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Biomedical subjects

J Tommassen

Publications and source records attributed to J Tommassen.

At least 163 records · Page 9Linked to original sources
PubMed1986

Topology of outer membrane pore protein PhoE of Escherichia coli. Identification of cell surface-exposed amino acids with the aid of monoclonal antibodies.

Monoclonal antibodies which recognize the cell surface-exposed part of outer membrane protein PhoE of Escherichia coli were used to select for antigenic mutants producing an altered PhoE protein. The selection procedure was based on the antibody-dependent bactericidal action of the complement system. Using two distinct PhoE-specific monoclonal antibodies, seven independent mutants with an altered PhoE protein were isolated. Among these seven mutants, five distinct binding patterns were observed with a panel of 10 monoclonal antibodies. DNA sequence analysis revealed the following substitutions in the 330-residue-long PhoE protein: Arg-201----His (three isolates), Arg-201----Cys, Gly-238----Ser, Gly-275----Ser and Gly-275----Asp. It is argued that amino acid residues 201, 238, and 275 are most likely directly involved in antibody binding and, therefore, exposed at the cell surface. Together with Arg-158, which was previously shown to be cell surface exposed as it is changed in phage TC45-resistant phoE mutants, these four positions show a remarkably regular spacing, being approximately 40 residues apart. A model is suggested in which the PhoE polypeptide repeatedly traverses the outer membrane in an antiparallel beta-pleated sheet structure, exposing eight areas to the outside which are all separated by approximately 40 residues.

Alleles↗
PubMed1986

Periplasmic accumulation of truncated forms of outer-membrane PhoE protein of Escherichia coli K-12.

In order to localize the information within PhoE protein of Escherichia coli K-12 required for export of the protein to the outer membrane, we have generated deletions throughout the phoE gene. Immunocytochemical labelling on ultrathin cryosections revealed that the polypeptides encoded by the mutant alleles are transported to, and accumulate in, the periplasm. These results show that, except for the signal sequence, there is no specific sequence within the PhoE protein that is essential for transport through the cytoplasmic membrane. The overall structure of the protein, rather than a particular sequence of amino acids, seems to be important for assembly into the outer membrane.

Autoradiography↗
PubMed1986

Nucleotide sequence of the exclusion-determining locus of IncI plasmid R144.

The exclusion-determining locus (exc) of IncI plasmid R144 has been proposed to contain two overlapping genes. The nucleotide sequence of this locus, as presently determined, reveals an open reading frame with a coding capacity of 220 amino acids (aa) and with a promoter located upstream of the translation-initiation region. Consistent with the proposed overlapping gene arrangement, a second putative promoter was found within this coding region. Thus, a polypeptide identical to the 147 C-terminal aa of the larger polypeptide can be expressed from this second promoter. The 3'-noncoding region contains a sequence that is representative for a Rho-independent transcription terminator.

Amino Acid Sequence↗
PubMed1986

O-antigenic chains of lipopolysaccharide prevent binding of antibody molecules to an outer membrane pore protein in Enterobacteriaceae.

The accessibility of outer membrane pore protein PhoE to antibody molecules at the cell surface of intact cells of various Enterobacteriaceae was investigated. Significant antibody binding was observed for only two of the nine strains tested. Analysis of the lipopolysaccharide by sodium dodecyl sulphate gel electrophoresis revealed a clear correlation between the presence of an O-antigenic side chain and the inability to bind PhoE protein-specific antibodies. As mutants that lack the O-antigen chain appeared to have acquired the ability to bind antibody, it must be concluded that the presence of O-antigenic chains of lipopolysaccharide prevents binding of antibodies to PhoE protein at the surface of intact cells. The relevance of this conclusion for the potential use of enterobacterial outer membrane pore proteins as vaccine components is discussed.

Antibodies, Monoclonal↗
PubMed1986

Effects of divalent cations and of phospholipase A activity on excretion of cloacin DF13 and lysis of host cells.

Induction of cloacin DF13 synthesis in Escherichia coli harbouring plasmid CloDF13 results in the release of cloacin DF13, inhibition of growth and ultimately in lysis of the host cells. Expression of the pCloDF13-encoded protein H is essential for both the release of cloacin DF13 and the lysis of the cells. The divalent cations Mg2+ and Ca2+ interfered with the mitomycin C-induced protein H-dependent lysis, but hardly affected the release of cloacin DF13. Essentially all of the bacteriocin was released from the cells before a detectable degradation of the peptidoglycan occurred, independent of the presence of mitomycin C. Experiments with phospholipase A mutants revealed that activation of detergent-resistant phospholipase A was essential for the export of cloacin DF13 across the outer membrane and the lysis of induced cells. Transport of cloacin DF13 across the cytoplasmic membrane was mainly dependent on protein H. A revised model for the excretion of cloacin DF13 is presented.

