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The phosphorylation of Escherichia coli isocitrate dehydrogenase in intact cells.

ABSTRACT: The isocitrate dehydrogenase of Escherichia coli ML308 can be reversibly activated by addition of pyruvate to cells growing on acetate [Bennett & Holms (1975) J. Gen. Microbiol. 87, 37-51]. By using cells pulse-labelled with [32P]Pi we showed that the activation and inactivation of the enzyme in these conditions correlate with its dephosphorylation and rephosphorylation respectively. Incubation of cell extracts prepared during an activation/inactivation cycle with purified isocitrate dehydrogenase phosphatase confirmed that the pyruvate-induced activation of the dehydrogenase goes essentially to completion. The results show that the reversible changes in the activity of the dehydrogenase in cells grown on acetate are solely due to phosphorylation/dephosphorylation. Inactive 32P-labelled isocitrate dehydrogenase was isolated from cells incubated with [32P]Pi in the presence of acetate. Both this material and purified enzyme phosphorylated in vitro were digested with chymotrypsin, and the phosphopeptides were isolated and analysed. Only one phosphopeptide was observed in each case; the results show that the residue phosphorylated in vivo is identical with that phosphorylated by purified isocitrate dehydrogenase kinase in vitro.

SUBMITTER: Borthwick AC

PROVIDER: S-EPMC1144244 | biostudies-other | 1984 Sep

REPOSITORIES: biostudies-other

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Project description:Sequences of the icd gene, encoding isocitrate dehydrogenase (IDH), were obtained for 33 strains representing the major phylogenetic lineages of Escherichia coli and Salmonella enterica. Evolutionary relationships of the strains based on variation in icd are generally similar to those previously obtained for several other housekeeping and for invasion genes, but the sequences of S. enterica subspecies V strains are unusual in being almost intermediate between those of the other S. enterica subspecies and E. coli. For S. enterica, the ratio of synonymous (silent) to nonsynonymous (replacement) nucleotide substitutions between pairs of strains was larger than comparable values for 12 other housekeeping and invasion genes, reflecting unusually strong purifying selection against amino acid replacement in the IDH enzyme. All amino acids involved in the catalytic activity and conformational changes of IDH are strictly conserved within and between species. In E. coli, the level of variation at the 3' end of the gene is elevated by the presence in some strains of a 165-bp replacement sequence supplied by the integration of either lambdoid phage 21 or defective prophage element e14. The 72 members of the E. coli Reference Collection (ECOR) and five additional E. coli strains were surveyed for the presence of phage 21 (as prophage) by PCR amplification of a phage 21-specific fragment in and adjacent to the host icd, and the sequence of the phage 21 segment extending from the 3' end of icd through the integrase gene (int) was determined in nine strains of E. coli. Phage 21 was found in 39% of E. coli strains, and its distribution among the ECOR strains is nonrandom. In two ECOR strains, the phage 21 int gene is interrupted by a 1,313-bp insertion element that has 99.3% nucleotide sequence identity with IS3411 of E. coli. The phylogenetic relationships of phage 21 strains derived from sequences of two different genomic regions were strongly incongruent, providing evidence of frequent recombination.

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The phosphorylation of Escherichia coli isocitrate dehydrogenase in intact cells.

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