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U Henning

Publications and source records attributed to U Henning.

At least 55 records · Page 3Linked to original sources
PubMed1988

Internal deletions in the gene for an Escherichia coli outer membrane protein define an area possibly important for recognition of the outer membrane by this polypeptide.

A series of overlapping deletions has been constructed in the ompA gene which encodes the 325-residue Escherichia coli outer membrane protein OmpA. Immunoelectron microscopy showed that the OmpA fragments were either located in the periplasmic space or were associated with the outer membrane. Apparently an area between residues 154 and 180 is required for this association; all proteins missing this area were found to be periplasmic. The nature of this association remained unknown; no membrane-protected tryptic fragments could be identified for any of these polypeptides. Hybrid genes were constructed encoding parts of the periplasmic maltose binding protein and an area of the ompA gene coding for residues 154-274. The corresponding proteins were not localized to the outer membrane but remained attached to the outer face of the plasma membrane, possibly because the normal mechanism of release from this membrane was impaired. In the OmpA protein the conspicuous sequence Ala180-Pro-Ala-Pro-Ala-Pro-Ala-Pro187 exists. Frameshift mutants were constructed to eliminate this sequence. There was no effect on the incorporation of the mutant proteins into the outer membrane. Thus, this "hinge" region is not involved in sorting. A proposal suggesting the existence of a sorting signal common to several outer membrane proteins (Benson, S. A., Bremer, E., and Silhavy, T. J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3830-3834) was subsequently rejected (Bosch, D., Leunissen, J., Verbakel, J., de Jong, M., van Erp, H., and Tommassen, J. (1986) J. Mol. Biol. 189, 449-455; Freudl, R., Schwarz, H., Klose, M., Movva, N. R., and Henning, U. (1985) EMBO J. 4, 3593-3598). Although it is not known whether or not the outer membrane association observed represents a step in the normal sorting mechanism, it is concluded that it remains an open question whether or not a sorting signal, as proposed originally, exists in outer membrane proteins.

Amino Acid Sequence↗
PubMed1988

The influence of amino substitutions within the mature part of an Escherichia coli outer membrane protein (OmpA) on assembly of the polypeptide into its membrane.

The membrane part of the 325-residue outer membrane protein OmpA of Escherichia coli encompasses residues 1-177. This part is thought to cross the membrane eight times in antiparallel beta-strands, forming four loops of an amphipathic beta-barrel. With the aim of gaining some insight into the mechanism of sorting, i.e. the way the protein recognizes and assembles into its membrane, a set of point mutants in the ompA gene has been generated. Selection for toxicity of ompA expression following mutagenesis with sodium bisulfite yielded genes with multiple base pair substitutions, the majority of which resulted in amino acid substitutions in the membrane moiety of the protein. None of the altered proteins was blocked in membrane incorporation. A proline residue exists at or near each of the presumed turns at the inner side of the outer membrane. Using oligonucleotide-directed mutagenesis, each of them was replaced by a leucine residue which is thought to be a turn blocking residue. None of these proteins had lost the ability to be incorporated into the membrane. Apparently, leucine residues are tolerated at turns in this protein. To interfere with the formation of antiparallel beta-strands, four double mutants were prepared: ompA-ON3 (Ala11----Pro, Leu13----Pro), -ON4 (Ala11----Asp, Leu13----Pro), -ON5 (Gly160----Val, Leu162----Arg), and -ON6 (Leu164----Pro, Val166----Asp). The former three proteins and even quadruple mutants consisting of a combination of ompA-ON2 or -ON4 with -ON5 were not defective in membrane assembly. In contrast, the OmpA-ON6 protein was translocated across the plasma membrane but could not be incorporated into the outer membrane. It is concluded that at least one rather small area of the polypeptide is of crucial importance for the assembly of OmpA into the outer membrane.

Amino Acid Sequence↗
PubMed1988

Alterations to the signal peptide of an outer membrane protein (OmpA) of Escherichia coli K-12 can promote either the cotranslational or the posttranslational mode of processing.

