Metabolism of serine and glycine in E. coli K12. I. The role of formate in the metabolism of serine-glycine auxotrophs

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1 Metabolism of serine and glycine in E. coli K12. I. The role of formate in the metabolism of serine-glycine auxotrophs ELAINE B. NEWMAN Departnler~t of'biologica1 Sciences, Sir George Williams University, Montreal, Qrte. Received January 23, 1970 NEWMAN,E.B Metabolism of serine and glycine in E. coli K12. I.The role of formate in the metabolism of serine-glycine auxotrophs. Can. J. Microbiol. 16: Certain auxotrophs of E. colik12 have a nutritional requirement for serine, glycine, or sodium formate. Formate is not a precursor of either serine or glycine; serine is a precursor of some but not all glycine. These findings do not fit in with the phosphorylated pathway of serine biosynthesis frequently postulated A hypothesis is made that formate reactivates an otherwise nonfunctional enzyme of serine biosynthesis. This is only one of several possible hypotheses. Complications of intermediary metabolism of serine and glycine are considered in some detail. The conclusion is reached that this area of metabolism is less well understood than is frequently thought. Introduction sential for the phenomenon studied. For studies of adaptation, cultures in log phase were taken from a 37 "C The pathway of serille bios~nthesis has resisted shaker, chilled immediately in salt ice, washed in cold understanding longer than those of most other medium or water, and resuspended in preincubated flasks. amino acids. Recently, however, considerable Met,lorls evidence has for a pathway Purines and protein were isolated according to the methphosphoglycerate via phosphohydroxypyruvate ods of Revel and Magasanik (8). Proteins were washed and phosphoserine in E. coli and S. typhimuriurn with acidified ethanol, and with ether, hydrolyzed in 6 N (7, 11, 12, 13). ~ l would ~ then be ~ formed i HC1 ~ for 14 ~ h at 115 C, dried in a desiccator over potassium hydroxide, and analyzed by the courtesy of the from serine by a enzyme, serine Department of Microbiology, Western Reserve Univerhydroxymeth~lase (2). This could then account sity, on a Beckman model 120 amino acid analyzer equipfor the entire biosynthesis of serine and glycine. ped with a Nuclear Chicago counter model 6350 and This paper is an account of three auxotrophs flow cell model RNA was determined by the orcinol derived from ~. K12 ~ ~ do not l fit ~ in with method and protein by the Folin method (3). Radioactive compounds were purchased from Calithe above scheme in any obvious manner. The fornia Biochemicals Gorp. nutritional requirements of these three strains (HM-100, -119, -129) can be met not only by Defi"itiorrs For purposes of the exposition of the results of growth serine and glycine but by sodium formate experiments it will be ~lnderstood that glucose minimal (6)- Two of these strains (HM-119, -129) have medium is always provided: "cells grown in glycine" been shown to lack the capacity to reduce phos- means cells grown in the above medium supplemented phoglycerate (3). However, the ability to grow with glycine. This is abbreviated further to "SX-grown" in the presence offormate is not readily explained meaning cells grown in serine and xanthine. "SX lag" refers to the fact that cells grown in serine and xanthine in terms an to 'Onvert phosphoglyc- when subcultured in glycine show a lag of 4 h or more (6). erate to phosphohydroxypyruvate. Thus it was The term C-1 unit refers to any carbon atom available considered of interest to study the role of for- for biosynthesis, most frequently in the form of a derivmate further, particularly with respect to the ative tetrahydrofolic acid. biosynthetic origin of serine and glycine in formate-grown cells. Results Materials and Methods 1. Nutritional Requiremeiits At 37 C strains HM , and -129 are Cultures auxotrophs responding to 1-serine, glycine, or Three mutants of E. coif strain K12 (HM-100, -119 sodium formate. At 28 they grow as protoand -129) and their parent K12 were obtained from Dr. H. E. Umbarger. trophs, but are nonetheless absolute auxotrophs at 37 C. At 42 C the ability to use formate is Growth Studies lost; the strains respond only to serine or gly- The growth medium and methods for following growth were described previously (6). The medium is buffered at cine, but are not inhibited when formate is also ph 6.4 and it is considered possible that this ph is es- present.

