Biomedical subjects

B Oudega

Publications and source records attributed to B Oudega.

At least 37 records · Page 2Linked to original sources

PubMed1998

The Escherichia coli SRP and SecB targeting pathways converge at the translocon.

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.

Adenosine Triphosphatases↗

PubMed1998

Optimization of bacteriocin release protein (BRP)-mediated protein release by Escherichia coli: random mutagenesis of the pCloDF13-derived BRP gene to uncouple lethality and quasi-lysis from protein release.

Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli periplasm into the culture medium. However, high-level expression of BRP causes apparent lysis of the host cells in liquid cultures (quasi-lysis) and inhibition of growth on broth agar plates (lethality). To optimize BRP-mediated protein release, the pCloDF13 BRP gene was subjected to random mutagenesis by using PCR techniques. Mutated BRPs with a strongly reduced capacity to cause growth inhibition on broth agar plates were selected, analyzed by nucleotide sequencing, and further characterized by performing growth and release experiments in liquid cultures. A subset of these BRP derivatives did not cause quasi-lysis and had only a small effect on growth but still functioned in the release of the periplasmic protein beta-lactamase and the periplasmic K88 molecular chaperone FaeE and in the release of the bacteriocin cloacin DF13 into the culture medium. These BRP derivatives can be more efficiently used for extracellular production of proteins by E. coli than can the original BRP.

Amino Acid Sequence↗

PubMed1997

The complete genome sequence of the gram-positive bacterium Bacillus subtilis.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Bacillus subtilis↗

PubMed1997

Membrane association of FtsY, the E. coli SRP receptor.

FtsY, the Escherichia coli homologue of the eukaryotic SRP receptor (SR alpha), is located both in the cytoplasm and in the inner membrane of E. coli. Similar to SR alpha, FtsY consists of two major domains: a strongly acidic N-terminal domain (A) and a C-terminal GTP binding domain (NG) of which the crystal structure has recently been determined. The domains were expressed both in vivo and in vitro to examine their subcellular localization. The results suggest that both domains associate with the membrane but that the nature of the association differs.

Bacterial Proteins↗

PubMed1997

Nascent membrane and presecretory proteins synthesized in Escherichia coli associate with signal recognition particle and trigger factor.

The Escherichia coli signal recognition particle (SRP) and trigger factor are cytoplasmic factors that interact with short nascent polypeptides of presecretory and membrane proteins produced in a heterologous in vitro translation system. In this study, we use an E. coli in vitro translation system in combination with bifunctional cross-linking reagents to investigate these interactions in more detail in a homologous environment. Using this approach, the direct interaction of SRP with nascent polypeptides that expose particularly hydrophobic targeting signals is demonstrated, suggesting that inner membrane proteins are the primary physiological substrate of the E. coli SRP. Evidence is presented that the overproduction of proteins that expose hydrophobic polypeptide stretches, titrates SRP. In addition, trigger factor is efficiently cross-linked to nascent polypeptides of different length and nature, some as short as 57 amino acid residues, indicating that it is positioned near the nascent chain exit site on the E. coli ribosome.

Bacterial Proteins↗

PubMed1996

The Escherichia coli K99 periplasmic chaperone FanE is a monomeric protein.

The monomeric or dimeric nature of the K99 periplasmic chaperone FanE was examined. The gene encoding FanE was subcloned in a pINIIIA1 derivative expression vector. A complementation experiment showed that the subcloned FanE was biologically functional. The protein was purified from the periplasm of cells harbouring the constructed plasmid. Automated Edman degradation experiments confirmed the predicted N-terminal amino acid sequence of FanE. A polyclonal mouse antiserum was raised against the FanE chaperone. The monomeric or oligomeric nature of the protein in the periplasm was studied by gel filtration, immunoblotting and chemical cross-linking experiments. The results indicated that FanE is a monomeric protein, in contrast to the K88 periplasmic chaperone.

Animals↗

PubMed1996

Isolation and characterization of chromosomal mTn 10 insertion mutations affecting K88 fimbriae production in Escherichia coli.

mTn 10 transposon mutagenesis of Escherichia coli producing K88 fimbria was carried out in order to identify host factors involved in the regulation of the fae (K88) operon and the production of K88 fimbriae. Five independent chromosomal insertion mutants were obtained which showed an increased expression of K88 fimbriae. Inverse PCR and nucleotide sequencing were carried out to characterize the mutations. One insertion affected the Ipp gene, encoding the major outer membrane lipoprotein. Another mutation was found to be located in the Irp gene, encoding the 'global' regulatory protein Lrp (leucine responsive regulatory protein). A third mutant was found to affect the expression of rfaF, encoding heptosyltransferase II, which resulted in a partially wild-type and partially Re-Rd1 type of LPS. A fourth mutation affected sseB, a gene involved in serine-sensitivity of E. coli cells. Another mutant contained an insertion in an unknown region of the E. coli genome. The mutants were further characterized with respect to K88 as well as K99 fimbriae production.