The signal sequence of the precursor of the Escherichia coli outer membrane protein OmpA was altered by oligonucleotide insertions into the corresponding gene. In one case, OmpA-S1, the hydrophobic core of the signal peptide, is reduced from 12 to 10 residues, and one positive charge is added near the NH2-terminus. In another case, OmpA-P1, the hydrophobic core is extended from 12 to 16 residues. The pro-OmpA protein is normally processed partially co- and partially posttranslationally. Processing of the pro-OmpA-S1 protein was entirely posttranslational and that of the pro-OmpA-P1 protein strictly cotranslational. Evidence is presented which strongly suggests that posttranslational processing reflects posttranslational translocation across the plasma membrane. The generation times of cells expressing pro-OmpA-P1 or pro-OmpA-S1 were identical and the pro-OmpA-S1 polypeptide could be chased into the mature protein in the absence of protein synthesis. Hence, it does not matter which mode of processing, or rather translocation, is used. The same oligonucleotides were inserted into the ompA gene of plasmid pRD87; a plasmid which leads to overproduction of the protein and to massive accumulation of both the mature protein and the precursor. In the OmpA signal sequence encoded by pRD87-P2 the hydrophobic core is extended from 12 to 20 residues. This peptide was also rapidly removed. Therefore, regardless of whether the hydrophobic core contains 12, 16, or 20 lipophilic residues, not only does the signal sequence always function correctly to mediate export, but in each case, the cleavage site is always accessible to the signal peptidase.

Amino Acid Sequence↗
PubMed1987

A unique amino acid substitution in the outer membrane protein OmpA causes conjugation deficiency in Escherichia coli K-12.

The outer membrane protein OmpA of E. coli K-12 can serve as a receptor for phages and is required for stabilizing mating aggregates during F'-mediated conjugation. Selection for resistance to OmpA-specific phages yields mutants with alterations in the protein at four cell surface exposed sites. It is shown that conjugation deficiency can be caused by apparently only one type of amino acid substitution at one of these sites, the replacement of glycine-154 by aspartic acid. This suggests that, in contrast to binding of phages, a ligand of the donor cell recognizes only a very small area of the protein.

Amino Acid Sequence↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1987

The signal sequence of an Escherichia coli outer membrane protein can mediate translocation of a not normally secreted protein across the plasma membrane.

The distal part of the long tail fibers of the Escherichia coli phage T4 consists of a dimer of protein 37. A fragment of the corresponding gene, encoding 253 amino acids, was inserted into several different sites within the cloned gene for the 325-residue outer membrane protein OmpA. In plasmid pTU T4-5 the fragment was inserted once and in pTU T4-10 tandemly twice between the codons for residues 153 and 154 of the OmpA protein. In pTU T4-22 two fragments were present, in tandem, between the codons for residues 45 and 46 of this protein. In pIN T4-6 one fragment was inserted into the ompA gene immediately following the part encoding the signal sequence. The corresponding mature proteins consist, in this order, of 605, 860, 835, and 279 amino acid residues. All precursor proteins were processed and translocated across the plasma membrane. Hence, not only can the OmpA protein serve as a vehicle for export of a nonsecretory protein, but the signal sequence alone can also mediate export of such a protein. Export of the pro-OmpA protein depends on the SecA protein. Export of the tail fiber fragment expressed from pIN T4-6 remained SecA dependent. Thus, the secA pathway in this case is chosen by the signal peptide. It is proposed that a signal peptide can mediate translocation of nonsecretory proteins as long as they are export-compatible. The inability of a signal sequence to mediate export of some proteins appears to be due to export incompatibility of the protein rather than to the absence of information, within the mature part of the polypeptide, which would be required for translocation.

Amino Acid Sequence↗
PubMed1987

T-even-type bacteriophages use an adhesin for recognition of cellular receptors.

Protein 38 of the Escherichia coli phage T4 is thought to be required catalytically for the assembly of the long tail fibers of this phage. It is shown that this protein of phage T2 and the T-even-type phage K3 and Ox2 act differently. It was found that NH2-terminal fragments of the protein, expressed from cloned fragments of gene 38 of phage K3, bind to gene 38 amber mutants of phage T2. Such phage or T2 gene 38 amber mutants, grown on a non-permissive host, possess a complete set of six tail fibers but are non-infectious. Both types of non-infectious phage could be repaired by incubation with an extract of cells harboring a cloned gene 38 of a host range mutant of phage K3, K3hx. The repaired phages had the host range of K3hx and not of T2. Immuno-electron microscopy showed that protein 38 is located at the free ends of the long tail fibers of phages T2, K3 and Ox2. The protein serves the recognition of the cellular receptor, i.e. it acts as an adhesin.