2 934 CANADIAN JOURNAL OF MI( :ROBIOLOGY. VOL. 16, 1970 None of the following compounds fulfills the nutritional requirements of strain HM 100: bicarbonate, amino acids other than serine and glycine, glyoxylate, glycollate, adenine, or xanthine. Formic acid distilled and neutralized with sodium hydroxide supports growth, as does sodium formate from any of a number of different commercial sources. 2. Yield of Cell Material Related to Nutrient Coizcentration To show that a strain is dependent for growth on a certain metabolite it is usual to show a proportionality between the amount of that metabolite provided and the yield of cell material. This is done by inoculating small numbers of cells grown previously on the metabolite to be tested into a series of flasks containing varying amounts of the metabolite, and determining the final optical density when growth has ceased. The proportionality expected can be shown for glycine and for serine (Fig. 1). Growth is not proportional to the concentration of formate at any level. Below a critical level, about 150 yg /ml (2.2 ymole /ml) no growth is seen even after several days. Above this level, the same high optical density is seen in all cases and the cultures appear to be limited by factors other than formate concentrations. Checks were routinely made to ensure that the final cultures consisted entirely of mutant cells, i.e. that prototrophs were not selected in the course of the experiment. On the other hand, in this experiment it did appear that the growth rate might be a function of formate concentration. Thus, although the final level of growth reached was the same for all concentrations from 150 to 700 yg/ml, this level was reached sooner at higher concentrations. To verify this, log phase cells pregrown on formate (500 yg/ml )were subcultured into medium containing varying amounts of formate (Fig. 2). Concentrations of 50, 200, and 300 yg/ml supported no growth, 400 yg/ml allowed extremely slow growth, 500 yg/ml permitted optimal growth with an apparent division time of 80 min, and higher concentrations appeared to be inhibitory. Thus formate is absolutely required for growth. No growth is seen in its absence, yet there is no proportionality between formate concentration and growth yield. The optimum concentration for formate (7.5 ymole/ml) is extremely high in view of the fact that serine is usually provided at 1.2 ymole /ml, and glucose, though carbon and energy source, at only 11 ymole/ml. This may indicate that the cell is poorly permeable to formate. Alternatively, the great difference in response to 400 and 500 yg/ml may mean that the cell has various ways of converting formate to a non-useful form and that these are saturated at concentrations about 400 yg/ml. 3. Incorporation of l4c-fornzate into Cell Components The fact that formate supports growth of a serinelglycine-auxotroph might suggest that RG. 1. Cell yield related to nutrient concentrations. FIG. 2. Growth rate related to forrnate concentration. Strain HM-100 pregrown on serine (0). glycine (o), and Strain HM-100 grown in forrnate 500 pg/rnl was subforrnate (A) was subcultured into varying concentrations cultured in sodium forrnate 300 (o), 400 (A), 500 (A), of the same nutrient and incubated at 37 C until growth 1000 (a), and 2000 (0) pg/rnl. Optical density was folceased. lowed at 420.

