Product induction of glycerol kinase in Escherichia coli

J. Mol. Biol. (1965) 14, 515-521

Product Induction of Glycerol Kinase in Escherichia coli SHIN·lom HAYAsmt AND E. C. C. LIN

Department of Biological Chemistry, Harvard Medical School Boston, Mass. 02115, U.S.A . (Received 2 July 1965, and in revised form 8 September 1965) Glycerol kinase can be induced in Escherichia coli K12 when the cells are grown in the presence of either glyo erol or L.IX.glyoerophosphate. A mutant has been obtained which is capable of produoing a protein immunochemioally indistinguishable from crystallized glycerol kinase, but which has very slight enzymic activity. In this mutant, the cross-reacting protein is inducible by L·IX.glycero. phosphate but not by glycerol. It is, therefore, concluded that in wild-type cells the induotion ofthe kinase by glycerol depends on its con ver sion to the phosphorylated oompound by the sm all amount of the kinase which is present in non-induced cells .

1. Introduction Glycerol and L.oc.glycerophosphate can be adaptively utilized by Escherichia coli Kl2 according to the following scheme (Hayashi, Koch & Lin , 1964; Koch, Hayashi & Lin , 1964; Hayashi & Lin, 1965):

Cyto pla sm

Medium Free diffusion Glycer o l

Glycerol

j

Act ive t ransport system

L-a-GP

L-

G lycerol kina se

a-GP

1

L-a-GP dehydrogenase

Dihydroxyacetone phosphate

~

Cells grown on either glycerol or L.O(.GPt are induced in all three functional units: the kinase, the transport system and the dehydrogenase. A single mutation can also lead to simultaneous de-repression of all three, indicating that they share a single regulator gene. These units, however, do not belong to a oommon operon as revealed

t Present address: Laboratory of Molecular Biology, National Institute of Arthritis and Metabolic Diseases, U.S . Public Health Service, Bethesda, Md. 20014, U.S.A. ~ Abbreviation used: GP, glycerophosphate. 515

516

S. HAYASHI AND E. C. C. LIN

by their differential sensitivity to catabolite repression and by results from genetic recombination (Cozzarelli, Hayashi & Lin, 1965). The question of whether glycerol and L-oc-GP act independently as inducers has been answered with respect to the transport system and the dehydrogenase by the use of mutants lacking glycerol kinase activity. In these mutants glycerol no longer could induce the remaining two units, although their induction by L-oc-GP was unaffected. Thus the capacity of glycerol to initiate induction in wild-type cells must be attributed to the action of the basal glycerol kinase, shown to be present at low but measurable levels. In view of the existence of a single repressor gene controlling all three functional units, it was argued that the kinase itself was likewise induced by L-oc-GP and not directly by glycerol. The experiments to be reported here were designed to test this postulate.

2. Materials and Methods (a) Bacteria All of the strains used in the present work were derived from E. coli K12 (CavalliSforza, 1950). Strain 60 was isolated from the above as a lactose-negative mutant, which in turn gave rise to strain 61, lacking glycerol kinase activity but capable of producing cross-reacting material upon induction. The origins of strain 4, a mutant which is negative both in glycerol kinase activity and in cross-reacting material, and strain 7, a constitutive mutant producing high levels of glycerol kinase, have been previously described (Koch et al., 1964). The mutants mentioned above were all isolated by Dr J. P. Koch following mutagenesis with ethyl methanesulfonate (Lin, Lerner & Jorgensen, 1962). (b) Chemicals Glycerol was obtained from Merck and Company, DL-cx-GP from Sigma Chemical Company, and vitamin-free casein acid hydrolysate from Nutritional Biochemicals Corporation. (c) Growth of cells and preparation of extracts The basal medium contained inorganic salts and tris-HCI buffer at pH 7·5 as previously described (Garen & Levinthal, 196()'). Inorganic phosphate was added at a concentration of 0·6 mx to repress the formation of alkaline phosphatase (Horiuchi, Horiuchi & Mizuno, 1959; Torriani, 1960). In all the experiments 1 % casein hydrolysate, found not to have appreciable catabolite repressive effect on our systems, was used as the major source of carbon. When tested for inducer activity, glycerol was added at 0·02 M and DL-cx-GPat 0·04 M. Previous studies have shown that the n-isomer is not a substrate for the L-cx-GP transport system, and, moreover, cannot be utilized by cells without alkaline phosphatase (Hayashi et al., 1964; Lin, Koch, Chused & Jorgensen, 1962). Therefore D-cx-GP can be considered biologically inactive for the purpose of the present study. Additional details concerning the growth of cells and the preparation of their extracts have been reported elsewhere (Koch et al., 1964). (d) Assay of activities of enzymes and of transport All enzyme assays were carried out on freshly prepared extracts. Glycerol kinase activity was measured by coupling the phosphorylation reaction to the reduction of NAD by the action of t-c-G'P dehydrogenase from rabbit muscle (Wieland & Suyter, 1957). The detailed conditions found optimal for the E. coli enzyme have been reported in another paper (Lin, 1962). The units of activity are expressed as ,.moles of L-CX·GP formed per minute at 25°C. The L-cx-GP dehydrogenase of E. coli is not NAD·linked, but its action can be followed by measuring the reduction of a tetrazolium dye to its formazan (Lin, Koch, Chused & Jorgensen, 1962). The units of activity are expressed as ,.moles of L-cx-GP dehydrogenated per min at 25°C. The active transport of L-cx-GP by suspended cells was measured by the uptake of 14C_ labeled substrate (Hayashi et al., 1964), and the units of activity are given in mumoles of substrate accumulated per min at 15°C.

