Search PubMed⌕ Search

Biomedical subjects

J Tommassen

Publications and source records attributed to J Tommassen.

At least 37 records · Page 2Linked to original sources
PubMed2000

Identification of a chitin-binding protein secreted by Pseudomonas aeruginosa.

One of the major proteins secreted by Pseudomonas aeruginosa is a 43-kDa protein, which is cleaved by elastase into smaller fragments, including a 30-kDa and a 23-kDa fragment. The N-terminal 23-kDa fragment was previously suggested as corresponding to a staphylolytic protease and was designated LasD (S. Park and D. R. Galloway, Mol. Microbiol. 16:263-270, 1995). However, the sequence of the gene encoding this 43-kDa protein revealed that the N-terminal half of the protein is homologous to the chitin-binding proteins CHB1 of Streptomyces olivaceoviridis and CBP21 of Serratia marcescens and to the cellulose-binding protein p40 of Streptomyces halstedii. Furthermore, a short C-terminal fragment shows homology to a part of chitinase A of Vibrio harveyi. The full-length 43-kDa protein could bind chitin and was thereby protected against the proteolytic activity of elastase, whereas the degradation products did not bind chitin. The purified 43-kDa chitin-binding protein had no staphylolytic activity, and comparison of the enzymatic activities in the extracellular medium of a wild-type strain and a chitin-binding protein-deficient mutant indicated that the 43-kDa protein supports neither chitinolytic nor staphylolytic activity. We conclude that the 43-kDa protein, which was found to be produced by many clinical isolates of P. aeruginosa, is a chitin-binding protein, and we propose to name it CbpD (chitin-binding protein D).

Amino Acid Sequence↗
PubMed1999

The C-terminal domain of the Pseudomonas secretin XcpQ forms oligomeric rings with pore activity.

The Pseudomonas secretin XcpQ forms an oligomeric complex, which is involved in the translocation of proteins across the outer membrane via the type II secretion pathway. Pseudomonas aeruginosa produces only small amounts of this complex, 50 to 100 copies per bacterium, and overexpression is lethal to these cells. However, overexpression of Pseudomonas alcaligenes XcpQ could be achieved in the P. alcaligenes mutant strain 537. Protease protection experiments with P. alcaligenes XcpQ showed that the C-terminal domain of XcpQ, which is conserved in all the different members of the secretin family, is largely resistant to proteinase K. This protease-resistant fragment is embedded in the membrane and remains a stable complex, indicating that this domain is involved in complex formation. Both the intact and the protease-protected XcpQ complex showed a tendency to form two-dimensional crystal-like structures. Electron microscopic analysis of these structures showed that the overall oligomeric rings of the intact and of the protease-resistant complex are highly similar. The central cavity of the intact XcpQ complex contains structured mass. Both the intact and the protease-protected XcpQ complex showed pore-forming activity in planar lipid bilayers, consistent with their role as a translocation channel. However, the single-channel conductances observed were not uniform. Together, these results demonstrate that the C-terminal secretin homology domain of XcpQ is the structural domain that forms the channel through which macromolecules are being transported.

Bacterial Proteins↗
PubMed1999

Sequence variability of the meningococcal lactoferrin-binding protein LbpB.

The lactoferrin receptor of Neisseria meningitidis consists of two proteins, LbpA and LbpB. LbpB is considered a promising vaccine candidate, and therefore its sequence variability was studied. LbpB from five different strains exhibited 70-80% mutual identity at the amino acid level. Most sequence variability was found in two stretches with a high content of negatively charged amino acids. These stretches were sequenced from six additional strains. One of the stretches is of variable length and is missing in some of the strains. The other stretch is present in all strains, but varies considerably in its exact amino acid sequence. The high degree of variability is disadvantageous for vaccine development, but may be useful for epidemiological studies.

Amino Acid Sequence↗
PubMed1999

Molecular organization of the xcp gene cluster in Pseudomonas putida: absence of an xcpX (gspK) homologue.