Amino Acid Sequence↗
PubMed1987

DNA sequence of genes 38 encoding a receptor-recognizing protein of bacteriophages T2, K3 and of K3 host range mutants.

Genes 38, which code for a receptor-recognizing protein present at the tip of the long tail fibers, have been sequenced from phages T2, the T-even-type phage K3 and its host range mutants K3hx, K3h1 and K3h1h. The genes from phages T2 and K3 code for proteins consisting of 262 and 260 amino acid residues, respectively. Fifty amino-terminal and 25 carboxy-terminal residues are highly conserved. The amino-terminal amino acids are most likely involved in binding to the neighboring protein 37. Between residues 116 and 226 of the T2 protein and residues 116 and 223 of the K3 protein, sequences exist that are similar to sequences present in Escherichia coli outer membrane proteins and which serve as phage receptors. Most likely, all of these regions in the latter proteins are exposed on the cell surface and are part of their phage receptor areas. In the phage proteins, these sequences are flanked by stretches rich in glycine, perhaps providing an increased flexibility for the polypeptide at these sites; some "wobble" may be required during the protein 38-receptor interaction. The mutational alterations in the host range mutants were found in gene 38. In the K3hx protein, a duplication of six base-pairs caused the wild-type sequence -Gly163-Lys-Leu-Ile- to be changed to -Gly163-Lys-Leu-Lys-Leu-Ile-. In the K3h1 protein, a glutamic acid residue at position 203 was substituted by a lysine. Both alterations occurred within areas similar to outer membrane proteins. Mutant K3h1h, derived from K3h1, exhibits an extended host range as compared to K3h1. No mutational alteration, in addition to that found in K3h1, was found in g38 nor was the part of gene 37 that encodes the carboxy-terminal moiety of the protein altered. K3h1h may represent a "trigger-happy" phage. The results of this and other work show that the phage-phage receptor systems under study represent a primitive immune system.

Amino Acid Sequence↗
PubMed1987

Nucleotide sequence of the gene for the peptidoglycan-associated lipoprotein of Escherichia coli K12.

During attempts to clone the gene for the Escherichia coli outer membrane protein III another gene was recovered. Its nucleotide sequence was determined and the deduced amino acid sequence showed that the gene does not encode protein III. It codes for a 173-residue polypeptide; 21 NH2-terminal residues are typical for a signal peptide. The sequence around the putative site (Ala-Cys) for removing this peptide, Ala-Ile-Ala-Ala-Cys-Ser-Ser-Asn, is highly homologous to that of the major cell envelope lipoprotein (Braun lipoprotein) surrounding its processing site; it is also homologous to the consensus pentapeptide Leu-Leu-Ala-Gly-Cys present in other lipoproteins of gram-negative bacteria. It could be shown that the gene expresses a lipoprotein with all the properties, including the amino acid composition, of the peptidoglycan-associated lipoprotein (PAL) [Mizuno, T. (1979) J. Biochem. (Tokyo) 86, 991-1000]. Therefore, the cloned gene is the pal gene. The protein does not contain hydrophobic regions which would serve as a membrane anchor. Tandemly repeated amino acid sequences exist at and near the NH2-terminus of the mature protein which are homologous to such repeats in the Braun lipoprotein, suggesting a common origin of this part of the two proteins. Attempts to place a transposon into the pal gene were unsuccessful. Hence the complete absence of the protein may be lethal and its function remains unknown.

Amino Acid Sequence↗
PubMed1987

The signal sequence suffices to direct export of outer membrane protein OmpA of Escherichia coli K-12.

We studied whether information required for export is present within the mature form of the Escherichia coli 325-residue outer membrane protein OmpA. We had previously analyzed overlapping internal deletions in the ompA gene, and the results allowed us to conclude that if such information exists it must be present repeatedly within the membrane part of the protein encompassing amino acid residues 1 to 177 (R. Freudl, H. Schwarz, M. Klose, N. R. Movva, and U. Henning, EMBO J. 4:3593-3598, 1985). A deletion which removed the codons for amino acid residues 1 to 229 of the OmpA protein was constructed. In this construct the signal sequence was fused to the periplasmic part of the protein. The resulting protein, designated Pro-OmpA delta 1-229, was processed, and the mature 95-residue protein accumulated in the periplasm. Hence, information required for export does not exist within the OmpA protein.

