ROLE OF a,e-diaminopimelic ACID IN THE CELLULAR INTEGRITY OF ESCHERICHIA COLI' LIONEL E. RHULAND Research Laboratories, The Upjohn Company, Kalamazoo, Michigan Received for publication December 17, 1956 a, e-diaminopimelic acid was first isolated from acid hydrolyzates of Corynebacterium diphtheriae by Work (1951). Subsequent studies by Dewey and Work (1952) and by Davis (1952) have demonstrated that a, e-diaminopimelic acid (DAP) can be decarboxylated to lysine and that the path of lysine synthesis apparently involves DAP as an intermediate. Davis (1952) reported that a mutant strain of Escherichia coli (173-25), isolated by the penicillin method (Lederberg and Zinder, 1948; Davis, 1948), had an absolute requirement for DAP and a relative requirement for ilysine. Growth in the absence of lysine was slow and there appeared to be a minimum requirement of lysine for rapid growth in the presence of DAP. The work reported here is an investigation of the role of diaminopimelic acid in the nutrition of the DAP auxotroph E. coli strain 173-25. MATERIALS AND METHODS E. coli strain 173-25 (Obtained from Dr. B. Davis, New York University) was maintained at 4 C after monthly transfer on a stock agar containing: K2HHPO4, 0.3 g; yeast extract, 0.1 g; vitamin-free casein hydrolyzate, 20 ml; agar, 1.5 g; and water, 80 ml. Glucose and DAP were added aseptically to a final concentration of 8 mg/ml and 20,g/ml, respectively. All growth experiments were conducted in a basal medium consisting of K2HPO4, 7.0 g; KH2PO4, 3.0 g; (NH4)2SO4, 1.0 g; MgSO4-7H20, 0.1 g; lysine, 20 mg; water to 1 L. Five ml of the 2 x concentrated basal medium was dispensed in 18 by 195 mm tubes. Glucose and lysine were added to a final concentration of 8 mg/ml and 20,g/ml. The tubes were diluted to a total vol- ' While this manuscript was in preparation Dr. Work informed me that she and Dr. Meadow had observed the lysis of Escherichia coli strain 173-25 in the absence of DAP. An abstract of this work has subsequently appeared (Meadow and Work, 1957). ume of 10.0 ml after all additions were made. When aeration of the culture was required, air was bubbled through these tubes to which a colorimeter tube had been fused (Cohen and Barner, 1955). Turbidity determinations were made in a Beckman Model B spectrophotometer at 660 m,u. All ultraviolet spectral determinations were made in a Cary recording spectrophotometer. Plate counts were made by spreading 0.1 ml of an appropriate dilution over the surface of the agar with a glass rod. The counting medium consisted of the basal medium described above containing in addition 20,ug/ml DAP, 2.5 mg/ml acid hydrolyzed casein (Difco), 8 mg/ml glucose, and agar to 1.5 per cent. The inoculum for all growth experiments was prepared by transferring the culture from stock agar to 10 ml of the liquid basal medium supplemented with 200,ug of DAP and 80 mg of glucose. The incubation time was 16-18 hr at 37 C. The cells were removed from the medium by centrifugation, washed once in distilled water and the cell suspension adjusted to 2.0 ml. Unless otherwise indicated, all tubes were inoculated with 0.1 ml of the concentrated cell suspension. The DAP was a synthetic material prepared as previously described (Rhuland et al., 1955). RESUIIrS In preliminary experiments to determine the growth response of E. coli to varying amounts of DAP, it was observed that, after an initial phase of rapid growth, lysis of the cells occurred. The change in optical density due to lysis was preceded by signs of clumping and formation of foam, suggesting the release of soluble proteins into the medium. This phenomenon appeared to be related to the concentration of DAP and did not occur when nonlimiting levels of DAP were used. Subsequent experiments demonstrated that lysis occurred in a predictable manner when the culture was grown in the presence of limiting amounts of DAP providing a number of factors 778
