Biomedical subjects

J Tommassen

Publications and source records attributed to J Tommassen.

At least 73 records · Page 4Linked to original sources

PubMed1996

Cloning of the fomA gene, encoding the major outer membrane porin of Fusobacterium nucleatum ATCC10953.

The major outer membrane protein, FomA, of the Gram-negative human oral pathogen Fusobacterium nucleatum functions as a porin and is assumed to act as a receptor protein in coaggregation with other oral pathogenic bacteria such as Streptococcus sanguis and Porphyromonas gingivalis. We describe here the cloning of fomA from F. nucleatum in E. coli. Using pGEM3Zf(+), three recombinant plasmids were carrying parts of the fomA gene, but none of these contained regions upstream of the coding sequence. From these plasmids a clone was constructed which contained the whole fomA gene. The ATCC 10953 fomA gene was cloned under the phosphate limitation-inducible phoE promoter, using a vector derived from pACYC184. The protein was found to be incorporated into the outer membrane of the host in an apparently normal manner, as judged by heat-modifiability, trypsin-accessibility, and accessibility to antibodies to the protein in a whole cell enzyme-linked immunosorbent assay. The cloned FomA was found to exhibit pore-forming activity.

Bacterial Outer Membrane Proteins↗

PubMed1996

The qmeA (ts) mutation of Escherichia coli is localized in the fabI gene, which encodes enoyl-ACP reductase.

The phenotypes of temperature-sensitive qmeA and fabI mutants of Escherichia coli appear to be very similar. Furthermore, the qmeA mutation could be complemented by the fabI gene on a plasmid, and the fabI allele derived from the qmeA mutant strain harbours a nucleotide substitution identical to that from a previously characterized fabI mutant. These results show that the qmeA gene is, in fact, identical to the fabI gene, which encodes enoyl-ACP reductase, involved in fatty acid elongation.

Enoyl-(Acyl-Carrier Protein) Reductase (NADPH, B-S↗

PubMed1996

Role of the propeptide in folding and secretion of elastase of Pseudomonas aeruginosa.

Elastase of Pseudomonas aeruginosa is synthesized as a pre-proprotein. The propeptide has been shown to inhibit the enzymatic activity of elastase. In this study, we investigated a possible additional role of the propeptide in the folding and secretion of the enzyme. When elastase was expressed in Escherichia coli without its propeptide, no active elastase was produced. The enzyme was poorly released from the cytoplasmic membrane and, depending on the expression level, it was either degraded or it accumulated in an inactive form in the cell envelopes, probably as aggregates. Since proper folding is required for the release of translocated proteins from the cytoplasmic membrane and for the acquirement of a stable and active conformation, these results suggest that the propeptide is involved in the proper folding of the elastase and that it functions as an intramolecular chaperone. When mature elastase was expressed without its propeptide in P. aeruginosa, the enzyme was not secreted, and it was degraded. Therefore, proper folding of mature elastase appears to be required for secretion of the enzyme. Expression of the propeptide, as a separate polypeptide, in trans with mature elastase resulted in the formation of active elastase. This active enzyme was secreted in P. aeruginosa. Apparently, the propeptide can also function as an intermolecular chaperone.

Bacterial Proteins↗

PubMed1996

Quaternary structure of a carrier protein influences antigenicity and immunogenicity of an inserted T cell determinant.

To study the influence of the quaternary structure of the outer membrane protein PhoE of Escherichia coli on the presentation of an inserted T cell epitope, an epitope comprising amino acid residues 72-85 of myelin basic protein (MBP) was inserted at different sites in PhoE. This sequence is the critical T cell epitope in experimental autoimmune encephalomyelitis (EAE) in Lewis rats. The antigenicity and immunogenicity of two different conformational forms of the chimeric PhoE constructs, i.e. the denatured monomeric form and the native trimeric form, were studied. It appeared that the monomeric form, but not the native trimeric form of such PhoE constructs induced proliferation of the MBP72-85-specific T cell line Z1a. This conformational discrepancy was independent of the site in PhoE in which the epitope was inserted. Immunization with the monomeric form of PhoE constructs resulted in the priming of MBP72-85-specific T cells. In contrast, the trimeric form of these constructs was much less efficient in priming such cells. The differences between the monomeric and trimeric forms were most apparent when induction of EAE was studied. The monomeric form was encephalitogenic while the trimeric form was not. Furthermore, the antigen fine specificity, Vbeta usage and encephalitogenicity of T cells triggered by immunization with a monomeric PhoE construct appeared to be the same as those of T cell line Z1a, which was obtained after immunization with MBP, indicating that similar cells are triggered by immunization with the epitope either in PhoE or in its native context.

