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J C Cortay

Publications and source records attributed to J C Cortay.

At least 37 records · Page 2Linked to original sources
PubMed1991

Regulation of the acetate operon in Escherichia coli: purification and functional characterization of the IclR repressor.

Growth of Escherichia coli on acetate requires operation of the anaplerotic sequence known as the glyoxylate bypass. In this pathway three different enzymes are activated: malate synthase, isocitrate lyase and isocitrate dehydrogenase kinase/phosphatase which are encoded by genes aceB, aceA and aceK, respectively. These three genes are clustered, in that order, in the same acetate (ace) operon whose expression is under the transcriptional control of the iclR gene located downstream from aceK. We have cloned the iclR gene in the pKK233-2 vector which allows optimization of both transcription and translation initiation. The IclR repressor has been overproduced, then purified to homogeneity in a one-step procedure by cation exchange chromatography after ammonium sulfate fractionation. Its specific interaction with the operator/promoter region of the ace operon has been analyzed by gel retardation and DNase I footprinting experiments. The IclR repressor has been shown to recognize a 35 bp palindromic sequence which largely overlaps the -35 recognition site of RNA polymerase. Moreover, the formation of the complex between IclR and the operator/promoter region has been found to be impaired by phosphoenol pyruvate but insensitive to acetate, acetyl-CoA, pyruvate, and oxaloacetate. These results are discussed in terms of primary regulation of the expression of the ace operon.

Acetates↗
PubMed1989

Relationship between guanosine tetraphosphate and accuracy of translation in Salmonella typhimurium.

In bacteria a high level of mistranslation is observed in amino acid starved rel-, but not rel+, strains, and mistranslation can be studied qualitatively by means of "stuttering" experiments in two-dimensional protein gels. It has been suggested that the low level of mistranslation that occurs in rel+ strains is assured by guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a nucleotide whose intracellular concentration greatly increases in rel+ cells under amino acid starvation. In the present study the relationship between level of ppGpp and mistranslation was analyzed by performing stuttering experiments in amino acid starved bacteria that contained either high or low levels of ppGpp. Three strains of Salmonella typhimurium were used in these experiments: a relA+ hisT+ strain (TA997), a relA+ hisT strain (TA1001), and a relA hisT strain (PD2). These strains were first characterized with respect to macromolecular syntheses and ppGpp levels under exponential growth and under amino acid starvation. Both rel+ strains exhibited stringent control over RNA synthesis. ppGpp accumulated to high levels when TA997 was starved for either of three amino acids. Starvation of TA1001 for histidine did not cause accumulation of ppGpp, whereas starvation for lysine and arginine produced high levels of ppGpp. Extracts from the three strains, obtained either under exponential growth or under amino acid starvation, were then subjected to two-dimensional electrophoretic anaylsis: mistranslation was observed whenever ppGpp was absent. In particular, starvation of TA1001 for histidine resulted in high mistranslation frequencies, while under lysine and arginine starvation mistranslation was undetectable, regardless of whether the cells were rel+ or rel-.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1988

Inaccurate protein synthesis in a mutant of Salmonella typhimurium defective in transfer RNA pseudouridylation.

Protein synthesis was studied comparatively in a wild-type strain of Salmonella typhimurium and in hisT mutant cells defective in the pseudouridylation of transfer RNA. From a quantitative point of view, no significant differences between the two types of strain was observed when measuring the rate of protein synthesis during either exponential growth or starvation for histidine. In contrast, the qualitative analysis of proteins by two-dimensional gel electrophoresis showed that histidine-starved hisT cells mistranslate the genetic program at a higher frequency than exponentially growing hisT cells or either starved or unstarved hisT+ cells.

Bacterial Proteins↗
PubMed1988

Promoter vectors with restriction-site banks.

New vectors harboring the promoter for the chloramphenicol acetyl transferase gene (cat promoter) have been constructed. These vectors are all derived from pJRD184 [Heusterspreute et al., Gene 39 (1985) 299-304], which contains a restriction-site bank. The cat promoter has been inserted at various positions and in reverse orientations so that almost all the restriction sites originally present on JRD184 can be used in cloning experiments. The expression of the aceK gene of Escherichia coli cloned under the control of the cat promoter has been tested. A large increase in the synthesis of the isocitrate dehydrogenase kinase, the aceK gene product, has demonstrated the efficiency of the newly constructed vectors.

Acetyltransferases↗
PubMed1988

Nucleotide sequence and expression of the aceK gene coding for isocitrate dehydrogenase kinase/phosphatase in Escherichia coli.