Amino Acid Sequence↗
PubMed1987

An open reading frame in the Escherichia coli bacteriophage lambda genome encodes a protein that functions in assembly of the long tail fibers of bacteriophage T4.

Assembly of the long tail fibers of the Escherichia coli bacteriophage T4 requires the catalytic action of two auxiliary proteins. It was found that a gene of the entirely unrelated phage lambda codes for a protein which can substitute for one of these T4 polypeptides, protein 38. The lambda gene was designated tfa (tail fiber assembly). Protein 38 consists of 183 residues, and the Tfa protein consists of 194 residues; the two polypeptides are about 40% homologous. Although the tfa gene is dispensable for the growth of phage lambda, these results indicate that it may have a function in lambda morphogenesis.

Amino Acid Sequence↗
PubMed1986

DNA sequence of the tail fiber genes 37, encoding the receptor recognizing part of the fiber, of bacteriophages T2 and K3.

The DNA sequences of genes 37 of bacteriophages T2 and K3 are presented and compared with that of phage T4. The corresponding proteins constitute, as dimers, the part of the long tail fibers that recognizes the bacterial receptor. The CO2H termini of the polypeptides are located at the free ends of the fibers. Morphologically, the three phages are essentially identical, but they use different receptors. The genes from phages T4, T2 and K3 encode proteins consisting of 1026, 1341 and 1243 amino acid residues, respectively. DNA-DNA hybridizations had shown earlier that genes 37, in contrast to the gene for the major capsid protein, of a number of T-even type phages are highly polymorphic. The deduced amino acid sequences now show that this polymorphism extends to the protein primary structures. About 50 NH2-terminal residues are conserved and are probably required for binding to the adjacent protein 36. This area is followed by more or less irregularly spaced regions of non-homology, partial homology or complete homology. The heterogeneity is most prominent in a region encompassing about 600 CO2H-terminal residues of the T2 or K3 proteins. Nevertheless, the amino acid compositions of the three proteins are very similar and all are rich in glycine. It has been found that the receptor specificities of phages K3 and T2 are determined by protein 38, a polypeptide required for the efficient dimerization of protein 37 of phage T4. Proteins 38 of phages K3 and T2 are functionally interchangeable, those of T4 and T2 or K3 are not. Proteins 37 of phages K3 and T2 possess a conserved sequence of 160 CO2H-terminal residues. This area is missing in the T4 protein. This region may serve as a binding site for polypeptides 38 of phages K3 and T2. The overall picture of the protein primary structures of the three phages strongly suggests that the evolution of genes 37, which was most likely driven by selection for variations in receptor recognition specificities, has not been a steady process but has involved loss and gain of segments of DNA.

Amino Acid Sequence↗
PubMed1986

Morphogenesis of the long tail fibers of bacteriophage T2 involves proteolytic processing of the polypeptide (gene product 37) constituting the distal part of the fiber.

Gene 37 of phage T2 codes for a protein that, as a dimer, constitutes the most distal, receptor-recognizing part of its long tail fibers. It was found that, from a plasmid carrying a fragment of gene 37 that lacked a region of the gene encoding 87 CO2H-terminal amino acid residues, a protein was expressed that was slightly larger than that present in the phage. This size difference could not be accounted for. The missing region of gene 37 and also gene 38 (which codes for the auxiliary protein required for dimerization of protein 37) were cloned. Plasmids were constructed with gene 37, or gene 37 together with gene 38, under inducible control. Independent of the presence of the latter gene, a protein was produced that had the same size as protein 37 in the phage. A pulse-chase experiment revealed that a precursor of protein 37 is synthesized and processed such that approximately 120 amino acid residues, most likely CO2H-terminal, are removed. Therefore, the protein produced from the truncated gene was larger because it cannot be processed. This fact also solved an old puzzle: an amber fragment of protein 37 of phage T2 had been found to be larger than the mature protein. The amber codon could be located 24 codons away from the normal stop codon. Obviously, the fragment cannot be processed. The existence of this fragment demonstrates that processing occurs during phage maturation.