(I) z w a DQA.P. CURVE A Downloaded from http://jb.asm.org/ B. on August 26, 2018 by guest 2 4 6 8 10 2 4 6 8 O TIME IN HOURS TIME IN HOURS Figure 1. The effect of diaminopimelic acid on the growth and lysis of a diaminopimelic acid requiring mutant. Escherichia coli strain 173-25. Basal medium, 5.0 ml; 8 mg/ml glucose; 20 pg/ml lysine. Adjuncts added as indicated, H20 to 10.0 ml. Turbidity determined at 660 mp in a Beckman Model B, spectrophotometer, aeration, temperature 37 C. Curve A, 1,g/ml DAP added at 0 hr; Curve B, 10,ug/ml DAP added at time indicated; Curve B lower right, 1 Ag/ml DAP plus 2.5 mg/ml casein hydrolyzate added at 0 hr. 779
780 RHULAND [VOL. 73 >- 0.b6-7.8 W ton -J 04~~~~~~~ 4 0 00.2 7.0 z~~ 44 ~T 2 D.A. H URVES C 7 4 addefgued7.cre,1,u/lda *de 16.6 2 4 ~~~6 8 TIME IN HOURS The prevention of lysis of Escherichia Figure 2. coli strain 173-25 by diaminopimelic acid. Conditions as per figure 1. Curve A, 1 jug/ml DAP added at 0 hr; Curve B optical density; Curve C viable count. DAP added at 10lg/ml at 4 hr as indicated. such as inoculum size, aeration rate, and temperature were controlled. Although the phenomenon of cell lysis due to depletion of a specific nutrient is not a common occurrence, Toennies and Gallant (1949) reported that cultures of Streptococcus faecalis will lyse when grown in the presence of limiting amounts of lysine. In addition, Schaefer et al. (1949) recorded a similar observation with Mycobacterium tuberculosis if adenine was used as the sole source of nitrogen. In the cases reported by the above authors, the cells appeared to lyse at a relatively slow rate and it appears that the situation described for E. coli was quite unique. The role of DAP in this phenomenon was examined in more detail. Diaminopimelic acid was added at various time periods during the growth cycle and the results clearly illustrate (figure 1) that nionlimiting amounts of DAP added late in the growth cycle can prevent lysis. In addition, when DAP is added after lysis occurs, there is an immediate resumption of growth at the same rate as initially observed. The addition of acid hydrolyzed casein, while resulting in an initial rapid rate of growth, did not prevent the cells from lysing, suggesting that, with respect to amino acids, the effect of DAP in maintaining the integrity of the cell is quite specific. Microscopic examination of stained preparations of cells removed from the culture at various times during its growth indicated no aberration of cell size or shape. However, to eliminate the possibility of protoplasmic synthesis without cell division, viable cell counts were made and clearly demonstrated (figure 2) that an increase in cell numbers occurs. A decrease in the number of living cells occurred before the turbidity of the culture decreased, suggesting that loss of ability to multiply occurs before complete disintegration of the cell takes place. The data described above suggest that DAP is essential for maintaining the integrity of the cell, probably due to its involvement in structural protein. If this thesis is correct, it should be possible to effect protoplasmic synthesis and prevent lysis of the cell. Since lysine is required for maximum growth, the lysine content of the medium was modified and the effect on growth and lysis of the cell determined. In the absence of lysine, E. coli grew slowly, and lysis was not observed during the course of the experiment. However, when suboptimal levels of lysine were used (figure 3), the lysis phenomenon could be affected without altering the initial growth rate. These data support the theory that the structural I.- z Lo: a00. 4 0 2 4 6 8 10 TIME IN HOURS Figure S. The effect of sub-maximum amounts of lysine on Escherichia coli strain 173-25. Conditions as per figure 1. All adjuncts added at start of experiment.
