The phosphorylated proteins of Escherichia coli, radioactively labeled with [32P]orthophosphate, have been analyzed by the O'Farrell gel technique and autoradiography. The effects of various culture conditions on the pattern of protein phosphorylation have been studied, including growth on different carbon sources in either exponential or stationary phase, treatment of cells with ethanol, heat shock and amino acid starvation. A total number of 128 different phosphoproteins, labeled to a varying extent, have been detected and each of them has been characterized by both its molecular mass and isoelectric point. These proteins are located mainly in the cytosoic fraction of cells, none of them being present within either ribosomes or nucleoids, and only three being associated with membranes. Analysis of their phosphoamino acid content has shown that they are phosphorylated mostly at serine residues and, less frequently, at threonine and tyrosine residues.
The flow of isocitrate through the glyoxylate bypass in Escherichia coli is regulated via the phosphorylationdephosphorylation of isocitrate dehydrogenase mediated by a bifunctional enzyme: isocitrate dehydrogenase kinase/phosphatase. The aceK gene coding for this enzyme is part of the polycistronic ace operon, which also includes the aceB and aceA genes coding, respectively, for malate synthase and isocitrate lyase, the two glyoxylate bypass enzymes. The complete nucleotide sequence of a 2,214-base-pair DNA fragment containing the aceK gene and its 5' flanking region has been determined. In vivo experiments based on gene expression in a miniceil system and protein fusion with j-galactosidase, as well as in vitro assays with a plasmid-directed transcription-translation coupled system, have shown that the aceK gene extends over 1,731 nucleotides encoding a 66,528-dalton protein. The 5' flanking region presents an unusual intercistronic structural pattern consisting of two consecutive long dyad symmetries, almost identical in sequence, which can yield very stable stem-loop units. These structures are probably responsible for the drastic downshifting in expression observed in acetate-grown bacteria between the aceK gene and the aceA gene located immediately upstream in the ace operon.Growth of Escherichia coli on acetate as the sole source of carbon and energy requires operation of the anaplerotic sequence known as the glyoxylate bypass (16). In this pathway two specific enzymes, isocitrate lyase and malate synthase, are activated by bacteria to divert isocitrate from the Krebs cycle and prevent the quantitative loss of the acetate carbons as carbon dioxide (3,35,36). The flow of isocitrate through the glyoxylate cycle is regulated via the phosphorylation-dephosphorylation of isocitrate dehydrogenase, the Krebs cycle enzyme which competes for a common substrate with isocitrate lyase (12, 13). When bacteria are grown on glucose, isocitrate dehydrogenase is fully active and unphosphorylated. Conversely, when cells are cultured on acetate, the activity of the enzyme declines drastically, concomitant with its phosphorylation at multiple sites (9, 28). Such a reversible phosphorylation reaction is mediated by a bifunctional enzyme, isocitrate dehydrogenase kinase/phosphatase, which contains both modifying and demodifying activities on the same polypeptide (19,27).The genes coding for isocitrate lyase (aceA) and malate synthase (aceB) are in the same ace operon located at 90 min on the E. coli K-12 linkage map (2, 5), with aceA downstream from aceB (14). The expression of the ace operon is under the transcriptional control of two genes: the iclR gene, which is adjacent to the ace operon, and the fadR gene, which maps at 25 min and is also involved in the regulation of the fatty acid degradation (fad) regulon (5, 32). Recently, it has been demonstrated that the kinase and phosphatase activities which regulate isocitrate dehydrogenase are encoded by a single gene (aceK), which has been cloned and shown to be essential for ...
1. The effects of infection with the filamentous phage M13 on the phosphorylation of Escherichia coli proteins were studied. Phosphorylated proteins were labeled with [32P]orthophosphate and analyzed by the O'Farrell twodimensional gel technique and autoradiography.2. Phage infection was shown to induce significant changes in the pattern of protein phosphorylation. At least eight different proteins were found to be phosphorylated to a larger extent while seven others were, by contrast, much less labeled than in uninfected bacteria.3. Labeling experiments with [35S]methionine demonstrated that these quantitative changes in protein phosphorylation were not connected, in any case, with changes in the amount of protein synthesized. They rather seemed to result from a variation of the phosphorylating capacity of the relevant protein kinase(s).4. The individual proteins, whose phosphorylation was affected by phage infection, were characterized by both their molecular mass and isoelectric point. One of them, whose phosphorylation was increased by a factor of 7, was identified as the dnaK protein which is necessary for both cellular and phage DNA replication.5. The chemical analysis of the phosphorylated moiety of dnaK protein showed that it was modified exclusively at serine residues during normal growth of cells, and mostly at threonine residues after phage infection. These results were discussed in terms of stimulation of the protein activity by phosphorylation.The phosphorylation of proteins is a well-known process of post-translational modification. It was originally discovered in eukaryotes and shown to play a key role in the control of several cellular activities (for reviews, see [l -31). Recently its occurrence in prokaryotes has also been demonstrated, first in Escherichia coli [4, 51 and Salmonella typhimurium [6], then in other bacterial species 17-lo]. In the search for the physiological significance of protein phosphorylation in bacteria, the effects of various environmental conditions have been examined including amino acid starvation, heat shock and growth in different media [ l l -131. In a few cases the role of this chemical modification in the regulation of metabolism has thus been elucidated. In particular, by varying the nature of the carbon source in the culture medium, it has been shown that the activity of the NADP-dependent isocitrate dehydrogenase in E. coli cells is controlled by its reversible phosphorylation in connection with the partition of carbon flux between the Krebs cycle and the glyoxylate bypass [14, 151. Also, in gram-positive strains the uptake of carbohydrates mediated by the phosphotransferase system is regulated through the reversible phosphorylation of the HPr protein [8, 161. Infection with bacteriophage T7 is another situation that promotes the specific phosphorylation of some E. coli proteins that are not naturally phosphorylated in uninfected cells [17].
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