Genetic control of isocitrate lyase activity in Escherichia coli.

Isocitrate lyase (EC 4.1.3.1) plays a necessary role in the growth of Escherichia coli on acetate as sole carbon source (1). Pyruvate and phosphoenolpyruvate both inhibit the activity and repress the synthesis of this enzyme (5). Mutants in which the synthesis of isocitrate lyase is no longer subject to control by these C3-compounds, and which thus form isocitrate lyase constitutively, have been obtained as secondary mutants from parent organisms which lack the ability either to carboxylate phosphoenolpyruvate (9) or to form phosphoenolpyruvate from pyruvate (5). It is the purpose of this communication to report the location on the genome of E. coli K-12 of genes which specify the structure (icl) and constitutivity (iciR) of isocitrate lyase. After treatment of a variety of strains of E. coli with ethylmethane sulfonate (7) and selection with penicillin (4) for inability to grow on acetate, four independently isolated mutants were affected in their ability to form isocitrate lyase; the genotypes of these organisms are listed in Table 1. By periodic interruptions of conjugation, it was established that all the icl alleles lay about 2 min from the argHBCE gene cluster, indistinguishably close to the metA marker and about 1 min from pgi (3); all ici mutants were thus mapped at 78 mi on the linkage map of Taylor and Trotter (8). The genes affected were located more precisely, with reference to the metBF, argHBCE, metA, and pgi markers, by phage Plkc-mediated transduction. All icl mutants were cotransducible with well over 80% frequency with metA (Fig. 1), which is in good agreement with the findings of Vanderwinkel and de Vlieghere (10). The occurrence of icl+ recombinants from genetic crosses among different iclmutants (which differed also in their met or arg alleles) showed that these mutants were not identical. Thus, of 886 arg+ transductants obtained from the cross icl-i X icl-2, 10 were icl+; of 941 arg+ from icl-i X icl-3, 13 were icl+; and of 833 arg+ from icl-2 X icl-3, 2 were icl+. Thus, these results also support the order of these three icl mutations given in Fig. 1; icl-4 has not yet been located to this degree of precision. Similar procedures were used for the location of the marker which conferred constitutivity of isocitrate lyase synthesis on a variety of mutants. Such iciR mutants were obtained from ppsparents and were recognized by their ability to grow on lactate, but neither on pyruvate nor on acetate plus pyruvate as carbon sources; that isocitrate lyase was formed constitutively was confirmed by enzymatic assay of sonic extracts of such iciR mutants (6).