Relation Between Protein Synthesis and Phospholipid Synthesis and Turnover in Escherichia coli

Turnover of phospholipids occurred continuously in a fatty acid auxotroph of Escherichia coli, but in the wild-type parent strain significant turnover occurred only under conditions where protein synthesis was inhibited but lipid synthesis continued. Both strains gave a stringent response of ribonucleic acid accumulation to amino acid starvation, but only in the wild type was lipid synthesis also inhibited. A revertant strain of the auxotroph resembled the wild type in this respect. The phospholipid that accumulates in the culture medium as a result of the lipid turnover appears to be part of a loosely bound low-density complex arising from the cell envelope.     

The Quiescent-Cell Expression System for Protein Synthesis in Escherichia coli

The quiescent-cell expression system is a radical alternative to conventional fermentation for protein overproduction in Escherichia coli. It is dependent on the controlled overexpression of a small RNA called Rcd in hns mutant strains to generate nongrowing, quiescent cells which are not nutrient limited. Quiescent cells no longer produce biomass and have their metabolic resources channelled toward the expression of plasmid-based genes. The biosynthetic capacity of the system is demonstrated by its ability to express chloramphenicol acetyltransferase to more than 40% of total cell protein. Quiescent cells may provide an ideal environment for the expression of toxic as well as benign proteins.     

Effect of Elevated Temperatures on Protein Synthesis in Escherichia coli

Upon a temperature shift from 30 C to 42 to 44 C, Escherichia coli experiences a transient slowdown in rate of optical density increase, uridine incorporation, and amino acid incorporation. The data show that the primary target for thermal inactivation is a molecule(s) required for the initiation of protein synthesis. The effect on ribonucleic acid synthesis is a secondary effect.     

Effect of Bacteriophage Ghost Infection on Protein Synthesis in Escherichia coli

The rate of protein synthesis by Escherichia coli markedly decreased within 1 min after phage T4 infection, whereas a complete cessation of protein synthesis was observed within at least 25 sec after T4 ghost infection. The cellular level of amino acids and aminoacyl-transfer ribonucleic acid (tRNA) did not change drastically upon infection with ghosts, indicating that the inhibition of protein synthesis took place at a step(s) beyond aminoacyl-tRNA formation. The host messenger RNA remained intact and still bound to ribosomes shortly after ghost infection. Kinetic studies of the effect of ghosts on host protein synthesis revealed that nascent peptide chains on ribosomes were not released upon ghost infection.     

Protein Synthesis and Deoxyribonucleic Acid-Membrane Attachment During Thymineless Death in Escherichia coli

The proteins synthesized during thymineless death in Escherichia coli B and B/r were analyzed by polyacrylamide gel elctrophoresis. It was found that the amount of a protein of molecular weight 80,000 to 88,000 is greatly increased during thymineless death compared to the amounts of other cell proteins. A technique for the isolation of cell membrane-deoxyribonucleic acid (DNA)-nascent ribonucleic acid (RNA) complex on detergent crystals was used to determine whether DNA might be detached from the cell membrane as a result of thymineless death. It was found that under no conditions of thymineless death or immunity to thymineless death was there any change in the attachment of DNA or pulse-labeled RNA to cell membrane.     

Protein export in Escherichia coli.

The export of protein from Escherichia coli has been studied by genetic, biochemical and biophysical techniques. These studies have defined a number of steps in the export pathway and have identified the cellular components required for the translocation process. New information is presented on the function of some of these components.     

Thiamine-Binding Protein of Escherichia coli

The ability to transport thiamine in Escherichia coli was reduced by osmotic shock treatment with a concomitant release of a thiamine-binding protein; its formation was repressed by thiamine added to the growth medium.     

