Journal ofApplied Bacteriology 1977, 43, 391-398

Diaminopimelic Acid Synthesis in Cultures of an Escherichia cob Double Auxotroph: Effects of Cultural Conditions J. SH. ABDULLAH", A. K. GOEL?

AND

A. N. HALL

Department of Chemistry andApplied Chemistry, University of Salford, Salford M5 4 W , Lancashire, England Received28 March 1977 and accepted 2 August 1977 The metabolic response to L-lysine of Escherichia coli ATCC 13002, a lysine-histidine double auxotroph, has been examined in a synthetic medium containing sucrose. In shaken cultures largest amounts of extracellular D A P were produced with an initial lysine concentration of 7.5 mg/l and in static cultures of 2.5 mg/l. Considerably smaller amounts of D A P accumulated under stationary conditions. In cultures shaken for 20 and 43 h there was an overall decrease in the yields of DAP, expressed in terms of cell biomass and of sucrose consumed, as the initial concentration of lysine was increased from 0.75 mg/l in steps up to 25 mg/l. The regulatory effect of lysine on D A P production was also observed when lysine was supplied to cultures at a constant rate employing diffusion capsules.

DIAMINOPIMELIC ACID (DAP) was isolated from Corynebacterium diphtheriae and Mycobacterium tuberculosis (Work 1951) and later identified as a component of the mucopeptide layer of certain bacterial cell walls (Salton 1956) and as an immediate biosynthetic precursor of lysine (Dewey & Work 1952; Davis 1952). DAP accumulates in cultures of lysine-requiring mutants of bacteria which are deficient in DAP decarbox ylase. A mutant of Escherichia coli (ATCC 13002; Huang et al. 1960) is doubly auxotrophic for lysine and histidine and is suitable for an investigation of the control and regulation of amino acid biosynthesis because of the lower probability of reversion to the wild-type compared to a mutant with a single requirement for lysine. This paper is concerned with the effects of exogenous lysine on DAP synthesis in cultures of this organism.

Materials and Methods Organism Escherichiu coli strain ATCC 13002 was obtained from the American Type Culture Collection, Rockville, Maryland, U.S.A. Stock cultures were maintained on Nutrient Agar (OXOID CM3) and stored at 4 OC. The identity of the strain was confirmed by demonstrating that L-lysine and L-histidine were required for growth in Sucrose-Salts medium. Medium The growth medium (Sucrose-Salts medium) (Davis & Mingioli 1950) had the following composition (g/l): K,HPO,, 7.0; KH,PO,, 3.0;MgS0,.7H,O, 0.10; (NH,), SO,, 1.0;

* Present address: Ministry of Health, State of Bahrain, P.O. Box 12, Bahrain, Arabian Gulf. t Present address: Department of Biological Sciences, Madurai University, Madurai 62502 1, India. [3911

392

J. SH. ABDULLAH, A. K. GOEL AND A. N. HALL

sucrose, 20.0; L-lysine and L-histidine, as indicated. In certain of the experiments with diffusion capsules, (NH$,SO, (2 g/l) was incorporated in Sucrose-Salts medium to ensure that sufficient nitrogen was available for the high yields of DAP produced. The pH of the solution containing inorganic salts and amino acids was adjusted to 7.0 with NaOH. Medium components were sterilized by autoclaving (1 15 OC for 20 min). Sucrose was sterilized separately in concentrated solution, and added at the time of inoculation.