19571 ROLE OF DIAMINOPIMELIC ACID IN E. COLI 781 TABLE 1 The effect of various DAP analogs on the lysis of Escherichia coli strain 173-26 Reactants Optical Density 4 hr 5 hr Basal medium...0....041 0.032 Basal medium + 1lAg/ml DAP.. 0.398 0.194 + a-aminopimelic acid.. 0.349 0.137 + a-amino-,j-methylpimelic acid... 69...0.469 4 0.201 ± diaminoadipic acid.. 0.377 0.143 + a-methylglutamic acid 0.284 0.097 ± a,-y-diaminoheptanoic acid 0.377 0.119 + piperidine-2,6-dicooh. 0.420 0.137 + pyridine-2,6-dicooh 0.4320.4 0.131 + cadaverine.0.482 0.201 + homocysteine...398 0.102 + j3-methyllanthionine*. 0.181 t 0. 061 t + a,a'-dimetbyldiaminopimelic acid*... 0. 155t + 6-hydroxydiaminopimelic acid*...398t 0.658t ± y-methyldiaminopimelic acid 0...0 0.824 + lanthionine*...0....310 0.620 + cystathionine*... 0.456 0.585 + djenkolic acid*... 0.585t 0.495t Basal medium: plus lysine 20 Ag/ml and glucose 8 mg/ml. All adjuncts added at 50,ug/ml unless indicated. Incubation at 37 C with aeration. Optical density determined at 660 m,u in a Beckman model B spectrophotometer. * 10,g/ml. t 5 and 6 hr readings. integrity of the cell can be maintained, even when the exogenous source of DAP has been eliminated, by slowing down protoplasm synthesis. It appeared of interest, therefore, to examinecompounds structurally related to DAP and to determine their effect on the phenomenon described above. The compounds selected for this study were first examined for their growth-supporting capacity for E. coli in the absence of DAP. Their effect on the lysis phenomenon was determined by adding them to a rapidly growing culture of E. coli containing 1,g/ml DAP. It may be seen from the optical density measurements shown in table 1 that of the compounds tested, cystathionine, lanthionine, a,e-diaminoj3-hydroxypimelic acid and a,e-diamino-'ymethylpimelic acid altered the lytic pattern of E. coli. The results with the -y-methyldiaminopimelic acid and the 13-hydroxydiaminopimelic acid were not surprising since preliminary growth studies indicated that they had growth supporting capacities for the DAP requiring mutant. The finding that lanthionine and cystathionine prevented disruption of the cells was unexpected since contrary to the observation of Siebert et al. (1954) these compounds did not support the growth of E. coli. Subsequent experiments revealed that exceedingly small amounts of DAP were sufficient to initiate the growth of the DAP requiring mutant in the presence of cystathionine or lanthionine. Effect of lanthionine on the growth of E. coli. The role of lanthionine in the system described above was investigated further and additional studies demonstrated that an optimum relationship existed between the amount of DAP required to initiate growth, and the amount of lanthionine necessary to prevent lysis. For example, with 10 ug/ml of DL-lanthionine and 0.5,ug/ml DAP, lysis of the cells could be prevented, whereas, addition of smaller amounts of lanthionine resulted in destruction of the cell in the same pattern as exhibited with limiting amounts of DAP. Viable cell counts revealed that the increase in optical density was due to an increase in cell numbers (figure 4) for at least two generations, although microscopic examination of these cells indicated that they were more coccoid and much larger than the cells observed under normal conditions of growth. These data suggest at least two hypotheses which might explain the apparent substitution of lanthionine, and by implication, cystathionine, for DAP in the growth of E. coli: (1) both compounds are incorporated directly into the cell protein to form a modified structural protein which is still functional; (2) both compounds are being degraded and resynthesized to DAP. In view of the strict requirement of E. coli for DAP and since there is no indication of a change in its growth rate in the presence of added lanthionine or cystathionine, the latter explanation appears quite unlikely. However, the possibility of degradation and resynthesis was considered and various combinations of possible degradation products, such as cysteine, alanine and homoserine were examined for their effect on the lysis phenomenon. In all cases the lytic pat-
