Protonated triplex DNA in E. coli cells as detected by chemical probing

The triplex structure in vitro is well established; however, no direct evidence has been available concerning its existence in the cell. Using the direct chemical probing here we show that the triplex H structure can exist in E. coli cells at acidic intracellular pH values; this structure differs in some details from that observed in vitro.     

Small molecule inhibitors of E. coli primase, a novel bacterial target

Bacterial primase is essential for DNA replication in Gram-positive and Gram-negative bacteria. It is also structurally distinct from eukaryotic primases, and therefore an attractive, but under-explored, target for therapeutic intervention. We applied virtual screening to discover primase inhibitors, and subsequently several commercially available analogs of these initial hits showed potent primase inhibition and in vitro antibacterial activity. This work provides a 3D pharmacophore for primase ligands, SAR trends, and leads that can be further optimized.     

The transfer of a bacterial transmembrane function to eukaryotic cells.

This communication reports our preliminary studies on the reconstitution of the bacterial dicarboxylate transport system into rat myoblasts and mouse L-cells. Purified dicarboxylate membrane transport components (SBP 1 and SBP 2) from Escherichia coli K12 were added to rat myoblasts and mouse L-cells. These components were readily incorporated into the cell membranes. The rat myoblasts, as well as the mouse L-cells, were unable to transport succinate by themselves, or in the presence of either one of the transport components. However, when both components were added to the cells, the latter acquired the ability to transport succinate. There was a direct relationship between the amount of transport components added and the rate of succinate uptake. The newly acquired dicarboxylate transport system exhibited similar substrate affinity and specificity as the E. coli dicarboxylate transport system. The above findings suggest that it is possible to transfer a bacterial transmembrane function into eukaryotic cell membrane, and that these proteins can function normally in a foreign environment.     

The spread of plasmids as a function of bacterial adaptability

Abstract The horizontal spread of plasmids among natural bacterial populations serves as an evolutionary function for adaptation to the ups and downs in nature. Recent evolutionary challenges are the introduction of antibiotics and the creation of new environmental conditions in agriculture and medical care. As a consequence, surviving bacterial populations have acquired new genetic determinants which enable the colonisation and maintenance in distinct ecological niches. The acquisition of new genetic determinants can take place rather rapidly because of the plasmids' biology: their self-transferability and their ability to pick up genes. As an example of horizontal gene transfer, from an ecological and evolutionary viewpoint, the emergence of resistance to streptothricins ( sat genes) is described.     

Periplasmic production of native human proinsulin as a fusion to E. coli ecotin

Native proinsulin belongs to the class of the difficult-to-express proteins in Escherichia coli. Problems mainly arise due to its small size, a high proteolytic decay, and the necessity to form a native disulfide pattern. In the present study, human proinsulin was produced in the periplasm of E. coli as a fusion to ecotin, which is a small periplasmic protein of 16 kDa encoded by the host, containing one disulfide bond. The fusion protein was secreted to the periplasm and native proinsulin was determined by ELISA. Cultivation parameters were studied in parallel batch mode fermentations using E. coli BL21(DE3)Gold as a host. After improvement of fed-batch high density fermentation conditions, 153 mg fusion protein corresponding to 51.5mg native proinsulin was obtained per L. Proteins were extracted from the periplasm by osmotic shock treatment. The fusion protein was purified in one step by ecotin affinity chromatography on immobilized trypsinogen. After thrombin cleavage of the fusion protein, the products were separated by Ni-NTA chromatography. Proinsulin was quantified by ELISA and characterized by mass spectrometry. To evaluate the influence of periplasmic proteases, the amount of ecotin-proinsulin was determined in E. coli BL21(DE3)Gold and in a periplasmic protease deficient strain, E. coli SF120.     