PRODUCT INDUCTION OF GLYCEROL KINASE

517

(e) Preparation oj crystalline glycerol kinase

Glycerol kinase was purified from strain 7, a constitutive mutant which could produce the enzyme to the extent of 5% of the total soluble protein. The final preparation, which had been crystallized twice, was homogeneous in its sedimentation pattern. Methods for the isolation of this protein and some of its physical and enzymic characteristics will be described in another report. (f) Preparation oj specific antiserum

Rabbits were immunized against glycerol kinase by two sets of subcutaneous injections separated by an interval of a month. Each dose consisted of 5 mg of the crystalline enzyme dissolved in 1 ml. of saline and emulsified with 1 ml. of Freund's adjuvant. In the second immunization, Mycobacterium butyricum was omitted from the adjuvant. Ten days after the second injection, blood was collected by heart puncture, incubated at 37°C for 2 hr and then kept at O°C for 1 day. The serum was separated from the clot by decantation and then centrifuged to remove the remaining red cells. To eliminate antibodies which might react with bacterial proteins other than glycerol kinase, the antiserum was treated with a crude extract of cells of strain 4 grown on casein hydrolysate without inducer. This mutant is not only glycerol-kinase negative, but is also incapable of producing cross-reacting material even in the presence of inducer. Nine ml. of the antiserum were incubated with the crude bacterial extract containing 90 mg of protein for 2 hr at 25°C and then kept for a day at O°C.The slightly turbid solution was centrifuged at 30,000 g for 15 min and the clear supernatant fraction was used for immunochemical studies. (g) Detection and estimation oj glycerol kinase protein For the qualitative demonstration of the protein, the double-diffusion technique (Ouchterlony, 1958) was employed. The gel consisted of 1 % agar in 0·5 M-sodium azide, 0·15 M·NaCI and 0·01 M.potassium phosphate at a final pH of 7·0. The diffusion took place at 25°C. For quantitative estimations, precipitin reactions were carried out (Kabat & Mayer, 1961). A mixture of 0·4 ml. of the partially purified antiserum and 0·3 ml, of the bacterial extract was incubated at 25°C for 2 hr and then kept at O°C for 1 day before the precipitate was collected by centrifugation at 10,000 g for 10 min. The pellet was resuspended in approximately 2 ml. of 0·15 M-NaCI in 0·01 M.potassium phosphate buffer at pH 7, after which centrifugation was again carried out. The final sediment was suspended in a total volume of 2·0 ml. in the same buffer and the turbidity was measured at 550 miL' The amount of glycerol kinase or that of cross-reacting material contained in the sample was calculated with the aid of a standard curve constructed with known amounts of the crystalline enzyme (Fig. 1). The validity of this assay is supported by the finding that the amount of precipitate per unit of enzyme activity obtained with crude extracts of induced wild-type cells was the same as that obtained with the pure enzyme. 0-5~------------'

0-4

?

.,---.

~ 0-3

/ /.

:

0-2

o

OIV/" o

10

20

30

40

50

60

Crystalline glycerol kinase (Jig)

FIG. 1. Estimation of glycerol kinase protein by the precipitin reaction.