Culture techniques Cultures were grown at 26 OC, which is in the optimum range for DAP synthesis by E. coli ATCC 13002 (Huang et al. 1960), in Erlenmeyer flasks (250 ml) containing 25 ml of medium (except when diffusion capsules were used). Shaken cultures were agitated at 250 rev/min in an orbital incubator (Gallenkamp Ltd., London). An inoculum was prepared from a 24 h shaken culture in Nutrient Broth (OXOID CM1); this culture was always derived from a single colony grown on nutrient agar. Cells were harvested by centrifugation at 2300 g for 5 min, washed twice with sterile saline and finally resuspended in the same volume of saline. Flasks were inoculated with 0.5 ml of suspension. Diffusion capsules (Pirt 1971; L. H. Engineering Co. Ltd., Stoke Poges, Buckinghamshire) were used in certain experiments to supply lysine to cultures at a constant rate. The procedure was to inoculate 100 ml amounts of Sucrose-Salts medium containing L-histidine (25 mg/l) and L-lysine (1 mg/l) with 2 ml of washed cell suspension and to incubate for 24 h, with shaking, during which time only slight growth occurred owing to the very low concentration of lysine provided. Sterilized diffusion capsules, fitted with two or three membranes, and containing lysine monohydrochloride solution ( 0 . 2 4 7 5 % w/v), were then added to flasks and incubation continued. Biomass determinations Bacterial growth was followed by measuring the optical density of suitably diluted cultures in an EEL absorptiometer fitted with a neutral filter (Evans Electroselenium Ltd., Halstead, Essex). Cell dry weight was estimated by reference to a calibration curve. This curve was constructed after drying, to constant weight, duplicate samples of an aqueous cell suspension for 3 h at 70-80 "C and subsequently over P,O, in uacuo at room temperature. Preparation of samplesfor analysis Culture samples were centrifuged at 2300 g for 5 min and the supernatant fluids removed for analysis. All analyses were performed in duplicate on samples from duplicate flasks. Standard errors did not exceed k 14% of the mean value. Extracellular DAP determination The estimation of DAP was based on the yellow colour which develops when the amino acid is treated with ninhydrin under acidic conditions either by the method of Gilvarg (1958) or the low temperature procedure of Work (1963). Sucrose determination Residual sucrose in the medium was determined by the anthrone method of Morris (1948).

DIAMINOPIMELATE IN E . COLI MUTANT CULTURES

393

Rates of difSusion of lysine from capsules The diffusion of lysine from capsules into 100 ml water in Erlenmeyer flasks (250 ml) shaken under normal culture conditions was measured by estimating lysine according to the method of Yemm & Cocking (1955). Combinations of several concentrations of lysine monohydrochloride with two or three membranes in capsules were tested.

Results Effect of exogenous lysine on the accumulation of DAP in shaken cultures The yields of DAP (Fig. 1) and of bacterial biomass (Fig. 2) were determined at intervals in cultures grown with different initial concentrations of L-lysine. Accumulation of DAP was clearly enhanced by growth-limiting concentrations of lysine, 7.5 mg/l of L-lysine being the most favourable for DAP synthesis in shaken cultures. The data do not permit a precise assessment of the times of maximum accumulation of extracellular DAP but it appears likely that in the medium with 7-5 mg/l of lysine this would have occurred between 43 and 70 h. A histogram (Fig. 3) was constructed showing the relationships between initial lysine concentration and cell dry weight, DAP concentration and DAP yields expressed on the basis of sucrose consumed and of cell dry 000 -

600 -

E -r,

4

\

01

3.

0

.O

400-

.-E

n

0

.E .-

a

200 -

I 160

Time (h)

Fig. 1. Production of extracellular diaminopimelic acid in shaken cultures of Escherichia coli ATCC 13002 in Sucrose-Salts medium supplemented with L-histidine (25 mg/l) and the following additions of L-lysine (mg/l): 0 , 0 . 7 5 ; A, 2.5; Q 7 . 5 ; .,25; A, 75; m, 250.

394

J. SH. ABDULLAH, A. K. GOEL AND A. N. HALL

Time (h)

Fig. 2. Biomass of Escherichia coli ATCC 13002 in shaken cultures in Sucrose-Salts medium supplemented with L-histidine (25 mg/l) and the following additions of L-lysine (mg/l): 0, 0.75; 42.5; @, 1.5; O , 2 5 ; A, 75;M, 250.

weight. In terms of mg DAP/mg bacterial biomass the yield was greatest at both 20 and 43 h in cultures supplied with the lowest lysine concentration (0.75 mg/l). On the basis of mg DAP/mg sucrose consumed the best yield appeared to be in the medium with 2 . 5 mg/l of lysine at 20 h but this may be an over-estimate as sugar consumption at 20 h in media with 0.75 and 2.5 mg/l of lysine was only just detectable and could not be determined accurately.

n

41 5

- '

20 h

075

25

5

L- lvsine

(mall)

43 h

i

n

375 1

25

75

25

-

70 h

Fig. 3. Metabolic response of Escherichia coli ATCC 13002 to L-lysine in cultures shaken in Sucrose-Salts medium supplemented with L-histidine (25 mg/l). 0, bacterial biomass (mg dry wt/ml); 3,mgDAP/ml; N, mgDAP/mg sucrose consumed; M,mg extracellular DAP accumulated/mg cell mass.

