of similar experiments involving biochemical mutants of Escherichia coli (Simmonds, 1948; Gots and Koh, 1950; Davis and Mingioli, 1950)

Transcription

1 METHIONINE BIOSYNTHESIS IN ESCHERICHIA COLI1,2 EDWIN B. KALAN3.' AND JOSEPH CEITHAML Department of Biochemistry, University of Chicago, Chicago, Illinoi8 The first nonsulfur containing compound implicated in the biosynthesis of the amino acid methionine in microorganism was reported by Teas et al. (1948). These investigators found that a single amino acid, -homoserine, would support the growth of a biochemical mutant of Neurospora crassa which required both methionine and threonine for growth. They concluded that L-homoserine condensed with cysteine to form cystathionine which then underwent cleavage to yield homocysteine, the immediate precursor of methionine, as shown in the following scheme: Received for publication March 1, 1954 of similar experiments involving biochemical mutants of Escherichia coli (Simmonds, 1948; Gots and Koh, 195; Davis and Mingioli, 195) are in accord with the scheme for Neurospora shown above. The present paper reports the results of a study of the pathway of methionine formation in mutants of E. coli which require methionine for growth. The results of this study are in harmony with the existing scheme for the biosynthesis of methionine in E. coli but in addition implicate other nonsulfur containing amino acids beside HOCH2CH2CHNH2COOH + HSCHECHNH2COOH - HOOCCHNH2CH2CH2SCH2CHNH2COOH i-homoserine L-cysteine i-cystathionine I CHsCHOHCNHN2COOH 3 carbon fragment i-threonine + HOOCCHNH2CH2CH2SCHE +CHs 4 HOOCCHNH*CH2CH2SH i-methionine L-homocysteine Fling and Horowitz (1951) later reported that both D-homoserine and j3-hydroxy-l-homoserine could satisfy the methionine but not the threonine requirement of this same homoserineless mutant. It was proposed that the four-carbon moiety of methionine is derived from the fourcarbon chain of -homoserine. Reports of results 1 This investigation was aided by a grant from the Dr. Wallace C. and Clara A. Abbott Memorial Fund of the University of Chicago. 2 Parts of this report are taken from a thesis submitted by Edwin B. Kalan in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of Chicago, August, ' Aided by a Predoctoral Fellowship from the United States Public Health Service, National Institute of Microbiology. 4Present address: United States Public Health Service, Tuberculosis Research Laboratory, Cornell University Medical College, New York 21, New York. D,L-homoserine and -hydroxy-ihomoserine as possible precursors of the four-carbon moiety of the methionine molecule. MATERAL AND METHODS I8o ion of mutants. Methionineless mutants were isolated by the penicillin method following ultraviolet irradiation of E. coli strain W (ATC', 9637), (Davis, 1948; Lederberg and Zind r, 1948). Stock cultures were stored at C on 1.5 per cent agar slants (Difco), supplemented.ith minimal medium (Davis and Mingioli, 195) and.2 per cent casamino acids (Difco). Compounds tested. The compounds listed in table 1 were tested individually and in combinations to determine their ability to support growth of the various methionineless mutants. Unless otherwise indicated, the compounds were commercial preparations. Those substances likely to be destroyed in solution by autoclaving were 293 Downloaded from on December 7, 218 by guest

