Ulf Henning

Aperiplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins

A search was performed for a periplasmic molecular chaperone which may assist outer membrane proteins of Escherichia coli on their way from the cytoplasmic to the outer membrane. Proteins of the periplasmic space were fractionated on an affinity column with sepharose‐bound outer membrane porin OmpF. A 17kDa polypeptide was the predominant protein retained by this column. The corresponding gene was found in a gene bank; it encodes the periplasmic protein Skp. The protein was isolated and it could be demonstrated that it bound outer membrane proteins, following SDS‐PAGE, with high selectivity. Among these were OmpA, OmpC, OmpF and the maltoporin LamB. The chromosomal skp gene was inactivated by a deletion causing removal of most of the signal peptide plus 107 residues of the 141‐residue mature protein. The mutant was viable but possessed much‐reduced concentrations of outer membrane proteins. This defect was fully restored by a plasmid‐borne skp gene which may serve as a periplasmic chaperone.

The Major Proteins of the Escherichia coli Outer Cell Envelope Membrane

A procedure is described that from one batch of cells allows the isolation of all major proteins of the outer cell envelope membrane of Escherichia coli B/r. The method involves differential extraction of cell envelopes with ionic and non-ionic detergents with and without Mg2+ present, and the proteins are finally separated by molecular sieve chromatography in the presence of sodium dodecylsulfate. From 200 g cell paste in ten days (including the five days chromatography) approximately 120 mg protein I (molecular weight approximately 38,000), approximately 110 mg protein II* (molecular weight approximately 33,000), approximately 50 mg protein III (molecular weight approximately 17,000), and approximately 30 mg protein IV (molecular weight approximately 7,000) are obtained in pure state, and these yields are near the expected ones assuming quantitatve recoveries. Protein II* is a heat-modifiable protein (perhaps due to complete unfolding and/or binding of sodium dodecyl-sulfate only at higher temperatures), and the isolated protein is completely in its unmodified form. Protein IV, Brauns lipoprotein, in the cell envelope exists in two forms, one covalently bound to the murein layer and the other not. The isolated protein IV represents the free form of the protein that so far had not been isolated; its protein part dies not differ substantially from that of the bound form.

Cell surface exposure of the outer membrane protein OmpA of Escherichia coli K-12

The 325-residue OmpA protein is one of the major outer membrane proteins of Escherichia coli K-12. A model, in which this protein crosses the membrane eight times in an antiparallel beta-sheet conformation and in which regions around amino acids 25, 70, 110 and 154 are exposed at the cell surface, had been proposed. Linkers were inserted into the ompA gene with the result that OmpA proteins, carrying non-OmpA sequences between residues 153 and 154 or 160 and 162, were synthesized. Intact cells possessing these proteins were treated with proteases. Insertion of 15 residues between residues 153 and 154 made the protein sensitive to proteinase K and the sizes of the two cleavage products were those expected following proteolysis at the area of the insertion. Addition of at least 17 residues between residues 160 and 162 left the protein completely refractory to protease action. Thus, the former area is cell surface exposed while the latter area appears not to be. The insertions did not cause a decrease in the concentration of the hybrid proteins as compared to that of the OmpA protein, and in neither case was synthesis of the protein deleterious to cell growth. It is suggested that this method may serve to carry peptides of practical interest to the cell surface and that it can be used to probe surface-located regions of other membrane proteins.

The Major Proteins of the Escherichia coli Outer Cell Envelope Membrane. Characterization of Proteins II* and III, Comparison of All Proteins

Protein II*, one of the major Escherichia coli outer cell envelope membrane proteins has been characterized. The protein is heat-modifiable and perhaps due to complete unfolding and/or binding of sodium dodecylsulfate only at higher temperatures the modified protein exhibits a higher apparent molecular weight (33,000) than the non-modified form (28,000). Protein-chemical evidence as well as the behavior of two mutant proteins II* very strongly suggest that this protein consists of a single polypeptide chain and that in the strains studied there is no other major protein with similar characteristics. For another outer membrane protein, protein III (molecular weight 17,000), it has not yet been established if it should be classified as a major protein. Protein III consists of one or perhaps two polypeptide chains. The possibility existed that protein III is bound covalently to lipopolysaccharide, and this has been ruled out. Also, the lipopolysaccharide of the E. coli strains studied does not carry covalently bound protein in amounts anywhere near stoichiometry. N-on-protein substituents were neither found in protein II* nor in protein III. It is concluded that in E. coli B/r and the E. coli K12 strains used there are three major proteins: I, II, and IV; protein III may also belong to this class. There are not more major proteins than these. All four proteins are compared and discussed regarding their unknown functions and their relation to E. coli outer membrane proteins studied by other authors.

Mutants of Escherichia coli K12 lacking all ‘major’ proteins of the outer cell envelope membrane

The outer cell envelope membrane of/~: coli, and very likely of Gram-negative bacteria in general, contains a set of so-called major proteins in a mol. wt range between 33 000 and 38 000 (references see table 1). These proteins and their designations used by several authors are listed in table 1 ; horizontal lines compare proteins that are likely to be identical. Proteins I and II* can be produced in rather large quantities, and it has been calculated that about l 0 s copies of each protein are present per cell [4,6]. Also, several lines of evidence strongly indicate that these proteins are orderly arranged in the outer membrane [6,11 ]. On the basis of these and other observations [12] we had considered the possibility that the proteins in question belong to a self-assembly system which participates in the expression of cellular shape. Recently, a number of mutants have been isolated exhibiting various types of abnormal protein composi t ion of the outer membrane. Such mutants miss one or another major protein [8,10,13] or harbor decreased amounts of several of these proteins [7,14]. Also, it appears that the relative amounts of these proteins can differ substantially depending on growth conditions [ 10]. All these facts indicated that at least a rather precise, stoichiometric arrangement of the outer membrane proteins in question is not a requirement for an indispensible cellular function. We wished to clarify the issue especially regarding the determination of cellular shape, and here we describe the isolation and some properties of mutants that miss all of these major outer membrane proteins.

Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12

The 325-residue outer membrane protein OmpA of Escherichia coli has been proposed to consist of a membrane-embedded moiety (residues 1 to about 170) and a C-terminal periplasmic region. The former is thought to comprise eight transmembrane segments in the form of antiparallel β-strands, forming an amphiphilic β connected by exposed turns. Several questions concerning this model were addressed. Thus no experimental evidence had been presented for the turns at the inner leaflet of the membrane and it was not known whether or not the periplasmic part of the polypeptide plays a role in the process of membrane incorporation. Oligonucleotides encoding trypsin cleavage sites were inserted at the predicted turn sites of the ompA gene and it was shown that the encoded proteins indeed become accessible to trypsin at the modified sites. Together with previous results, these data also show that the turns on both sides of the membrane do not possess specifically topogenic information. In two cases one of the two expected tryptic fragments was lost and could be detected at low concentration in only one case. Therefore, bilateral proteolytic digestion of outer membranes can cause loss of β-strands and does not necessarily produce a reliable picture of protein topology. When ompA genes were constructed coding for proteins ending at residue 228 or 274, the membrane assembly of these proteins was shown to be partially defective with about 20% of the proteins not being assembled. No such defect was observed when, following the introduction of a premature stop codon, a truncated protein was produced ending with residue 171. It is concluded that (1) the proposed β-barrel structure is essentially correct and (2) the periplasmic part of OmpA does not play an active role in, but can, when present in mutant form, interfere with membrane assembly.

Receptor-recognizing proteins of T-even type bacteriophages: Constant and hypervariable regions and an unusual case of evolution

Proteins 38 of bacteriophages T2, K3, Ox2 and M1 are located at the free ends of their long tail fibers and function as adhesins, i.e. they mediate binding to the bacterial receptors. The latter three phages use the Escherichia coli outer membrane protein OmpA as a receptor, while T2 uses the outer membrane proteins OmpF or Ttr. The DNA sequences of genes 38 of phages Ox2 and M1 have been determined and are compared with those known for T2 and K3. The genes encode 262(T2), 260(K3), 266(Ox2) and 262(M1) amino acid residues. Three domains are distinguishable in these proteins. There are two conserved regions encompassing about 120 NH2-terminal and about 25 CO2H-terminal residues, respectively. The area between these was found to be hypervariable, and it is shown that a very large number of amino acid substitutions, deletions and/or insertions have occurred. Glycine-rich stretches are present within and flanking these areas. Their positions are essentially conserved, indicating an important structural role in receptor recognition. The hypervariability, most likely caused by a constant struggle with bacterial phage-resistant mutants, is so drastic that one cannot discern that T2 uses different receptors from those of the other phages. The partially known sequence of gene 38 of phage T4 has been completed. The gene encodes a protein consisting of 183 amino acid residues. The amino acid composition and sequence of this protein is completely different from those of phages T2, K3, Ox2 and M1. Also, the protein is functionally unrelated to the other proteins 38: it is not present in phage T4 and, unlike the other proteins 38, is required for the efficient dimerization of protein 37. All phages under study are of the same morphology and the genomic organization of the tail fiber genes is identical, with genes 36, 37 and 38 most likely representing, in this order, a transcriptional unit. Sequence similarities between the CO2H-termini of genes 37 of the non-T4 phages and gene 38 of phage T4 were found; this part of gene 37 does not exist in T4. It is suggested that gene 38 of phage T4 originated from a segment of gene 37 of a T2-type phage. Gene 38 of phage T4 is not unique, DNA-DNA hybridization experiments indicated that two other T-even type phages, TuIa and TuIb, possess a T4-type gene 38.

Superinfection exclusion by T-even-type coliphages

When a bacterial cell is infected with a T-even coliphage, immunity to a superinfecting phage is rapidly established (superinfection exclusion). Two phage-encoded proteins, Imm and Sp, are responsible for this exclusion: Imm blocks DNA transfer across the plasma membrane and partially inhibits release of DNA from the superinfecting virion, and Sp inhibits local degradation of bacterial murein by a phage-associated lysozyme.

Major proteins of the outer cell envelope membrane ofEscherichia coli K-12: Multiple species of protein I

SummaryProtein 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.

Radioimmunological screening method for specific membrane proteins.

Abstract A simple and sensitive radioimmunoassay is described which can detect insoluble membrane proteins in single colonies of Escherichia coli K-12. The method involves transfer of colonies onto filter paper, extraction with organic solvents, exposure to radioiodinated specific immunoglobulin, and autoradiography. Some 30,000 colonies can easily be screened within a week. The method should be applicable for shot-gun-type cloning experiments aiming at genes for insoluble membrane proteins and when selection for a corresponding wild-type allelc is not possible.