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AMP Receptor Protein Proc. Natl. Acad. Sci. USA Vol. 77, No. 10, pp. 5799-5801, October 1980 Biochemistry Catabolite repression in Escherichia coli mutants lacking cyclic AMP receptor protein (glucose effect/transcription termination/rho factor) CHANTAL GUIDI-RONTANI*, ANTOINE DANCHINt, AND AGNES ULLMANN* *Dpartement de Biochimie et Genetique Moleculaire, Institut Pasteur, 28, rue du Docteur Roux, 75015 Paris, France; and tInstitut de Biologie Physico-Chimique, 13, rue Pierre et Marie Curie, 75005 Paris, France Communicated by Roger Y. Stanier, July 10, 1980 ABSTRACT Pleiotropic carbohydrate-positive pseudo- repression. This result indicates strongly that catabolite re- revertants have been isolated from a specific class of rho-crp pression can take place in strains lacking functional CAP pro- double mutants of Escherichia coli carrying both defective vided that they are also deficient in rho transcription termination protein, rho, and cyclic AMP receptor protein. protein. The modulation of catabolite repression of ft-galac- tosidase, amylomaltase, and tryptophanase has been studied MATERIALS AND METHODS in the pseudorevertants. It has been found that these mutants Strains and Growth Media. The exhibit catabolite repression. Because catabolite-sensitive op- following E. coli K-12 erons can be expressed in the absence of functional cyclic AMP strains were used: PP7811 (F-, argH, his); PP7812, isogenic receptor protein, this would suggest on the one hand that the with PP7811 except for the rho tsl5 allele (8); PP7810 (F-, ilvA, cyclic AMP-receptor protein complex is not the exclusive me- argH, his); and G61 (Hfr G6, aroB, his). All mutants derived diator of catabolite repression anKon the other hand that rho from these strains are described in the text. Strains were grown might be involved in the regulation of catabolite-sensitive op- in LB or 63 minimal medium (9) supplemented with the re- erons. quired amino acids, thiamine, and glucose or succinate as car- bon sources. It is generally believed that, in Escherichia coil, adenosine 3',- 5'-cyclic monophosphate (cAMP) and its receptor protein (CAP) Enzymatic Assays. f-Galactosidase (EC 3.2.1.23) was as- are specifically required for sayed according to Pardee et al. (10), amylomaltase (EC transcription initiation of cata- 2.4.1.25) according to Schwartz (11), and tryptophanase (EC bolite-sensitive operons. Mutants deficient for adenylate cyclase to Suelter et al. in (cya) and for CAP (crp) are unable to metabolize a great 4.1.99.1) according (12) toluenized bacterial number of carbohydrates, and the expression of catabolite- suspensions. Galactokinase (EC 2.7.1.6) was assayed as described sensitive operons is reduced. by Wetekam et al. (13) in sonicated bacterial extracts. One unit severely Several lines of evidence is the amount of enzyme that converts 1 nmol of substrate per suggested, however, that cAMP is not the unique regulator of min at 280C. catabolite repression (1, 2). Furthermore, expression of the lactose and cAMP Assay. Stationary-phase cultures were diluted to ob- arabinose operons independent of cAMP and CAP tain 300,gg (dry weight) of bacteria per ml. The bacterial sus- has been described (3, 4) and shown to be due to specific pensions were heated for 5 min at 100'C, and the total amount mutations in the regulatory regions of the operons. Another of cAMP was determined by a standard radioimmunoassay. category of mutants (alt mutants affecting the a subunit of Reagents. They were obtained from the following com- RNA polymerase) (5, 6) has been obtained as Ara+ revertants panies: ['4C]galactose from Amersham, cAMP reagent kit from from a cya strain synthesizing reduced levels of cAMP. These Institut Pasteur Production, isopropyl-f3-D-thiogalactoside, mutants partially overcome cAMP deficiency for ,B-galacto- D-fucose, and cAMP from Sigma, and all other chemicals from sidase synthesis at low temperature. However, pleiotropic de- Merck. repression of catabolite-sensitive operons in the absence of functional CAP has never been described. This kind of study would require, in the first place, obtaining pleiotropic carbo- RESULTS hydrate-positive revertants (Lac+, Mal+ Ara+, Tna+) from crp Isolation of rho-crp Mutants. Our former studies have mutants. Such mutants have not, as yet, been found. The ra- shown that rho-mediated natural polarity can be overcome by tionale to search for these kinds of mutants was provided by our the cAMP-CAP complex (7). The growth thermosensitivity of recent results (7) that show that the cAMP-CAP complex is strains carrying the rho tsl5 allele is usually ascribed to the involved in transcription termination, suggesting a functional absence of transcription termination (8). We reasoned that, if relationship between this complex and the transcription ter- cAMP-CAP is relieving spontaneous termination, mutants mination protein rho. Therefore, we argued that if we could defective in the production of this complex might be protected obtain appropriate mutants deficient in both rho and CAP against the deleterious effects of the rho