2 294 E. B. KALAN AND JOSEPH CEITHAML [VOL. 68 TABLE 1 Compounds tested as growth factors for methionineless mutants of Escherichia coli strain W Na2SO, NaHSOs Na,S24 Na282s Na2,S9H2 Flowers of sulfur NasCO, CH,OH HCHO L-Cysteic acid HCOOH L-Taurine L-Alanine DL-Alanine DL-Valine L-Tyrosine Glycine DL-Allothreonine L-Cystine L-Cysteine HCl L-Lysine*HCI L-Serine DL-Leucine DL-Threonine DL-Homoserine D-Methioiiine L-Methionine DL-Methionine DL-a-Amino butyric acid DL-Phenylalanine,y-Amino butyric acid (S. Mandeles)t DL-Homocysteine DL-Homocysteine thiolactone.hcl (N. Horowitz) DL-Cystathionine* LL-Cystathionine (N. Horowitz) Folic acid Choline chloride Vitamin Bi, p-aminobenzoic acid Creatin Acetyl sarcosine (K. Bloch) Sarcosine (K. Bloch) Thymine Creatin Ba-3-phosphoglycerate Na pyruvate Na butyrate Oxalacetic acid Crotonic acid DL-a-Hydroxy butyric acid Na acetoacetate Na-a-Keto butyrate (D. Harris) DL-Na-,e-hydroxy butyrate (S. Barkulis) D-N-Acetyl glucosamine (S. Roseman) D-Glucosamine*HCl D-Glucosaminic acid (S. Roseman) D-Galactosamine*HCl (S. Roseman) Ca gluconate DL-Threo 1,2 dihydroxy butyric acid (J. Glattfeld) DL-Erythro 1,2 dihydroxy butyric acid (J. Glattfeld) 'y-lactone of DL 1,3 dihydroxy butyric acid (J. Glattfeld) DL 1,2 dihydroxy isobutyric acid (J. Glattfeld) 'y-lactone of erythronic acid (J. Glattfeld) Trihydroxy isobutyric acid (J. Glattfeld) 'y-lactone of DL 2,3 dihydroxy butyric acid (J. Glattfeld) * Prepared according to the methqd of duvigneaud et al. (1942), contaminated with DL-homocystine t Name in parentheses following a compound indicates donor to whom we are indebted for the sample' sterilized by filtration through ultrafine, porous ass filters.?7rowth assay method. Growth tests were perfoi aed by streaking aqueous suspensions of mu 'nt strains upon 1.5 per cent agar petri plates containing minimal medium plus the compound. Each of the compounds listed in table 1 was tested individually at more than one concentration and frequently on more than one occasion. In addition 75 combinations of 2 or 3 of these compounds also were tried. Positive responses in all cases were verified by further tests. Agar plates were observed after 24, 48, and 72 hours of inctubation at 37 C, and the growth response was determined by visual inspection and was recorded as heavy (+ + +) moderate (+ +), light (+), or no growth at all (). Although this method is only semiquantitative, it has a number of advantages (Davis and Mingioli, 195): the response to smal inocula is more uniform; slight growth is recognized with more certainty; and the growth of the mutant strain is readily distinguished from that of occasional back mutations, thus permitting long periods of incubation and observation. A suspension of wild type E. coli was streaked on every plate as a control. In all experiments the mutant strains were streaked on minimal plates as well as on the supplemented plates in order to test for back mutations to the wild type. When Downloaded from on December 7, 218 by guest

3 1954] METHIONINE BIOSYNTHESIS IN E. COLI 295 TABLE 2 Growth responses of methionineless mutants MUTANT NO. 1. F-25 M-1 CW-143 CW F-11 CW-1o CW-19 CW N-96 M-5 CW-136 CW-182 Wild type DL-MKTH- IONINE 2 pg/mi I+++ DL-HOMOCYS- TErINZ TEIO- LACTONz *HCI 8 Ag/mi LL-CYSTA- THIONINE 2 pg/ml I+++ VITAMIN Bn 1 img/ml +++ (heavy growth), ++ (moderate growth), + (light growth), (no growth). Petri plates were incubated at 37 C for 72 hours; solid media consisted of 15 ml of 2 per cent agar, 4 ml of 5X minimal medium supplemented with 2 ml of a stock solution of compounds tested. Minimal plates supplemented with 2 ml of H,O were used as controls to detect reversions to wild type. Aqueous suspensions of mutant strains were streaked on the surface of the solid medium. A suspension of wild type E. coli, strain W, also was streaked on each plate as a positive control. Upon each plate some 2 individual streaks were made. Responses shown here refer to a 24 hour period of incubation except negative responses which refer to a 72 hour period. Responses to all other compounds tested (table 1) were negative. Mutants in each group were isolated independently but showed the same growth responses and probably represent the same mutant strain. The purity of the amino acids reported in this table was verified by paper chromatographic analysis. growth of a mutant strain occurred on the minimal control plate, reversion to wild type was suspected and the results for that particular strain were not considered. When growth of a mutant strain appeared only on the supplemented plate, a sample of these bacteria from the plate was transferred to.5 ml of sterile water, and this suspension was then restreaked on minimal and supplemented plates to verify that the original growth had been that of the mutant and not that of a chance back mutation on the supplemented plate. RESULTS The methionine requiring strains of E. coli used in these studies can be divided into four different groups. Three of these are similar to those reported by Davis and Mingioli (195) who also used E. coli. The growth responses of these three groups of mutants are summarized in table 2. A summary of the results obtained with the fourth group of mutants appears in table 3. It should be noted that tables 2 and 3 indicate only the growth responses to compounds which were found to be active in one case or another. However, all the other compounds listed in table 1 also were tested and were found to be inactive. Members of the fourth group of mutants can utilize the nonsulfur containing amino acids, D,L-alanine, D,L-homoserine, D,L-a-aminobutyric acid, D, -valine, and D,L-isoleucine, as well as the previously mentioned sulfur containing amino acids. However, a comparison of the growth of these mutants on plates containing methionine with that on plates supplemented with the effective nonsulfur containing amino acids makes it apparent that these mutants of group 4 are primarily methionineless, and most probably they utilize the effective nonsulfur-containing amino acids to supply the four-carbon moiety of the methionine molecule. Moreover, although vitamin B12 alone will not support growth of these mutants, in its presence growth on effective supplements is accelerated. DISCUSSION Members of the first group of mutants grew only in the presence of added methionine and therefore appear to be blocked in an as yet unknown terminal reaction in the biosynthesis of methionine. The second group of mutants grew on either methionine or vitamin B12 and upon no other compounds tested. These mutants therefore probably are blocked in the reaction involving the production of methyl groups or their incorporation into the methionine molecule. In the third group are found the mutants which could utilize cystathionine, homocysteine, or methionine. These mutants resemble those reported by Simmonds (1948) for E. coli, strain K-12, and by Gots and Koh (195) for E. coli strain B, as well as by Davis and Mingioli (195) for E. coli strain W. Members of this third group of Downloaded from on December 7, 218 by guest

