen- enzyme was used. As a control on the stereospecific- tration of L-alanine was 50 mg/liter. Incorporation of ity of the enzyme, commercial samples of DL- and the radioactivity in murein was tested by measuring L- ["C ]glutamic acid were also tested as the radioac- the lysozyme-degradable fraction of the acid-precipi- tive substrate. table material. Enzyme assays. Procedures for the assay of UDP- Degradation with lysozyme. After incubation in MurNAc: L-alanine ligase (ADP) (EC 6.3.2.8) (L-ala- CWSM-I, 2.0 ml of the suspension was cooled and nine adding enzyme), D-glytamic acid adding en- washed twice with and suspended in one volume of zyme); UDP-MurNAc-L-Ala-D-Glu: meso-diamino- 10-' M tris(hydroxymethyl)aminomethane (Tris)- pimelic acid (m-Dpm) ligase (ADP) (m-Dpm adding hydrochloride, pH 7.0. After ultrasonic disintegration enzyme, and UDP-MurNAc-L-Ala-D-Glu-m-Dpm: (4), the suspension was heated for five min at 100 C, D-Alanyl-D-alanine ligase (ADP) (D-alanyl-D-alanine cooled, and incubated with and without lysozyme adding enzyme) have been described previously (Fig. (100 jg/ml) for one hour at 37 C. The acid-precipita- 1) (3). The radioactivity of the "4C-labeled amino ble radioactivity of 0.2 ml of the washed cell suspen- acid or dipeptide that was bound to the appropriate sion before and after lysozyme treatment was deter- uridine nucleotide precursor was the measure for mined. Under these conditions between 20 and 40% enzyme activity. D- [4C ]Alanine was the substrate of the murein was found to be resistant to lysozyme for D-alanine: D-alanine ligase (ADP; EC 6.3.2.4) treatment (4). (D-alanyl-D-alanine synthetase). The enzyme was Uridine nucleotides. Accumulation and purifica- assayed in the presence of UDP-MurNAc-tripeptide. tion of reference uridine nucleotides was carried out The product of D-alanine: D-alanine ligase was im- as described previously (5, 9), except the accumula- mediately coupled to UDP-MurNAc-tripeptide be- tion of UDP-MurNAc-pentapeptide. This nucleotide cause the specific activity of the D-alanyl-D-alanine was accumulated in Bacillus cereus T growing under adding enzyme is about eightfold higher than that of vigorous aeration at 37 C in CGPY medium. When D-alanine: D-alanine ligase. This procedure prevents the optical density was 0.7 (0.37 mg [dry wt] per ml inhibition of D-alanine: D-alanine ligase by its prod- suspension), chloramphenicol was added to a final uct (3). L-Alanine:D-alanine racemase (EC 5.1.1.1) concentration of 100 Ag/ml. Fifteen minutes later, (alanine racemase) was assayed essentially as D- vancomycin (10 gg/ml) was added. After further alanine: D-alanine ligase, except that L- [4C ]alanine incubation for 40 min, the cells were cooled and was the labeled substrate. This procedure could be harvested (9). With this procedure, the yield of crude used because the racemase activity is about 20 times uridine nucleotides was about 300 ,umol/8 liters of lower than that of D-alanine: D-alanine ligase (3). All culture, which is many times higher than that substrates and products were separated by chroma- obtained with the method described in a previous tography, detected by autoradiography, and counted. paper (9). Details of the assay procedures were described previ- Radioactive uridine nucleotide precursors were ously (3). isolated and identified as described previously (5). For the assay of D-alanine: 2-oxo-glutarate amino- The conditions for growth and labeling were the same transferase (EC 2.6.1.6; D-alanine aminotransferase) as described for murein synthesis. L- ["C]Alanine or two methods were tested. Method A was the colori- L- ["C ]glutamic acid were present in final concentra- metric determination of pyruvate with salicylalde- tions of 2 MCi/ml. After incubation for 40 or 60 min at hyde in alkali, as described by Martinez-Carrion and 37 C, the cells of a 2.0-ml suspension were harvested, Jenkins (10). Either sonically disrupted cells or suspended in distilled water, and heated for 15 min soluble protein after ammonium sulfate precipitation at 100 C to liberate the precursors into the superna- (3) was used as the enzyme source in a final protein tant fluid. The charcoal-adsorbable material was concentration of 2 mg/ml. Ketoglutarate was omitted isolated (5). from the control. The mixtures were incubated for 1 h Chromatography. Samples were applied to What- at 37 C. Method B was a microassay, based on the man 1 chromatography paper and chromatographed conversion of radioactive 2-ketoglutarate to gluta- in isobutyric acid-i M ammonia (5:3; vol/vol). Hy- mate. The assay mixture contained (i) [2-"Clketo- drolysates were chromatographed for 12 to 16 h, and glutarate (2 uCi/ml); (ii) D-alanine (50 mM); (iii) charcoal-adsorbable material was chromatographed pyridoxal-5'-phosphate (0.4 mM); (iv) Tris-hydro- for 90 h. Radioactive areas were detected by autora- chloride (100 mM), final pH of 7, 8, or 9; (v) MnCl, (2 diography (4). R, values were: glutamate, 0.42; mM); (vi) adenosine-5'-triphosphate (4 mM); (vii) 2-ketoglutarate, 0.35; UDP-MurNAc-L-Ala, 0.28; UDP-MurNAc-L-Ala (0.4 mM); and (viii) enzyme as UDP-MurNAc-dipeptide, 0.2; UDP-MurNAc-tripep- used in method A.
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