Molec. gen. Genet. 140, 81--90 (1975) © by Springer-Verlag 1975

Catabolite Repression in Escherichia coli K12 Mutants Defective in Glucose Transport Vladimir N. Gershanovitch, N a t a l y a V. Y o u r o v i t s k a y a , L n d m i l a V. Komissarova, T a t y a n a N. Bolshakova, R a i s a S. Erlagaeva, a n d Genrich I. B o u r d Laboratory of Genetical Regulation of Biochemical Processes in Bacteria, The Gamaleya Institute for Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow Received May 7, 1975

Summary. The phenomenon of glucose catabolite repression was studied in Escherichia coli mutants unable to transport this carbohydrate. The pts I,H mutant P34 was much less sensitive to permanent and transient repressive effect of glucose on fl-galactosidase synthesis than parental type. The 1103 mutant with lack of enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (ptsI) behaves as well as P34 mutant after addition of glucose to casamino acids mineral medium. But in minimal medium with succinate as the sole source of carbon cells of the 1103 mutant (in accordance with the data of Perlman and Pastan, 1969) show hightened sensibility to transient glucose repression. The effect of hypersensibil!ty disappears when the la¢I mutation rendering the fl-galactosidase synthesis to costitutivity is introduced in 1103 mutant. It is shown that the hightened sensibility of fl-galactosidase synthesis to glucose transient repression in 1103 m u t a n t is not an effect of the pts mutation and most probably is due to "inducer exclusion" of the lac operon. It is also shown that if one introduces the P34 mutation in strain devoided of one of the enzymes II for glucose (gptA) (and due to this resistant to glucose catabolite repression) then the level of resistance in double mutant does not increase in spite of considerable supression of 1~C glucose accumulation. It is discussed the role of separate components of Escherichia coli K12 glucose transport system in realization of the phenomenon of catabolite repression.

Introduction The transfer of glucose into cells of E. coli is carried out b y P T S 1 on the principle of group t r a n s l o c a t i o n (Roseman, 1969). I t m e a n s t h a t t r a n s p o r t of glucose is t i g h t l y coupled with its p h o s p h o r y l a t i o n i n m e m b r a n e s at the expence of p h o s p h o e n o l p y r n v a t e a n d is accompanied with f o r m a t i o n of glucose-6-phosphate. Therefore the E. cell K12 m u t a a t s lacking even one c o m p o n e n t of the glucose phosphotransferase system fail to utilize this carbohydrate. This fact was d e m o n s t r a t e d i n our l a b o r a t o r y (Bourd, Andreeva, Shabolenko, Gershanovitch, 1968; Bourd, Shabolenko, Andreeva, K l u t c h e v a , Gershanovitch, 1969) a n d i n some foreign ones (Morse, P e n b e r t h y , Morse, 1971 ; Kornberg, 1973) on pts m u t a n t s with the defect i n e n z y m e I or H p r - p r o t e i n or both i n e n z y m e I a n d Hpr. i PTS = phosphoenolpyruvate-dependent phosphotransferase system; cAMP = cyclic adenosine-3",5'-monophosphate, IPTG = isopropyl-fl-n-thiogalactoside. Genetic symbols are given in accordance with Taylor and Trotter (1972). gptA and gptB = genes coded for glucose specific enzymes II of the PTS (Epstein, Curtis, 1972). 6