The E. coli multitest: a set of strains to characterize diverse genotoxic effects

A set of E. coli strains was developed by Toman et al. (1985) to study the effects of chemical and physical agents on forward mutation, homologous recombination and induction of the SOS system. New tester strains have been constructed to improve this test system in order to explore quantitative genotoxicity spectra. Through the use of these strains: (i) SOS induction can be specifically detected without interference from mutagenesis; (ii) SOS-dependent and SOS-independent mutational events can be distinguished; (iii) the sensitivity of the recombination system has been considerably increased.     

N-formylmethionine as a N-terminal group of E. coli ribosomal protein

Summary Enzymatic digestion of³⁵S ribosomal protein with pronase yielded 0.14 molar % of the amino acids as N-formylmethionine. Analysis showed that approximately two polypeptide chains in the protein of the 30 S subparticle and approximately nine in the 50 S subparticle start with N-formylmethionine.     

Beta-galactosidase from E. coli as a marker in immunoenzyme analysis

In the present review the authors analyze factors influencing sensitivity of the enzyme immunoassay (EIA). beta-Galactosidase from E. coli was chosen as a marker enzyme. Physico-chemical properties of the enzyme, detection methods and various ways of obtaining chemical conjugates with antigens and antibodies are discussed. Some examples of using beta-galactosidase as a label in homogeneous and heterogeneous EIA are given which enables different compounds to be analyzed with a high sensitivity. New approaches employing gene engineering for constructing "fusions" between beta-galactosidase and antigens (alternative to chemical conjugates) are discussed that can be used in the near future.     

Transmembrane signaling by bacterial chemoreceptors: E. coli transducers with locked signal output

Methyl-accepting chemotaxis proteins (MCPs) function as transmembrane signalers in bacteria. We isolated and characterized mutants of the E. coli Tsr protein that produce output signals in the absence of overt stimuli and that are refractory to sensory adaptation. The properties of these "locked" transducers indicate that MCP molecules are capable of generating signals that actively augment clockwise and counter-clockwise rotation of the flagellar motors. Transitions between MCP signaling states can be influenced by amino acid replacements in many parts of the molecule, including the methylation sites, at least one of the two membrane-spanning segments, and a linker region connecting the receptor and signaling domains. These findings suggest that transmembrane signaling may involve direct propagation of conformational changes between the periplasmic and cytoplasmic portions of the MCP molecule.     

Using a targeted chemical nuclease to elucidate conformational changes in the E. coli 30S ribosomal subunit

Determining the detailed tertiary structure of 16S rRNA within 30S ribosomal subunits remains a challenging problem. The particular structure of the RNA which allows tRNA to effectively interact with the associated mRNA during protein synthesis remains particularly ambiguous. This study utilizes a chemical nuclease, 1, 10-o-phenanthroline-copper, to localize regions of 16S rRNA proximal to the decoding region under conditions in which tRNA does not readily associate with the 30S subunit (inactive conformation), and under conditions which optimize tRNA binding (active conformation). By covalently attaching 1,10-phenanthroline-copper to a DNA oligomer complementary to nucleotides in the decoding region (1396-1403), we have determined that nucleotides 923-929, 1391-1396, and 1190-1192 are within approximately 15 A of the nucleotide base-paired to nucleotide 1403 in inactive subunits, but in active subunits only cleavages (1404-1405) immediately proximal to the 5' end of the hybridized probe remain. These results provide evidence for dynamic movement in the 30S ribosomal subunit, reported for the first time using a targeted chemical nuclease.     

Test-strains of E. coli for detection of chemical mutagens]

Two temperature-sensitive mutants--AP 16 and AP 18 were isolated after the treatment of E. coli AB2500 strain with two mutagens (acridine orange and 5-bromuracil). The mutants obtained proved to be sensitive and formed revertants when treated with the following agents: N-methyl-N'-nitro-N-nitrosoguanidine, hydroxylamine, nitrous acid, sodium metabisulfite, methylmethansulfonate, and proflavine. Introduction into the mentioned strains of additional mutation causing elevation of their sensitivity to crystal violet increased somewhat their capacity to form revertants under the effect of proflavine and methylmethansulfonate.