A DNA fragment containing xcp (gsp) gene homologues, required for extracellular protein secretion by the general secretory pathway (GSP) in various Gram-negative bacteria, was cloned from Pseudomonas putida (Pp) strain WCS358 and sequenced. The results presented here and those previously reported (de Groot, A., Krijger, J.-J., Filloux, A., Tommassen, J., 1996. Characterization of type II protein secretion (xcp) genes in the plant growth-stimulating Pseudomonas putida, strain WCS358 Mol. Gen. Genet. 250, 491-504) complete the sequence of the xcp gene cluster of Pp. Unlike that of Pseudomonas aeruginosa (Pa), the xcp gene cluster of Pp contains a gspN homologue. More surprisingly, in contrast to all known gsp gene clusters, the xcpX (gspK) homologue is not found. In addition, genes flanking the xcp cluster of Pp are not related to those flanking the xcp genes of Pa. Overall, the xcp gene products of Pp are as much related to those of Pa as to gsp gene products of enterobacterial species, suggesting that the xcp clusters of Pp and Pa have evolved separately.

Bacterial Proteins↗
PubMed1999

Specificity of the lipase-specific foldases of gram-negative bacteria and the role of the membrane anchor.

Folding of lipases that are secreted by Pseudomonads and other gram-negative bacteria via the type II secretion pathway is facilitated by dedicated chaperones, called lipase-specific foldases (Lifs). Lifs are membrane-anchored proteins with a large periplasmic domain. The functional interaction between the Lif and its cognate lipase is specific, since the Pseudomonas aeruginosa Lif was found not to substitute for Lifs from Burkholderia glumae or Acinetobacter calcoaceticus. However, the P. aeruginosa Lif was able to activate the lipase from the closely related species P. alcaligenes. Hybrid proteins constructed from parts of the P. aeruginosa and B. glumae Lifs revealed that the C-terminal 138 amino acids of the B. glumae Lif determine the specificity of the interaction with the cognate lipase. Furthermore, the periplasmic domain of the B. glumae Lif was functional when cloned in frame with a cleavable signal sequence, which demonstrates that the membrane anchor is not essential for Lif function in vivo. However, the recombinant Lif was released into the medium, indicating that the function of the membrane anchor is to prevent secretion of the Lif together with the lipase.

Acinetobacter calcoaceticus↗
PubMed1999

Affinity of the periplasmic chaperone Skp of Escherichia coli for phospholipids, lipopolysaccharides and non-native outer membrane proteins. Role of Skp in the biogenesis of outer membrane protein.

The Skp protein of Escherichia coli has been proposed to be a periplasmic molecular chaperone involved in the biogenesis of outer membrane proteins. In this study, evidence is obtained that Skp exists in two different states characterized by their different sensitivity to proteases. The conversion between these states can be modulated in vitro by phospholipids, lipopolysaccharides and bivalent cations. Skp is able to associate with and insert into phospholipid membranes in vitro, indicating that it may associate with phospholipids in the inner and/or outer membrane in vivo. In addition, it interacts specifically with outer membrane proteins that are in their non-native state. We propose that Skp is required in vivo for the efficient targeting of unfolded outer membrane proteins to the membrane.

Bacterial Outer Membrane Proteins↗
PubMed1999

Identification of positively charged residues of FomA porin of Fusobacterium nucleatum which are important for pore function.

FomA porin is the major outer-membrane protein of Fusobacterium nucleatum. It exhibits the functional properties of a general diffusion porin, but has no sequence similarity to other porins. According to the proposed topology model, each monomer of this trimeric protein is a beta-barrel consisting of 16 transmembrane segments with eight surface-exposed loops. Several conserved charged residues are proposed to extend from the beta-barrel wall into the aqueous channel lumen, and may contribute to a transverse electric field similar to that at the pore constriction of porins with known structure. The goal of our study was to identify particular basic residues contributing to such an electric field in FomA. Several arginines and lysines were replaced by negatively charged glutamates or uncharged alanines. The mutated FomA porins were expressed in Escherichia coli, and the effects on pore function were studied in vivo, by assaying the uptake rate of beta-lactam antibiotics, and in vitro after reconstitution of the purified proteins in lipid bilayer membranes. Some of the point mutations had a significant impact on the channel properties. The substitution R92A produced a 130% increased permeability of the zwitterionic beta-lactam cephaloridine, and the cation selectivity of R92E increased by 70%. The effects of the R90E substitution on channel properties were similar. Most of the point mutations had a minor effect on the voltage gating of the FomA channel, resulting in an increased sensitivity, except for K78E, which showed a decreased sensitivity. The latter mutation had no effect on cation selectivity, but the K78A substitution improved the uptake rate of cephaloridine. The results presented here indicate that arginines 90 and 92 are probably part of the constriction zone of the FomA porin, and lysine 78 and arginines 115 and 117 are probably in close proximity to this region as well.