Amino Acid Sequence↗

PubMed1996

Binding of host iron-binding proteins and expression of iron-regulated membrane proteins by different serotypes of Pasteurella multocida causing haemorrhagic septicaemia.

Pasteurella multocida strains of serotype B: 2,5, B: 3,4 and E: 2,5 are associated with haemorrhagic septicaemia in domestic and feral ruminants. These strains were investigated for their ability to bind transferrin, lactoferrin and haemoglobin and for their ability to use these host iron-binding proteins as a source of iron. All strains bound haemoglobin, none of the strains bound lactoferrin, whereas transferrin binding was restricted to serotype B: 2,5 strains. Growth experiments indicated that transferrin (serotype B: 2,5) and haemoglobin could restore bacterial growth under iron-depleted conditions. Two distinct serotype-independent profiles of iron-regulated membrane proteins were expressed in vitro as well as in vivo.

Animals↗

PubMed1996

The N-terminal beta-barrel domain of the Escherichia coli K88 periplasmic chaperone FaeE determines fimbrial subunit recognition and dimerization.

The K88 periplasmic chaperone FaeE is a homodimer, whereas the K99 chaperone FanE is a monomer. The structural requirements for dimerization of the K88 fimbrial periplasmic chaperone and for fimbrial subunit-binding specificity were investigated by analysis of mutant chaperones. FaeE contains a C-terminal extension of 19 amino acid residues when compared to FanE and most other fimbrial chaperones. A C-terminal truncate of the K88 chaperone FaeE was constructed that lacked 19 C-terminal amino acid residues. Expression and complementation experiments revealed that this C-terminal shortened chaperone was still functional in binding the K88 major subunit FaeG and K88 biosynthesis. Two hybrid chaperones were constructed. Each hybrid protein contained one beta-barrel domain of FaeE and the other beta-barrel domain of FanE (Fae/FanE or Fan/FaeE, respectively). Expression and complementation experiments revealed that the Fae/FanE but not the Fan/FaeE hybrid chaperone was functional in the formation of K88 fimbriae. The Fan/FaeE hybrid chaperone was active in the bio-synthesis of K99 fimbriae. The truncated FaeE mutant chaperone and the hybrid Fae/FanE chaperone were able to form stable periplasmic protein complexes with the K88 major fimbrial subunit FaeG. Cross-linking experiments suggested that the C-terminal shortened chaperone and the Fae/FanE hybrid chaperone were homodimers, as is the wild-type K88 chaperone. Altogether, the data suggested that the N-terminal beta-barrel domain of a fimbrial chaperone determines subunit specificity. In the case of the K88 periplasmic chaperone, this N-terminal domain also determines dimerization of the protein.

Amino Acid Sequence↗

PubMed1996

Molecular and structural aspects of fimbriae biosynthesis and assembly in Escherichia coli.

Fimbriae are long filamentous polymeric protein structures located at the surface of bacterial cells. They enable the bacteria to bind to specific receptor structures and thereby to colonise specific surfaces. Fimbriae consist of so-called major and minor subunits, which form, in a specific order, the fimbrial structure. In this review emphasis is put on the genetic organisation, regulation and especially on the biosynthesis of fimbriae of enterotoxigenic Escherichia coli strains, and more in particular on K88 and related fimbriae, with ample reference to well-studied P and type 1 fimbriae. The biosynthesis of these fimbriae requires two specific and unique proteins, a periplasmic chaperone and an outer membrane located molecular usher ('doorkeeper'). Molecular and structural aspects of the secretion of fimbrial subunits across the cytoplasmic membrane, the interaction of these subunits with periplasmic molecular chaperone, their translocation to the inner site of the outer membrane and their interaction with the usher protein, as well as the (ordered) translocation of the subunits across the outer membrane and their assembly into a growing fimbrial structure will be described. A model for K88 fimbriae is presented.

