The effect of small concentrations of antibiotics blocking the synthesis of bacterial cell wall on the permeability of Escherichia coli cell wall for plasmid DNA

Short treatment of Escherichia coli cells with antibiotics disturbing synthesis of bacterial cell wall in small concentrations renders the cells capable of absorbing foreign plasmid DNA. A novel express-method for transformation of E. coli cells by plasmid DNA has been developed on the basis of the results obtained. The whole procedure can be performed at room temperature. Depending on cell strain and the plasmid size, the efficiency of transformation can vary from 1.10(4) to 5.10(5) transformants per 1 mkg of DNA. The method suggested improves significantly the every-day work aimed at constructing plasmids.     

Heptose-deficient mutants ofEscherichia coli K12 deficient in up to three major outer membrane proteins

Molecular Genetics and Genomics - Mutants ofEscherichia coli K12, deficient in up to three major outer membrane proteinsb,c andd have been constructed. Mutants that lack the lipopolysaccharide...     

Isolation and characterization of outer membrane permeability mutants in Escherichia coli K-12.

Escherichia coli normally requires the lamB gene for the uptake of maltodextrins. We have identified and characterized three independent mutations that allow E. coli to grow on maltodextrin in the absence of a functional lamB gene by allowing maltodextrins with a molecular weight greater than 1,000 to cross the outer membrane barrier. Two of the mutations map to the structural gene for the outer membrane porin OmpF, and the remaining mutation maps to the structural gene for the second major outer membrane porin, OmpC. These mutations increase the permeability of the outer membrane to small hydrophilic substances, antibiotics, and detergents. These mutations alter the electrophoretic mobility of the respective porin proteins.     

Information within the mature LamB protein necessary for localization to the outer membrane of E coli K12

It has been proposed that the efficient localization of the outer membrane protein LamB requires a functional signal sequence and at least two additional regions contained within the mature protein. We define these regions more precisely by deletion analysis, and we describe methods for cloning deleterious lacZ fusions onto high-copy-number plasmids and generating in-frame deletions. Analysis of the effects of a series of internal lamB deletions on the export of a LamB-LacZ hybrid protein and of the LamB protein itself indicates that necessary informational signal(s) required for localization lie at the amino-terminal end of the protein. In addition, our analysis indicates that there is a region of information close to or within the fusion joint of the largest lamB-lacZ fusion that increases the efficiency of the export process. A unique deletion that removes a protein segment from amino acid 70 to 200 appears to prevent proteolytic removal of the signal sequence. Nevertheless, the mutant protein is exported to the outer membrane.     

The role of the gene umuC and plasmid muc genes in mutagenesis induced by dioxidine in E. coli K12

The mutability induced by dioxidine in E. coli cells has been shown to be stringently dependent on a function of chromosomal umuC+ gene. Suppression of an umuC mutation by plasmids pKM101 or ColIb, restoring the dioxidine induced mutability, proves the possibility of umuC gene functional complementation by the plasmid muc+ genes.     

The role of outer membrane proteins in peptide uptake by Escherichia coli

Abstract The influence of various outer membrane proteins on peptide penetration through the outer membrane in Escherichia coli was assessed by determining peptide transport kinetics in wild type and outer membrane protein-deficient strains. Peptide uptake was measured in whole cells by using a fluorescamine-based assay to monitor continuously the removal of peptides from the medium. Transport data were collected and processed using a microcomputer to give overall K _m and V _max values for peptide transport in each strain. In the mutants, K _m values were changed more markedly then V _max values reflecting an alteration in diffusion through the envelope. This approach shows that porins are involved in facilitating peptide penetration and that the OmpF channel appears to be more important than either OmpC or PhoE proteins. The loss of OmpA protein also decreases outer membrane permeability towards peptides, although whether this protein forms pores itself or exists more to maintain the functional integrity of other proteins is not known.     

Polyamines decrease Escherichia coli outer membrane permeability.

The permeability of the outer membranes of gram-negative bacteria to hydrophilic compounds is mostly due to the presence of porin channels. We tested the effects of four polyamines (putrescine, cadaverine, spermidine, and spermine) on two processes known to depend on intact porin function: fluxes of beta-lactam antibiotics in live cells and chemotaxis. In both cases, inhibition was observed. Measurements of the rate of permeation of cephaloridine and of chemotaxis in swarm plates and capillary assays were used to determine the concentration dependence of this modulation. The effective concentration ranges depended on the nature of the polyamine and varied from submillimolar for spermine to tens of millimolar for cadaverine. Both OmpC and OmpF porins were inhibited, although the effects on OmpC appeared to be milder. These results are in agreement with our observations that polyamines inhibit porin-mediated ion fluxes in electrophysiological experiments, and they suggest that a low-affinity polyamine binding site might exist in these porins. These results reveal the potential use of porins as targets for blocking agents and suggest that polyamines may act as endogenous modulators of outer membrane permeability.     