Allergens↗

PubMed1996

Delta mu H+ dependency of in vitro protein translocation into Escherichia coli inner-membrane vesicles varies with the signal-sequence core-region composition.

Signal sequences frequently contain alpha-helix-destabilizing amino acids in the hydrophobic core. Nuclear magnetic resonance studies on the conformation of signal sequences in membrane mimetic environments revealed that these residues cause a break in the alpha-helix. In the precursor of the Escherichia coli outer membrane protein PhoE (pre-PhoE), a glycine residue at position -10 (Gly -10) is thought to be responsible for the break in the alpha-helix. We investigated the role of this glycine residue in the translocation process by employing site-directed mutagenesis. SDS-PAGE analysis showed drastic variations in the electrophoretic mobilities of the mutant precursor proteins, suggesting an important role of the glycine residue in determining the conformation of the signal sequence. In vivo, no drastic differences in the translocation kinetics were observed as compared with wild-type PhoE, except when a charged residue (Arg) was substituted for Gly -10. However, the in vitro translocation of all mutant proteins into inverted inner-membrane vesicles was affected. Two classes of precursors could be distinguished. Translocation of one class of mutant proteins (Ala, Cys and Leu for Gly -10) was almost independent of the presence of a delta mu H+, whereas translocation of the other class of precursors (wild type or Ser) was strongly decreased in the absence of the delta mu H+. Apparently, the delta mu H+ dependency of in vitro protein translocation varies with the signal-sequence core-region composition. Furthermore, a proline residue at position -10 resulted in a signal sequence that did not prevent the folding of the precursor in an in vitro trimerization assay.

Amino Acid Sequence↗

PubMed1996

Demonstration of a folded monomeric form of porin PhoE of Escherichia coli in vivo.

The porins in the outer membranes of gram-negative bacteria are trimeric proteins. A folded monomeric form of the Escherichia coli porin PhoE, with a higher electrophoretic mobility than that of the denatured protein, has recently been detected in in vitro folding studies. To investigate the possible biological significance of the folded monomer, we attempted to detect this form in vivo. After pulse-labeling, folded monomers could be detected by immunoprecipitation. Furthermore, folded monomers were detected in a preparation of mutant PhoE porins, in which the subunit interactions were weakened by a E-66-->R substitution. Together, these results show that the folded monomer is not an in vitro folding artifact but an integral part of the native trimer.

Electrophoresis, Gel, Pulsed-Field↗

PubMed1995

A conserved histidine residue of Escherichia coli outer-membrane phospholipase A is important for activity.

Escherichia coli outer-membrane phospholipase A (OMPLA) is thought to be a member of the class of serine hydrolases, having a classical Asp-His-Ser catalytic triad [Horrevoets, A. J. G., Verheij, H. M. & de Haas, G. H. (1991) Eur. J. Biochem. 198, 247-253]. To identify the histidine residue that is important for catalytic activity, the four histidine residues in E. coli OMPLA that are conserved in other enterobacterial OMPLA enzymes were replaced by cysteine residues using PCR-directed, site-specific mutagenesis. The resulting mutant proteins were all well expressed and displayed heat modifiability, indicating that they were properly folded. Enzyme assays showed that only the His142Cys mutant protein was lacking enzymatic activity. In addition, a His142Gly mutant protein appeared to be inactive. These results show that His142 is important for the enzymatic activity of OMPLA.

Amino Acid Sequence↗

PubMed1995

In vitro folding of Escherichia coli outer-membrane phospholipase A.

