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A J Driessen

Publications and source records attributed to A J Driessen.

At least 55 records · Page 3Linked to original sources
PubMed1999

Complementary and overlapping selectivity of the two-peptide bacteriocins plantaricin EF and JK.

Plantaricin EF and JK are both two-peptide bacteriocins produced by Lactobacillus plantarum C11. The mechanism of plantaricin EF and JK action was studied on L. plantarum 965 cells. Both plantaricins form pores in the membranes of target cells and dissipate the transmembrane electrical potential (Deltapsi) and pH gradient (DeltapH). The plantaricin EF pores efficiently conduct small monovalent cations, but conductivity for anions is low or absent. Plantaricin JK pores show high conductivity for specific anions but low conductivity for cations. These data indicate that L. plantarum C11 produces bacteriocins with complementary ion selectivity, thereby ensuring efficient killing of target bacteria.

Anions↗
PubMed1999

Temporal expression of the Bacillus subtilis secA gene, encoding a central component of the preprotein translocase.

In Bacillus subtilis, the secretion of extracellular proteins strongly increases upon transition from exponential growth to the stationary growth phase. It is not known whether the amounts of some or all components of the protein translocation apparatus are concomitantly increased in relation to the increased export activity. In this study, we analyzed the transcriptional organization and temporal expression of the secA gene, encoding a central component of the B. subtilis preprotein translocase. We found that secA and the downstream gene (prfB) constitute an operon that is transcribed from a vegetative (sigmaA-dependent) promoter located upstream of secA. Furthermore, using different independent methods, we found that secA expression occurred mainly in the exponential growth phase, reaching a maximal value almost precisely at the transition from exponential growth to the stationary growth phase. Following to this maximum, the de novo transcription of secA sharply decreased to a low basal level. Since at the time of maximal secA transcription the secretion activity of B. subtilis strongly increases, our results clearly demonstrate that the expression of at least one of the central components of the B. subtilis protein export apparatus is adapted to the increased demand for protein secretion. Possible mechanistic consequences are discussed.

Adenosine Triphosphatases↗
PubMed1999

Preprotein translocation by a hybrid translocase composed of Escherichia coli and Bacillus subtilis subunits.

Bacterial protein translocation is mediated by translocase, a multisubunit membrane protein complex that consists of a peripheral ATPase SecA and a preprotein-conducting channel with SecY, SecE, and SecG as subunits. Like Escherichia coli SecG, the Bacillus subtilis homologue, YvaL, dramatically stimulated the ATP-dependent translocation of precursor PhoB (prePhoB) by the B. subtilis SecA-SecYE complex. To systematically determine the functional exchangeability of translocase subunits, all of the relevant combinations of the E. coli and B. subtilis secY, secE, and secG genes were expressed in E. coli. Hybrid SecYEG complexes were overexpressed at high levels. Since SecY could not be overproduced without SecE, these data indicate a stable interaction between the heterologous SecY and SecE subunits. E. coli SecA, but not B. subtilis SecA, supported efficient ATP-dependent translocation of the E. coli precursor OmpA (proOmpA) into inner membrane vesicles containing the hybrid SecYEG complexes, if E. coli SecY and either E. coli SecE or E. coli SecG were present. Translocation of B. subtilis prePhoB, on the other hand, showed a strict dependence on the translocase subunit composition and occurred efficiently only with the homologous translocase. In contrast to E. coli SecA, B. subtilis SecA binds the SecYEG complexes only with low affinity. These results suggest that each translocase subunit contributes in an exclusive manner to the specificity and functionality of the complex.

Adenosine Triphosphatases↗
PubMed1999

Sulfate transport in Penicillium chrysogenum: cloning and characterization of the sutA and sutB genes.

In industrial fermentations, Penicillium chrysogenum uses sulfate as the source of sulfur for the biosynthesis of penicillin. By a PCR-based approach, two genes, sutA and sutB, whose encoded products belong to the SulP superfamily of sulfate permeases were isolated. Transformation of a sulfate uptake-negative sB3 mutant of Aspergillus nidulans with the sutB gene completely restored sulfate uptake activity. The sutA gene did not complement the A. nidulans sB3 mutation, even when expressed under control of the sutB promoter. Expression of both sutA and sutB in P. chrysogenum is induced by growth under sulfur starvation conditions. However, sutA is expressed to a much lower level than is sutB. Disruption of sutB resulted in a loss of sulfate uptake ability. Overall, the results show that SutB is the major sulfate permease involved in sulfate uptake by P. chrysogenum.

Amino Acid Sequence↗
PubMed1999

Functional identification of the product of the Bacillus subtilis yvaL gene as a SecG homologue.

Protein export in Escherichia coli is mediated by translocase, a multisubunit membrane protein complex with SecA as the peripheral subunit and the SecY, SecE, and SecG proteins as the integral membrane domain. In the gram-positive bacterium Bacillus subtilis, SecA, SecY, and SecE have been identified through genetic analysis. Sequence comparison of the Bacillus chromosome identified a potential homologue of SecG, termed YvaL. A chromosomal disruption of the yvaL gene results in mild cold sensitivity and causes a beta-lactamase secretion defect. The cold sensitivity is exacerbated by overexpression of the secretory protein alpha-amylase, whereas growth and beta-lactamase secretion are restored by coexpression of yvaL or the E. coli secG gene. These results indicate that the yvaL gene codes for a protein that is functionally homologous to SecG.