518

S. HAYASHI AND E. C. C. LIN

3. Results To determine whether glycerol could act as an inducer for the kinase without prior conversion to L-Ot-GP, we searched for a mutant capable of producing a protein immunochemically recognizable but enzymically inactive. As a preliminary step, mutants capable of utilizing L-Ot-GP, but not glycerol, were grown on casein in the presence of both glycerol and DL-Ot-GP. Extracts of cells which contained very low activities of glycerol kinase were further screened for the presence of cross-reacting material. A mutant, strain 61, was thus obtained, which could produce full amounts of the crossreacting protein. Cells of strain 61 were next grown under three conditions: on casein hydrolysate alone, with glycerol in addition, and with DL-Ot-GP in addition. Their extracts were examined for the presence of cross-reacting material with the Ouchterlony doublediffusion technique using an antiserum prepared against a crystallized preparation of glycerol kinase. As shown in Plate I, only the extract of cells grown in the presence of DL-Ot-GP was found to produce cross-reacting material. Glycerol, which induced the enzymically active protein in the parental strain 60, did not act as an inducer for the cross-reacting material in the mutant. The induced cross-reacting material in the extract of strain 61, moreover, is immunochemically identical with glycerol kinase, since a continuous band without spur was formed in the agar between the central well charged with antiserum and the adjacent wells inoculated, respectively, with the extract of strain 60 induced with glycerol, the crystallized enzyme, and the extract of strain 61 induced with L-Ot-GP. The appearance of a well-defined single band of precipitation also shows that the antiserum is monospecific and that the antigen in each well is homogeneous. The possibility that the cross-reacting substance in the extract of strain 61 cells induced with L-Ot-GP might be attributable to L-Ot-OP dehydrogenase or proteins associated with the L-Ot-GP transport system was excluded with the aid of another glycerol kinase-negative mutant, strain 4. When cells of this strain were grown in the presence of L-Ot-GP, full induction of the transport system and the dehydrogenase occurred. Yet extracts from such cells showed no reaction with the antiserum. The induction of the cross-reacting material by L-Ot-GP and not by glycerol was further substantiated in a more extensive experiment in which the response to induction of all three functional units-the kinase protein, the L-Ot-GP transport system and L-Ot-GP dehydrogenase-was compared in strains 60 and 61. In this experiment the precipitin reaction was used to measure the amount of the kinase protein or its crossreacting material. In addition, the extracts were assayed for the activity of glycerol kinase. The results, summarized in Table 1, fully confirm the notion that the induction of each of the three units by glycerol is contingent upon its conversion to L-cx-GP. The results, however, cannot exclude the possibility that a trace of glycerol is required to act in concert with L-Ot-OP as a co-inducer, because even though alkaline phosphatase was repressed under the experimental conditions, slow hydrolysis of L-Ot-GP would still occur in the presence of other non-specific phosphatases (Lin, 1962; Neu & Heppel, 1964). Finally, it might be noted from the Table that the cross-reacting material produced by strain 61 was not altogether devoid of enzymic activity and that even with extracts of non-induced cells, a very slow but definite rate of phosphorylation of glycerol could be observed. The failure of glycerol to exhibit any inducing activity in this leaky

PLATE I. Detection of cro ss -reacting material in ce ll-free ex t ract s by the Ouc hterlo ny techniq ue . The ce nte r well co n tains the antiserum ; well no . 1, extract of strain 60 grown in the presence of glycero l ; we ll no . 2, crystalline glycerol kinase (200 fLg ); well no . a, extract of st rain 61 grown in t he pre se nc e of DL- iX-G P ; well no. 4, ex t ra ct of st ra in 61 grown in t he pres en ce of glycerol; well n o. 5, ext rac t of stra in 61 grown on casein hyd rol ysate alone.

[facing p _ ,)J 8

PRODUCT INDUCTION OF GLYCEROL KINASE

519

mutant would indicate that a relatively high level of intracellular L-ot-GP is required for induction. The significance of this requirement will be examined in the context of functional demands imposed on the control system for glycerol metabolism. TABLE

1

Responseof glycerol kinase-positive and glycerol kinase-negative cells to glycerol and L-ot- GP as inducers

Strain

Addition to growth medium']

Glycerol kinase or cross-reacting materialt (/Lg/mg protein)

60

None Glycerol DL-o:-GP

<

0·1 4·9 3·9

61

None Glycerol DL-o:-GP

< <

0·1 0·1 8·2

Glycerol kinase activity (unitajmg protein)

< <

Activity of the L-o:-GP L-o:-GP dehydrogenase transport system activity (unitsjmg (units/mg dry cells) protein)

0-006 0·59 0·34

0·047 0·79 0·29

0·010 0·15 0·064

0-0004 0·0002 0·005

0·046 0·050 0·73

0·009 0·012 0·072

t All contained 1 % casein hydrolysate. t Measured by the precipitin reaction.