Amino Acid Sequence↗
PubMed1999

Functional activities and epitope specificity of human and murine antibodies against the class 4 outer membrane protein (Rmp) of Neisseria meningitidis.

Antibodies against the class 4 outer membrane protein (OMP) from Neisseria meningitidis have been purified from sera from vaccinees immunized with the Norwegian meningococcal group B outer membrane vesicle vaccine. The human sera and purified antibodies reacted strongly with the class 4 OMP in immunoblots, whereas experiments with whole bacteria showed only weak reactions, indicating that the antibodies mainly reacted with parts of the class 4 molecule that were not exposed. The purified human anti-class 4 OMP antibodies and the monoclonal antibodies (MAbs) were neither bactericidal nor opsonic against live meningococci. Three new MAbs against the class 4 OMP were generated and compared with other, previously described MAbs. Three linear epitopes in different regions of the class 4 OMP were identified by the reaction of MAbs with synthetic peptides. The MAbs showed no blocking effect on bactericidal activity of MAbs against other OMPs. However, one of the eight purified human anti-class 4 OMP antibody preparations, selected from immunoblot reactions among sera from 27 vaccinees, inhibited at high concentrations the bactericidal effect of a MAb against the class 1 OMP. However, these antibodies were not vaccine induced, as they were present also before vaccination. Therefore, this study gave no evidence that vaccination with a meningococcal outer membrane vesicle vaccine containing the class 4 OMP induces blocking antibodies. Our data indicated that the structure of class 4 OMP does not correspond to standard beta-barrel structures of integral OMPs and that no substantial portion of the OmpA-like C-terminal region of this protein is located at the surface of the outer membrane.

Adult↗
PubMed1999

Bacteriocin release protein triggers dimerization of outer membrane phospholipase A in vivo.

Bacteriocin release protein is known to activate outer membrane phospholipase A (OMPLA), which results in the release of colicin from Escherichia coli. In vivo chemical cross-linking experiments revealed that the activation coincides with dimerization of OMPLA. Permeabilization of the cell envelope and dimerization were characterized by a lag time of 2 h.

Alkaline Phosphatase↗
PubMed1999

Structural characterization of the lactoferrin receptor from Neisseria meningitidis.

The meningococcal lactoferrin receptor is composed of the integral outer membrane protein LbpA and the peripheral lipoprotein LbpB. Homooligomeric complexes of LbpA and heterooligomers consisting of LbpA and LbpB were identified. Furthermore, five cell surface-exposed loops of LbpA were identified, which partially confirms a previously proposed topology model.

Amino Acid Sequence↗
PubMed1999

A novel lipolytic enzyme located in the outer membrane of Pseudomonas aeruginosa.

A lipase-negative deletion mutant of Pseudomonas aeruginosa PAO1 still showed extracellular lipolytic activity toward short-chain p-nitrophenylesters. By screening a genomic DNA library of P. aeruginosa PAO1, an esterase gene, estA, was identified, cloned, and sequenced, revealing an open reading frame of 1,941 bp. The product of estA is a 69.5-kDa protein, which is probably processed by removal of an N-terminal signal peptide to yield a 67-kDa mature protein. A molecular mass of 66 kDa was determined for (35)S-labeled EstA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The amino acid sequence of EstA indicated that the esterase is a member of a novel GDSL family of lipolytic enzymes. The estA gene showed high similarity to an open reading frame of unknown function located in the trpE-trpG region of P. putida and to a gene encoding an outer membrane esterase of Salmonella typhimurium. Amino acid sequence alignments led us to predict that this esterase is an autotransporter protein which possesses a carboxy-terminal beta-barrel domain, allowing the secretion of the amino-terminal passenger domain harboring the catalytic activity. Expression of estA in P. aeruginosa and Escherichia coli and subsequent cell fractionation revealed that the enzyme was associated with the cellular membranes. Trypsin treatment of whole cells released a significant amount of esterase, indicating that the enzyme was located in the outer membrane with the catalytic domain exposed to the surface. To our knowledge, this esterase is unique in that it exemplifies in P. aeruginosa (i) the first enzyme identified in the outer membrane and (ii) the first example of a type IV secretion mechanism.

Amino Acid Sequence↗
PubMed1998

The secretion apparatus of Pseudomonas aeruginosa: identification of a fifth pseudopilin, XcpX (GspK family).

