Gene,86 (1990) 27-33 Elsevier

21

GENE 03378

Cyclic AMP synthesis in E~&er~&u coli strains bearingknowndeletionsin the pts phosphotransfkase operon (Phosphoenolpyruvate; adenylate cyclase; reverse genetics)

Sophie L&y*, Guo-Qing Zing and Antoine Danchin R&dation de I’Exptesssion G&u!tique,InstitutPasteur,28. rue du Docteur Roux, k5724 Paris Cedex 15 (Pine) Received by J.-P. Lecocq:9 May 1989 Revised: 2 October 1989 Accepted: 3 October 1989

SUMMARY

A series of isogenic strains harboring known deletions in the pkr operon of Esciterichiucolihave been constructed by reverse genetics. Strains bearing deletions for the whole pts operon failed to grow on maltose or on carbon sources of the same class. In these strains the total CAMP synthesis was significantly lower than in a strain deleted only for the cn gene. This indicated that enzyme I or phosphorylated histidine-containing phosphotransferase protein in addition to its role in phosphorylating enzyme III”rc, is involved in adenylate cyclase (AC) activation or CAMP excretion. It was further shown that deletions in the pts operon do not affect synthesis of AC.

INTRODUCTION

Uptake and phosphorylation of many carbohydrates are catalyzed in Escclericirlucofi by the phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS). In addition to its role in the transport of carbohydrates (named PTS carbohydrates), the PTS controls the activity of proteins involved in carbon metabolism. The uptake of PTS Corwpxdezce to: Dr. A. Danchin, Unit&R.E.G.,28, rue du Doctsur Roux, 75724 Paris Cedex 15 (France) Tel. (1)45688442; Fax (1)40560125. ILPresentaddress:Laboratoirede GCnCtique IBV,Centrede Recherche de Vitry, 13 quai Jules Guesde, 94400 Vitry s/Seine (France) Tel. 45 7378 97. Abbreviations:aa, amino acid(s);Ab, antibiotic;AC, adenylatecyclase; Ap, ampicillin;Bu,butyrosin;CAP,catabolite-activatingprotein;CAMP, cyclic AMP; err, gene encoding enzyme III”% d, deletion; Fm, fosfomycin; HPr, histidine-containingphosphotransferaseprotein; Km, kanamycin;: :, novel joint (insertion);ori, origin of DNA replication;@, gene fusion; PTS, phosphotransferasesystem; pts,genes encoding components of PTS; R, resistance/resistant;resp, respectively;Tc, tetracycline; wt. wild type. 03784119/90/$03.500 1990Elsevier Science Publishers B.V.(BiomedicalDivision)

carbohydrates (i) inhibits certain non-PTS carbohydrate transport systems, such as those for maltose, melibiose,and glycerol, thus preventing entry or building up of inducer intracellular concentration (inducer exclusion) and (ii) modulates AC activity, thus controlling the expression of CAPcyclic AMP-dependent operons. The growth of pts mutants suggested that phosphorylated enzyme III”‘c activated AC: (i) in err strains of E. coli (lacking enzyme III”‘c) the CAMP level is low (Feucht and &tier, 1980);(ii) in Salmonella typhSmurium mutants deleted for the p&H and ptsf genes, the CAMP level is also low (Feucht and Saier, 1980);(iii)in both types of mutants, the growth defect is restored either by exogenous addition of CAMP or by a crp* mutation (which directs synthesis of a CAP protein which is active in the absence of CAMP) (Scholte and Postma, 1980).These results have led Saier and Roseman (1976) and Nelson et al. (1982) to propose that phosphorylated enzyme IIIOrc could regulate the CAMP level by activating AC. Several physiologicalobservations as well as biochemical data on AC are, however, difficult 6n correlate with this ,ofsky (1981)to model. The study of ptsl mutant.

28 propose that AC activity was increased by the phosphorylated form of enzyme I. Further studies with toluenized cells (Liberman et al., 1985) and with bacterial extracts (Reddy et al., 1985) led to the conclusion that the combined action of HPr, enzyme I and enzyme III ° ~ activated AC; however, no precise role of any of these proteins could be assigned. In order to have better indications of the physiological role of HPr, enzyme I and enzyme IIl °~c in activating AC, a series ofisogenic strains harboring known deletions in the pts operon were constructed, using a technique that easily permitted the replacement of a dispensable gene on the chromosome using a copy disrupted by a cassette coding for an antibiotic resistance (Ab e) gene.