Amino Acid Sequence↗

PubMed1996

Molecular characterization of a heme-binding protein of Bacteroides fragilis BE1.

An iron-repressible 44-kDa outer membrane protein plays a crucial role in the acquisition of heme by the anaerobic bacterium Bacteroides fragilis. The DNA sequence of the gene encoding the 44-kDa protein (hupA) was determined. The hupA gene encodes a protein of 431 amino acid residues with a calculated molecular mass of 48,189 Da. The hupA gene is preceded by an open reading frame of 480 bp that probably encodes a protein with a calculated molecular mass of 18,073 Da. hupA and this open reading frame are likely organized in an operon, and a sequence homologous to the Escherichia coli consensus Fur box was present in the putative promoter region of the operon. Heme-binding studies showed that HupA binds heme. Analysis of the deduced amino acid sequence revealed signature heme-binding consensus motifs, characteristic of heme lyases. Subcellular localization studies in E. coli revealed that HupA was mainly found in the cytoplasmic membrane but not in the outer membrane of E. coli. This suggested that B. fragilis uses another strategy for the translocation of this outer membrane protein across its cell envelope than E. coli does. HupA did not have significant homology with other putative bacterial heme receptors.

Amino Acid Sequence↗

PubMed1995

Early events in preprotein recognition in E. coli: interaction of SRP and trigger factor with nascent polypeptides.

In Escherichia coli, components of a signal recognition particle (SRP) and its receptor have been identified which appear to be essential for efficient translocation of several proteins. In this study we use cross-linking to demonstrate that E. coli SRP interacts with a variety of nascent presecretory proteins and integral inner membrane proteins. Evidence is presented that the interaction is correlated with the hydrophobicity of the core region of the signal sequence and thereby with its ability to promote transport in vivo. A second E. coli component, which is identified as trigger factor, can be efficiently cross-linked to all tested nascent chains derived from both secreted and cytosolic proteins. We propose that SRP and trigger factor act as secretion-specific and general molecular chaperone respectively, early in protein synthesis.

Amino Acid Isomerases↗

PubMed1995

The stable bacteriocin release protein signal peptide, expressed as a separate entity, functions in the release of cloacin DF13.

The pCloDF13 encoded bacteriocin release protein (BRP) plays a role in the release of the bacteriocin cloacin DF13. The BRP signal peptide is stable after cleavage, and accumulates in the cytoplasmic membrane. A BRP which is correctly targeted by the unstable murecin lipoprotein signal peptide (Lpp-BRP) is not capable of inducing the release of cloacin DF13. To investigate the role of the stable BRP signal peptide in the release of cloacin DF13, the stable BRP signal peptide and the Lpp-BRP were expressed in trans in cells also producing cloacin DF13. Expression and release experiments indicate that the stable signal peptide can complement the Lpp-BRP in the release of cloacin DF 13.

Bacterial Proteins↗

PubMed1995

Optimization of bacteriocin-release-protein-induced protein release by Escherichia coli: extracellular production of the periplasmic molecular chaperone FaeE.

Expression of the pCloDF13-encoded bacteriocin-release protein (BRP) results in the release of periplasmic proteins into the culture medium. The BRP-mediated release of a periplasmic protein was investigated and optimized. As a periplasmic model protein, the 50-kDa dimeric E., coli fimbrial molecular chaperone FaeE was used. Plasmids were constructed for the simultaneous expression of the BRP and FaeE, controlled by independently inducible promoters. The efficiency of FaeE release increased when the BRP was targeted by the unstable murein lipoprotein signal peptide, instead of by its own stable signal, peptide. Furthermore, optimal efficacy of FaeE release was found when cells of E. coli strain C600 were used, which harboured one plasmid encoding both FaeE and BRP instead of two separate plasmids and which were cultured at 37 degrees C in broth supplemented with MgCl2. Maximal production levels of 21 mg FaeE/l culture were obtained.

Bacterial Proteins↗

PubMed1995

Expression of the pCloDF13 encoded bacteriocin release protein or its stable signal peptide causes early effects on protein biosynthesis and Mg2+ transport.

The effect of the pCloDF13 encoded bacteriocin release protein (BRP) on Escherichia coli cell lethality was studied. Induction of the BRP resulted in a strong inhibition of the incorporation of radioactive labeled amino acids and affected the transport of Mg2+ ions. Similar effects were obtained when the BRP stable signal peptide was expressed as a separate entity. Kinetic studies revealed that these effects occurred prior to quasi-lysis and release of cloacin DF13. The results indicated that the BRP induced cell lethality is caused by early effects on protein synthesis and Mg2+ transport, due to the accumulation of stable BRP signal peptides in the cytoplasmic membrane.

