AMP - iIrsensitive fructose bisphosphatase in Escherichia coli and its consequences ( aflosteric enzyme / metabolism )

Inhibition of fructose bisphosphatase (EC 3.1.3.11) by AMP is thought to be an important control mechanism, and, for the Escherichia coli enzyme, is the only control known. We here report on a mutant E. coli fructose bisphosphatase almost insensitive to this inhibition. The purified enzyme is normal in other respects. A strain with this enzyme instead of the wild-type enzyme grows normally in a variety ofmedia. A strain with a very high level of the wild-type enzyme also grows normally. A strain with the very high level of mutant enzyme, however, does show growth abnormalities, but they are not clearly associated with the AMP insensitivity. Fructose bisphosphatase (fructose-1,6-P2 1-phosphohydrolase, EC 3.1.3.11) is thought to be a key enzyme in regulation of gluconeogenesis (1-3). It also has an essential biosynthetic role in the growth of microorganisms on noncarbohydrate carbon sources (4, 5). The enzymes from various sources are almost all inhibited by 5'-AMP, and the eukaryotic enzyme is also inhibited by fructose-2,6-P2. Various other processes, including phosphorylation (6), proteolysis (7, 8), and repression, may also affect the activity (5, 9). In eukaryotic cells, evaluation of the role of AMP inhibition offructose bisphosphatase is particularly difficult in view of the several types of control. The problem should be easier to assess in Escherichia coli, in which the enzyme is also AMP sensitive (10) but not clearly subject to any of the other controls. Even in E. coli, however, the physiological role of AMP inhibition of this reaction is not obvious. Thus, one might expect AMP concentration to be lower in growth on glycerol, when the enzyme activity is required, than on glucose, but the contrary result was found (11), and the actual levels would be expected to inhibit the enzyme. Labeling experiments bearing on the question of whether the activity functions at all during growth on glucose have been inconclusive (11, 12). We here report on a mutant E. coli fructose bisphosphatase that is almost insensitive to inhibition by AMP. A strain containing this enzyme instead of the wild-type enzyme seems to grow normally, and so does a strain with a very high level of the wild-type enzyme. A strain with the high level of mutant enzyme, however, is somewhat impaired in its growth. MATERIALS AND METHODS Bacterial Strains and Plasmids. All strains are isogenic to DF1001 (Hfr C relAl spoT pit-10 tonA22 phage T2-resistant phage A-). DF656 contains a deletion of the 1bp region [dellfbp (ref. 13), now designated Afbp287 (B. Bachmann, personal communication)] linked to the TnlO insertion zjg920: :TnlO (misnamed in ref. 13 as zVe: :TnlO). DF672 (fbp-S zjg920::TnlO) has the Jbpallele from pJS51 as a single chromosomal copy, replacing fbp'. DF672 was constructed by a variation of the allele-exchange protocol described earlier (13). Strain SY634 (polA) was transformed with pJS51 with selection for resistance to ampicillin (Ampr). One Ampr clone was grown in the absence of ampicillin, and a segregant containing AMP-insensitive fructose bisphosphatase (i.e., presumptive replacement of Jbp+ of SY634 by Jbp-5) was identified by assay. After reversion ofpolA, the region was transduced into DF1001 by employing linkage with zVg920::TnlO; transductants were screened by enzyme assay. The resultant strain, DF672, had fructose bisphosphatase insensitive to AMP but present in an amount typical of the chromosomal enzyme. To test fidelity of the exchange, the Jbpallele of DF672 was reisolated on a plasmid and tested for the original phenotype, as follows. DF672 was transformed with pJS24 [Afbp287 (ref. 13)] and conjugated with an Fstreptomycin-resistant (Strr) strain carrying the same deletion chromosomally [DF659 (ref. 13)], selecting Ampr Strr exconjugants. The expectation was that in the merozygote a plasmid carrying the fbp allele from DF672 could be formed by recombination. Indeed, temperature-sensitive exconjugants were obtained, whose phenotype resembled the one conferred by the original Jbp-S plasmid, pJSS1, as confirmed by a further transformation. pJS33 is a derivative of pBR322 carrying fbp+ in a 4kilobase-pair (kbp) insert; the flp gene is known to be contained in a 1.7-kbp fragment [pJS35 (ref. 13)] ofthis insert. pJS51 was derived by in vivo nitrosoguanidine mutagenesis of pJS33 as described (13). pJS52 is a derivative of pJS51 from which a 0.6-kbp EcoRV fragment has been deleted (Fig. 1). pJS33 and pJS51 were converted from Ampr to kanamycin resistance (Kanr by introduction of the 1.1-kbp Pst I fragment from pUC71K (14) into the Pst I site in the pBR322 portion of the two plasmids, giving pJS54 (fbp+ KanT), and pJS53 (fbp-S Kanr), respectively. Genetic Techniques and Media. As before (13), genetic constructions employed LB medium, and growth experiments employed minimal medium 63. Minimal medium always also contained thiamin'HCl at 1 pg/ml and the specified carbon source, usually at 0.2%. Antibiotics were ampicillin at 200 gg/ml, tetracycline at 20 ug/ml, and kanamycin at 40 gg/ml. Standard genetic and cloning techniques were used (15, 16). All incubations were aerobic. Fructose Bisphosphatase. Assay (at 250C) and purification were as reported for wild-type enzyme (10); 1 unit (U) hydrolyzes 1 ;Lmol/min. The mutant enzyme was purified Abbreviations: Ampr, Strr, and Kanr, resistance to ampicillin, streptomycin, and kanamycin, respectively; Fbp, no growth on gluconeogenic compounds but normal growth on sugars; U, enzyme unit; kbp, kilobase pair(s). *Present address: Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139. tPermanent address: Departamento de Quimica, Facultad de Ciencias, Universidad de Chile, Santiago, Chile. 1656 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 83 (1986) 1657