The xcp gene products in Pseudomonas aeruginosa are required for the secretion of proteins across the outer membrane. Four of the Xcp proteins, XcpT, U, V and W, present sequence homology to the subunits of type IV pili at their N-termini, and they were therefore designated pseudopilins. In this study, we characterized the xcpX gene product, a bitopic cytoplasmic membrane protein. Remarkably, amino acid sequence comparisons also suggested that the XcpX protein resembles the pilins and pseudopilins at the N-terminus. We show that XcpX could be processed by the prepilin peptidase, PilD/XcpA, and that the highly conserved glycine residue preceding the hydrophobic segment could not be mutated without loss of the XcpX function. We, therefore, classified XcpX (GspK) as the fifth pseudopilin of the system.

Amino Acid Sequence↗
PubMed1998

Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is able to translocate proteins across both membranes of the cell envelope. Many of these proteins are transported via the type II secretion pathway and adopt their tertiary conformation in the periplasm, which implies the presence of a large transport channel in the outer membrane. The outer membrane protein, XcpQ, which is involved in transport of folded proteins across the outer membrane of P. aeruginosa, was purified as a highly stable homomultimer. Insertion and deletion mutagenesis of xcpQ revealed that the C-terminal part of XcpQ is sufficient for the formation of the multimer. However, linker insertions in the N-terminal part can disturb complex formation completely. Furthermore, complex formation is strictly correlated with lethality, caused by overexpression of xcpQ. Electron microscopic evaluation of the XcpQ multimers revealed large, ring-shaped structures with an apparent central cavity of 95 A. Purified PilQ, a homologue of XcpQ involved in the biogenesis of type IV pili, formed similar structures. However, the apparent cavity formed by PilQ was somewhat smaller, 53 A. The size of this cavity could allow for the transport of intact type IV pili.

Amino Acid Sequence↗
PubMed1998

Molecular characterization of LbpB, the second lactoferrin-binding protein of Neisseria meningitidis.

The lbpA gene of Neisseria meningitidis encodes an outer membrane lactoferrin-binding protein and shows homology to the transferrin-binding protein, TbpA. Previously, we have detected part of an open reading frame upstream of lbpA. The putative product of this open reading frame, tentatively designated lbpB, showed homology to the transferrin-binding protein TbpB, suggesting that the lactoferrrin receptor, like the transferrin receptor, consists of two proteins. The complete nucleotide sequence of lbpB was determined. The gene encodes a 77.5 kDa protein, probably a lipoprotein, with homology, 33% identity to the TbpB of N. meningitidis. A unique feature of LbpB is the presence of two stretches of negatively charged residues, which might be involved in lactoferrin binding. Antisera were raised against synthetic peptides corresponding to the C-terminal part of the putative protein and used to demonstrate that the gene is indeed expressed. Consistent with the presence of a putative Fur binding site upstream of the lbpB gene, expression of both LbpA and LbpB was proved to be iron regulated in Western blot experiments. The LbpB protein appeared to be less stable than TbpB in SDS-containing sample buffer. Isogenic mutants lacking either LbpA or LbpB exhibited a reduced ability to bind lactoferrin. In contrast to the lbpB mutant, the lbpA mutant was completely unable to use lactoferrin as a sole source of iron.

Amino Acid Sequence↗
PubMed1998

Secretion of elastinolytic enzymes and their propeptides by Pseudomonas aeruginosa.

Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. The signal sequence is cleaved off during transport across the inner membrane and, in the periplasm, proelastase is further processed. We demonstrate that the propeptide and the mature elastase are both secreted but that the propeptide is degraded extracellularly. In addition, reduction of the extracellular proteolytic activity led to the accumulation of unprocessed forms of LasA and LasD in the extracellular medium, which shows that these enzymes are secreted in association with their propeptides. Furthermore, a hitherto undefined protein with homology to a Streptomyces griseus aminopeptidase accumulated under these conditions.

Bacterial Proteins↗
PubMed1997

Role of the constriction loop in the gating of outer membrane porin PhoE of Escherichia coli.

Porins form voltage-gated channels in the bacterial outer membrane. These proteins are composed of three identical subunits, each forming a 16-stranded beta-barrel. In this study, the role in voltage gating of a loop that forms a constriction within the pore was studied. The channel characteristics of mutant PhoE porins, in which the tip of the constriction loop was connected to the barrel wall, were determined. Whereas the properties of several mutant channels were changed, all of these channels could still be closed at high potential, showing that a gross movement of the constriction loop within the channel is not implicated in voltage gating.

Anti-Bacterial Agents↗