Bacterial Proteins↗
PubMed1985

Demonstration of a bacteriophage receptor site on the Escherichia coli K12 outer-membrane protein OmpC by the use of a protease.

The Escherichia coli K12 outer-membrane proteins OmpA, OmpC, OmpF, PhoE, and LamB (all of transmembrane nature) can serve as phage receptors. We have shown previously that one OmpA-specific phage, Ox2, can give rise to the host range mutants Ox2h10 and Ox2h12, with the latter being derived from the former [Morona, R. & Henning, U. (1984) J. Bacteriol. 159, 579-582]. Unlike Ox2, both host range phages can use the OmpA and OmpC proteins as receptors and Ox2h12 is better adapted to the OmpC protein than Ox2h10. In a search for the site(s) of OmpC protein involved in phage recognition, it was found that proteinase K is able to cleave all of the proteins mentioned above. OmpC protein (Mr = 38306) could be cleaved from outside the cell by proteinase K resulting in two fragments of Mr approximately equal to 21000 and Mr approximately equal to 17500. The use of OmpC-PhoE hybrid proteins allowed us to assign the approximately equal to 21000-Mr fragment to the CO2H-terminal moiety of the protein. Proteinase K treatment of intact cells abolished their activity to neutralize the OmpC-specific phage Tulb and reduced this ability towards phage Ox2h12. The OmpA, OmpF, PhoE and LamB proteins were cleaved by the protease not in intact cells but only when acting on cell envelopes. The sizes of the OmpC protein fragments and the results obtained with the hybrid proteins very strongly suggest that the protein is cleaved from outside the cell at a region involving amino acid residues 150-178 of the 346-residue protein, which shows homology to two regions of the OmpA protein which are involved in its phage receptor site (loc. cit.). These areas also exhibit some homology to a region of the LamB protein which is thought to be part of this protein's receptor site [Charbit et al. (1984) J. Mol. Biol. 175, 395-401]. This suggests that there is a common denominator for proteinaceous phage receptor site because the LamB-specific phage lambda and phage Tulb are of completely different nature. We conclude that the region of the OmpC protein in question is cell-surface-exposed and acts as a phage receptor site.

Bacterial Outer Membrane Proteins↗
PubMed1985

Monoclonal antibodies directed against the cell-surface-exposed part of PhoE pore protein of the Escherichia coli K-12 outer membrane.

Six monoclonal antibodies directed against the trimeric form of outer-membrane pore protein PhoE of Escherichia coli K-12 were isolated and characterized. All six antibodies bind to PhoE protein in intact cells and as isolated trimers, but not to dodecyl-sulphate-denatured monomers. Cross-reaction with the related pore proteins OmpF protein and OmpC protein was not observed. A hybrid pore protein in which the amino-terminal 74 amino acids of PhoE protein have been replaced by the corresponding part of OmpF protein is able to bind the six monoclonal antibodies. Five of the antibodies bind to the PhoE proteins of thirteen different Enterobacteriaceae when expressed in E. coli K-12, whereas the other antibody recognizes PhoE protein from nine of these strains. Four monoclonal antibodies are able to block adsorption of the PhoE-protein-specific phage TC45 to its receptor on whole cells. None of the antibodies has any effect on the uptake rate of the antibiotic cefsulodin through PhoE protein pores. Five antibodies are able to direct the complement-mediated killing of PhoE-protein-carrying cells. It is concluded that the six monoclonal antibodies recognize at least three distinct cell-surface-exposed epitopes whose specificity is determined by the carboxy-terminal 256 amino acids of PhoE protein.

Antibodies, Monoclonal↗
PubMed1985

Failure of E. coli K-12 to transport PhoE-LacZ hybrid proteins out of the cytoplasm.

A phoE-lacZ hybrid gene encoding the N-terminal 300 amino acid residues of pre-PhoE protein, fused to an almost complete beta-galactosidase molecule was constructed in vitro. Cell fractionation experiments suggested that the hybrid gene product is transported to the outer membrane. However, by using immuno-cytochemical labelling on ultra-thin cryosections it was shown that the hybrid protein accumulated in the cytoplasm. Thus, it appears that: (i) data on the localization of hybrid proteins merely based on cell fractionation experiments are not reliable, and (ii) either the C-terminal 15% of PhoE protein contain information which is essential for transport, or PhoE-LacZ hybrid proteins can never be transported out of the cytoplasm. The implications of these results for current models on the translocation of outer membrane proteins are discussed.

Bacterial Proteins↗
PubMed1985

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.

Amino Acid Sequence↗
PubMed1985

Relationship between the proteins encoded by the exclusion determining locus of the IncI plasmid R144 and the cellular localization of these proteins in Escherichia coli K-12.