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One can t h i n k t h a t pts m u t a n t s due to their i n a b i l i t y to t r a n s p o r t a n d utilize glucose will loss the sensibility to repressive effect of glucose on t r a n s c r i p t i o n of the catabolical genes. I n other words, pts m u t a t i o n m u s t lead to resistance to glucose catabolite repression. This was shown b y us on P34 m u t a n t (Gershanovitch, Y o u r o v i t s k a y a , S a p r y k i n a a n d K l u t c h e v a , 1970) a n d b y Tyler a n d Magasanik (1970) on GN2 m u t a n t . So we accepted with surprise the c o m m u n i c a t i o n of P e r l m a n a n d P a s t a n (1969) t h a t pts m u t a n t s possess h e i g h t e n d sensitivity of fl-galactosidase synthesis to glucose t r a n s i e n t repression. I n present article we m a d e a n a t t e m p t to reveal the cause of the " h y p e r s e n s i t i v i t y " of pts m u t a n t s described b y P e r l m a n a n d P a s t a n . Besides we succeeded i n elucidation of the role of separate components of the glucose t r a n s p o r t system i n realization of the catabolite repression. Materials and Hethods The experiments were carried out on Escherichia coli K12 strains listed in Table 1. Some strains were constructed using following bacteria: H/r H (thi strs); W1895D1 (H]r, met bgl+ gptA str s) (Pastan, Perlman, 1969; Curtis, Epstein, 1971); CHE 25 (H/r KL16, par C bgl+ thi strs); H/r 3300 (H/r H, lacI thi strs), W1655 (F', lac+/met strS). Strain JD31 (F-, pur C gptA str r) was isolated in mating CHE25 × JD3. Bacteria 11001 and 11031 are pro, T7 phage resistant derivatives of 1100 and 1103 (Anderson, 1970). Strain J623 (F-, pro trp pur C str r) is his + pur C, recombinant from cross CITE25 × J62. J623/(F-, trp par C str r) was obtained as pro + lac+ recombinant in mating H/rH × J623. Table 1. Escherichia coli K12 strains used in experiments Strain

Genotype

J62

~'-, pro his trp lac str r

P34

F - , ptsI, H pro his trp lac str r

References

Bourd, Andreeva, Shabolenko and Gershanovitch (1968)

J621

F-, pro his lac str r

trp + recombinant HfrH a x J62

P341

F-, pro his lac str r

trp + recombinant HfrH a X P34

J62/

F-, his trp str r

pro+ la~+ recombinant W1655 a x J62

F-, his gptA str r

resistant to glucose catabolite repression trp + recombinant W1895D1 a x J62/

JD3

JD3134

F-, ptsI,H his gptA strr

purC+ pts transductant P34 × JD31 a

P3462

F-, ptsI,H pro trp lac str r

purC + pts transductant P34 × J623 a

P3462/

F-, ptsI,H trp str r

purC + pts transductant P34 × J623/a

1100

F-, thi strs

Pastan, Perlman (1969)

1103

F-, ptsI thi strs

Pastan, Perlman (1969)

ll00i

F-, thi lacI str s

pro+ lacI recombinant Hfr 3300 a × 11001a

1103i

F-, ptsI lacl thi strs

pro + lacI recombinant Hfr 3300 × 11031a

a See Materials and Methods.

Catabolite Repression in Escherichia coli K12 Mutants

83

Cultures were grown for 3.5-4 hours with aeration at 37 ° in minimal medium M-9 (Clowes, Hayes, 1968). Succinate or lactate or casamino acids (Difco) were used as the source of carbon (0.4%), Cells were adapted to the appropriate substrate during night growth. Measurements of fl-galactosidase and L-tryptophanase in toluenized cells were performed as it was described by Pardee, Jacob and Monod (1959), Pardee, Prestidge (1961). Bacteria were induced with 10-a M IPTG or 1.5 × 10-a M L-tryptophane. Cells for studying the glucose accumulation were collected on filters, washed with salt medium M-9 at room temperature and resuspended in M-9 medium without additions (protein concentrations~ 0.05-0.1 mg/ml). Suspensions had an equal optical density at 2 ~ 650 nm. The cultures were placed in water bath at 37 ° where they were incubated with labelled sugar (1~C glucose, Amersham, specific activity 3 mC/mM). 2 ml of suspension were removed in 1 rain of incubation and cells were collected on membrane filters HAWP (Millipore), washed with 7-10 ml of M-9 medium at the temperature of incubation. The filters were dried and their radioactivity was measured in liquid scintillator counter SL-20 (Intertechnique). The growth rates of bacteria were studied using biophotometer B10-LOG I I (Jouan). Permanent repression was studied on cultures grown at presence of 0.4% glucose. Inducer was added after the 4.0 hours of growth. The time of induction of fl-galactosidase was 30 rain, L-tryptophanase--60 rain. The induction was interrupted using chloramphenicol (0.05 mg/ml) and the enzyme activities were measured, The transient repression of fl-galactosidase synthesis was measured on cultures at log growth phase, and glucose was added together with inducer and after 20 rain of induction chloramphenicol was added. Matings and Plkc mediated transductions were performed using standard methods (Clowes, Hayes, 1968; Epstein, Devies, 1970). Results