782 RHULAND [vol. 73 TIME IN HOURS Figure 4. The effect of lanthionine on the growth and lysis of Escherichia coli strain 173-25. Conditions as per figure 1. Curve A, 1 isg/ml DAP added at 0 hr; Curve B, optical density; Curve C, viable count. Lanthionine added at 10lg/ml at 4 hr as indicated to tube containing 1,ug/ml DAP at 0 hr. tern was not altered. In addition, these compounds either alone or in combinations could not support the growth of E. coli in the absence of DAP. These data suggest that cystathionine or lanthionine are not degraded and subsequently resynthesized to DAP, even though the problem of penetration has not been answered. The thesis that lanthionine or cystathionine is incorporated can best be tested by the use of labeled lanthionine and will be the subject of future investigation. However, preliminary attempts were made to demonstrate differences in DAP content of cells grown with minimal DAP plus lanthionine as compared with cells grown in the presence of excess DAP. The data on the amount of DAP present, as determined by a microbiological assay in the acid hydrolyzates of these cells, suggest that there is less DAP in the cells grown with lanthionine. The effect of y-methyldiaminopimelic acid on the growth of E. coli. The demonstration that y- methyldiaminopimelic acid and #-hydroxydiaminopimelic acid support the growth of E. coli in the absence of DAP represents the first exceptions in the specificity of the above mutant for DAP. The supply of 3-hydroxydiaminopimelic acid was limited which prevented any extensive study of this material, however, a comparison of the growth response of the DAP auxotroph to DAP and the 7y-methyl derivative revealed that thirty times more of the methyl derivative was required to obtain an initial growth rate comparable to that with DAP. The lysis pattern of the culture grown on -y-methyldiaminopimelic acid was similar to that obtained with DAP and in addition, the growth response to the methyl derivative was slow in the absence of lysine. The similar growth response of E. coli to -y-methyldiaminopimelic acid coupled with the close structural relationship between the two compounds is quite compatible with the idea that y-methyldiaminopimelic acid, and by analogy (-hydroxydiaminopimelic acid, can substitute for DAP in the cellular protein. Examination of cell lysates of E. coli. In order to examine the medium for the presence of lysing inducing substance, 0.5-ml aliquots of clear supernatant from a lysed culture were added to E. coli at various stages during its growth cycle. Under these conditions, the growth and lysis of the culture was not changed. Concentrations of cell lysates were prepared and were equally ineffective under the same conditions, thus eliminating the possibility of lysis inducing substances in the medium. In addition, these data demonstrated that free DAP was not liberated into the medium unless at a level less than 0.1,ug per 500 Ag of cell lysate. The ultraviolet absorption spectrum of the culture supernatant was determined on aliquots removed from the culture at 0, 3, 4, 5, and 6 hr. At 5 hr, just prior to lysis of the culture, a material absorbing at 266 m, was formed. At the sixth hr after the culture had lysed, a marked change in the ultraviolet absorption spectrum occurred with a major peak, presumably nucleic acid, forming at 258 m,u. The nature of the 266 m,u absorbing material has not been examined in any detail. DISCUSSION The data described herein illustrates the essential role of DAP in maintaining the cellular integrity of E. coli, apparently by acting as a structural component of the cell wall. It appears that under conditions of limiting DAP during growth, synthesis of cell wall material either stops or an incomplete cell wall is made. This results in rupture of the cell wall since synthesis of cytoplasmic material continues.
