JOURNAL OF BACTERIOLOGY, May 1973, p. 666-673 Vol. 114, No. 2 Copyright 0 1973 American Society for Microbiology Printed in U.S.A. Effect of Methionine Sulfoximine and Methionine Sulfone on Glutamate Synthesis in Klebsiella aerogenes JEAN E. BRENCHLEY Department of Microbiology, The Pennsylvania State University, University Park, Pennsylvania 16802 Received for publication 1 December 1972 At least two pathways exist in Klebsiella aerogenes for glutamate synthesis. A mutant blocked in one pathway due to the loss of glutamate dehydrogenase (gltD) does not require glutamate and has the same growth characteristics as the parent strain in most media; however, its growth is inhibited by the analogues methionine sulfoximine and methionine sulfone. Wild-type Klebsiella is resist- ant to 0.1 M methionine sulfoximine or methionine sulfone, whereas the gltD mutant is sensitive to 1 mM concentrations. Either glutamate or glutamine is effective in overcoming this inhibition. Activities of both glutamine synthetase and glutamate synthetase, two enzymes involved in the second pathway of glutamate synthesis, are inhibited by methionine sulfoximine and methionine sulfone. The primary effect of methionine sulfoximine appears to be the prevention of glutamine production necessary for subsequent glutamate synthe- sis via glutamate synthetase enzyme. Recently it has been found (8) that at least ley, Prival, and Magasanik, manuscript in prep- two pathways are involved in the assimilation of aration). ammonia and the synthesis of glutamate. One Interest in this "low ammonia assimilatory" pathway functions by the glutamate dehydrogen- pathway was furthered when it was found that ase (GltD) (reduced form of nicotinamide-ade- in K. aerogenes catabolite repression of histi- nine dinucleotide [NADPH ]EC 1.4.1.4) reaction dase activity (but not ,B-galactosidase) by glu- converting a-ketoglutarate to glutamate. Am- cose could be overcome during nitrogen limita- monia can be further assimilated by the produc- tion. It was also found that this relief from tion of glutamine from glutamate and ammonia repression is correlated with high glutamine by glutamine synthetase (GlnS). synthetase activities and low glutamate dehy- drogenase activities. To determine whether the GltD a-ketoglutarate Glutamate (1) absence of glutamate dehydrogenase alone NH,-N could be responsible for increased levels of GlnSG glutamine synthetase, mutants lacking gluta- Glutamate mate dehydrogenase activity were constructed NH, and characterized. One mutant, MK-275, lacks GltS Glutamine - > Glutamate (2) glutamate dehydrogenase (gltD) activity and a-ketoglutarate yet surprisingly appears to grow as well as the parental strain under all conditions tested The second pathway involves the reaction of (Brenchly and Magasanik, manuscript in prep- a-ketoglutarate and glutamine by glutamine aration). The normal growth and glutamine (amide): 2-oxoglutarate amido-transferase ox- synthetase level show that glutamate dehy- ido-reductase (NADP) (glutamate synthetase, drogenase is not necessary for growth or regula- GltS) to form glutamate. Because the gluta- tion of catabolite repression during nitrogen mine synthetase has a lower Km for ammonia limitation. Thus it appeared that, despite its than glutamate dehydrogenase, it is able to high level of regulation, glutamate dehydrogen- react with ammonia at much lower concentra- ase has no important function in K. aerogenes. tions. In Klebsiella this second pathway is the However, it was felt that it might be possible to major route utilized during growth in media use analogues which would specifically inhibit containing less than 1 mM ammonia (Brench- glutamate synthesis in the gltD strain, and so 66 VOL. 114, 1973 INHIBmON OF GLUTAMATE SYNTHESIS 667 the effects on growth of MK-275 by a number of used as inocula for growth curves in minimal medium analogues were examined. Growth inhibition were grown in the same medium prior to use. Growth occurred with methionine sulfone and methio- temperature was 37 C. Cells to be used for extract nine sulfoximine and these were studied fur- preparation were harvested with rapid cooling and centrifugation when cultures reached a value of 100 5 ther. Since the inhibition by methionine sulfox- Klett units (no. 42 filter, approximately 9 x 108 imine is more pronounced, its effects were cell/ml). The cells were washed twice with cold 0.85% studied in more detail. NaCl and stored as pellets at 0 to 4 C for 8 to 12 h be- Previous work with methionine sulfoximine fore sonic treatment. The cells were suspended to demonstrated its inhibitory effect on Leuconos- 1/100 their original volume in a buffer containing 10 toc mesenteroides (3, 9) and suggested its in- mM imidazole-hydrochloride (pH 7.15), 10 mM volvement in inhibition of glutamine synthesis. MnCl2, and 0.14 ml of mercaptoethanol per liter. This This inhibitor also has been identified as a buffer is the one used for the glutamine synthetase as- powerful convulsant agent in a number of says, and since this was the enzyme of primary in- animals (4, 