Glucose Repression at pts Mutants Our i n v e s t i g a t i o n we b e g a n w i t h verification of t h e results o b t a i n e d b y P e r l m a n a n d P a s t a n . Really, if to grow cells of 1103 m u t a n t in m i n i m a l m e d i u m w i t h succinate as t h e sole source of carbon, t h e effect of "hypersensitivity" to t r a n s i e n t glucose repression is c o m p l e t e l y r e p r o d u c e d . H o w e v e r , we n o t i c e d t h a t on succinate t h e 1103 m u t a n t h a d t h e t i m e of g e n e r a t i o n 3 t i m e s higher t h a n t h e p a r e n t a l wild t y p e strain. W e suggested t h a t t h e effect of "hypersensitivity" in some occasions is connected w i t h t h e r e d u c e d g r o w t h r a t e of t h e m u t a n t on succinate. I n d e e d , we failed to o b t a i n t~ypersensitivity to glucose t r a n s i e n t repression if cells were g r o w n in M-9 m i n i m a l m e d i u m w i t h casamino acids, where t h e m u t a n t a n d p a r e n t a l s t r a i n s h a v e a n equal g r o w t h rates (Fig. 1). I t is k n o w n t h a t in E. eoli u n d e r t h e conditions of p o o r i n d u c t i o n of fl-galactoside p e r m e a s e one observes a n effect of " i n d u c e r e x c l u s i o n " of lac operon u n d e r t h e influence of glucose (Magasanik, 1970; Jones-Mortimer, K o r n b e r g , 1974). I n o u t w a r d a p p e a r a n c e it m a n i f e s t s in a m p l i f i c a t i o n of c a t a b o l i t e repression of fl-galactocidase s y n t h e s i s w i t h glucose b u t a c t u a l l y t h e r e occurs repression of i n d u c e r influx (Cohn, H o r i b a t a , 1959; Magasanik, 1970). Since t h e inducer exclusion d i d n o t h a v e place in strains w i t h c o n s t i t u t i v e synthesis of fl-gMactoside p e r m e a s e (Cohn, H o r i b a t a , 1959), we isolated lacI d e r i v a t i v e s of 1100 a n d 1103 strains. W e underline t h a t t h e c o n s t i t u t i v e synthesis of fl-galactosidase in lacI m u t a n t s is also s u b j e c t e d to glucose c a t a b o l i t e repression (Magasanik, 1970). T h e d a t a r e p r e s e n t e d in T a b l e 2 a n d Fig. 2 show t h a t laeI v a r i a n t of 1103 m u t a n t does n o t e x i b i t e t h e " h y p e r s e n s i t i v i t y " to t r a n s i e n t a n d p e r m a n e n t repression in succinate m i n e r a l m e d i u m . I t is even s o m e w h a t m o r e r e s i s t a n t to

V.N. Gershanovitch et al.

84 I00

80

2O 0 ----~/,

~_ 1 s

[

f

]0-a 1{3-3 Otucose (M)

I

10-z

I

10-1

Fig. 1. Transient repression of fl-galactosidase synthesis in 1100 (--A--) and 1103 (~-'--) in casamino acids medium. The results are represented as % of control (cultures without glucose) Table 2. Permanent glucose repression of fl-galactosidase and n-tryptophanase syntheses in p~s mutants and their parental strains Strains

Source of carbon

Enzyme

Enzyme synthesis % a

J62/ P3462/ J62I P34I 1100 1103 1100i 1103i

lactate lactate casamino acids casamino acids succinate succinate succinate succinate

fi-galaetosidase fi-galactosidase L-tryptophanase L-tryptophanase fl-galactosidase fl-galactosidase fl-galactosidase /~-galactosidase

2.1 64 10 121 9 25 19 37

a The results are represented as % of control cultures without glucose.