Autoradiography↗
PubMed1986

An outer membrane protein (OmpA) of Escherichia coli K-12 undergoes a conformational change during export.

Pulse-chase experiments were performed to follow the export of the Escherichia coli outer membrane protein OmpA. Besides the pro-OmpA protein, which carries a 21-residue signal sequence, three species of ompA gene products were distinguishable. One probably represented an incomplete nascent chain, another the mature protein in the outer membrane, and the third, designated imp-OmpA (immature processed), a protein which was already processed but apparently was still associated with the plasma membrane. The pro- and imp-OmpA proteins could be characterized more fully by using a strain overproducing the ompA gene products; pro- and imp-OmpA accumulated in large amounts. It could be shown that the imp- and pro-OmpA proteins differ markedly in conformation from the OmpA protein. The imp-OmpA, but not the pro-OmpA, underwent a conformational change and gained phage receptor activity upon addition of lipopolysaccharide. Utilizing a difference in detergent solubility between the two polypeptides and employing immunoelectron microscopy, it could be demonstrated that the pro-OmpA protein accumulated in the cytoplasm while the imp-OmpA was present in the periplasmic space. The results suggest that the pro-OmpA protein, bound to the plasma membrane, is processed, and the resulting imp-OmpA, still associated with the plasma membrane, recognizes the lipid A moiety of the lipopolysaccharide. The resulting conformational change may then force the protein into the outer membrane.

Centrifugation, Density Gradient↗
PubMed1986

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.

Amino Acid Sequence↗
PubMed1986

New locus (ttr) in Escherichia coli K-12 affecting sensitivity to bacteriophage T2 and growth on oleate as the sole carbon source.

The nature of resistance to phage T2 in Escherichia coli K-12 was investigated by analyzing a known phage T2-resistant mutant and by isolating new T2-resistant mutants. It was found that mutational alterations at two loci, ompF (encoding the outer membrane protein OmpF) and ttr (T-two resistance), are needed to give full resistance to phage T2. A ttr::Tn10 mutation was isolated and was mapped between aroC and dsdA, where the fadL gene (required for long-chain fatty acid transport) is located. The receptor affected by ttr was the major receptor used by phage T2 and was located in the outer membrane. Phage T2 was thus able to use two outer membrane proteins as receptors. All strains having a ttr::Tn10 allele and most of the independently isolated phage T2-resistant mutants were unable to grow on oleate as the sole carbon and energy source, i.e., they had the phenotype of fadL mutants. The gene fadL is known to encode an inner membrane protein. The most likely explanation is that fadL and ttr are in an operon and that ttr encodes an outer membrane protein which functions in translocating long-chain fatty acids across the outer membrane and also as a receptor for phage T2.

Bacterial Outer Membrane Proteins↗
PubMed1985

The nature of information, required for export and sorting, present within the outer membrane protein OmpA of Escherichia coli K-12.

Information, in addition to that provided by signal sequences, for translocation across the plasma membrane is thought to be present in exported proteins of Escherichia coli. Such information must also exist for the localization of such proteins. To determine the nature of this information, overlapping inframe deletions have been constructed in the ompA gene which codes for a 325-residue major outer membrane protein. In addition, one deletion, encoding only the NH2-terminal part of the protein up to residue 160, was prepared. The location of each product was determined by immunoelectron microscopy. Proteins missing residues 4-45, 43-84, 46-227, 86-227 or 160-325 of the mature protein were all efficiently translocated across the plasma membrane. The first two proteins were found in the outer membrane, the others in the periplasmic space. It has been proposed that export and sorting signals consist of relatively small amino acid sequences near the NH2 terminus of an outer membrane protein. On the basis of sequence homologies it has also been suggested that such proteins possess a common sorting signal. The locations of the partially deleted proteins described here show that a unique export signal does not exist in the OmpA protein. The proposed common sorting signal spans residues 1-14 of OmpA. Since this region is not essential for routing the protein, the existence of a common sorting signal is doubtful. It is suggested that information both for export (if existent) and localization lies within protein conformation which for the former process should be present repeatedly in the polypeptide.

Bacterial Outer Membrane Proteins↗