3 NEWMAN: FORMATE AND SERINE METABOLISM 935 formate is a precursor of one or the other. To investigate this possibility, strain HM 100 was grown from a negligibly small inoculum to midlog phase in the presence of 14C-formate ( c.p.m./ymole; 500 yg/ml). Protein was isolated and chromatographed as described. The specific activity of certain amino acids is given in Table I. No amino acid was derived to any significant extent from formate. The highest incorporation is into arginine (5300 c.p.m. /ymole) and glutamic acid (3000 c.p.m. /ymole), probably because of conversion of formate to carbon dioxide. Serine is almost totally unlabeled and therefore must be synthesized from glucose. Glycine (750 c.p.m./ymole) must also be made largely from glucose. However, since there is considerably more 14C in glycine than in serine, it appears that at least some of the glycine is made by a pathway in which serine is not an intermediate. Thus, in the presence of formate, strain HM- 100, though a serinelglycine-auxotroph, has the capacity to make both amino acids, and to make glycine by a pathway not involving serine. Formate is a precursor of neither, though its presence seems to be required for their synthesis to occur. Formate is, however, heavily incorporated into purines. In the experiment just described, the purine bases were isolated and chromatographed, and their specific activity determined : guanine c.p.m. Iymole and adenine c.p.m. lymole. Thus the C-1 units for purine biosynthesis are extensively derived from formate (cf. ref. 6). Were the formation of purines the principal role of formate in this strain, TABLE I Incorporation of formate-14c into amino acidsa Amino acid Specific activity Arginine 5300 Glutamic 3000 Proline 3100 Lysine 2700 Threonine 2200 As~artic 2000 ~licine Serine Histidine -Protein from cells of strain HM-100 grown at 37 C with fonnate c.p.m. / vmole was isolated, hydrolyzed, and chromatographed as described. the purines themselves should support growth and they do not (Results (1)). 4. Eflect of Incubation at Elevated Temperature Formate does not support growth at 40 C or above. When flasks of strain HM-100 in log phase on formate (500 yg/ml) at 37 C are transferred to 42 C, growth stops immediately as does RNA and protein synthesis (as judged by orcinol and folin reactions on KOH hydrolysates). The viable count, however, remains constant for at least 24 h. Although growth appears to be suspended at 42 C, incubation at 42 C for periods of up to 3 h does not alter the ability of the cells to grow when returned to 37 C. Thus cells of strain HM- 100 pregrown at 37 C on formate (500 yg/ml) were inoculated into a series of flasks containing medium supplemented with formate 500 yglml prewarmed to 42 C. These were placed at 37 C after varying periods of 42 C and their optical density followed in the Klett (Fig. 3). In all cases growth began almost immediately on transfer back to 37 C and at much the same rate as cells which had never been incubated at the higher temperature. In some (but not all) experiments, longer incubation at 42 C affects the cells such that on replacement at 37 C no further growth is seen. In such cases the viable count as judged by plating on complex media is unchanged. Moreover, when serine is added, the cells start to grow Time (Min) FIG. 3. Effect of incubation at 42 C on growth on formate at 37 C. Strain HM-100 grown on formate 500 pg/ml 37 C was inoculated into the same medium and incubated for 0 (e), 30 (a), 90 (o), 180 (a, 360 (p) min at 42 C before incubation at 37 C. Opt~cal dens~ty was followed at 420.

4 936 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 16, 1970 However, once cells are "deadapted" by incubation at 42 C, there seems to be no way in which the cells can readapt when given only formate. Unfortunately such experiments are highly variable and the conditions for this "deadaption" as yet are unclear. Cells grown in SX show a lag on transfer to glycine (7), and this lag can be avoided by adding formate with glycine. This effect of formate in overcoming the lag is also temperature-sensitive and does not occur at 42 C but does at 40 C. Thus at 40 C formate overcomes the SX-lag but does not support growth. This may indicate either that formate has two roles in cell metabolism, or that the minimal amounts of some product made at 40 C suffice for one purpose but not for another. 