repressive influence of glucose t h a n the parental strain. I n t r o d u c t i o n of lac operon constitutivity in strain 1103 did not change the growth rate in suceinate medium. These results are in good accordance with the observation of Tyler and Magasanik (1972) t h a t the constitutive/~-galactosidase synthesis in pts m u t a n t is resistant to transient repression. Our data indicate t h a t the " h y p e r s e n s i t i v i t y " of 1103 to transient repression on succinate is due most likely to an effect of inducer exclusion t h a n is the consequence of pts mutation, and w h a t is more the mutational damage of the pts loci leads to some or other degree of the resistance of enzyme inducible synthesis to glucose action. I t is well illustrated with the results obtained b y us on pts I , H m u t a n t P34. The a c t i v i t y of enzyme I in P34 is completely absent a n d there are detected only traces of Hpr-protein in it, so this m u t a n t is n o t " l e a k y " (Bourd, Shabolenko, Andreeva, K l u t c h e v a and Gershanovitch, 1969; Bourd, Andreeva, Shabolenko and Gershanovitch, 1968; W. Epstein, personal communication). This eircum-

Catabolite Repression in Escherichia coli K12 Mutants

85

80 60 40

20

j~

I i0-s

r

E

I0-4 10-3 Glucose (M)

I

[

i0-2

i0-I

Fig. 2. Transient repression of fl-galactosidase synthesis in ll00i ( + ) and 1103i (--,--) in succinate medium. The results are represented as % of control (cultures without glucose)

stance makes much of interest the stydying of glucose repression of enzyme syntheses at 1)34. Fig. 3 illustrates the data about the transient repression of fl-galactosidase synthesis at lac+ derivative of P34. I t is clear that this pts m u t a n t does not manifest "hypersensitivity" to transient repression when grown in lactate or in casamino acids mineral medium. I t is more resistant to glucose repression than the parental strain. I n Table 2 we represent the data concerning the permanent glucose repression of fl-galactosidase and L-tryptophanase synthesis in pts mutants. I t is evident t h a t fl-galactosidase synthesis is much less sensitive to permanent repression with glucose in m u t a n t than in parental strains and synthesis of L-tryptophanase in m u t a n t is not repressed at all under the same conditions. These data are in good agreement with our results we have published earlier (Gershanovitch, Yourovitskaya, Saprykina and Klutcheva, 1970). Thus P34 m u t a n t does not exibit "hypersensitivity" of enzyme synthesis to glucose transient repression, but on the contrary it possesses the lesser sensitivity to repressive action of the carbohydrate. This pts I , H m u t a n t behaves as the resistant strain under the conditions of glucose permanent repression.

Glucose Catabolite Repression at pts, gptA Mutants I t is known t h a t pts mutants fail to transport glucose. But the analysis of results represented in Table 2 and on Fig. 3 shows t h a t fl-galactosidase synthesis in studied pts mutants is still affected (though partially) with glucose repression. I t means that in spite of absence of phosphoenolpyruvate-dependent phosphorylation the transfer of glucose in P34 cells is sufficient for the partial repression of fl-galactosidase synthesis. As it is seen from Fig. 4, P34 and 1103 mutants accumulate glucose, although the level of accumulation is greately decreased in comparison with t h a t in wild type cells. 7

Molec. gen. Genet.

86

V.N. Gershanovitch et al. 100

'°

) 0~

b) {

10-5

[

10-4

I

I

10-3

10-2

1

~

:

I

10-1 10-5 Gtucose (M)

I 10-4

~,~,

I

I'

10-3

10-2

I 10-1

Fig. 3a and b. Transient repression of fligalaetosidase synthesis in J62l ( • ) and P34621 (--c---) in lactate (a) and casamino acids (b) media. The results are represented as % of contro] (cultures without glucose) 100 °h

B06040200 --7//

I

10-s

I

10-~ Glucose (M)

I

10-3

I

10-2

Fig. 4.14C glucose accumulation in P34 (--o--) 1103 ( ~ ) , JO3134 (--~--) and JI)3 (--~--). The results are represented as % of accumulation rates in 1100 (for 1103) or in J62 (for other strains)

Here one must recall t h a t E. coli probably possesses two glucose specific enzymes I I of the P T S (Curtis, Epstein, 1971; Epstein, Curtis, 1972; Kornberg, Reeves, 1972). One of it has high affinity to methyl-~-D-glucoside but is lesser specific to glucose. I n extracts of wild type cells 40 % of glucose phosphotransfcrase activity is due to this enzyme II. Gene gptA coded for this enzyme I I is located at 24 rain of E. coli chromosome. Curtis and Epstein (1971) suppose t h a t gptA mutation is identical to cat mutation described b y group of Magasanik (Tyler, Wishnow, Loomis and Magasanik, 1969). Mutation in gptA loci as well as cat mutation leads to resistance of inducible enzyme synthesis to repressive effect of glucose (Curtis, Epstein, 1971). Strains LA12 and W1895D1 possess such mutation (Curtis, Epstein, 1971; Magasanik, 1970).