1957] ROLE OF DIAMINOPIMELIC ACID IN E. COLI 783 The effect on the lysis phenomenon resulting when the exogenous lysine is maintained at a suboptimal level is compatible with the latter thesis. The ability of,b-hydroxydiaminopimelic acid and -y-methyldiaminopimelic acid to substitute for DAP is also in agreement with this postulate. It appears quite unlikely on the basis of the growth response by the DAP auxotroph that the latter compounds are converted to DAP and it is most probable that they are used in the structural proteins of the cell. The sulfur amino acids, lanthionine and cystathionine, appear to substitute for DAP, but only after growth has been initiated by the amino acid. Although further experiments need to be done, it would appear that both amino acids owe their activity to their incorporation into cell wall protein rather than by permitting more efficient use of DAP. The failure of a, e-diamino-a,e-dimethylpimelic acid to substitute for DAP in the E. coli auxotroph might be assumed to reside in the steric effects due to the methyl groups on the alpha carbons. However, it would appear that minor modifications on the carbon skeleton, other than on the a carbon, have no effect on a compound's usability in this system. ACKNOWLEDGMENTS The author wishes to acknowledge many stimulating discussions with Dr. S. S. Cohen and the technical assistance of Frank T. Maida. The a, E-diamino-3-hydroxypimelic acid was kindly supplied by Dr. John Stewart, Rockefeller Institute, the a,a'-diamino-a,a'-dimethylpimelic acid by Dr. Carl Pfister, Merck & Co., Inc., the 3-methyllanthionine by Dr. Gordon Alderton, U. S. Dept. of Agriculture, and the a,s-diamino-y-methylpimelic acid was prepared by M. F. Murray, Research Division, The Upjohn Company. SUMMARY Escherichia coli strain 173-25, an a,e-diaminopimelic acid (DAP) requiring mutant, lyses when grown in the presence of limiting amounts of DAP. The lysis can be prevented by the addition of noilimiting amounts of DAP, lanthionine, cystathionine, y-methyldiaminopimelic acid or 3-hydroxydiaminopimelic acid. Use of sub-maximum amounts of lysine decreases the rate of lysis. The thesis is advanced that DAP is found only in cell wall material, the synthesis of which stops when the exogenous supply of DAP is exhausted. REFERENCES COHEN, S. AND BARNER, H. 1955 Enzymatic adaptation in a thymine requiring strain of Escherichia coli. J. Bacteriol., 69, 59-66. DAVIS, B. 1948 Isolation of biochemically deficient mutants of bacteria by penicillin. J. Am. Chem. Soc., 70, 4267. DAVIS, B. 1952 Biosynthetic interrelations of lysine, diaminopimelic acid, and threonine in mutants of Escherichia coli. Nature, 169, 534-536. DEWEY, D. L. AND WORK, E. 1952 Diaminopimelic acid and lysine diaminopimelic acid decarboxylase. Nature, 169, 533-534. LEDERBERG, J. AND ZINDER, N. J. 1948 Concentration of biochemical mutants of bacteria with penicillin. J. Am. Chem. Soc., 70, 4267-4268. MEADOW, P. AND WORK, E. 1957 Interrelationships between diaminopimelic acid, lysine and their analogs in mutants of Escherichia coli. Biochem. J. (London), 64, accepted for publication. RHULAND, L. E., WORK, E., DENMAN, R. F., AND HOARE, D. S. 1955 The behavior of the isomers of alpha, epsilon, diaminopimelic acid on paper chromatograms. J. Am. Chem. Soc., 77, 4844-4846. SCHAEFER, W. B., MARSHAK, A., AND BURKHART, B. 1949 The growth of Mycobacterium tuberculosis as a function of its nutrients. J. Bacteriol., 58, 549-563. SIEBERT, F., SOTO-FIGUEROA, E., MILLER, E., AND SEIBERT, M. 1954 Comparison of the amino acid composition of alcoholic extracts of neoplastic tissues with similar extracts of normal muscle. Growth, 18, 145-165. TOENNIES, G. AND GALLANT, D. L. 1949 Bacterimetric studies. II. The role of lysine in bacterial maintenance. J. Biol. Chem., 177, 831-839. WoRE, E. 1951 The isolation of a, e-diaminopimelic acid from Corynebacterium diphtheriae and Mycobacterium Tuberculosis. Biochem. J. (London), 49, 17-23.