Polysome Turnover During Amino Acid Starvation in Escherichia coli

The experiments presented in this paper support earlier evidence that ribosomes are released from polysomes when they encounter a codon for which no charged transfer ribonucleic acid is available. However, it is further shown that these ribosomes then reinitiate and resume translation. The size and the level of polysomes during deprival of an amino acid is a function of the frequency with which that particular amino acid appears in cellular proteins. Polysomes from starved cells are more stable than those from growing cells, and, moreover, polysomes from starved relaxed strains are more stable than those from starved stringent strains.     

Expression of Outer Membrane Proteins in Escherichia coli Growing at Acid pH

It is generally accepted for Escherichia coli that (i) the level of OmpC increases with increased osmolarity when cells are growing in neutral and alkaline media, whereas the level of OmpF decreases at high osmolarity, and that (ii) the two-component system composed of OmpR (regulator) and EnvZ (sensor) regulates porin expression. In this study, we found that OmpC was expressed at low osmolarity in medium of pH below 6 and that the expression was repressed when medium osmolarity was increased. In contrast, the expression of ompF at acidic pH was essentially the same as that at alkaline pH. Neither OmpC nor OmpF was detectable in an ompR mutant at both acid and alkaline pH values. However, OmpC and OmpF were well expressed at acid pH in a mutant envZ strain, and their expression was regulated by medium osmolarity. Thus, it appears that E. coli has a different mechanism for porin expression at acid pH. A mutant deficient in ompR grew slower than its parent strain in low-osmolarity medium at acid pH (below 5.5). The same growth diminution was observed when ompC and ompF were deleted, suggesting that both OmpF and OmpC are required for optimal growth under hypoosmosis at acid pH.     

Amino Acid Antagonist Death in Escherichia coli

Six analogues of amino acids killed corresponding auxotrophs of Escherichia coli. With all but one analogue, protein synthesis was required for lethality.     

Evidence for the Existence of Two Arginyl-Transfer Ribonucleic Acid Synthetase Activities in Escherichia coli

Two arginyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.13, arginine: ribonucleic acid ligase adenosine monophosphate) activities were found in extracts of Escherichia coli strains AB1132 and NP2. The two arginyl-tRNA synthetase activities in extracts of strain AB1132 were found to be separable by diethylaminoethyl-cellulose column chromatography, Sephadex column fractionation, and by sucrose density gradient centrifugation. In addition, in the standard assay using extracts of strain AB1132 there were two pH optima for arginyl-tRNA synthetase activity. Furthermore, when arginyl-tRNA synthetase of strain NP2 was fractionated by hydroxylapatite column chromatography, two activities were observed which were similar to those of strain AB1132.     

Amino Acid Differences in a 30S Ribosomal Protein from Two Strains of Escherichia coli

The 30S ribosomal proteins of the K-12 and B strains of Escherichia coli differ in at least one protein component. This component, which is allelic in the two strains, has been isolated from both organisms. Amino acid analyses show that the protein from strain B contains between 20 and 28 more amino acids than does the analogue protein from strain K-12.     

Basic Amino Acid Transport in Escherichia coli: Properties of Canavanine-Resistant Mutants

A mutant of Escherichia coli strain CanR 22 has been isolated which is resistant to growth inhibition by canavanine, an analogue of arginine. The properties of this strain and of another canavanine-resistant mutant, JC182-5 (isolated by Celis et al. [5]), were studied. The mutation is pleiotropic in that it results in a reduction in the activity of two distinct permeases, the arginine-specific and lysine-arginine-ornithine transport systems. The lesion maps at min 56 of the E. coli linkage map, at or near the argP locus. Although strain CanR 22 excretes arginine, this excretion appears to result from reduced ability to concentrate arginine, rather than the loss of transport ability being the result of excretion. This conclusion is based on findings with a canavanine-resistant strain auxotrophic for arginine, which exhibits transport properties similar to those of the prototrophic strains. Additionally, growth in the presence of arginine or ornithine results in a repression of the activity of the two basic amino acid transport systems. Neither the arginine-specific nor the lysine-arginine-ornithine binding proteins of the mutant cells show significant alterations in terms of amount, physical properties, or kinetic parameters. These observations lead to the proposal of a model for the two basic amino acid transport systems in which two carrier proteins with different specificities interact with a common energy coupling mechanism. A lesion in the gene (or one of the genes) for this coupling mechanism can confer canavanine resistance.     

Ultrastructure of Escherichia coli Depleted of an Amino Acid

Particles accumulating during amino acid starvation of Escherichia coli C600 are described and illustrated.     

Radiation Inhibition of Amino Acid Uptake by Escherichia coli

The inhibition of macromolecular synthesis in Escherichia coli by ionizing radiation has been investigated. The survival of the ability to incorporate arginine, leucine, isoleucine, histidine, uracil, and glucose after various doses of gamma radiation, deuteron and alpha particle bombardment has been measured. All amino acids are incorporated by processes which show the same radiation sensitivity. The sensitivity of uracil corresponds to a volume which is roughly spherical, of radius about 160A, whereas the amino acids possess sensitive regions which are long and thin in character. The uptake of glucose is concerned with a smaller, roughly spherical unit. The possible identification of the radiation-sensitive targets with cellular constituents is discussed. The long thin character observed for amino acids suggests that the sensitive region affected by radiation is an unfolded form of a ribosome, or alternatively a long nucleic acid molecule. For uracil the sensitive region fits with a 70S ribosome, while for glucose a smaller particle would fit the data.     