DIAMINOPIMELATE IN E. COLI MUTANT CULTURES

395

EfSect of exogenous lysine on the accumulation of DAP in static cultures The level of extracellular D A P production (as pg DAP/ml) was considerably lower in static than in shaken cultures owing to the reduced growth. At the times examined, greatest amounts of DAP in static cultures (as pg DAP/ml) were found to be produced with lower initial lysine concentrations (2.5 mg/l, Fig. 4) than in shaken cultures (7.5 mg/l of lysine, Fig. 3). It is noteworthy that considerably higher yields, as mg DAP/mg cell mass, were obtained in static (max. f a . 3-9) than in shaken cultures (max. ca. 1.8). Accumulation of DAP in shaken cultures supplied with lysine at defined rates The rate of diffusion of L-lysine from capsules had a marked effect on DAP synthesis (Table 1). In media containing 1 g/l of (NH,),SO, the best yields of DAP were obtained with lysine diffusion rates of 1-30 and 2.60 pg ml-' h-l. With a more generous supply of L-lysine (3 36 pg ml-I h-l) the DAP yield was much reduced. At the optimum rates of lysine diffusion for D A P yield (1.30 and 2.60 pg ml-' h-l) into media with either 1 or 2 g/l of (NHJ,SO,, D A P was produced in amounts which approached the theoretical conversion of 1000 pglml of (NH,),SO, to 1400 pg/ml of DAP. The highest yield of DAP realized in shaken cultures not receiving a continuous supply of lysine, in the same Sucrose-Salts medium, was ca. 800 ,ug/ml from 1000,ug/ml of (NH4),S0, (Fig. 1). TABLE1

Diaminopimelic acid production by Escherichia coli A TCC I3002 in shaken cultures supplied with lysine from digusion capsules. Sucrose-Salts medium initially contained L-histidine (25 mgll) and L-lysine (1 rngll) L-lysine monohydrochloride (NH,),SO, s o h in in medium capsule (g/l) (% w/v) b

1

0.15 2

DAP* Rate of lysine diffusion g ml-I h

Cuglml)

Hours after addition of capsule A

f-

I)

24

0.50 1.30 2.60

600 i 29 546 i 0 757 i 104

3.36 1.30 2.60

80 k 0 546 f 0 739 k 85

\

48

64

84

410 +_ 38 1290 k 18

-

1365 11 863 2 13

310 2 25 1230 i 35 2093 & 93 -

290 2 5 -

2387

41

-

2044 f 142 2051 i 24

* Data are the means of analyses f S.E.of the mean on samples from two or three replicate flasks.

Discussion It is known from investigations of the metabolism of bacterial auxotrophs with a requirement for lysine only that the biosynthesis of DAP in cultures of such mutants is regulated by the concentration of lysine in the medium, and that optimum DAP synthesis occurs when the concentration of lysine is growth-limiting (Angulo et al. 1960; Huang 1964; Hagino & Nakayama 1970). The results presented in Figs 3 and 4 show that the synthesis of DAP in shaken and stationary cultures of E. cofi ATCC 13002, which is doubly auxotrophic for lysine and histidine, is also regulated by lysine.

J. SH. ABDULLAH, A. K. GOEL A N D A. N. HALL

396

4.0

3.c

2. c

0.0

Ll - -d

0

2

5.0 7.5

0

d

d

5 0 7.5

2

2

L - lysine (mg/L)

24 h

48 h

72 h

.,

Fig. 4. Growth of Escherichia coli ATCC 13002 and production of diarninopimelic acld in static cultures in Sucrose-Salts medium supplemented with L-histidine (25 mg/l) and four lysine concentrations. 0, bacterial biomass (mg dry wt/ml); 0, mgDAP/ml; mg extracellular DAP accumulated/mg cell mass.

