Major proteins of the Escherichia coli outer cell envelope membrane as bacteriophage receptors.

Three Escherichia coli phages, TuIa, TuIb, and TuII, were isolated from local sewage. We present evidence that they use the major outer membrane proteins Ia, Ib, and II, respectively, as receptors. In all cases the proteins, under the experimental conditions used, required lipopolysaccharide to exhibit their receptor activity. For proteins Ia and II, an approximately two- to eightfold molar excess of lipopolysaccharide (based on one diglucosamine unit) was necessary to reach maximal receptor activity. Lipopolysaccharide did not appear to possess phage-binding sites. It seemed that the lipopolysaccharide requirement reflected a protein-lipopolysaccharide interaction in vivo, and lipopolysaccharide may thus cause the specific localization of these proteins. Inactivation of phage TuII by a protein II-lipopolysaccharide complex was reversible as long as the complex was in solution. Precipitation of the complex with Mg2+ led to irreversible phage inactivation with an inactivation constant (37 degrees C)K = 7 X 10-2 ml/min per microgram. With phages TuIa and TuIb and their respective protein-lipopolysaccharide complexes, only irreversible inactivation was found at 37 degrees C. The activity of the three proteins as phage receptors shows that part of them must be located at the cells surface. In addition, the association of proteins Ia and Ib with the murein layer of the cell envelope makes this pair trans-membrane proteins.     

Major proteins of the outer cell envelope membrane of Escherichia coli K-12: multiple species of protein I.

Summary Protein I, one of the major outer membrane proteins ofE. coli, in a number of strains exists as two electrophoretically separable species Ia and Ib. Two phages, TuIa and TuIb, have been found which use, as receptors, proteins Ia and Ib, respectively. Selection for resistance to phage TuIb yielded mutants still possessing protein Ia and missing protein Ib (Ia⁺ Ib^-). Selection in this background, for resistance to phage TuIa yielded one class of mutants missing both species of protein I and another synthesizing a new species of protein I, polypeptide Ic.Tryptic fingerprints of Ia and Ic are very similar and the sequence of 8 N-terminal amino acids is identical for Ia and Ic. Yet, Ic showed an entirely different pattern of cyanogen bromide fragments than that of protein Ia. With another example (cyanogen bromide fragments of protein II^*, with and without performic acid oxidation) it is shown that protein modification can lead to gross changes of the electrophoretic mobility of cyanogen bromide fragments. It is not unlikely that all protein I species observed so far represent in vivo modifications of one and the same polypeptide chain.A genetic analysis together with data from other laboratories revealed that at least 4 widely separated chromosomal loci are involved in the expression of the protein I species known to date.     

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.     

Action of a major outer cell envelope membrane protein in conjugation of Escherichia coli K-12.

Protein II, a major outer cell envelope membrane protein, was found together with lipopolysaccharide to stoichiometrically inhibit conjugation in Escherichia coli K12.     

Antigenic and immunogenic properties of cyanogen bromide peptides from a serotype 5 gonococcal outer membrane protein I

Treatment of a gonococcal major outer membrane protein IB (serotype 5) with cyanogen bromide (CNBr) resulted in cleavage of PIB into three major fragments of apparent molecular weight of 15, 13, and 8 kD. The location of these peptides in the intact protein was determined by analysis of partial cleavage products. The 8 kD peptide (CB2) was found to be located in the central region of the protein. Chymotrypsin cleavage of PIB revealed a cleavage site near one of the CNBr cleavage sites. Trypsin was found to cleave the protein, either in outer membranes (OMC) or in detergent micelles, in the central CB2 fragment. These result suggest that CB2 is a part of the surface exposed region of PIB.Immunization of mice with purified PIB (serotype 5) induced antibodies against all three CB-peptides. Absorption of the sera with homologous intact OMC resulted in a complete removal of antibodies against CB2, supplying further evidence for its surface exposed nature. Antibodies against the 13 kD peptide (CB1) could not be absorbed with intact OMC, suggesting that this peptide is buried within the outer membrane.Antisera raised against CB2 of serotype 5 demonstrated a considerable cross-reactivity with heterologous outer membranes. On the contrary, intact OMC induced mainly type-specific antibodies. These data demonstrate the presence of conserved epitopes on the surface exposed CB2 peptide. These conserved epitopes are generally not very immunogenic when present in intact OMC.     

Diploidy for a structural gene specifying a major protein of the outer cell envelope membrane from Escherichia coli K-12.