Catabolite Repression in Escherichia coli K12 Mutants 8O

87

80

%

50

1.3

//// ////

//// 1///

"///, ////

///A

/ / I /

ihYA ;;;; J 621

P 346Zt

JD3

JD 313/~

Fig. 5. Sensitivity of fl-galactosidase synthesis to glucose (10-8 M) repression in J62/, P3462l, JD3 and JD3134

Another glucose specific enzyme I I of the PTS, which has high affinity to glucose is coded for the gene gptB (Curtis, Epstein, 1971 ; Epstein, Curtis, 1972). In wild type cell extracts 60 % of glucose phosphotransferase activity is due to this enzyme II. The precise localization of gptJB gene on E. coli chromosome is not yet established. It is supposed, that it is located near 30th min (Fraenkel, VinopM, 1973). Mutation in gptA gene does not affect glucose transport considerably (Epstein, Curtis, 1972), but as it was mentioned above it leads to highly sizable resistance to glucose catabolite repression. So, gptA mutation produces an effect which is contrast with the effect of pts mutation : if in the first case insignificant disturbance in glucose transport is accompanied b y appearance of resistance of enzyme s3mthesis to repressive effect of this carbohydrate, but in the case of pts mutation 80-90 % depression of the rate of transfer of glucose does not affect so considerably the ability of this carbohydrate to repress fl-galactosidase induction. In connection with this it was very interesting to investigate the behaviour of the double m u t a n t - - w i t h gptA and pts mutations. As it is shown on Fig. 4 gptA mutation does not reinforce the disturbance in glucose transport produced by pts mutation. But at the same time gptA mutation appreciably increases resistance of fl-galactosidase synthesis to glucose repression in strain with lesion in the PTS (Fig. 5). However the level of resistance of the double mutant JD3134 does not exeed the level of resistance in JD3 mutant which has only gptA mutation. Discussion

The molecular mechanism of glucose catabolite repression in E. coli has been ascertained not long ago (Ullmann, 1971 ; Pastan, Perlman, 1970). As it has been established glucose decreases the intracellnlar concentration of cAMP (Peter-

88

V.N. Gershanoviteh et al.

kofsky, Gazdar, 1971), and this leads to repression of mRNA synthesis on the stage of initiation (Varmus, Perlman, Pastan, 1970). As far as now it is not clear how does glucose affect intracellular cAMP concentration. But it is clear at present that to produce an effect glucose must permeate into the cell. This conclusion follows from the fact that pts mutants (defective in system of p h o s p h o ~ t t p r generation and because of this unable to transport glucose) become to some extent insensitive to influence of this carbohydrate. There are described mutations (gptA, tgl) when transport and glucose phosphorylation are slightly changed but the resistance to repressive effect on enzyme induction is manifested much stronger (Curtis, Epstein, 1971; Epstein, Curtis, 1972; Board, Erlagaeva, Bolshakova and Gershanovitch, 1974, 1975). Apparently it may be so that the main part in establishment of catabolite repression has that very pool of glncose which is formed after permeation via system coded for gptA (or tgl). I t is clear from this that the level of repression of fl-galactosidase synthesis remained in pts mutants is provided with the system determined by gptA (or tgl) gene(s). If pts and gptA mutations are combined together in one strain, the fl-galactosidase synthesis in such bacteria remains completely insensitive to glucose action. I t is clear that pts mutation blocking the system of phospho ~-~ttpr generation also leads to resistance to glucose repression of inducible enzyme synthesis : phospho ~ H p r is necessary for the phosphorylation of glucose transported via system coded for gptA. In all probability even those amounts of phospho ~ H p r , which synthesized in " l e a k y " pts mutants are quite enough for the formation of a small pool of glucose-6-phosphate necessary for represion. One must not exclude the possibility that at high concentrations of carbohydrate (10-9 M and 5 × 10-9 M) when the levels of accumulation of 14C glucose at pts mutants reach considerable quantities, enzyme I I can transport the carbohydrate in free form, without coupling with phosphoenolpyruvate-dependent phosphorylation. Such mode of enzyme I I function has already been proposed in some articles (Tanaka, Fraenkel and Lin, 1967; Gachelin, 1970; Bourd, Erlagaeva, Bolshakova and Gershanovitch, 1975). One can suppose also that at high concentrations of carbohydrate the system coded for gene gptA functions using some other donator of phosphate (e.g. ATP). I t remaines unclear why inducible synthesis of fl-galactosidase in P34 is only partially resistant to glucose, while L-tryptophanase induction is completely resistant. Probably it is connected with the different necessity of several inducible systems to cAMP concentrations in bacteria. Recently that was shown on ara and lac operons of E. coli K12 (Lis, Schleif, 1973). And finaly the main question which leads from our observations: in what manner do the products of glucose catabolism (in particular-glucose-6-phosphate) influence on the cAMP maintenance in bacterial cell ? We shall t r y to answer this question in our further studies.