Turnover of Phospholipids in an Unsaturated Fatty Acid Auxotroph of Escherichia coli

The membrane phospholipids of an unsaturated fatty acid auxotroph of Escherichia coli were found to undergo turnover. These phospholipids were excreted into the culture medium, and were replaced in the cell with newly synthesized phospholipids. Phospholipids of growing cells supplemented with elaidic acid underwent rapid turnover, while those of cells supplemented with oleate, or cis-vaccenate plus palmitoleate, underwent slow turnover. Starvation for required amino acids stimulated this turnover in the latter two cases. Protein was also lost from growing cells. However, after amino acid starvation this loss ceased while phospholipid turnover continued. Electron micrographs of growing cells indicated that large pieces of membrane-like material were separating from the cell surface.     

Turnover of phosphatidylglycerol in Escherichia coli

In growing cultures of Escherichia coli, the nonacylated glycerol of phosphatidylglycerol (PG) is labeled more rapidly than is the acylated glycerol. This is, in part, due to a rapid exchange reaction of the nonacylated glycerol. Only some of the PG molecules undergo this reaction while others are stable. Using a mutant unable to make glycerophosphate, we have shown that the nonacylated glycerol of PG can exchange with non-phosphorylated glycerol.     

Two Amino Acid Residues of Transposase Contributing to Differential Transposability of IS1 Elements in Escherichia coli

Escherichia coli W3110 contains four types of IS1 elements in the chromosome. Using an insertion element entrapping system, we collected 116 IS1 plasmid insertion mutants, which resulted from a minimum of 26 independent IS1 insertion events. All of them had insertions of IS1 of the IS1A (IS1E and IS1G) type. Inspection of the transposase sequences of the four IS1 types and the IS1 of the resistance plasmid R100 showed that two amino acid residues, His-193 and Leu-217 of transposase, might contribute to differential transposability of IS1 elements in W3110. The two amino acid residues of the transposase in IS1A (IS1E and IS1G) were altered separately by site-directed mutagenesis, and each mutant was found to mediate transposition at a frequency about 30-fold lower than that of IS1A (IS1E and IS1G). Thus, the assumption that His-193 and Leu-217 of transposase contribute to differential transposability of IS1 elements in W3110 was confirmed.     

Fibrous Aggregates of Short Peptides Containing Two Distinct Aromatic Amino Acid Residues

The aim of the study was the assessment of the ability of short peptides to form aggregates under physiological conditions. The dipeptides studied were derived from different aromatic amino acids (heteroaromatic peptides). Tripeptides were obtained from two distinct aromatic amino acids and cysteine or methionine residue in the C‐terminal, N‐terminal, or central position. The ability of the peptides to form fibrous aggregates under physiological conditions was evaluated using three independent methods: the Congo Red assay, the Thioflavin T assay, and microscopic examinations using normal and polarized light. Materials potentially useful for regenerative medicine were selected based on their cytotoxicity to the endothelial cell line EA.hy 926 and physicochemical properties of films formed by peptides. The required parameters of biocompatibility were fulfilled by H?PheCysTrp?OH, H?PheCysTyr?OH, H?PheTyrMet?OH, and H?TrpTyr?OH.     

Amino Acid Residues Involved in the Functional Integrity of Escherichia coli Methionine Aminopeptidase

Amino acid residues in the metal-binding and putative substrate-binding sites of Escherichia coli methionine aminopeptidase (MAP) were mutated, and their effects on the function of the enzyme were investigated. Substitution of any amino acid residue at the metal-binding site resulted in complete loss of the two cobalt ions bound to the protein and diminished the enzyme activity. However, only Cys70 and Trp221 at the putative substrate-binding site are involved in the catalytic activity of MAP. Changing either of them caused partial loss of enzyme activity, while mutations at both positions abolished MAP function. Both residues are found to be conserved in type I but not type II MAPs.     

Systematic Nomenclature for GGDEF and EAL Domain-Containing Cyclic Di-GMP Turnover Proteins of Escherichia coli

In recent years, Escherichia coli has served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely used E. coli K-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 “degenerate” enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenic E. coli strains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling in E. coli, we now propose a general and systematic dgc and pde nomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains of E. coli in future studies.     

Amino Acid Control over Deoxyribonucleic Acid Synthesis in Escherichia coli Infected With T-Even Bacteriophage

Starvation for a required amino acid of normal or RC(str)Escherichia coli infected with T-even phages arrests further synthesis of phage deoxyribonucleic acid (DNA). This amino acid control over phage DNA synthesis does not occur in RC(rel)E. coli mutants. Heat inactivation of a temperature-sensitive aminoacyl-transfer ribonucleic acid (RNA) synthetase similarly causes an arrest of phage DNA synthesis in infected cells of RC(str) phenotype but not in cells of RC(rel) phenotype. Inhibition of phage DNA synthesis in amino acid-starved RC(str) host cells can be reversed by addition of chloramphenicol to the culture. Thus, the general features of amino acid control over T-even phage DNA synthesis are entirely analogous to those known for amino acid control over net RNA synthesis of uninfected bacteria. This analogy shows that the bacterial rel locus controls a wider range of macromolecular syntheses than had been previously thought.     

Identification of Two New Proteins in Spermidine Nucleoids Isolated from Escherichia coli

The Escherichia coli nucleoid contains DNA in a condensed but functional form. Analysis of proteins released from isolated spermidine nucleoids after treatment with DNase I reveals significant amounts of two proteins not previously detected in wild-type E. coli. Partial amino-terminal sequencing has identified them as the products of rdgC and yejK. These proteins are strongly conserved in gram-negative bacteria, suggesting that they have important cellular roles.