5. De novo Syntl~esis of Serine and Gfycine Formate-grown cells make serine and glycine from glucose at 37 C. This suggests the possibility that even glycine-grown cells might make all or part of their serine from glucose. The fact that the strain does not grow on formate at 42 C suggests that the glucose pathway is activated with difficulty, if at all, at 42 C. One might then expect that synthesis from glucose in glycine grown cells, if it takes place at all will be more extensive at 37 C than at 42 C. For this reason strain HM-100 was grown both at 42 C and at 37 C on glycine-i-1% and glycine-2-14c (i.e. four cultures). Protein hydrolysates were chromatographed as described. Incorporation into protein serine and glycine is shown in Table 11. At both temperatures, serine and glycine are in equilibrium, i.e. the specific activity of serine made from glycine-2-14c is twice that of the glycine indicating that the average molecule of serine is made from two molecules of glycine. TABLE I1 Incorporation of glycine-14c into protein serine and glycine" Specific activity of: Amino acid Growth protein protein supplied temp. serine glycine Glycine-1-14C Giycine-2-14C ~Medium containing plycine-1-14c or plycine-2-1 C was divided in two parts, autoclaved, and inoculated with strain HM-100. Protein from lop-phase cells was isolated and processed as described. However, both serine and glycine at 37 C show a specific activity only two-thirds of that seen at 42 C. This one-third dilution might represent either exchange of glycine with some unlabeled compound derived from glucose, or de novo synthesis of both amino acids. The hypothesis of exchange is sufficiently vague as to be difficult to test. On the other hand, the assumption that the dilution represents net synthesis entails other less evident assumptions as follows. De 1201~0 synthesis may involve either (a) glucose conversion to glycine and then serine or (6) glucose to serine to glycine, or else a fortuitous combination which results in an apparent equilibration. This latter possibility is disregarded here. A pathway of type a is in complete accord with the 14C data. kny glycine synthesized de lzovo would then dilute the whole pool equally. The objections to it are only (1) that SG biosynthesis is thought to proceed by the other route and that (2) glycine is thought to inhibit its own synthesis (9). Route b as stated is excluded by the following argument. Suppose glucose to be converted to serine, which is then broken down to glycine and C-1 units. At the same time exogenous glycine- 14C is taken into the cell and converted to 14C-1 units. Every molecule of serine then dilutes the C-1 pool not only directly by forming unlabeled C-1 but also indirectly by decreasing the specific activity of the glycine pool. When serine is resynthesized (as it must be if it is to contain any 14C) it must be less labeled in the P-carbon than in the other two. The ratio of the specific activity of serine derived from glycine-2-14c to that from glycine-1-14c will then be significantly less than 2, which is not what it is seen. Attributing numbers clarifies the argument. The cell requires for an arbitrary amount of cell material (6), 659 pmoles of glycine as C-1 and 535 as glycine. Assume that it makes 300 pmoles of serine de novo and puts it into the pool as 300 C-1 and 300 glycine. The cell then needs 359 and 235 pmoles of exogenous glycine-1". Three hundred of the 894 glycine molecules ( ) or about 30% will then be unlabeled, whereas the C-1 pool will be diluted by 300 cold and 359 X 4 or a total of 420 cold in 659, i.e. 65y0 dilution. The serine resynthesized will then contain 65% C-12 in the beta carbon and the ratio of the specific activities of serine derived

5 NEWMAN: FORMATE AND SERINE METABOLISM 937 from glycine-2- and 1-14C will be 1.5. Thus any amount of de nova synthesis which dilutes the glycine sufficiently to agree with the observed data on glycine will dilute the C-1 pool too much. One can avoid this problem by assuming that all serine is made from glucose but that none of it breaks down to form glycine. If it is then assumed that serine and glycine exchange freely, but only in the presence of C-1 (which in this case has to be derived from glycine) one can, from the preceding calculations, figure that 184 pmoles of serine by exchange could dilute the glycine by one-third. However, even with all the preceding assumptions (1) that serine is made extensively de novo (2) that 14C enters serine by a rapid exchange reaction iilvolving as obligatory partners both glycine and C-1 and (3) that the synthesis of C-1 from glycine is rapid: even with these, a further assumption is needed, i.e. that the cell is not freely permeable to glycine. As long as there is no net synthesis of glycine via serine, the total amount of serine made must be small compared to the amount of exogenous glycine present. If the cell were freely permeable to glycine, the C-12 from serine would exchange into a large pool of 14C both internal and external, and would barely dilute it. Since a contribution from C-12 is in fact seen, it seems that the glycine pool must be small, i.e. that the permeability of the cells to glycine must be limited, and this, at least. proves to be true (see below). 