MATERIALS AND METHODS

(a) DNA manipulations Plasmid D N A preparation, D N A restriction, separation of fragmen~ on agarose gels by electrophoresis, ligation

and bacterial transformation were performed according to the methods described by Maniatis et al. (1982). The plasmids used are listed in Table I. Plasmids pDIA4516, pDIA4519, pDIA4526, pDIA4527 and pDIA4701 were the result of a cloning strategy involving several steps. Only the resulting plasmids are described in Fig. 1. Chromosomal D N A was extracted from E. coli strains according to Silhavy et al. (1984). Southern blot analysis and labelling of D N A probes by nick translation were performed as described in Maniatis et al. (1982). (b) Chemicals Restriction enzymes, 1"4 D N A ligase and Klenow fragment of E. coli D N A polymerase I were used as recommended by the suppliers (Boehringer Mannheim, F.R.G., Pharmacia, Sweden, or Amersham, U.K.). Agarose type II for gel electrophoresis was from Litex (Denmark), Ap, Kin, sulfate, Tc and Fin were from Sigma. Bu was a kind gift of Rhone Poulenc and other chemicals were from Merck (Darmstadt, F.R.G.). [0c-32p]dCTP was from Amersham, U.K. [ ~'51]cAMP was from the Pasteur Institute.

TABLE ! Bacterial Escherichla coil strains Strain

Relevant properties n

FB8

F ' , prototrophic tbr.l, leuB6, tonA21, leuYI, supE44, nag£467, tkl.I, nsdRSIS, manXYZ, =~/225 ::'In !0 F-, thl.I, argO6, arsE3, hb.4, mtl.I, xyl.5, tsx.29, rpsL, d/acX74, nag£::TngO3 F ' , x.vl, arfHl, ,41acXT4,aroB, IIvA TP211 I, .4(ptDH, ptsl, cw), Km n TP211 I, A(p~H, ptsl, c,~), Kmn TP211I, .4(ptsH, ptsl, o'r), Kma T.V2:! 1, Ll(pr.,;,err), KmR TP211 I, .4(m.), KmR TP2111, ia¢ ÷ TP2865, iac + TP2862, manXYZ, zdj225::Tni0 TP2864, n~g£::Tn.90.~ TP2874, manXYZ + F - , xyl, argH l , @(cya-lacZ ), lacX74 T,~2065, ,J(ptsll, ptsl, err), Km n TP2065, d(ptsl, err), Km R TP2065, .4(m.), Km ~

IBPCA63 IBPC$42 TP2111 TP2807

TP2811 TP2815 TP2819 TP2865 TP2860 TP2862 TP2864 TP2874 TP2875 TP2065 TP28,I8 TP2871 TP2870

References

lJ,..i ct ai, (1977) J, Piumbridlle, unpublished J. PlumbridlN, unpublished Roy et ai. (1983) This work

This work This work This work This work This work This work This work This work Roy et ai. (1988) This work This work This work

" Generalizedtransductions were performed with PI phage as in Miller (1972), Strain TP2860(rasp TP2862) was obtained after PI transduction oflac + locus from strain FB8 into TP2111 (rasp, TP2865) and selection on minimum medium plates complemented with lactose, according to Miller (1972). Transduction of`pts deletions from TP281I, TP2819 and TP2865 into TP2065 and selection for Km~ led to TP2848, TP2871 and TP2870 respectively, Strain TP2864 was obtained after transduction of a manXYZ operon m.tation linked to TnlO born strain IBPCA63into strain TP2862; transductants were seZectedfor Tca and screened for absence of`growthon minimalmcdium supplementedwith mannose. Transduction of the aog£::Tn903 mutation from strain IBPCM2 into strain TP2864 and selection for butyrosinresistance c,m£erredby Trig03led to strain TP2874. Traneduction of`the wt manXl'Z locus from strain IBPCM2 into strain TP2874 and selection on minimal medium plates supplemented with mannuse led to strain TP2875. The usual growth media were the rich medium LB or the synthetic medium M63 supplemented with 0,4% carbon source and 100PS aa/mi when required, as described by Miller (1972). MacCo~,keyagar plates supplementedwith ! % carbohydrate were used for screeningthe strains' fermentation ability. Final concentrations of`Ab was for I00 PS Aplml, 25 ~8 Km/ml, 20 ~g Tc/mi, 40 pg Fm/ml and 50 PS Bu/ml.