Amino Acids↗

PubMed1995

The Escherichia coli K88 periplasmic chaperone FaeE forms a heterotrimeric complex with the minor fimbrial component FaeH and with the minor fimbrial component FaeI.

K88ab fimbriae are long polymeric protein structures mainly composed of FaeG proteins. The Escherichia coli K88 periplasmic chaperone FaeE is a homodimer and forms a heterotrimeric complex with the K88 major fimbrial component FaeG in the periplasm. In this study the direct interaction of FaeE and the minor K88 fimbrial subunits FaeH and FaeI were investigated. The faeH gene and the faeI gene were subcloned in a pINIIIA1-derivative vector containing the faeE gene. SDS-PAGE using normal and gradient gels and immunoblotting revealed that the subcloned genes were expressed in the periplasm. Analyses of periplasmic fractions by native gel electrophoresis and isoelectric focusing (IEF) showed that FaeE and FaeH, as well as FaeE and FaeI formed protein complexes. These complexes were isolated and purified by FPLC or IEF and native gel electrophoresis. The stoichiometry of the proteins in these complexes was studied by automated Edman degradation and gel image analysis. The results showed that FaeE and FaeH, and FaeE and FaeI formed heterotrimeric E2H and E2I complexes, respectively. In addition to the E2H complex, cells expressing FaeE and FaeH accumulated unbound FaeH in their periplasm. In contrast to the E2G complex, the purified E2H complex was not stable and was partly dissociated in the experimental conditions used, suggesting that the interaction between FaeE and FaeH is not as strong as the interaction of FaeE and FaeG.

Amino Acid Sequence↗

PubMed1995

Subcellular localization and topology of the K88 usher FaeD in Escherichia coli.

The subcellular localization of the K88 usher FaeD was studied in Escherichia coli whole cells by using isopycnic sucrose density gradient centrifugation of isolated membranes, the detergents Triton X-100 and sodium lauryl sarcosinate and immunoblotting with a specific FaeD antiserum. Cells containing the complete K88 operon, as well as cells containing the subcloned faeD gene in various expression vectors, were used. Most of the FaeD was present in the outer membranes in a detergent-resistant form. Agglutination experiments with E. coli cells expressing FaeD confirmed an outer membrane localization and indicated the presence of FaeD at the cell surface. Automated Edman degradation indicated that the mature FaeD contained 777 amino acid residues and confirmed that FaeD is synthesized with a rather long signal sequence of 35 amino acid residues. Twelve different FaeD-PhoA fusion proteins were prepared and characterized by nucleotide sequencing and immunoblotting. Most of these fusion sites were located in the amino-terminal and carboxyl-terminal regions of FaeD. Six amino-terminal fusion proteins were soluble proteins in the periplasm, whereas the other fusion proteins were associated with the outer membrane. The protease accessibility of FaeD and of the six outer membrane-bound FaeD-PhoA fusion proteins was studied using whole cells, cells with permeabilized outer membranes, and isolated membranes. Collagenase H, kallikrein, trypsin and proteinase K were used. Based on the results of these experiments and computer predictions, a model for the membrane topology of FaeD was developed in which FaeD contains a large central domain containing 24 membrane-spanning segments and two relatively large periplasmic regions, at the amino-terminal and carboxyl-terminal end of the protein, respectively.

Amino Acid Sequence↗

PubMed1995

Bacteriocin release proteins: mode of action, structure, and biotechnological application.

The mechanism by which Gram-negative bacteria like Escherichia coli secrete bacteriocins into the culture medium is unique and quite different from the mechanism by which other proteins are translocated across the two bacterial membranes, namely through the known branches of the general secretory pathway. The release of bacteriocins requires the expression and activity of a so-called bacteriocin release protein and the presence of the detergent-resistant phospholipase A in the outer membrane. The bacteriocin release proteins are highly expressed small lipoproteins which are synthesized with a signal peptide that remains stable and which accumulates in the cytoplasmic membrane after cleavage. The combined action of these stable, accumulated signal peptides, the lipid-modified mature bacteriocin release proteins (BRPs) and phospholipase A cause the release of bacteriocins. The structure and mode of action of these BRPs as well as their application in the release of heterologous proteins by E. coli is described in this review.

Amino Acid Sequence↗