Recombinant outer-membrane phospholipase A (OMPLA) of Escherichia coli was expressed without its signal sequence from the T7 phi 10 promoter. As a result of the cloning strategy the protein had an N-terminal extension of six amino acid residues. The protein accumulated in the cytosol in inclusion bodies. Conditions were established for the efficient folding of OMPLA in vitro in the presence of Triton X-100. After in vitro folding, the protein was present as a mixture of folded and unfolded forms. Ion-exchange chromatography was used for the purification of OMPLA and the separation of correctly folded, enzymically active enzyme from unfolded inactive protein. The final protein preparation was pure and fully heat-modifiable based on SDS/PAGE. The recombinant enzyme had a specific activity of 71 U/mg, which is similar to the value of the wild-type enzyme, purified from the membrane. The final yield of active enzyme was 35 mg protein/l culture of an A600 of 6. Circular dichroism spectroscopy revealed a high content of beta strand, in good agreement with a predicted beta-barrel structure of this outer-membrane protein.

Bacterial Outer Membrane Proteins↗

PubMed1995

The C terminus of SecA is involved in both lipid binding and SecB binding.

Using C-terminal deletion mutations in secA, we localized the previously proposed (Breukink, E., Keller, R.C. A., and de Kruijff, B. (1993), FEBS Lett. 331, 19-24) second lipid binding site on SecA. Since removal of these residues completely abolished the property of SecA to cause aggregation of negatively charged phosphatidyl-glycerol vesicles, we conclude that the C-terminal 70 amino acid residues of SecA are involved in lipid-binding. The C-terminal 70 amino acid residues of SecA are important for efficient in vitro translocation of the SecB-dependent precursor of PhoE across inverted inner membrane vesicles. Moreover, in vivo studies showed that this region is essential for growth. SecB and a SecB-precursor complex were shown to inhibit the SecA-mediated lipid vesicle aggregation, suggesting that the overall acidic SecB protein binds at or near the second lipid binding site on SecA. This together with the observation that the SecA mutant protein lacking the C-terminal 70 residues had a strongly reduced ability to mediate binding of SecB-precursor complexes to inverted inner membrane vesicles demonstrates that the C terminus of SecA is also involved in SecB binding.

Adenosine Triphosphatases↗

PubMed1995

Intracellular processing and presentation of T cell epitopes, expressed by recombinant Escherichia coli and Salmonella typhimurium, to human T cells.

Vaccines based on recombinant attenuated bacteria represent a potentially safe and effective immunization strategy. A carrier system was developed to analyze in vitro whether foreign T cell epitopes, inserted in the outer membrane protein PhoE of Escherichia coli and expressed by recombinant bacteria, are efficiently processed and presented via human leukocyte antigen (HLA) class I and II molecules by bacterial infected human macrophages. A well-defined HLA-B27-restricted cytotoxic T cell (CTL) epitope and an HLA-DR53 restricted T helper (Th) epitope of the fusion protein of measles virus were genetically inserted in a surface-exposed region of PhoE, and the chimeric proteins were expressed in E. coli and Salmonella typhimurium. Macrophages infected with both recombinant bacteria presented the Th epitope to the specific CD4+ T cell clone, but failed to present the CTL epitope to the specific CD8+ T cell clone. Presentation of the Th epitope by the infected macrophages was inhibited by cytochalasin D, indicating that phagocytic processing of intact bacteria within infected macrophages was essential for antigen presentation via HLA class II. Presentation of the Th epitope to the CD4+ T cell clone by infected macrophages was blocked by brefeldin A and cycloheximide, indicating the requirement of nascent HLA class II molecules for presentation. The efficiency of macrophages to process and present the inserted Th epitope was similar for both the recombinant E. coli and S. typhimurium strains.

Amino Acid Sequence↗

PubMed1995

Induction of the phoE promoter upon invasion of Salmonella typhimurium into eukaryotic cells.

Live attenuated Salmonella typhimurium strains expressing foreign antigens can be used for vaccination purposes. Due to deleterious effects of constitutive, high-level expression of the heterologous antigens, there is often strong selection pressure against plasmids encoding these antigens, resulting in rapid segregation in vivo. In vivo-inducible promoters may be a good alternative for constitutive promoters. The outer membrane protein PhoE of Escherichia coli is being used as a carrier for foreign antigenic determinants. Here we studied whether its expression from a plasmid is induced in S. typhimurium upon invasion of eukaryotic cells. This appeared to be the case. Furthermore, a S. typhimurium phoE mutant was constructed and the effects of the mutation on invasion, intracellular survival and virulence were studied. Survival in HEp-2 cells or in the macrophage-like cell line J744 was not, or only slightly, affected. Furthermore, the mutant appeared to be as virulent for mice as the wild-type strain.