29 RESULTS AND DISCUSSION

(a) in vitro construction of pts deletions a n d transfer to t h e ebrfk~o~ome

In contrast with other microorganisms, in either eukaryotes such as Saccharomyces cerevisiae or prokaryotes such as Bacillus subtilis, where DNA modified in vitro can easily be replaced at the homologous locus on the chromosome, E. coli does not permit easy exchange of its known markers using exogenous DNA. Several methods have, therefore, been proposed to transfer DNA modified in vitro onto the £. coil chromosome. Most methods necessitate strains harboring several mutations or special plasmids or bacteriophages (Gay, 1984; Jasin and Schimmel, 1984; Joyce and Grindley, 1984; Matsuyama and Mizushima, 1985). Since PTS proteins interact with many factors involved in cellular metabolism, we devised a method convenient for exchanging a wt gene with a mutated copy into wt E. coli strains. A somewhat similar method has been proposed by Kiei et ai. (1987) based on the natural instability of a plasmid. More recently Blunt et 8]. (1989) described a means of replacement of target sequences using phage Ml3mp vectors and a MI3 lysogen intermediate. The procedure described here requires two steps. In the first step, the cloned gene is disrupted on a plasmid by an AbR marker. The plasmid bearing the Ab~ gene, flanked by chromosomal DNA, is then deleted for its or/and circularized in vitro, This plasmid is used to transform a strain, selecting for Ab a. Bacteria that are able to grow have integrated the plasmid into their chromosome by homologous recombination. At this step the 'intermediary' strain contains a duplication of the locus containing the gene to be deleted, one copy having the wt genotype, the second copy corresponding to the mutation generated in vitro and harboring the Aba cassette. The second step of the procedure implies an internal recombination event between these two partially homologous regions. Screening or selection of the clones having the mutated phenotype permits isolation of those which have replaced the wt region by the in vitro mutated one. In case of a mutation which leads to an unknown phenotype, a derived procedure can be devised. In this case the plasmid used at step I must contain an additional gene that can be counterselected, such as the Tc R gene of TnlO (Maloy and Nunn, 1981). Screening for bacteria deleted for this latter gene will yield strains where step 2 has occurred. A check of each step can be performed by Southern blotting of the corresponding bacterial chromosome. A major advantage of this procedure is that the intermediary strain provides estimates of the frequency at which non-deleterious deletions of the target gene should occur and thus allows interpretation of a negative outcome. In the

examples presented, the second recombination event occurred at a frequency of 10 - 4 . If no deletion is recovered at this step, or if a ve~ low ner~ber cf ~.!ortcs ~ , . ...... "~, harboring rearrangements of the region) is obtained, it can be concluded that cells carrying the deletion are unable to survive. In the present study several deletions, replaced by the KraR cassette, were generated on plasmids, as described in Fig. I. Circularized DNA fragments, lacking the replication origin of pBR322 were then transformed into the recipient strain (TP2111) and KmR clones were selected. It was expected that deletions ofthe pts region would lower cAMP synthesis (Feucht and Saier, 1980), and since a low level of cAMP results in a FmR phenotype (Cordaro et al., 1976), selection on LB plates supplemented with Fm was subsequently performed. The frequency of recombinants obtained was about 10-4. This has to be compared to the about 10- 7 frequency of spontaneous Fm R mutants under similar conditions. It (A)lnl

~+

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__L.L_], p01A4Sle "