ts15 mutation. We activity, these might be good candidates for selecting pseudo- therefore selected growing colonies on McConkey maltose revertants. exhibiting catabolite repression in the absence of plates at nonpermissive temperature (44°C) from strain functional cAMP-CAP complex. In the present paper we show PP7812. When 108 colonies were incubated on such a plate, that a specific class of double mutants, rho-crp (displaying about 50 growing clones were selected. Among these, 10 were carbohydrate-negative character), can give rise to pleiotropic phenotypically Mal-. We analyzed in more detail 192 such carbohydrate-positive pseudorevertants exhibiting catabolite Mal- mutants. All were pleiotropic carbohydrate negative and five among them were characterized as cya mutants. All other The publication costs of this article were defrayed in part by page clones behaved like CAP- mutants. Several were mapped and charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: cAMP, adenosine 3',5'-cyclic monophosphate; CAP, this fact. cAMP receptor protein. 5799 5800 Biochemistry: Guidi-Rontani et al. Proc. Natl. Acad. Sci. USA 77 (1980) were found to be mutated at or near the crp locus. One such in most of the strains. The main feature of these experiments mutant, RCC2, was kept for further study and was used as the appeared when glucose was used as a carbon source: three parent strain in the present work. strains were sensitive to catabolite repression exerted by glu- Isolation of Pleiotropic Carbohydrate-Positive Pseudo- cose-i.e., the differential rates of enzyme synthesis were re- revertants from rho-crp Mutants. An overnight culture of duced (to different extents depending upon the strains) com- strain RCC2 grown in LB medium at 41'C was centrifuged, pared to those obtained in succinate. In one strain, CGR16, resuspended in 63 minimal medium, and plated on maltose glucose did not seem to exert a catabolite repression effect be- minimal agar plates supplemented with eosin/methylene blue cause enzyme levels were not significantly different in glucose (109 bacteria per plate). After 48 hr of incubation at 41'C, compared to succinate. growing clones appeared at a frequency of about 10-7. Ten In strains carrying wild-type CAP, cAMP antagonized to independent clones were tested for growth on lactose or arab- some extent catabolite repression exerted by glucose. As can be inose as carbon source. All exhibited Lac+ Ara+ Mal+ pheno- seen in Table 1, cAMP stimulated ,B-galactosidase synthesis in type. Four of such pleiotropic carbohydrate-positive pseudo- the two parental strains (PP7811 and PP7812), whereas in strain revertants will be described herein. RCC2 and its derivatives it had no effect on enzyme synthesis, Functional Analysis of Mutants. As will be shown below, as might be expected for CAP-defective mutants. the pseudorevertants retained their crp mutation. According Genetic Analysis of Mutants. Our conclusions are strongly to the usual model, it therefore becomes difficult to visualize dependent on the genetic characterization of the mutants- how they can express catabolite-sensitive operons in the absence namely, their crp and rho ts15 characters. They were controlled of functional CAP. We therefore measured the level of ex- as follows. pression of (3-galactosidase, amylomaltase, and tryptophanase, (i) crp mutation: Absence of cAMP stimulation of f3-galac- known to be sensitive to catabolite repression, under two ex- tosidase synthesis is circumstantial evidence suggesting that treme conditions: growth in the presence of glucose (condition CAP is lacking in the mutants. We have also measured cAMP of severe catabolite repression) and in the presence of succinate levels in the mutants. Indeed, it has been noted (14) that crp (catabolite-sensitive operons are fully derepressed). mutants overproduce cAMP by a factor of 3-10; this was con- Table 1 compares the original strains, PP7811 (rho+), PP7812 firmed in all our mutants (Table 2). (rho tsl5), and RCC2 (rho tsl5 crp), with the spontaneous P1 transduction into an aroB strain for AroB+ character re- carbohydrate-positive derivatives of RCC2. As expected, none sulted in 24-40% pleiotropic carbohydrate-negative bacteria. of the three catabolite-sensitive enzymes was expressed in strain This strongly supports our assumption that the crp character RCC2. In the carbohydrate-positive derivatives, these enzymes was still present in the mutants. Finally, with a sensitive and were expressed at various levels in succinate medium, attaining specific radioimmunoassay (15), no CAP could be detected in wild-type levels for tryptophanase expression in all strains and the extracts of our mutants. levels for and amylomaltase (ii) rho tslS mutation: We have checked the presence of the remarkably high f3-galactosidase rho ts15 allele by two independent criteria: measurement of Table 1. Expression of fl-galactosidase, amylomaltase, and relief of polarity in the gal operon and mapping of the mutation tryptophanase in pleiotropic carbohydrate-positive pseudo- with respect to the ilv locus.
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