▪ Abstract Most prokaryotic signal-transduction systems and a few eukaryotic pathways use phosphotransfer schemes involving two conserved components, a histidine protein kinase and a response regul...

Two-component signal transduction

A new minimal medium for enterobacteria has been developed. It supports growth of Escherichia coli and Salmonella typhimurium at rates comparable to those of any of the traditional media that have high phosphate concentrations, but each of the macronutrients (phosphate, sulfate, and nitrogen) is present at a sufficiently low level to permit isotopic labeling. Buffering capacity is provided by an organic dipolar ion, morpholinopropane sulfonate, which has a desirable pK (7.2) and no apparent inhibitory effect on growth. The medium has been developed with the objectives of (i) providing reproducibility of chemical composition, (ii) meeting the experimentally determined nutritional needs of the cell, (iii) avoiding an unnecessary excess of the major ionic species, (iv) facilitating the adjustment of the levels of individual ionic species, both for isotopic labeling and for nutritional studies, (v) supplying a complete array of micronutrients, (vi) setting a particular ion as the crop-limiting factor when the carbon and energy source is in excess, and (vii) providing maximal convenience in the manufacture and storage of the medium.

Culture Medium for Enterobacteria

Analysis of gene control signals by DNA fusion and cloning in Escherichia coli

Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu.

Escherichia coli is the organism of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of recombinant proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.

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Recombinant protein expression in Escherichia coli: advances and challenges.

Nitrogen regulation in Saccharomyces cerevisiae.

Transcription from nitrogen-regulated promoters, such as glnAp2, requires the glnG gene product, NRI, as well as the rpoN(glnF) gene product, sigma60, and is regulated by the glnL gene product, NRII We find that in a reaction mixture containing NRI, NRII, and ATP, NRII catalyzes the transfer of the gamma phosphate of ATP to NRI This covalent modification of NRI occurs concurrently with the acquisition of the ability by the reaction mixture to activate transcription from glnAp2 In the presence of PII, the product of glnB, NRII catalyzes the removal of the phosphate from NRI-phosphate This reaction occurs concurrently with the loss by the reaction mixture of the ability to activate transcription from glnAp2 On the basis of this evidence, we propose that NRI-phosphate activates transcription from nitrogen-regulated promoters and that the role of NRII is control of the formation and breakdown of NRI-phosphate in response to cellular signals of nitrogen availability

https://www.pnas.org/content/pnas/83/16/5909.full.pdf

Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli

https://www.cell.com/cell/pdf/0092-8674(86)90553-2.pdf

Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter.

The glutamine synthetase (GS) from Klebsiella aerogenes is similar to that from Escherichia coli in several respects: (i) it is repressed by high levels of ammonia in the growth medium; (ii) its biosynthetic activity is greatly reduced by adenylylation; and (iii) adenylylation lowers the pH optimum and alters the response of the enzymes to various inhibitors in the gamma-glutamyl transferase (gammaGT) assay. There are, however, several important differences: (i) the isoactivity point for the adenylylated and non-adenylylated forms in the gammaGT assay occurs at pH 7.55 in K. aerogenes and at pH 7.15 in E. coli; (ii) the non-adenylylated form of the GS from K. aerogenes is stimulated by 60 mM MgCl2 in the gammaGT assay at pH 7.15. A biosynthetic reaction assay that correlates well with number of non-adenylylated enzyme subunits, as determined by the method of Mg2+ inhibition of the gammaGT assay, is described. Finally, we have found that it is necessary to use special methods to harvest growing cells to prevent changes in the adenylylation state of GS from occurring during harvesting.

Biochemical parameters of glutamine synthetase from Klebsiella aerogenes.

We have shown that the purified glnF (ntrA) product of Escherichia coli binds to core RNA polymerase. Together these proteins initiated transcription at the nitrogen-regulated promoter glnAp2 on a supercoiled template. The initiation of transcription at glnAp2 on a linear template required in addition NRI, the product of glnG (ntrC), and NRII2302, the product of a mutant allele of glnL (ntrB). These results identify the glnF product as a new sigma factor specifically required for the transcription of nitrogen-regulated and of nitrogen-fixation promoters. We propose rpoN as the proper designation for glnF, and sigma 60 for its product. Our results indicate that sigma 60 RNA polymerase recognizes the nitrogen-regulated/nitrogen-fixation promoter consensus sequence C-T-G-G-Y-A-Y-R-N4-T-T-G-C-A. Initiation of transcription in the intact cell appears to require in addition the active form of NRI, the product of glnG. Conversion of NRI to its active form is apparently brought about by NRII, the product of glnL, in response to nitrogen deprivation.

Boris Magasanik

Papers

Nitrogen regulation in Saccharomyces cerevisiae.

Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli

Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter.

Biochemical parameters of glutamine synthetase from Klebsiella aerogenes.

Transcription of glnA by purified Escherichia coli components: core RNA polymerase and the products of glnF, glnG, and glnL