Homogenotes, heterogenotes, and intergeneric hybrids have been studied that are diploid for the structural gene of a major outer cell envelope membrane protein (protein II) from Escherichia coli. This protein can act as a phage receptor. In wild-type homogenotes, diploidy for the gene did not cause a gene dosage effect. It could be shown with two heterogenotes that both the chromosomal mutant and the episomal wild-type genes are expressed, and in each case more of the mutant than the wild-type protein species was found in the cell envelope. In on case of 21 phage-resistant mutants missing protein II was a trans effect observed of the mutant gene on the expression of the episomal wild type gene. Transfer of E. coli episomes carrying the protein II structural gene into Salmonella typhimurium and Proteus mirabilis resulted in intergeneric hybrids that became sensitive to the relevant phage and harbored the E. coli protein II in their cell envelopes. The results may be taken as suggestive evidence for a simple feedback mechanism for the regulation of synthesis of protein II, and they show that there are no highly specific requirements on protein primary structure for incorporation into an outer cell envelope membrane.     

Cell envelope and shape of Escherichia coli: multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins.

Starting with an Escherichia coli strain missing the outer membrane lipoprotein, multiple mutants were constructed than in addition to this defect miss the outer membrane proteins II, Ia and Ib, or Ia, Ib, and II. In contrast to all single mutants or strains missing the lipoprotein and polypeptides Ia and Ib, drastic influences on the integrity of the outer membrane and cell morphology were observed in mutants without lipoprotein and protein II. Such strains exhibited spherical morphology. They required increased concentrations of electrolytes for optimal growth, and Mg2+ or Ca2+ were the most efficient. These mutants were sensitive to hydrophobic antibiotics and detergents. Electron microscopy revealed abundant blebbing of the outer membrane, and it could clearly be seen that the murein layer was no longer associated with the outer membrane.     

Topographic labelling of pore-forming proteins from the outer membrane of Escherichia coli.

The topography of three pore-forming proteins from the outer membrane of Escherichia coli has been explored by using two labelling techniques. Firstly, the distribution of nucleophilic residues has been investigated by selective chemical modification using arylglyoxals (for arginine residues), isothiocyanates (for lysine residues), carbodi-imides (for carboxy residues) and diazonium salts. Secondly, the membrane-embedded domains have been investigated by labelling with photoactivatable phospholipid analogues and a reagent that partitions into the membrane. Few nucleophilic groups are found to be freely accessible to pore-impermeant probes reacting in the aqueous medium. More groups are accessible to small, pore-permeant probes, suggesting that several groups of each sort are contained within the pore. In addition, there appear to be a number of arginine, lysine, carboxyl and many tyrosine residues that are rather inaccessible and that react only with small, hydrophobic probes, if at all. Amongst these more deeply buried residues there are four arginine residues and an as-yet-undetermined number of carboxy residues that appear to be essential to the structural integrity of the oligomeric molecule.     

Eukaryotic precursor proteins are processed by Escherichia coli outer membrane protein OmpP.

A new specific endopeptidase that cleaves eukaryotic precursor proteins has been found in Escherichia coli K but not in E. coli B strains. After purification, protein sequencing and Western blotting, the endopeptidase was shown to be identical with E. coli outer membrane protein OmpP [Kaufmann, A., Stierhof, Y.-D. & Henning, U. (1994) J. Bacteriol. 176, 359-367]. Further characterization of enzymatic properties of the new peptidase was performed. Comparison of the cleavage specificities of the newly found endopeptidase and that of rat mitochondrial processing peptidase (MPP) showed that patterns of proteolytic cleavage on the investigated precursor proteins by both enzymes are similar. By using three mitochondrial precursor proteins, the specificity assigned to OmpP previously, a cleavage position between two basic amino-acid residues, was extended to a three amino-acid recognition sequence. Positions +1 to +3 of this extended recognition site consist of an amino-acid residue with a small aliphatic side chain such as alanine or serine, a large hydrophobic residue such as leucine or valine followed by an arginine residue. Additionally, structural motifs of the substrate seem to be required for OmpP cleavage.     

Studies on bacterial chemotaxis. I. Separation of proteins from cell envelope of Escherichia coli.

Radioactive proteins from Escherichia coli cell envelope fraction were separated by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis was carried out under several sets of conditions, and autoradiographs were obtained. Many of the proteins were separated at well-defined positions with good reproducibility. Some of the proteins moved relative to these stationary proteins depending at least two factors, i.e. the amount of proteins applied in the first dimension and the electric current applied in the second dimension. Among more than 200 spots, methyl-accepting chemotaxis protein and flagellin were identified by using labelled or cold preparations of these proteins as markers. Some of the spots were assigned to proteins from the outer membrane of the bacteria. The results provide a good foundation for comparative studies of membrane proteins from genetically altered strains of the bacteria.