4. Discussion The induction of catabolic enzymes by products or metabolites rather than by substrates has been observed in several other cases. Thus the inducer activity of lactose for the Lac operon in E. coli was shown to be dependent upon the transgalactosidation activity of ,8-galactosidase (Miiller-Hill, Rickenberg & Wallenfels, 1964; Burstein, Cohn, Kepes & Monod, 1965). The co-ordinate induction of tryptophan pyrrolase and formylkynurenine formamidase in Pseudomonas jluorescens by tryptophan was shown to result from its conversion to L-kynurenine (Palleroni & Stanier, 1964). Histidase and urocanase are induced by urocanate, but not by histidine, in Aerobacter aeroqenes (Scotto, Schlesinger & Magasanik, 1965). Several other enzymes were found to be inducible by their products, although the capacity of the substrates also to act directly as inducers was not investigated. These examples include the invertase of yeast (Euler & Cramer, 1913); tannase (Knudson, 1913) and lipase (Schenker, 1921) of Aspergillus niger; pectinesterase of Penicillium chrysogenum (Phaff, 1947); histidase of Pseudomonas fiuoreecens (Suda, Miyahara, Tomihata & Kato, 1952); amylases of Pseudomonas saccharophila (Thayer, 1953); and phenylalanine hydroxylase of Pseudomonas sp. (Guroff & Ito, 1963). For hydrolytic enzymes which act outside of the cell membrane on macromolecular substrates, product induction is most likely a matter of mechanistic necessity, since inducers must be able to enter the cell readily. For intracellular catabolic enzymes the substrates of which happen also to be biosynthetic intermediates, it was suggested that product induction offers a damping mechanism which would prevent premature enzyme synthesis in response to transient abundance of the substrate, a res-

520

S. HAYASHI AND E. C. C. LIN

ponse which might subsequently jeopardize the biosynthetic pool (Palleroni & Stanier, 1964). In the case of glycerol kinase, the substrate of which is not a normal biosynthetio intermediate, the utilization of product induction might be related to the fact that the cell membrane of E. coli is freely permeable to glycerol but not to L-a-GP. The scavenging power of the cell for glycerol resides in the kinase instead of in an active transport system (Hayashi & Lin, 1965). If glycerol were to be used as the inducer for the kinase, the K m of induction would have to be considerably lower than 10- 6 M, the K m of the enzyme, to permit the expression of the full scavenging capacity of the cell. Such a super-sensitive control system would, however, suffer from two important drawbacks: (1) the triggering of enzyme synthesis by low concentrations of the substrate even when the total amount of the compound available in the environment is too meager to warrant the biosynthetic investment; and (2) the futile synthesis of the enzyme in response to compounds which readily mimic the inducer. On the other hand, if L-a-GP serves as the inducer, the K m of induction need not be very low,'] since L-a-GP accumulates intracellularly as the autocatalytic induction proceeds. This would allow the cell to produce enzyme in response to an amplified signal, with the attendant advantages. Significant rates of enzyme synthesis would occur only if there is a continuous and adequate supply of glycerol to maintain a critically high intracellular level of L-a-GP. In addition, faulty induction is less likely to happen, because screening can be exercised at two levels: discrimination by glycerol kinase and by the apo-repressor. Induction by an amplified signal, of course, can take place without the intervention of intracellular enzymes, whenever there is a specific active transport system. Finally, a control mechanism which is even better equipped to discriminate against noise would be bivalent induction by substrate and product. Such a mechanism is likely to be operating in the benzoic acid oxidase system in Micrococcus ureae (Sanno, Tanaka, Hayashi & Suda, 1965). In summary, it should be stressed that although numerous induction patterns can be justified on teleonomie grounds, many examples must exist in which the specificity of induction simply reflects the historical development of the individual genes. In a multi-cistronio operon, the mechanism of induction is likely to have co-evolved initially with the primeval structural gene, and the type of control is likely to persist until a more advantageous alternative becomes available. The same may be said of a regulon (Maas & Clark, 1964) in which a common repressor is shared by more than one operon. Here not only historical events but also the economy of having a minimal number of repressor genes may be of decisive importance in establishing inducer specificity. We thank Gregory W. Siskind for valuable advice and generous assistance on immunochemical aspects of this study, and Nicholas R. Cozzarelli and Gordon M. Tomkins for helpful criticisms during the preparation of this paper. Part of this work was presented before the American Society of Biological Chemists, Atlantic City, New Jersey, April, 1965 (Cozzarelli et al., 1965). The support from the National Science Foundation (GB-722), the United States Public Health Service (GM-1l983 and GM-K3-17925) and the American Cancer Society is gratefully acknowledged.

t Since L-ot-GP is also an obligatory intermediate in the biosynthesis of lipids (E. P. Kennedy and M. Lubin, personal communications), and hence must be present at a minimal concentration under all conditions of growth, a relatively high value for the K m of induction is necessary in this particular case. Otherwise internal induction would readily occur.

PRODUCT INDUCTION OF GLYCEROL KINASE

521

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