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~m

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I I Fig. I. Restriction map of plasmids used for construction of strains bearing deletions in pu operon. Open boxes specify pBR322 (Bolivar et ai., 1977) DNA, hatched boxes specit;y pSA206 (Close et al., 1984) DNA, solid boxes specify chromosomal DNA derived from pDIA3206 (De Reuse et ai., 1984). The genes are represented by wide open boxes under the corresponding DNA segments. The notched end of a box and an apostrophe next to a gene symbol indicate that the gene is truncated. The direction of transcription is indicated by an arrow underneath each box. The symbol rep specifies the plasmid replication region. Restriction enzymes sites are as follows: A, Accl; B, BA,/II; C, Clal; E, £coRl; K, Kpnl; P, P ~ I ; X, Xhol; Xm, Xmnl. (The only Accl site specified is the one within the err gene used for plasmid construction; others are omitted). Restriction sites in brackets were lost during the construction.

30

P1 lkb B I

(a)

B I

E I

TP2111

ptsH ptsl crr E I .....

TP 2 8 0 7

! pt sH[' K[~m

B I

E I

n I

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TP2819

(b) Phenotype of strains bearing deletions in the pts operon

ptsHptsl' Km

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I

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TP 2111

I

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I

C

TP 2865

I

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(b)

A 9 C 0 E

size (kb)

F l;

size (kb)

¢

~.-8,4 • ~ - - 8,8 at .e-- 4,1 I t

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I

Two control experiments were carried out. First the deletion was transduced back into the parental strain, using the Ab R marker as the selective agent. Second, Southern blotting experiments on the mutated D N A were performed (Fig. 2). The pts regions of the constructed strains are shown in Fig. 2a. The deletions extend throughout: (i) the crr gene (strain TP2865); (ii) the ptsl and crr genes (TP2819) and ~di) the ptsH, ptsl and crr genes (TP2807, TP2811 and TP2815). The deletions start at different positions in the pts operon, but they all end at the same Xmnl restriction site overlapping the last codon of the crr gene.

q . . 4,1 4 . - 3,2 2,4

The main features of the strains thus constructed are summarized in Table II. Strains bearing either a A(ptsHptsl-cw) deletion or a A(p~l-cvr) deletion have the same growth pattern on various carbon sources. They do not ferment class-I carbon sources (maltose, glycerol and melibiose) unless exogenous cAMP is added to the growth medium. It seems worth noting that the phenotype of these strains differs from that of other E. coil (ptsH, crr) or (ptsl, crr) double mutants which have been shown to ferment class-I carbohydrates (Saier and Roseman, 1976 reviewed in Postma and Lengeler, 1985). As a control, the deletion present in strain TP2807 was introduced into the prototrophic strain FB8 by PI transduction. The resulting phenoTABLE 11 Phenotypes of strains bearing deletions in the pts operon Relevant genotype"

PTS

Class !

Class I1

+

+

+

-

+

+

4err

+

+

-

4 or + cAMP

+

+

+

Wt

Fig, 2. Physical evidence for chromosomal deletions of strains TP2807, TP281I, TP281$, TP2819 and TP2865. (a) Restriction map of chromosomai DNA from different strains (B, BamHl; C, Clal; E, EcoRl), Pl stands for the 3,2-kb Pall DNA frasment used to probe chromosomal DNA of strains TP2111, TP2807, TP2811, TP2815 and TP2819. P2 stands for the 4-kb BamHI.EcoRlDNA fragmentused to probe chromosomal DNA of strains TP211! and TP2865. (b) Hybridization analysis of chromosomal DNA digested by BamHl +EcoRl (lanes: A, TP2111; B, TP2807; C, TP2811, D, TP2815; E, TP2819) or by Clal (lanes: F, TP2111; G, TP2865). Pl probes two BamHI.EcoRl frasments for all chromosomal DNA; one (5.1 kb) upstream pts operon remains intact, another frasment contains part of the pts operon and changes size with strains. P2 probes two Clal fragments for all chromosomal DNA; one (8.4 kb) upstream pls operon remains intact, another frasment contains part of pts operon and changes size with strains. Note that BamHl + EcoRl digests of TP2111 and TP2807 chromosomal DNA are partial, standing for truncation ofthe 3' and ofa 8ene (also truncated box), e.g. cvr'; hatched boxes correspond to wt sesments of the pts genes, whereas open boxes stand for AbR cartridges.