6. Permeability of Strain Glycine That strain 100 is permeable to glycine is obvious from the fact that exogenous glycine-14c is incorporated into cell material. It remains possible, however, that strain HM-100 has little or no ability for active transport of glycine and that glycine enters the cell by diffusion. In this case one would expect that at higher external concentrations of glycine, the intracellular level would also be increased. This was tested in the following manner. Log phase cells of strain HM-100 growing on glycine 100 pg/ml were inoculated into medium containing glycine at concentrations from 0 to 3000 pg/ml. Growth rates were determined by following optical density. The apparent division time decreased markedly with increasing glycine concentration (Fig. 4). Glycine at a concentration between 1000 and 2000 pg/ml supports growth at ~lluch the same rate as serine (100 pg/ml). This concentration, which is optimal for growth rate, is over 10 times the amount needed for optimal yield. Increasing the serine concentration 10-fold is somewhat inhibitory. It would seem that the simplest explanation for stimulation of growth at high concentrations of glycine is that glycine enters the cell only by diffusion. Mutants impermeable to glycine have been previously reported (10). The following data are consistent with this hypothesis but not decisive. When the same cells used above were inoculated illto medium containing (in addition to glycine (100 pg/ml)) adenine, xanthine, tryptophane (each 20 pg /ml), thymine (10 pg /ml), and methioniile (50 pg/ml), the growth rate was again markedly increased (70 min). If the rate at which glycine enters the cell limits the growth rate, then anything sparing glycine should increase the growth rate. However, a slow conversion of glycine to some precursor of the compounds added would fit the data equally well. 7. Pervlenbilitj, and the SX-lag During the study of the SX-lag (6) it became important to show that SX-grown cells were not impermeable to glycine. The experiments reported above show that strain HM-100 is in fact permeable to glycine, but to a limited extent only. In case these facts might indicate some contradiction, cells pregrown in serine and xanthine were inoculated into glycine at a variety of concentrations. The lag seen is well over 4 h in I Time (Min) FIG. 4. Growth rate related to glycine concentration. Strain HM-100 grown on glycine 100 ~ g/ml was inoculated into 0 (e), 100 (A), 500 (o), 1000 (o), and 3000 (.) pg/ml glycine at 37 C. Optlcal denslty was followed at 420.

6 938 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 16, 1970 all cases from 100 to 1000 pg /ml. Thus relative impermeability to glycine reported here does not conflict with the previous report. 8. Adaptation between Alternative Growth Factors Although strain HM-100 can grow when supplemented with serine, glycine, or formate, these compounds are not equivalent, and growth is not always instantaneous on transfer from one to the other. If we consider the behavior of cells grown in medium supplemented with glucose and compound A and transferred to medium with glucose and compound B we find the following results. (i) Formate or glycine to serine: immediate growth at the usual rapid rate (g.t.50 min). No faster growth is ever seen for strain HM-100 in synthetic media. (ii) Serine to formate: immediate growth at a slow rate for 2-3 h, followed by an abrupt change to a more rapid rate. This is not altered by the simultaneous provision of serine at 1 pg/ml. When small inocula are used (less than 106 cells/ml) no adaptation occurs unless low levels of serine are provided. (iii) Serine to glycine: immediate slow growth. Growth rate is very markedly increased by the addition of serine 1 pg/ml. This "sparking" effect of serine exists even when glycine is provided at 1000 pg/ml. (iv) Formate to glycine: long lag of over 4 h similar in origin to the SXlag (see below). (v) Glycine to formate: immediate growth at rates slower than that of cells fully adapted to formate. These results are summarized in Fig Time (Min) FIG. 5. Adaptations between nutrients. Strain HM-100 previously grown on glycine (A), serine (B), and formate (C) was subcultured in serine (e), glycine (a), and formate (A) at 37 C. Optical density was followed at 420. Growth on serine is thus, so to speak, constitutive. The enzymes required are either always present or easily synthesized in the presence of serine. The cell always requires a source of serine, glycine, and C-1 units. When given glycine, it can derive its serine from either glucose or glycine. However, when given formate, the cell has to make everything from glucose. One would expect then that the transition to glycine would be simpler than that to formate. This is, however, not so. Discussion 1. Biosynthesis of Serirze and Glycirre Evidence for a biosynthetic pathway usually includes a demonstration of all the enzymes postulated in the prototrophic strain; mutants lacking each of these enzymes; and perhaps overall incorporation of the