Changes in Protein Synthesis as a Consequence of Heme Depletion in Escherichia coli

Two-dimensional gel electrophoresis and N-terminal amino acid sequence determination were used to compare the protein synthesis of exponentially growing Escherichia coli with heme-deficient cells. Mutation of the E. coli hemA gene encoding glutamyl-tRNA reductase resulted in the absence of detectable amounts of heme. As a consequence of heme deficiency, the induction of tryptophanase (trpA), citrate synthase (gltA), and aldehyde dehydrogenase (aldA) and the repression of enolase (eno) and phosphoglycerate kinase (pgk) were observed. All induced genes are under the control of the catabolite repressor protein Crp. The observed changes in gene expression as a consequence of heme depletion are discussed. Received: 19 March 1998 / Accepted: 28 April 1998     

Inhibition of Polyuridylic Acid-Directed Protein Synthesis by Aurintricarboxylate in Extracts of Escherichia coli

Aurintricarboxylic acid (ATA) inhibits protein synthesis directed by polyuridylic acid. All steps tested are inhibited by ATA. We conclude that inhibition of polyphenylalanine formation is an additive effect of inhibition of various steps in the protein synthetic machinery.     

Dissociation of tsl-tif-Induced Filamentation and recA Protein Synthesis in Escherichia coli K-12

In Escherichia coli, expression of the tif-1 mutation (in the recA gene) induces the "SOS response" at 40 degrees C, including massive synthesis of the recA(tif) protein, cell filamentation, appearance of new repair and mutagenic activities, and prophage induction. Expression of the tsl-1 mutation (in the lexA gene) induces massive synthesis of the recA protein and cell filamentation at 42 degrees C, although other SOS functions are not induced. In this paper we show that the septation inhibition induced in tif and tsl strains at 42 degrees C is not due to the presence of a high concentration of recA protein since (i) no recA mutants (相似文献   

Nucleic Acid and Protein Synthesis in Reconstituted Lyophilized Escherichia coli Exposed to Air

S ummary : Escherichia coli strain BT^-, freeze dried from distilled water, loses viability by exposure to air in the dry state, and decay curves of such bacteria were determined. The amounts of tritiated thymidine incorporated in the DNA of such 'dead', rehydrated bacteria are compatible with an assumption that the synthesis of DNA proceeds till the completion of its replication cycle, but not beyond. Induction of β-galactosidase served as an indicator of RNA and protein synthesis in the dead bacteria. Such induction occurred after a short lag after which it proceeded at a rate similar to that observed in lyophilized, untreated bacteria. The loss of viability of these bacteria cannot be accounted for by defects in the production of DNA or RNA and protein synthesis; it is most probably due to damage to the DNA initiation point.     

Analysis of Protein Synthesis Rates after Initiation of Chromosome Replication in Escherichia coli

The aim of this study was to investigate whether the synthesis rates of some proteins change after the initiation of replication in Escherichia coli. An intR1 strain, in which chromosome replication is under the control of an R1 replicon integrated into an inactivated oriC, was used to synchronize chromosome replication, and the rates of protein synthesis were analyzed by two-dimensional polyacrylamide gel electrophoresis of pulse-labeled proteins. Computerized image analysis was used to search for proteins whose expression levels changed at least threefold after initiation of a single round of chromosome replication, which revealed 7 out of about 1,000 detected proteins. The various synthesis rates of three of these proteins turned out to be caused by unbalanced growth and the synthesis of one protein was suppressed in the intR1 strain. The rates of synthesis of the remaining three could be correlated only to the synchronous initiation of replication. These three proteins were analyzed by peptide mass mapping and appeared to be the products of the dps, gapA, and pyrI genes. Thus, the expression of the vast majority of proteins is not influenced by the state of chromosome replication, and a possible role of the replication-associated expression changes of the three identified proteins in the cell cycle is not clear.     

Growth and Initiation of Protein Synthesis in Escherichia coli in the Presence of Trimethoprim

Escherichia coli grew exponentially at a reduced rate in the presence of 50 or 100 mug of trimethoprim/ml if the low-molecular-weight products of folate metabolism or their precursors (thymidine, purines, methionine, glycine, and pantothenate) were supplied in the medium. Folate metabolism was inhibited 99.9% by these concentrations of trimethoprim, but a low level of formylation of methionyl transfer ribonucleic acid (met-tRNA(F)) could be detected. However, in a medium containing all major amino acids, nucleosides, and vitamins, formylation of met-tRNA(F) was undetectable in the presence of trimethoprim. No other amino-masked amino acids were detected, and methionine remained a major amino-terminal amino acid of mature proteins. met-tRNA(F) was rapidly labeled with exogenous methionine and was associated with 30s ribosomal subunits and 70s ribosomes. It was concluded that initiation of protein synthesis can occur with unformylated met-tRNA(F) in E. coli. Changes in macromolecular composition were associated with the lack of formylation, in particular a fourfold increase in both met-tRNA(F) and ribosomal subunits. These changes would tend to compensate for the low specific rate of initiation with unformylated met-tRNA(F).     