High lysine concentrations are believed to suppress DAP synthesis through feed-back inhibition and repression of certain vital enzymes involved in the biosynthetic pathway (Cohen 1968). An interesting feature of the progress of DAP formation in shaken cultures is the decrease in the concentration of D A P observed in several of the cultures after 43 h. It seems likely that this decrease in DAP concentration might be ascribed to the development of lysine-independent (revertant) forms of the organism capable of decarboxylating DAP. In control flasks (to which no lysine had been added) no growth was visible in 43 h but after this time considerable growth occurred and this may have been due to the development of a revertant. Indeed, a lysine-independent organism was isolated from such a control flask and shown to utilise DAP rapidly when grown in Sucrose-Salts medium supplemented with L-histidine (25 mg/l) and DAP (250 mg/l). The development of lysine-independent forms of the single auxotroph, E. coli ATCC 12408, after exhaustion of lysine from the medium has been reported (Angulo et al. 1960; Bishop & Work 1965). Escherichia coli ATCC 13002 grown in shaken culture exhibits a diphasic-type growth curve in which a stationary phase of about 20 h follows the initial (presumably exponential) growth period and precedes a second growth phase. Diphasic growth has been reported in DAP-producing cultures of E. coli mutants singly auxotrophic for lysine by Angulo et al. (1960), Lilly et al. (1963) and Bishop & Work (1965) who suggest that diphasic growth is related to the formation of DAP. During the course of cultivation in stirred and aerated fermenters, these workers observed that DAP production started soon after completion of the exponential phase of growth and continued at a steady rate in the second growth phase until there was no further increase

DIAMINOPIMELATE IN E. COLI MUTANT CULTURES

397

in the opacity of the culture. In our comparable experiment, employing shaken cultures, the production of DAP had reached a good level (at 43 h) before the onset of secondary growth, less D A P synthesis being observed in this later phase. If it is correct to assign this secondary growth to the development of lysine-independent forms as suggested by Angulo et al. (1960) and Lilly et al. (1963), some utilization of DAP might be anticipated. The partial reversal at 70 h of the trend, observed at 20 and 43 h, for the yield of D A P (mg/mg cell mass) to decrease in shaken cultures of E. coli ATCC 13002 with increasing lysine concentration may be most reasonably ascribed to such secondary growth. Our data suggest that maximum DAP synthesis (in pglml) occurs in cultures of E. coli ATCC 13002 with lower initial concentrations of lysine (of the order 7.5 mg/l in shaken cultures and 2.5 mg/l in static cultures) than have been reported to be optimal for aerated cultures of E. coli ATCC 12408 by Angulo et a/. (1960; 100 mg/l) and Bishop & Work (1965; 48 mg/l), and of E. coli ATCC 13002 by Huang et a/. (1960; 200-400 mg/l). The extent to which cultures are aerated must affect the utilization of lysine for growth. Bishop & Work (1965) have reported a high oxygen requirement for DAP production by E. coli ATCC 12408; the increased biomass generated under these conditions would appear to be an important factor. Evidence of a requirement for oxygen in D A P synthesis may also be seen by comparing the yields of DAP (,ug/ml) in shaken and static cultures of E. coli ATCC 13002. That DAP synthesis was favoured by a lower initial lysine concentration in static (2.5 mg/l) than in shaken cultures (7-5 mg/l) confirms that the optimum concentration of lysine increases with the degree of aeration. It appears likely that, by stimulating growth, aeration effects a more rapid reduction of the lysine concentration to levels sufficiently low to permit efficient synthesis of DAP. It is of interest that although the amounts of DAP produced were lower in static than in shaken cultures the yields, expressed as mg extracellular DAP/mg cell mass, were on the whole considerably higher in static cultures, which may indicate a more efficient conversion of carbohydrate to DAP in the less oxygenated environment. The data for D A P production by E. coli ATCC 13002 when lysine was fed to cultures at a constant rate of either 1.30 or 2.60 pg ml-l h-' are of interest because the yields approached the theoretical conversion of (NH,),SO, to DAP in media containing either 1 or 2 g/1 of (NH,),SO,. These high yields of DAP, as compared with those obtained when the rate of lysine diffusion was 3.36 pg ml-I h-l, probably reflect both an enhanced synthesis of two key enzymes of the biosynthetic pathway, viz. aspartokinase 111 and aspartate semialdehyde dehydrogenase (Cohen & Patte 1963), and a reduced allosteric inhibition by L-lysine of aspartokinase I11 (Stadtman et a/. 1961) and of dihydrodipicolinate synthetase (Yugari & Gilvarg 1962) in the cultures supplied with lysine at the lower rates. This work was supported by Research Contract No. 936 of the International Atomic Energy Agency.