first specific precursor into the final product. The evidence for the phosphoglycerate pathway of serine biosynthesis in E. coli does not meet all these standards. The pathway is postulated to proceed as follows: phosphoglyceric acid... enzyme 1... phosphohydroxypyruvate... enzyme 2... phosphoserine... enzyme 3... serine. Incorporation of phosphoglycerate into phosphoserine (7) and serine (3) has been shown. It has, however, been difficult to assay enzyme I except by 14C exchange (7). The enzymes have not been assayed in mutants and their parent strains. Indeed only two of the three expected genetic loci have been demonstrated. Moreover, every mutant studied lacks enzyme 1. No mutant lacking either enzyme 2 or 3 has been reported. This could be explained if both enzymes 2 and 3 are wide-spectrum enzymes not specific to this pathway. On the other hand one, and only one, enzyme of a potential nonphosphorylated pathway has been shown in E. coli Crookes (1 I), which shows that the demonstration of a given enzyme is not enough evidence for a pathway. The finding of mutants with an alternative requirement for serine, glycine, or formate further questions this pathway, the more so as this is one of the mutants reported to lack enzyme 1. What might the role of formate be? It is clearly not a precursor of any amino acid normally

7 NEWMAN: FORMATE AND SERINE METABOLISM 939 found in protein. It is heavily incorporated into purines; but as purines do not themselves support growth, this cannot be its critical role in the cell. Formate could have a role unrelated to the provision of either serine or glycine. This possibility is less likely since the mutants were originally isolated as serinelglycine auxotrophs. It may, however, be necessary to reconsider this point. Formate has two kinds of metabolic effects. (1) It supports growth at 37 but not at 40 or above. (2) It overcomes the SX-lag (in the presence of glycine) even at 40 "C but not at 42 "C or above. Now SX-grown cells have been shown to be starved of C-1 when transferred to glycine (6). One could conclude then that formate provides a C-1 source, except that formate is not incorporated into serine. Suppose that the presence of formate allows serine to be formed even though it is not itself a precursor. This could be either an enzyme stabilization or a shift in the induction repression pattern of related enzymes. Thus formate might activate an otherwise nonfunctional enzyme of serine biosynthesis. The serine then formed could provide C-1 and this would equally explain growth on formate alone. This argument does not apply to a glycine biosynthetic enzyme, since glycine alone does not (immediately) support growth of SX-grown cells and reactivating a glycine-forming pathway would not overcome the lag. However, the argument would apply to the pathway forming C-1 from glycine, if glycine were made directly from glucose rather than from serine. In either case, the efficiency of the proposed activation would decrease with increasing temperature. At 28 C the enzyme functions; at 37 C it is present in the cytoplasm in nonfunctional form but can be activated by formate or derivative thereof. At 40 "C the enzyme functions less well even in the presence of formate and cannot support growth on its own. It can, however, allow the formation of enough serine to provide for the synthesis of the G to C-1 system. At still higher temperatures, the enzyme cannot be activated at all. The hypothesis that formate activates an enzyme of serine biosynthesis could be tested, if the identity of the enzyme involved were known. However, a different type of hypothesis can be advanced that the original parent K12 prototroph had two pathways associated with SG-biosynthesis. One of these would be temperature-sensitive even in the prototroph and converts glucose to serine. The second would not be temperature sensitive at all, and no evidence restricts its possible nature. The mutations in HM-100, and -129 would then all be in the non-temperature sensitive pathway. This possibility is now under investigation. 2. Shifts in Metabolic Patlzways as a Consequerzce of Formate Utilization Strain HM-100 makes serine and glycine at least partly by independent routes when growing on formate. Using the figures and calculations described earlier (6), we can calculate that when formate contributes C-1 to purines, the cell must make from glucose much more glycine than C-1. This means that formate-grown cells contain an excess of C- I. It is clear then by the reasoning used earlier (6) that the formate-grown cell does not contain enzymes to convert glycine to C-I. One would also expect that if there are alternative ways to form glycine (other than from serine), these would be used in cells growing on formate. One possible route would be via threonine aldolase (14), which one would expect to find in formategrown but not serine-grown cells. 