Acknowledgement. We thank Dr. W. Epstein and Dr. F. Fox (Chicago. USA) for the strains CHE25, 1100 and 1103.

Catabolite Repression in Escherichia coli K12 l~utants

89

References Anderson, C. W. : Spontaneous deletion formation in several classes of E. coli mutants deficient in recombination ability. Mutation Res. 9, 155 (1970) Bourd, G. I., Andreeva, I. V., Shabolenko, V. P., Gershanoviteh, V. N. : The absence of the phosphotransferase system in the mutant of E. coli K12 with damaged system of the carbohydrate transport. Molec. Biol. 2, 89-94 (1968) [in Russian] Bourd, G. I., Erlagaeva, R. S., Bolshakova, T.N., Gershanovitch, V. N. : Disconnection of transport and phosphorylation of ~-mcthylglucoside in a mutant of E. coli K12 resistant to glucose catabolite repression. Dokl. Akad. Nauk SSSR 2D5, 1243-1246 (1974) [in Russian] Bourd, G. I., Erlagaeva, R. S., Bolshakova, T. N., Gershanovitch, V. N.- Glucose catabolite repression in E. coli K12 mutants defective in methyl-~-D-glucoside transport. Europ. J. Biochem. 53, 419427 (1975) Bourd, G.I., Shabolenko, V.P., Andreeva, I.V., Klutchcva, V.V., Gershanovitch, V. N.The characteristics of the transport mutants of ~. coli with different lesions in Roseman's phosphotransferasc system. Molec. Biol. 3, 256-266 (1969) [in Russian] Clowes, R. C., Hayes, W. : Experiments in microbial genetics. Oxford-Edinburgh: Blackwell Scientific publications (1968) Cohn, M., Horibata, M. : Phisiology of the repression by glucose of the induced synthesis of the fl-galactoside-enzyme system of E. coli. J. Bact. 78, 624-635 (1959) Curtis, S. J., Epstein, W. : Two constitutive P N H p r : glucose phosphotransferases in E. coli K12. Fed. Proc. 30, 1123 (1971) DaM, R., Wang, R. J., Morse, M. L.: Effect of pleiotropic carbohydrate mutations (ctr) on tryptophan catabolism. J. Bact. 107, 513-518 (1971) Epstein, W., Curtis, S. J. : Genetics of the phosphotransferase system. "Role of membranes in secretory processes," Ed. L. Bolis. Amsterdam: North-Holland Publishing Co. (1972) Epstein, W., Davies, M. : Potassium-dependent mutants of B. coli K12. J. Bact. 191, 836-843 (1970) Fraenkel, D. G., Vinopal, R. T. : Carbohydrate metabolism in bacteria. Ann. Rev. Microbiol. 27, 69-100 (1973) Gaehelin, G. : Studies on the :¢-methylglucoside permease of E. coli. A two-step mechanism for the accumulation of a-methylglucoside-6-phosphate. Europ. J. Biochem. 16, 342-357 (1970) Gershanovitch, V. N., Yourovitskaya, N. V., Saprykina, T. P., Klutcheva, V. V. : Catabolite repression of enzyme synthesis in Escherichia coli mutants defective in carbohydrate transport. Dokl. Akad. Nauk SSSR 190, 1232-1234 (1970) [in Russian] Jones-Mortimer, M. C., Kornberg, H. L. : Genetic control of inducer exclusion in Escherichia coli. FEBS Letters 48, 93-97 (1974) Kornberg, H. L. : Carbohydrate transport by microorganisms. Proc. roy. Soc. B 183, 105-123 (1973) Kornberg, I-I. L., Reeves, R . E . : Inducible phosphoenolpyrnvate-dependent hexose phosphotransferase activities in E. coli. Biochem. J. 128, 1339-1344 (1973) Lis, G. T., Schleif, 1~.: Different cyclic AMP requirements for induction of the arabinose and lactose operons of E. coli, J. molec. Biol. 79, 149-162 (1973) Magasanik, B. : Glucose effect, inducer exclusion and repression. "The lactose operon." Eds. J. R. Beckwith and D. Zipser. New York: Cold Spring Harbor Lab. (1970) Morse, H. G., Penberthy, W.K., Morse, M.L.: Biochemical characterization of the ctr mutants of E. coli. J. Bact. 198, 690-694 (1971) Pardee, A. B., Jacob, F., Monod, J.: The genetic control and cytoplasmic expression of "inducibility" in the synthesis of fl-galactosidase by E. coll. J. molec. Biol. l, 165-178 (1959) Pardee, A. B., Prestidgc, L. S. : The initial kinetics of enzyme induction. Biochim. biophys. Aeta (Amst.) 49, 77-88 (1961) Pastan, I., Perlman, R. L. : Repression of fl-galactosidase synthesis by glucose in phosphotransferase mutants of E. coll. Repression in the absence of glucose phosphorylation. J. biol. Chem. 244, 5836-5842 (1969) Pastan, I., Perlm~n, R. L. : Cyclic adenosine monophosphate in bacteria. Science 169, 339-344