Cell Line↗

PubMed1995

Characterization of an Escherichia coli rotA mutant, affected in periplasmic peptidyl-prolyl cis/trans isomerase.

The rotA gene of Escherichia coli encodes a peptidyl-prolyl cis/trans isomerase (PPIase), which is supposed to catalyse protein folding in the periplasm. To investigate the importance of the enzyme, the rotA gene was cloned and a chromosomal deletion mutant was created. The rotA mutant was normally viable. No residual PPIase activity could be detected in the periplasmic fraction of the mutant. Comparison of the patterns of periplasmic and outer membrane proteins by SDS-PAGE revealed no differences in protein composition between the rotA mutant and its parental strain. Similarly, the kinetics of periplasmic protein folding and outer membrane protein assembly appeared unaffected by the rotA mutation. Our results show that the periplasmic PPIase of E. coli is not essential and that the protein does not play an important role in protein folding.

Amino Acid Isomerases↗

PubMed1995

The pho regulon of Shigella flexneri.

Growth of Escherichia coli K-12 in low-phosphate conditions results in the induction of the synthesis of many proteins, including the outer membrane porin PhoE, alkaline phosphatase, and the Pst system for the transport of phosphate (P1). This response is controlled by a two-component regulatory system of which PhoB and PhoR are the response-regulator and the sensor/kinase, respectively. When Shigella flexneri was starved for P1, neither PhoE nor alkaline phosphatase was produced. However, induction of the synthesis of the PstS protein was observed, indicating that S. flexneri contains a functional PhoB/PhoR regulatory system. Consistent with this notion, the introduction of the E. coli phoA gene in S. flexneri resulted in the induction of alkaline phosphatase synthesis under phosphate limitation. However, introduction of phoE on a plasmid did not lead to the expression of PhoE protein, indicating that S. flexneri PhoB does not recognize the phoE promoter region. The phoB gene was cloned and sequenced and in the deduced amino acid sequence two deviations from that of E. coli PhoB were detected. Site-directed mutagenesis revealed that one of these deviations, i.e. Leu-172, which is Arg in E. coli PhoB, is responsible for the lack of expression of the PhoE protein in S. flexneri.

Alkaline Phosphatase↗

PubMed1995

Signal transduction in bacteria: phospho-neural network(s) in Escherichia coli?

The molecular basis of many forms of signal transfer in living organisms is provided via the transient phosphorylation of regulatory proteins by transfer of phosphoryl groups between these proteins. The dominant form of signal transduction in prokaryotic microorganisms proceeds via so-called two-component regulatory systems. These systems constitute phosphoryl transfer pathways, consisting of two or more components. Most of these pathways are linear, but some converge and some are divergent. The molecular properties of some of the well-characterised representatives of two-component systems comply with the requirements to be put upon the elements of a neural network: they function as logical operators and show the phenomenon of autoamplification. Because there are many phosphoryl transfer pathways in parallel and because there also appears to be cross-talk between these pathways, the total of all two-component regulatory systems in a single prokaryotic cell may show the typical characteristics of a 'phospho-neural network'. This may well lead to signal amplification, associative responses and memory effects, characteristics which are typical for neural networks. One of the main challenges in molecular microbial physiology is to determine the extent of the connectivity of the constituting elements of this presumed 'phospho-neural network', and to outline the extent of intelligence-like behaviour this network can generate. Escherichia coli is the organism of choice for this characterization.

Escherichia coli↗

PubMed1995

Molecular characterization of FrpB, the 70-kilodalton iron-regulated outer membrane protein of Neisseria meningitidis.

The structural gene encoding the 70-kDa outer membrane protein FrpB of Neisseria meningitidis was cloned and sequenced. A mutant lacking FrpB was constructed. No difference in iron utilization between the mutant and the parental strain was observed. A minor effect of the mutation on serum resistance was observed. A topology model for FrpB in the outer membrane is proposed.