Fermentation of the sugars on MacConkey platesb

4( ptsH.ptsl,,m,) 4(ptsl-crr)

4(ptsl-crr)

J + cAMP

a All deletions have been tested in TP211! background. A total deletion for the pts operon transduced from TP2807 in the FB8 background led to the same phenotype (see Table I). e + or - indicates fermentation or non-fermentation al~er 24 h on MacConkey plates containing different compounds. PTS compounds tested were fructose, glucose, mannose, mannitol and sorbitol. Class-I compounds tested were maltose, melibiose and glycerol. Ciass-ll compound tested was rhanmose.

31 type appears to be the same as that of strain TP2807 (Table II). The failure to ferment class-I compounds in strains bearing a A(ptsH-ptsl-crr) deletion is therefore transducible with the deletion. This shows that the phenotypc is not due to a particular genetic background nor to an additional unlinked mutation. Strain TP2865 harboring a err gene deletion fermented class-I carbon sources (as already described for other err mutations; Lengeler et eLI., 1981). As previously observed with other mutants, none of the constructed strains were found to ferment c~.~ss-ll carbon sources in the absence of exogenous cAMP (Postma and Lengeler, 1985).

(c) Cyclic AMP levels from strains bearing deletions in the pts operon The carbohydrate non-fermenting phenotype of strains containing deletions for the whole pts operon could be a consequence of a particularly low level of cAMP, making them unable to ferment class-I compounds. The cAMP level was therefore measured in all mutants as reported in Table llI. Strains bearing either a A(ptsH-ptsl-crr) deletion or a A(ptsl-crr) deletion yielded the same cAMP level, i.e., approximately 30 times less than the parental strain level (TP2111). Strain TP2865, bearing a ~ r r deletion, gave 14-fold less cAMP than TP2111 and twofold more than strains with deletions extending into the ptsl gene.

(d) Effect of pts operon deletions in strains mutated in the

nqE gene It has been reported that two enzymes II, specific for /Y-glucosides (II n'l) and N-acetyi-glucosamine (ll~q), contain a C-terminal domain which is very similar to enzyme 111°1° in aa sequence (Bramley and Kornberg, 1987; Schnetz et al., 1987; Rogers et el., 1988). More recently, Vogler et al. (1988) have shown that the enzyme lll°S°-Iike domain of these enzymes II can replace enzyme III°~c in transport and phosphorylation of glucose. This was interpreted in a model involving a possible role of enzyme II N'e (and enzyme II "Ss when expressed, which is not the case in wt E. coil) in controlling AC activity. This model postulated that, in the absence of enzyme III °to, enzyme I could still phosphorylate enzyme II Nag, which would then activate adenylate cyclase. This additional role of phosphorylated enzyme II N~s might thus explain the different cAMP levels of strains bearing deletions in either the err gene or in the ptsl and err genes. This was tested by constructing a strain bearing a deletion in err and a mutation inactivating nagE (coding for enzyme llNaS). As seen in Table III inactivation ofthe nagE gene does not affect the cAMP level of a strain bearing a deletion in the crr gene.

TABLE I!! cAMP levels of strains bearing deletions in the per operon Strains"

Relevant properties"

cAMP level b

TP21 i i TP2807 TP2811 TP2815 TP2819 TP2865 TP2860 TP2862 TP2875

A/acX74 A/acX74, A(ptsH-ptsl-crr) A/acX74, A(ptsH.ptsi.or) A/acX74, d(/mH-ptd.cn.) A/acX74, d(ptsl-c~) d/acX74, dcrr

2890 ! 16 ! 14 103 102 211 3030 289 3! 8

lac + lae + , Acrr

lac +, Acrr, nagE:: Trig03

a see Table !. b cAMP levels are expressed as pmol/mg bacteria dry weight. Bacteria were first grown at 37°C during 20 h in minimal medium containing gluconate (0.4%), casaminoacids (0.2%) and shikimate (0.008%). Total cAMP from bacterial extract was then measured following a standard RIA as described by Joseph et 8.1.(1982)~ These authors have shown that the total cAMP content of a culture directly reflects the intraeelIular concentration of cAMP.