(1970)

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Peterkofsky, A., Gazdar, C.: Glucose and the metabolism of adenosine 3':5'-cyclic rochephosphate in E. coli. Prec. nat. Acad. Sei. (Wash.) 68, 2794-2798 (1971) Roseman, S. : The transport of carbohydrates by bacterial phosphotransferase system. J. gen. Physiol. 54, 138S-180S (1969) Tanaka, S., Fraenkel, I). G., Lin, E. C. : The enzymatic lesion of strain MM-6, a pleiotropic carbohydrate-negative mutant of E. coli. Bioehem. biophys. Res. Commun. 27, 63-67 (1967) Taylor, A. L., Trotter, C. D. : Linkage map of E. cell strain K12. Bac~. Rev. 36, 504-524 (1972) Tyler, B., Magasanik, B. : Physiological basis of transient repression of catabolic enzymes in E. coll. J. Bact. 192, 411-422 (1970) Tyler, B., Wishnow, R., Loomis, N. F., Magasanik, B. : Catabolite repression gene of E. c~li. J. Baet. 196, 809--816 (1969) Ullmann, A. : Repression catabolique 1970. Biochimie 53, 3-8 (1971) Varmus, H. E., Perlman, R. L., Pastan, I.: Regulation of lac messenger ribonucleic acid synthesis by cyclic adenosine 3',5'-monophosphate and glucose. J. biol. Chem. 245, 22592267 (1970) Note Added in Proo/. According to personal communication of Dr. W. Epstein (Chicago, USA) gene symbol gptB is changed to rapt. Gene mpt has been mapped by Dr. W. Epstein between ]abD and eda. Besides this, professor H. L. Kornberg (Leicester, England) has reported that gene TtsX identified by him as involved in fructose uptake is identical to rapt gene (Kornberg, H. L., Jones-Mortimer, M. C. : PtsX: a gene involved in the uptake of glucose and fructose by Escherichia cell). FEBS Letters 51, 1 4 (1975). C o m m u n i c a t e d by H. B6hme Dr. Vladimir N. Gershanovitch Dr. Natalya V. Yourovitskaya Dr. Ludmila V. Komissarova Dr. Tatyana N. Bolshakova Dr. Raisa S. Erlagaeva Dr. Genrich I. Bourd The Gamaleya Institute for Epidemiology and Microbiology Academy of Medical Sciences ~oscow D-98 USSR