Localization of functional domains in E. coli K-12 outer membrane porins.

The genes ompC and phoE of Escherichia coli K-12 encode outer membrane pore proteins that are very homologous. To study the structure-function relationship of these proteins, we have constructed a series of ompC-phoE hybrid genes in which the DNA encoding part of one protein is replaced by the corresponding part of the other gene. These hybrid genes were easily obtained by using in vivo recombination. The fusion sites in the hybrid genes were localized by restriction enzyme mapping. The hybrid gene products were normally expressed and they were characterized with respect to functions and properties in which the native OmpC and PhoE proteins differ, such as pore characteristics, the receptor activity for phages and the binding of specific antibodies. Three regions within the N-terminal 130 amino acids were localized which determine pore characteristics and a segment between residues 75 and 110 contains amino acids which determine specificity for PhoE phages. A major cell surface-exposed region is located between residues 142 and 267. This region contains residues which are required for the binding of monoclonal antibodies directed against the cell surface-exposed part of PhoE and residues which determine specificity for OmpC phages.     

Effect of the molecular weight of DNA on the effectiveness of plasmid transformation of E. coli K12

The level of plasmid transformation and transfection by the high molecular mass DNA was studied for Escherichia coli mutants having increased efficiency of plasmid transformation by low molecular mass DNA. Decreased level of plasmid transformation and transfection registered in some mutants as compared to the one in wild type strain suggests the specificity of Escherichia coli cells penetration for DNA of different molecular mass.     

Major proteins of the outer cell envelope membrane of Escherichia coli K12: multiple species of protein I differ in primary structure.

Summary Protein I, one of the major outer membrane proteins of E. coli in most K12 strains is represented by two very similar polypeptides Ia and Ib. Sequential mutations (involving selections for phage resistance) can lead to loss of proteins Ia and Ib. Among revertants of such Ia^- Ib^- mutants clones exist that instead of Ia or Ib produce a third species of protein I, polypeptide Ic.Ichihara and Mizushima [J. Biochem. 83, 1095–1100 (1978)] have shown that proteins Ia and Ib exhibit differences in primary structure. Here evidence is presented indicating that protein Ic also is not identical in primary structure with Ia or Ib. Thus, 3 very similar structural genes appear to exist for the protein I species known to date, and that for Ic normally is silent. Introduction of a functional Ic locus into a Ia⁺ Ib⁺ strain caused expression of all three proteins with a reduced rate of synthesis of protein Ia.     

Architecture of the outer membrane of Escherichia coli K12. IV. Relationship between outer membrane particles and aqueous pores.

The hypothesis that intramembraneous particles, observed in the outer membrane of Escherichia coli by freeze-fracture electron microscopy, are the morphological representation of aqueous pores, was tested. A mutant which is deficient in five major outer membrane proteins, b, c, d, e and the phage lambda receptor protein, contains a largely decreased number of intramembraneous particles and also shows a greatly decreased rate of uptake of several solutes. In derivatives of this strain which contain only one of these proteins in large amounts a strong decrease of the number of intramembraneous particles is observed, which is accompanied by a complete restoration of the rate of uptake of those solutes which use pores in which the protein in question is involved. The results provide strong evidence for the notion that an individual pore contains only one protein species, a property which has been found earlier for individual particles. The observed correlation between particles and equeous pores strongly supports the hypothesis that the particles are the morphological representation of pores. Implications of this hypothesis for the structure of the particles are discussed.     

Infrared dichroism of twisted beta-sheet barrels. The structure of E. coli outer membrane proteins

The infrared dichroic ratios of the amide bands from oriented beta-barrels yield an experimental value for the mean orientation, beta, of the beta-strands, relative to the barrel axis. For a barrel of n strands, this then gives the shear number, S, that characterizes the stagger of the beta-sheet. Combining values of beta and n specifies the barrel geometry by using the optimized model of Murzin, Lesk & Chothia for regular barrels. Application to published infrared data on the Escherichia coli outer membrane protein, OmpA yields S=9-10 (n=8), a barrel radius of 0.81(+/-0.01) nm, and an internal free volume of 0.031 nm(3) per residue, where the average twist of the beta-sheets is theta approximately 28 degrees, and their coiling angle is epsilon approximately 1 degrees. Hydrophobic matching of the 2.6 nm transmembrane stretch partly determines the shear number of the OmpA beta-barrel.