(e) Effect of pts operon deletions on cya expression The low level of cAMP detected in strains bearing deletions in the pts operon could be due to a decreased synthesis of adenylate cyclase or to the synthesis of AC of lower specific activity. Alternatively pts deletions could interfere with cAMP excretion: this can be excluded ifone takes into account the data showing that cAMP leaks out of the cell at a very high rate (Joseph et 8]., 1982). To distinguish between these possibilities, expression of the cya gene (coding for AC) was monitored in strain TP2065 which bears a chromosomal gene fusion between cya and lacZ (coding for p-galactosidasc) (Roy et al., 1988). "l'hc~ different deletions in the pts operon were inserted by Pl transduction on the chromosome of this strain. The expression level of the fusion, in the presence of a err deletion, a A(ptsl-crr) deletion or a A(ptsH.ptsl-crr) deletion was measured. In all strains the p-galactosidase activity measured (Pardee et al., 1959) was the same as in strain TP2065 (5 units per ms dry weight of bacteria). Therefore, it can be concluded that the deletions constructed do not affect the expression level of the cya gene but rather the specific activity of AC.

(f) Conclusions Construction of a series of E. coil strains with known deletions in the pts operon has enabled their phenotype to be correlated with their cAMP levels and with the expression of their cya gene. This implied three major observations.

32 (1) A deletion for the whole pts operon introduced on the E. coli chromosome results in lack of growth on maltose or carbohydrates of the same class. The phenotype of such deleted strains is thus different from that of published E. coli (ptsH, err) or (ptsl, err) double mutants (Saler and Roseman, 1976 reviewed in Postma and Lengeler, 1985) which were found to ferment class-I compounds. Deletions in the S. typhimurium pts operon, that have been selected after mutagenesis (Cordaro and Roseman, 1972; Cordaro et al., 1976), have been shown to ferment maltose but they have not been characterized further. The difference of phenotype reported here is probably due to a lower level of cAMP in the strains we constructed. In fact, it has already been pointed out that the cAMP level varies from strain to strain (Feucht and Saier, 1980). This observation emphasizes the importance of comparing strains originating from the same genetic background before drawing conclusions from differences in phenotypes. (2) A deletion in the err gene resulted in a 14-fold decrease in the cAMP level and an additional ptsl deletion induced a further decrease (two- to threefold) of the overall cAMP level. Thus, either enzyme I or phosphorylated HPr, in addition to its role in phosphorylating enzyme III °~¢, is involved in AC activation. As a mutation inactivating the nagE 8ene coding for enzyme II Nas does not affect the cAMP level of a strain bearing a deletion in the err gene, phosphorylated enzyme I1N~sis not an intermediate in the modulation of cAMP concentration mediated by enzyme I or phosphorylated HPr. (3) Irene accepts the observation of Joseph et ai, (1982) that cAMP almost instantaneously leaks out of the cell, thus indicating that cAMP excretion could hardly be increased, the effects, on the cAMP level, of deletions in the pts operon appear to operate only by modulation of ACspecific activity, As a final comment we stress that the strains presented here constitute the only set of precisely delimited deletions in the pts region existing in an isogenic E. coli background. Furthermore, these strains contain convenient genetic markers close to cya and crp loci, allowing introduction of secondary mutations. This series of deletions thus constitute an excellent tool for investigating the interactions of the different components involved in the glucose effects.

ACKNOWLEDGEMENTS

We thank H. De Reuse very much for constructive discussions and support. We are indebted to Dr. Agn~,s Ullmann for her useful advice and to R, Predeleanu for her expert technical help in cAMP assays. We thank J. Plumbridge for the generous gift of strains and B. Cameron for critical reading of the manuscript. We

thank B. George for quick and efficient typing. During this work S. L. was a recipient of a RhOne-Poulenc fellowship and G.Q.Z. was a recipient of a French Government fellowship. Financial support came from the Centre National de la Recherche Scientifique (UA1129), Institut Pasteur (13845) and the EC contract N ° ST2J-0478C.

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Cyclic AMP synthesis in Escherichia coli strains bearing known deletions in the pts phosphotransferase operon.

A series of isogenic strains harboring known deletions in the pts operon of Escherichia coli have been constructed by reverse genetics. Strains bearin...
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