4 296 E. B. KALAN AND JOSEPH CEITHAML [VOL. 68 COMOUNDS TABLE 3 Growth responses of methionineless mutants of group 4 (Mutant nos. F-22, CW-96, CW-1) -Bu2 +Bu (1.5 VxO/mL) Og/ml 24 hr 48 hr 72 hr 24 hr 4S hr 72 hr DL-Methionine DL-Homocysteine thiolactonehci LL-Cystathionine DL-Alanine DL-Homoserlne DL-a-Aniinobutyric acid DL-Valine DL-Isoleucine Vitamin B1 1 m.g/ml +++ (heavy growth), ++ (moderate growth), + (light growth), 1 (very light), (no growth). Petri plates were incubated at 37 C; solid media consisted of 15 ml of 2 per cent agar, 4 ml of 5X minimal medium supplemented with 2 ml of a stock solution of the compounds indicated. Minimal plates supplemented with 2 ml of HO were used as controls to detect reversions to wild type. Aqueous suspensions of mutant strains were streaked on the surface of the solid medium. A suspension of wild type E. coli also was streaked on each plate as a positive control. Upon each plate some 2 individual streaks were made. Responses shown are for compounds which support growth; all other compounds (table 1) tested were negative. The three mutants employed had been isolated independently but showed the same growth responses and may represent the same mutant strain. The purity of the amino acids reported in this table was verified by paper chromatographic analysis. mutants apparently are blocked in the reaction leading to the formation of cystathionine. Our fourth group of mutants is blocked earlier still in this sequence of reactions. These mutants appear to be blocked in the production or possibly in the utilization of the four-carbon precursor of methionine. The effective sulfur-containing and nonsulfur-containing amino acids then serve as an exogenous source of four-carbon particles. Once the four-carbon moiety is supplied, the trace factor, vitamin B12, accelerates the incorporation of this moiety into the methionine molecule. The function of vitamin B12 may be interpreted as being involved in methyl transfer or methyl group production which permits indirectly a more efficient utilization of the four-carbon precursor in the formation of methionine. Such a coenzyme function for vitamin B, has been suggested already by Davis and Mingioli (195) for E. coli and by Oginsky (195) for rats. It should be noted that Dubnoff (1952), working with E. coli, ascribed a different role to vitamin B12, namely one of maintaining homocysteine in the reduced state. The role of homoserine in methionine biosynthesis has been described in the introduction of this paper. It should be noted that although members of our fourth group of mutants could utilize homoserine, they consistently did not utilize aspartic acid under the conditions of our experiments. Recently, however, Black and Wright (1953) have described the reduction of #-aspartyl phosphate to homoserine by yeast extract while Hirsch and Cohen (1953) reported the conversion of -aspartic acid to -homoserine in a threonineless mutant of E. coli. The pathway by which the other effective amino acids contribute a four-carbon moiety is not yet known. However, Umbarger and Mueller (1951) did report that a-keto,,-hydroxy butyric acid, a-amino and a-keto butyric acids, threonine and isoleucine each was utilized by E. coli strain K-12 mutants. Tatum and Adelberg (1951) on the basis of isotopic evidence obtained from the analysis of valine and isoleucine intermediates accumulated by a mutant of Neurospora, postulated that the carbon skeleton of these two amino acids was formed by the condensation of a four-carbon particle with a molecule of acetic acid. Umbarger and Adelberg (1951) suggested Downloaded from on December 7, 218 by guest