The flow of isocitrate through the glyoxylate bypass in Escherichia coli is regulated via the phosphorylation-dephosphorylation 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 minicell system and protein fusion with beta-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.

Amino Acid Sequence↗
PubMed1987

Effect of bacteriophage M13 infection on phosphorylation of dnaK protein and other Escherichia coli proteins.

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 two-dimensional 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.

Bacterial Proteins↗
PubMed1986

Characterization of the phosphoproteins of Escherichia coli cells by electrophoretic analysis.

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 cytosolic 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.

Alkaline Phosphatase↗
PubMed1986

Phosphorylation of an Escherichia coli protein at tyrosine.

The analysis of protein phosphorylation in the bacterium Escherichia coli showed that, while most phosphoproteins are modified at serine and/or threonine residues, one of them is modified exclusively at tyrosine. This particular protein which has a molecular weight of 54,500 and a pHi value of 5.6 is found associated with the membrane/ribosome fraction of the cell.

Amino Acid Sequence↗
PubMed1984

Two-dimensional analysis of proteins phosphorylated in E. coli cells.

Proteins phosphorylated in Escherichia coli cells were analyzed by the O'Farrell two-dimensional gel technique. Cytoplasmic and ribosomal fractions were studied separately. Double labeling with [32P]orthophosphate and [35S]sulfate followed by selective autoradiographic detection of each radioisotope allowed precise location of 12 major phosphoproteins on the total protein pattern of bacteria. Both the molecular mass and isoelectric point of these phosphoproteins were determined.

Autoradiography↗
PubMed1983

A study of bacterial response to polypeptide antibiotics.

When Escherichia coli cells are treated with either polymixin or gramicidin at concentrations that block protein and RNA synthesis, they accumulate a significant amount of guanosine tetraphosphate ppGpp. Such accumulation occurs in stringent (relA+) as well as in relaxed (relA) strains and no guanosine pentaphosphate pppGpp is then detected within the cells. These observations suggest that polypeptide antibiotics elicit ppGpp formation through a mechanism different from the stringent control system triggered by amino acid starvation of bacteria. Experiments based on tetracycline action indicate, moreover, that the accumulation of ppGpp under polymixin or gramicidin treatment is connected with a strong restriction of the degradation rate of this nucleotide.

Anti-Bacterial Agents↗
PubMed1983

Effects of aminoglycoside antibiotics on the coupling of protein and RNA syntheses in Escherichia coli.

The interdependency of protein and RNA syntheses was studied comparatively in bacteria confronted with amino acid starvation or treated separately with various aminoglycoside antibiotics. By contrast with the concomitant inhibition of macromolecular syntheses in cells deprived of an essential amino acid, RNA production was found to continue in drug-treated cells while protein synthesis was arrested. Such uncoupling process was also observed in bacteria subjected simultaneously to amino acid starvation and treatment with certain antibiotics (neomycin, gentamicin, spectinomycin and kasugamycin) but not with others (streptomycin and kanamycin). These results were related to the intracellular concentration of guanosine polyphosphates, ppGpp and pppGpp. They were discussed in terms of interaction of aminoglycosides with ribosomes.

Amino Acids↗
PubMed1983

Functional aspects of bacterial polysomes during limited protein synthesis.

The effects of amino acid starvation on the metabolic behavior of polysomes and the size distribution of proteins have been studied in an otherwise isogenic pair of stringent (relA+) and relaxed (relA) strains of Escherichia coli. The stability of polysomes has been analyzed by using two different approaches. First, the process of their degradation has been followed after treating the cells with rifampicin, an inhibitor of the synthesis of all classes of RNA including messenger RNA. Secondly, the process of their assembly has been studied after their previous conversion to monosomes, as induced by glucose deprivation of cells. It is shown that, in either type of bacterial strain, polysomes are continually broken down and re-synthesized during amino acid starvation. However, such polysome turnover is then less rapid than in normally growing bacteria and, moreover, it seems amino acid specific since it occurs at a lower rate during arginine starvation than during histidine starvation, namely, in the relaxed strain. The molecular weight distribution of proteins has been determined after labeling of cells with radioactive methionine and separation of polypeptides by one-dimensional polyacrylamide gel electrophoresis. The average size of polypeptides synthesized in the stringent strain during starvation is quite similar to that measured during normal growth. By contrast, a significant shift towards smaller molecules is observed in the relaxed strain deprived of an essential amino acid. Here again, this reduction of the size of polypeptides seems amino acid specific since it is especially marked during arginine starvation. These results are discussed in terms of ribosomes translocation and premature peptide chain termination in connection with the accuracy of the translational process.

Bacterial Proteins↗