3. Adaptations We have seen that transitions to serine are always easily made but that transitions to formate are more difficult and to glycine even more so (Fig. 5). This can be understood by the hypothesis that formate reactivates a nonfunctioning enzyme of serine biosynthesis. However, Roberts (9) has shown that serine inhibits the de novo synthesis of both serine and glycine. One would therefore expect the biosynthetic enzymes to be repressed by growth in serine. One therefore must assume that the strain used here is not repressed, or that formate can act on the basal levels present in the repressed strain. The biphasic nature of the growth curve seen on transfer of cells from serine to formate seems to favor the latter possibility. 4. Serine and Protein Synthesis In an earlier paper (6), it was shown that growth in serine and xanthine represses the system for

8 940 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 16, 1970 making C-l units from glycine. The long lag on glycine was attributed to the fact that cells were starved of C-l units, but why this starvation should entail so long a lag was unexplained. This seems to indicate a role for C-l units in cell metabolism other than those indicated in the preceding discussion. A good possibility for this is the formation of formylmethionine. If protein synthesis really cannot occur without the initial formylation of methionine, one could well imagine an extremely long lag in the absence of C-1. This may be why no auxotroph has been isolated which requires only glycine but not serine. Such an auxotroph might be lethal. Experiments to test the role of serine in initiation of protein synthesis are currently underway. 5. General Observation Recent years have shown a remarkable flowering of metabolic control mechanisms and some very intricate experimentation. Thus where two pathways require the same initial enzyme, duplicate enzymes of similar activity but different controls have been demonstrated. Other mechanisms of this variety have also been described. But a point of metabolism such as serine and glycine synthesis might be expected to be immensely more complex even than this. The cell requires serine and glycine as protein constituents, as C-l donors for thymine, methionine, tyrosine, etc. and perhaps also as C-l donor for the very initiation of protein synthesis. Adaptation to new conditions would itself require serine since protein synthesis, even minimal protein synthesis, could not occur without it. Thus the complexity discussed in this paper should perhaps have been expected, rather than apparently unusual. Acknowledgments This work was begun at the Department of Biochemistry, Western Reserve University, Cleveland, Ohio, and supported by a training grant No. 5TI-GM given the department by the NIH. It is continuing under NRCC grant No. A6050. The author thanks Dr. J. W. Corcoran and Dr. H. Amos, for indispensable assistance in completing this work. The amino acid analyses were performed with particular efficiency by Miss Violet Forgach to whom the author is greatly indebted. 1. DEMEREC, M Bacterial genetics. Carnegie Inst. Wash. Year B. 54: HUENNEKENS, F. M., and M. J. OSBORN Folic acid coenzymes and one carbon metabolism. hz Advances in enzymology. Vol. 21. Edited by F. F. Nord. Interscience Publishers, Inc. pp LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, and R. RANDALL Protein measurement with the Folin reagent. J. Biol. Chem. 193: MONOD, J., J. P. CHANGEUX, and F. JACOB Allosteric proteins and cellular control systems. J. Mol. Biol. 6: MORSE, M. L., and C. E. CARTER The synthesis of nucleic acids in cultures of Escherichin coli strains B and B /R. J. Bacteriol. 58: NEWMAN, E. B., and B. MAGASANIK The relation of serine, glycine metabolism to the formation of single carbon units. Biochim. Biophys. Acta, 78: PIZER, L. I The pathway and control of serine synthesis in E. coli. J. Biol. Chem. 238: REVEL, H. B., and B. MAGASANIK Utilization of the imidazole carbon-2 of histidine for the biosynthesis of purines in bacteria. J. Biol. Chem. 233: ROBERTS, R. B., P. H. ABELSON, D. B. COWIE, E. T. BOLTON, and R. J. BRITTEN Studies of biosynthesis in E. coli. Carnegie Inst. Wash. Publ SIMMONDS, S., and D. A. MILLER Metabolism of glycine and serine in E. coli. J. Bacterio!.74: SMITH, R. A,, C. W. SHUSTER, S. ZIMMERMAN, and I. C. GUNSALUS Serine synthesis in Escherichia coli. Bact. Proc. p UMBARGER, H. R., and M. A. UMBARGER The biosynthetic pathway of serine in Salmonelln typhimurillttl. Biochim. Biophys. Acta, 62: UMBARGER. H. R.. M. A. UMBARGER. and P. M. L SIU ~ios~nihesis of serine in Escherichin coli. J. Bacteriol. 85: VAN LENTEN, E. J., and S. SIMMONDS Metabolic relations between I-threonine and glycine in E. coli. J. Biol. Chem. 240: WOOD. W. A Fermentation of carbohvdrates. In he bacteria. Vol. 11. Edited by I. C. ~Gnsalus, and R. Y. Stanier. Academic Press. p. 85.