References ANGULO,J., MAURINO,T. G. G., MUNICIO,A. M. & RIVERO,W. 1960 Biosynthesis of a,Ediaminopimelic acid by Escherichia coli. VI. Growth of a lysine auxotrophic mutant. Anales de la Real Sociedad Espanola de Fisica y Quimica (Madrid) 56B,413-426.

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BISHOP,D. G. & WORK,E. 1965 An extracellular glycolipid produced by Escherichia coli grown under lysine-limiting conditions. Biochemical Journal 96,567-5 76. COHEN,G. N. 1968 The Regulation of Cell Metabolism. New York: Holt, Rinehart and Winston, Inc. COHEN,G. N. & PATTE,J. C. 1963 Some aspects of the regulation of amino acid biosynthesis in a branched pathway. Cold Spring Harbor Symposia on Quantitative Biology 28,5 135 16. DAVIS,B. D. 1952 Biosynthetic interrelations of lysine, diaminopimelic acid and threonine in mutants of Escherichia coli. Nature, London 169, 534-536. DAVIS,B. D. & MINGIOLI,E. S. 1950 Mutants of Escherichia coli requiring methionine or vitamin B,2,Journal of Bacteriology 60, 17-28. DEWEY,D. L. & WORK, E. 1952 Diaminopimelic acid and lysine. Diaminopimelic acid decarboxylase. Nature, London 169,533-534. GILVARG,C. 1958 The enzymatic synthesis of diaminopimelic acid. Journal of Biological Chemistry 233, 1501-1504. HAGINO,H. & NAKAYAMA, K. 1970 Accumulation of a,&-diaminopimelic acid by lysine auxotrophic mutants of Brevibacterium ammoniagenes. Nippon Nogei-kagaku Kaishi (Japanese) 44,422-427. HUANG, H. T. 1964 Microbial production of amino acids. In Progress in fndustrial Microbiology Vol. 5 , ed. Hockenhull, D. J. D. London: Heywood-Temple Press Books Ltd. HUANG,H. T., GRIFFIN,J. M. & FRIED,J. H. 1960 U.S. Patent 2955986. LILLY,M. D., CLARK,P. H. & MEADOW,P. M. 1963 The accumulation of nucleotides by Escherichia coli. Journal of General Microbiology 32, 103-1 16. MORRIS,D. L. 1948 The quantitative determination of carbohydrates with Dreywood’s anthrone reagent. Science 107,254-255. PIRT, S. J. 1971 The diffusion capsule, a novel device for the addition of a solute at a constant rate to a liquid medium. Its application to metabolic regulation. Biochemical Journal 121, 293-297. SALTON, M. R. J. 1956 Bacterial cell walls. In Bacterial Anatomy 6th Symposium of the Society for General Microbiology, ed. Spooner, E. T. C. & Stocker, B. A. D. Cambridge: University Press. E. R., COHEN,G. N., LEBRAS,G. & DE ROBICHON SZULMAJSTEK, H. 1961 STADTMAN, Feedback inhibition and repression of aspartokinase activity in Escherichia coli and Saccharomyces cereuisiae. Journal of Biological Chemistry 236,2033-2038. WORK,E. 195 1 The isolation of a,&-diaminopimelicacid from Corynebacterium diphtheriae and Mycobacterium tuberculosis. Biochemical Journal 49, 17-23. WORK,E. 1963 a,&-Diaminopimelicacid. In Methods in Enzymology Vol. 6 ed. Colowick, S. P. & Kaplan, N. 0. New York & London: Academic Press. YEMM,E. W. & COCKING,E. C. 1955 The determination of amino acids with ninhydrin. Analyst, London 80,209-2 13. YUGARI,Y. & GILVARG, C. 1962 Co-ordinated end-product inhibition in lysine synthesis in Escherichia coli. Biochimica et Biophysica Acta 62,612-614.