Synthesis of papain in Escherichia coli

We have transferred the cloned papain genetic information into an expression vector (pT7-7) regulated by the T7-promoter and have obtained in vitro expression as well as expression in Escherichia coli. In Western blots the proteins produced are immunologically recognizable as papain. Multiple forms of specific but differing sizes are detected, suggesting either that initiation can occur at more than one of the upstream methionines, or that the enzyme is processed after synthesis.     

Control of Protein Synthesis in Escherichia coli: Analysis of an Energy Source Shift-Down

The energy source shift-down described in the preceding paper (Molin et al., J. Bacteriol. 131: 7-17, 1977) was used to study the effects of shift-down on protein synthesis. The overall rate of protein synthesis was reduced immediately, and to the same extent, in stringent and relaxed strains. The primary effect of the shift was a slowing down of the polypeptide chain growth rate, a finding not previously reported. In stringent strains the normal, preshift rate was reestablished within 2 to 3 min, whereas in relaxed strains the chain growth rate remained low for about 20 min before slowly returning to the normal value, which was reestablished some 50 to 60 min after the shift. Throughout this transition, the stability of messenger ribonucleic acid (mRNA) remained unchanged in both strains. We interpret these findings as evidence of the more rapid reduction of the mRNA pool in the stringent strain after shift-down: we believe that very soon after the shift, the stringent strain reduces its pool of mRNA and with it the number of ribosomes engaged in protein synthesis. In this manner the number of active ribosomes is adjusted to the availability of energy and carbon. The relaxed strain cannot rapidly reduce its mRNA pool, which thus remains large enough to engage a near-preshift number of ribosomes during a prolonged period; as a consequence its ribosomes must work at a reduced rate. The possibility that ppGpp is involved in the control of mRNA production is discussed. After shift-down, the initial part of beta-galactosidase (the auto-alpha fragment) was produced at a higher rate than complete beta-galactosidase in the relaxed strain, as expected when translation is impeded.     

Synthesis of Ribonucleic Acid and Protein in Plasmid-Containing Minicells of Escherichia coli K-12

Unlike the deoxyribonucleic acid (DNA)-deficient minicells produced by F(-) parents, minicells produced by plasmid-containing strains contain significant amounts of plasmid DNA. We examined the ability of plasmid-containing minicells to synthesize ribonucleic acid (RNA) and protein. In vivo, minicells produced by F(-) parents are unable to incorporate radioactive precursors into acid-insoluble RNA or protein, whereas minicells produced by F', R(+), or Col(+) parents are capable of such synthesis. Using a variety of approaches, including polyacrylamide gel analysis of the RNA species produced and electron microscope autoradiography, we demonstrated that the synthesis observed in minicell preparations is a property of the plasmid-containing minicells and not a result of the few cells (approximately 1 per 10(6) minicells) contaminating the preparations. That the observed synthesis is of biological importance is suggested by the ability of plasmid-containing minicells to yield viable phage upon infection with T4.     

Low-Temperature-Induced DnaA Protein Synthesis Does Not Change Initiation Mass in Escherichia coli K-12

Expression of the dnaA gene continues in the lag phase following a temperature downshift, indicating that DnaA is a cold shock protein. Steady-state DnaA protein concentration increases at low temperatures, being twofold higher at 14 degrees C than at 37 degrees C. DnaA protein was found to be stable at both low and high temperatures. Despite the higher DnaA concentration at low temperatures, the mass per origin, which is proportional to the initiation mass, was the same at all temperatures. Cell size and cellular DNA content decreased moderately below 30 degrees C due to a decrease in the time from termination to division relative to generation time at the lower temperatures. Analysis of dnaA gene expression and initiation of chromosome replication in temperature shifts suggests that a fraction of newly synthesized DnaA protein at low temperatures is irreversibly inactive for initiation and for autorepression or that all DnaA protein synthesized at low temperatures has an irreversible low-activity conformation.