11), and extensive biochemical terest it was chosen for all extract preparations. The specific activity of glutamate dehydrogenase was the studies have been reported with glutamine same for extracts prepared with this buffer as for one synthetase purified from sheep brain by Meister prepared in a tris(hydroxymethyl)aminomethane and co-workers (7, 10, 12). They demonstrated buffer. It is not known whether the imidazole buffer that methionine sulfoximine binds glutamine affected the glutamate synthetase stability. synthetase tightly and causes irreversible inhi- The cells resuspended in the imidazole buffer were bition of activity. The results reported here are sonically treated (Bronwill Biosonik Ill sonic oscilla- consistent with methionine sulfoximine inhibit- tor with a needle probe, 25% power setting) three ing the glutamine synthetase from K. aerogenes times for 15 s with 20-s cooling intervals. This fraction was centrifuged for 20 min at 17,000 x g, and the but suggest that the primary growth inhibition supernatant fluids were saved. Extracts were stored in is due to the inability of the glutamate dehy- an ice bath, and the enzymes were assayed routinely drogenase-negative mutant to produce gluta- within 8 h after extract preparation. Protein determi- mate for growth. The effect of methionine nations were made by the method of Lowry et al. (5). sulfoximine and methionine sulfone on the Enzyme assays. Glutamate dehydrogenase and newly reported glutamate synthetase is also glutamate synthetase activities were measured by investigated in these strains. following the rate of NADPH oxidation as described by Meers et al. (8). It was found during these studies that the glutamate synthetase activity is unstable in MATERIALS AND METHODS the reaction mixture unless both glutamine and Strains and media. K. aerogenes mutants were a-ketoglutarate are present. The usual procedure for derived from strains isolated by MacPhee et al. (6). this assay had been to follow the endogenous rate of Strain MK-247 is used as the wild type and has all NADPH oxidation prior to glutamine addition and three enzymes involved in glutamate synthesis. Strain the measurement of the glutamine-dependent gluta- MK-189 lacks glutamate synthetase and cannot as- mate synthetase activity. Most of this activity, how- similate ammonia at concentrations less than 1 mM. ever, is lost within 10 min at 37 C (Fig. 1), but when Strain MK-275 has no glutamate dehydrogenase ac- the reaction mixture contains glutamine and a-keto- tivity and was constructed from a glutamate-requir- glutarate (both are necessary) the activity is stable for ing strain lacking both glutamate dehydrogenase and at least 1 h. Under these conditions the reaction was glutamate synthetase (gItD and gitS). Transductants started by NADPH addition, and the endogenous of this double mutant were selected for their ability to oxidation was determined in separate experiments. grow on limiting ammonia. One transductant, Other experiments demonstrated that about 90% of MK-275, had regained the glutamate synthetase ac- the glutamate synthetase activity is lost when the tivity, but not glutamate dehydrogenase activity, and crude extract is frozen for 18 h. Since the activity is did not require glutamate. Strain MK-8 lacks uroca- more stable in cells stored as a pellet at 4 C (loss of 20 nase, the second enzyme involved in histidine degra- to 25% activity), they were kept in this manner when dation, and cannot form glutamate from histidine (1). the assays could not be completed immediately. Minimal medium contained per liter of distilled Whenever specific activities of different extracts were water (NH4)2SO4, 2 g; Na2HPO4, 6 g; KH2PO4, 3 g; to be compared, the extract preparation conditions NaCl, 3 g; Na.SO4, 11 mg; MgCl2, 0.2 g; CaCl2, 27 were kept as similar as possible to prevent differential mg; and FeCl, .6H20, 0.2 mg, pH 7.0. Carbon sources loss of activity from influencing the results. were added at 0.4%. When histidine was used as a The glutamine synthetase assay was the non-bio- nitrogen source, the (NH4)2SO4 was omitted ard 0.2% synthetic transferase reaction described by Stadtman histidine was substituted. et al. (14). Blanks without adenosine diphosphate and Complex medium was Luria broth (LB) and con- arsenate were included for every assay condition, and tained per liter of distilled water: tryptone, 10 g; yeast the values were subtracted from those obtained with extract, 5 g; NaCl, 10 g; and glucose, 1 g. Agar was the reaction mixture. added at 1.5% to make either minimal or LB agar. Materials. L-Methionine-DL-sulfoximine, DL-methi- Culture conditions and extract preparation. onine sulfoxide, L-methionine sulfone, and DL-a- Stock cultures were kept on LB agar slants. Cells to be methyl glutamic acid were obtained from Sigma 668 BRENCHLEY J. BACTERIOL. ' ' 'A^MK-275 could be due to the inability of the cell to synthesize adequate quantities of glutamine >. loC >A' Seither to supply glutamine itself for protein A synthesis or supply glutamate made from gluta- mine the reaction.