A region of the IncI plasmid R144, determining and controlling exclusion (exc), codes for two proteins, designated 13K and 19K after their apparent molecular weights (respectively 13,000 and 19,000). Both proteins were simultaneously affected by various mutations that resulted in exclusion deficiency. In this paper the relationship between these proteins as well as their cellular location is reported. We found no indications that the 19K protein is a precursor form of the 13K protein. Analysis of gene products of recombinant plasmids carrying exc as well as of several derivatives, however, provided a strong indication that the proteins result from overlapping genes. Besides, evidence was obtained that the 19K protein is essential for exclusion. Localization studies revealed that this protein exists in a membrane-bound form, associated at the periplasmic side of the inner membrane, and in a soluble form residing in the cytoplasm.

Bacterial Proteins↗
PubMed1984

Physical mapping of the gene for aminopeptidase N in Escherichia coli K12.

The pepN gene has been cloned into the multicopy plasmid pBR322. The restriction map of the insert was established and the gene was localized. By comparison with the restriction map of the plasmid pJP30 bearing the ompF region, it has been possible to order the ompF, asnS, and pepN genes. The ompF and asnS genes are contiguous and pepN is separated from asnS by a DNA fragment of about 1.6 kb.

Aminopeptidases↗
PubMed1984

Gene encoding a hybrid OmpF--PhoE pore protein in the outer membrane of Escherichia coli K12.

To study the structure-function relationship of outer membrane pore proteins of E. coli K12, a hybrid gene was constructed in which the DNA encoding amino acid residues 2-73 of the mature PhoE protein is replaced by the homologous part of the related ompF gene. The product of this gene is incorporated normally into the outer membrane. It was characterized with respect to its pore activity and its phage receptor and colicin receptor properties. It is concluded that the preference of the PhoE protein pore for negatively charged solutes is partly determined by the amino terminal 73 amino acids, that part of the receptor site of PhoE protein for phage TC45 is located in this part of the protein, that colicin N uses OmpF protein as (part of) its receptor, that the specificity of OmpF protein as a colicin N receptor is completely located within the 80 amino terminal amino acid residues, whereas the specificity of this protein as a colicin A receptor is completely located within the 260 carboxy terminal amino acid residues, and that the amino terminal 73 amino acid residues of PhoE protein span the membrane at least once.

Bacterial Outer Membrane Proteins↗
PubMed1984

Cloning of phoM, a gene involved in regulation of the synthesis of phosphate limitation inducible proteins in Escherichia coli K12.

Like the synthesis of alkaline phosphatase, the synthesis of outer membrane PhoE protein is shown to be dependent on the phoM gene product in phoR mutants of E. coli K12. This phoM gene has been cloned into the multi-copy vector pACYC184 using selection for alkaline phosphatase constitutive synthesis in a phoR background. The gene was localized on the hybrid plasmids by analysis of deletion plasmids constructed in vitro and of mutant plasmids generated by gamma delta insertions. Interestingly, two of the selected hybrid plasmids contained the entire phoA-phoB-phoR region of the chromosome, as a multiple copy state of these genes results in the constitutive synthesis of alkaline phosphatase. The presence of multiple copies of the phoM gene hardly influences the level of expression of alkaline phosphatase and PhoE protein in a pho+ strain, but significantly increases the levels of these proteins in a phoR mutant strain.

Alkaline Phosphatase↗
PubMed1984

Cloning and expression in Escherichia coli K-12 of the structural gene for outer membrane PhoE protein from Enterobacter cloacae.

In Escherichia coli K-12, the phoE gene encodes an outer membrane pore protein, which is induced by phosphate starvation. The corresponding gene of Enterobacter cloacae was transferred to E. coli K-12 by using RP4::mini Mu plasmid pULB113 and selecting for R-prime plasmids that carry the genes proA and proB, which are closely linked to phoE in E. coli K-12. The phoE gene was subcloned into the multicopy vector pACYC184, and the location of the gene was determined by analysis of in vitro constructed deletion plasmids and mutant plasmids generated by gamma delta insertions. The E. cloacae phoE gene is normally expressed in E. coli K-12, and the regulation of the expression is similar to that of the E. coli phoE gene. Functionally, the products of the phoE genes of E. coli K-12 and E. cloacae behave very similarly since they form pores in the outer membrane with a recognition site for negatively charged compounds and they serve as (part of) the receptor for phage TC45.

Bacterial Outer Membrane Proteins↗
PubMed1984

Amino terminus of outer membrane PhoE protein: localization by use of a bla-phoE hybrid gene.

Expression of a recently constructed bla-phoE hybrid gene results in synthesis and incorporation into the outer membrane of PhoE protein containing an amino-terminal extension of 158 amino acid residues of beta-lactamase (Tommassen et al., EMBO J. 2:1275-1279, 1983). As the PhoE protein part of this hybrid protein is apparently normally incorporated into the outer membrane, the beta-lactamase part of the protein can be considered as a label of the amino terminus of PhoE protein. By using trypsin accessibility experiments, this beta-lactamase part was shown to be located at the periplasmic side of the membrane. Therefore, the amino terminus of PhoE protein most likely faces the periplasm.

Amino Acids↗