5 1954] 194METHIONINE BIOSYNTHESIS IN E. COLI 297 that the four-carbon compound was a-hydroxyf-keto butyric acid, which would be in tautomeric equilibrium with a-keto,,-hydroxy butyric acid reported by Umbarger and Mueller (1951) for E. coli, strain K-12, and mentioned above. The a-keto,,-hydroxy butyric acid has not been tested upon members of our fourth group of mutants. However, a variety of di- and trihydroxy butyric acids listed in table 1 failed to promote growth. Moreover, it should be noted that a-keto butyric acid and pyruvic acid were also inactive while their aminated counterparts, a-amino butyric acid and alanine, both promote growth. Failure of these keto acids to support growth may be due to permeability factors or it may suggest that a preformed amino group is required by these mutants. The utilization of alanine by these mutants suggests a relationship between this compound and homoserine similar to the one between glycine and serine (Sakami, 1948). Hift and Mahler (1952) have demonstrated an enzyme in beef liver which catalyzes the aldol condensation between pyruvate and formaldehyde, giving rise to a-keto, y-hydroxy butyric acid, the a-keto analogue of homoserine. A similar reaction involving a onecarbon compound plus alanine to form homoserine might obtain in our mutant strain of E. coli. It is interesting to note that since the mutants in our fourth group are capable of utilizing nonsulfur containing amino acids in the biosynthesis of methionine, they obviously must secure the sulfur for this same biosynthesis from the minimal medium. The sole source of sulfur in this medium is inorganic sulfate. These mutants therefore can incorporate this inorganic sulfur into the methionine molecule provided a supply of appropriate four-carbon units is available. SUMMARY Four different groups of methionine requiring mutants of Escherichia coli, strain W, have been isolated by the penicillin method. The first of these requires methionine for growth and can utilize no other supplement tested. The second group can utilize either vitamin Ba2 or methionine, while the third group grows when the minimal medium is supplemented with methionine, homocysteine, or cystathionine. Mutants in the fourth group can grow on nonsulfur-containing amino acids including alanine, homoserine, valine, isoleucine, and a-amino butyric acid, as well as on methionine, homocysteine, and cystathionine. The growth of this fourth group of mutants on the effective supplements is accelerated by the addition of trace amounts of vitamin Bn. These results are consistent with the hypothesis that a four-carbon, a-amino acid is a precursor of methionine in E. coli. REFERENCES BLACK, S., AND WRIGHT, N. G Enzymatireduction of -aspartyl phosphate to homoserine. J. Am. Chem. Soc., 75, DAVIS, B. D Isolation of biochemically deficient mutants of bacteria by penicillin. J. Am. Chem. Soc., 7, DAVIs, B. D., AND MINGIOL, E. S. 195 Mutants of Eacherichia coli requiring methionine or vitamin B,2. J. Bacteriol. 6, DUBNOFF, J. W The role of B12 in methionine synthesis in E. coli. Arch. Biochem. and Biophys., 37, DUVIGNEAUD, V., BROWN, G. B., AND CHANDLER, J. P The synthesis of LL-s-(famino-,B-carboxyethyl) homocysteine and the replacement by it of cystine in the diet. J. Biol. Chem., 143, FLNG, M., AND HOROWITZ, N. H Threonine and homoserine in extracts of a methionine-less mutant of Neuro8pora. J. Biol. Chem., 19, GOTS, J. S., AND KOH, W. Y. 195 Methionine synthesis in Escherichia coli. Bacteriol. Proc Hirr, H., AND MAHLER, H. R The enzymatic condensation of pyruvate and formaldehyde. J. Biol. Chem., 198, HIRSCH, M. L., AND COHEN, G. N Transformation of L-aspartic acid to L-threonine via L-homoserine. Comp. rend., 236, LEDERBERG, J., AND ZINDER, N Concentration of biochemical mutants of bacteria with penicillin. J. Am. Chem. Soc., 7, OGINSKY, E. L. 195 Vitamin B,2 and methionine formation. Arch. Biochem., 26, SAKAMI, W The conversion of formate and glycine to serine and glycogen in the intact rat. J. Biol. Chem., 176, SIMMONDS, S Utilization of sulfur-containing amino acids by mutant strains of Escherichia coli. J. Biol. Chem., 174, Downloaded from on December 7, 218 by guest

6 298 E. B. KALAN AND JOSEPH CEITHAML [VOL. 68 TATUM, E. L., AND ADELBERG, E. A Origin of the carbon skeletons of isoleucine and valine. J. Biol. Chem., 19, TEAS, H. J., HOROWITZ, N. H., AND FLING, M Homoserine as a precursor of threonine and methionine in Neurospora. J. Biol. Chem., 172, UMBARGER, H. E., AND ADELBER, E. A The role of a-keto-o-ethylbutyric acid in the biosynthesis of isoleucine. J. Biol. Chem., 192, UMBARGER, H. E., AND MUELLER, J. H Isoleucine and valine metabolism of E8cherichia coli. I. Growth studies on amino aciddeficient mutants. J. Biol. Chem., 189, Downloaded from on December 7, 218 by guest