Amino Acid Sequence↗

PubMed1994

Localization of functional domains in the Escherichia coli coprogen receptor FhuE and the Pseudomonas putida ferric-pseudobactin 358 receptor PupA.

Transport of ferric-siderophores across the outer membrane of gram-negative bacteria is mediated by specific outer membrane receptors. To localize the substrate-binding domain of the ferric-pseudobactin 358 receptor, PupA, of Pseudomonas putida WCS358, we constructed chimeric receptors in which different domains of PupA were replaced by the corresponding domains of the related ferric-pseudobactin receptors PupB and PupX, or the coprogen receptor FhuE of Escherichia coli. None of the chimeric proteins composed of pseudobactin receptor domains facilitated growth on any of the original substrates, or they showed only an extremely low efficiency. However, these receptors enabled cells of Pseudomonas BN8 to grow on media supplemented with uncharacterized siderophore preparations. These siderophore preparations were isolated from the culture supernatant of WCS358 cells carrying plasmids that contain genes of Pseudomonas B10 required for the biosynthesis of pseudobactin B10. Hybrid proteins that contained at least the amino-terminal 516 amino acids of mature FhuE were active as a receptor for coprogen and interacted with the E. coli TonB protein. A chimeric PupA-FhuE protein, containing the amino-terminal 94 amino acids of mature PupA, was also active as a coprogen receptor, but only in the presence of Pseudomonas TonB. It is concluded that the carboxy-terminal domain of ferric-pseudobactin receptors is important, but not sufficient, for ligand interaction, whereas binding of coprogen by the FhuE receptor is not dependent on this domain. Apparently, the ligand-binding sites of different receptors are located in different regions of the proteins. Furthermore, species-specific TonB binding by the PupA receptor is dependent on the amino-terminal domain of the receptor.

Bacterial Outer Membrane Proteins↗

PubMed1994

Detergent-induced folding of the outer-membrane protein PhoE, a pore protein induced by phosphate limitation.

The folding of the in vitro synthesized outer-membrane protein PhoE, a protein induced by phosphate limitation, has previously been studied using immunoprecipitation experiments with monoclonal antibodies that recognize conformational epitopes [De Cock, H., Hendricks, R., de Vrije, T. & Tommassen, J. (1990) J. Biol. Chem. 265, 4646-4651]. A folded monomer of the protein was detected in this way, while the addition of outer membranes was required to induce trimerization. In this study, we demonstrate that the folding of the in vitro synthesized PhoE protein did not occur spontaneously, but was dependent on the detergent that was present in the immunoprecipitation buffer. A remarkable specificity of phenyl-containing detergents on the efficient in vitro folding of PhoE molecules was observed. Furthermore, trimerization was detected in the absence of outer membranes when such detergents were present. However, the rate of trimerization was increased by the addition of crude cell envelopes containing outer membranes. The outer membranes probably enhanced trimerization by concentrating the folded PhoE molecules.

Cell Membrane↗

PubMed1994

Effect of mutations in the -10 region of the phoE promoter in Escherichia coli on regulation of gene expression.

The phoE promoter region in Escherichia coli contains a -10 region, typical of sigma 70-dependent promoters and, instead of a normal -35 region, a so-called pho box, to which the transcriptional activator phospho-PhoB binds under low phosphate conditions. A second pho box is present upstream of the first one and is required for full expression of phoE during phosphate starvation. To determine whether the lack of expression under high phosphate conditions is due solely to the absence of a genuine -35 box, the -10 region was further optimized towards the consensus -10 sequence and promoter activity was measured using alkaline phosphatase as a reporter. The mutations resulted in a drastic increment in the basal level of expression under high phosphate conditions, indicating that the deviations from consensus in the -10 region also play a role in determining the poor expression of the wild-type promoter under these conditions. The expression under high phosphate conditions was partly dependent on the presence of the phoB gene, showing that a small amount of active PhoB must be present under these circumstances. During phosphate starvation, the activity of the mutant promoters was further induced. The upstream pho box was not required for full expression from the mutant promoters under these conditions. Apparently, the wild-type phoE promoter is carefully balanced by deviations from the optimal Pribnow box sequence that reduce expression under high phosphate conditions and by the presence of several copies of the pho box, which enhance expression under phosphate starvation.

Base Sequence↗