Amino Acid Sequence↗
PubMed1999

Differential dependence of levansucrase and alpha-amylase secretion on SecA (Div) during the exponential phase of growth of Bacillus subtilis.

SecA, the translocation ATPase of the preprotein translocase, accounts for 0.25% of the total protein in a degU32(Hy) Bacillus subtilis strain in logarithmic phase. The SecA level remained constant irrespective of the demand for exoprotein production but dropped about 12-fold during the late stationary phase. Modulation of the level of functional SecA during the exponential phase of growth affected differently the secretion of levansucrase and alpha-amylase overexpressed under the control of the sacB leader region. The level of SecA was reduced in the presence of sodium azide and in the div341 thermosensitive mutant at nonpermissive temperatures. Overproduction of SecA was obtained with a multicopy plasmid bearing secA. The gradual decrease of the SecA level reduced the yield of secreted levansucrase with a concomitant accumulation of unprocessed precursor in the cells, while an increase in the SecA level resulted in an elevation of the production of exocellular levansucrase. In contrast, alpha-amylase secretion was almost unaffected by high concentrations of sodium azide or by very low levels of SecA. Secretion defects were apparent only under conditions of strong SecA deprivation of the cell. These data demonstrate that the alpha-amylase and levansucrase precursors markedly differ in their dependency on SecA for secretion. It is suggested that these precursors differ in their binding affinities for SecA.

Adenosine Triphosphatases↗
PubMed1999

Protein targeting to the bacterial cytoplasmic membrane.

Proteins that perform their activity within the cytoplasmic membrane or outside this cell boundary must be targeted to the translocation site prior to their insertion and/or translocation. In bacteria, several targeting routes are known; the SecB- and the signal recognition particle-dependent pathways are the best characterized. Recently, evidence for the existence of a third major route, the twin-Arg pathway, was gathered. Proteins that use either one of these three different pathways possess special features that enable their specific interaction with the components of the targeting routes. Such targeting information is often contained in an N-terminal extension, the signal sequence, but can also be found within the mature domain of the targeted protein. Once the nascent chain starts to emerge from the ribosome, competition for the protein between different targeting factors begins. After recognition and binding, the targeting factor delivers the protein to the translocation sites at the cytoplasmic membrane. Only by means of a specific interaction between the targeting component and its receptor is the cargo released for further processing and translocation. This mechanism ensures the high-fidelity targeting of premembrane and membrane proteins to the translocation site.

Bacterial Outer Membrane Proteins↗
PubMed1998

Plantaricin A is an amphiphilic alpha-helical bacteriocin-like pheromone which exerts antimicrobial and pheromone activities through different mechanisms.

Production of bacteriocins by lactic acid bacteria is in some cases regulated by a quorum sensing mechanism that involves a secreted bacteriocin-like peptide pheromone. In the case of Lactobacillus plantarum C11, this pheromone, the 26-mer plantaricin A (PlnA), has the interesting property of having both bacteriocin and pheromone activities. To gain insight into how PlnA functions as a pheromone and as a bacteriocin, the L- and D-enantiomers of an N-terminally truncated form of PlnA were synthesized (PlnA-22L and PlnA-22D; PlnA-22L has full biological activity). With circular dichroism, it was shown that the two peptides are unstructured in aqueous solution, but they adopt mirror-image amphiphilic helical structures in the presence of trifluoroethanol and membrane-mimicking entities such as micelles of dodecylphosphocholine and negatively charged Ole2GroPGro liposomes, but not in the presence of zwitterionic Ole2GroPCho liposomes. Thus, the negative charge on the membrane is important for structuring of the (positively charged) PlnA peptides. In terms of in vivo antimicrobial activity, PlnA-22L and PlnA-22D behaved almost identically. Likewise, the peptides dissipated the membrane potential and the transmembrane pH gradient in sensitive cells equally effectively. PlnA-22L induced bacteriocin production in L. plantarum C11 (i.e., displayed pheromone activity), the level of induction being clearly dose-dependent. PlnA-22D did not display pheromone activity, but, at high concentrations, was able to inhibit the pheromone activity of PlnA-22L. The results indicate that the antimicrobial activity of PlnA does not require chiral interactions and is mediated through the formation of a strongly amphiphilic alpha-helical structure. In contrast, PlnA's pheromone activity is dependent on a chiral interaction between the amphiphilic helix (PlnA-22L) and a receptor protein. One may speculate that PlnA is an evolutionary intermediate between a true bacteriocin and a pheromone.

Amino Acid Sequence↗
PubMed1998

Biochemical characterization of the SecA protein of Streptomyces lividans--interaction with nucleotides, binding to membrane vesicles and in vitro translocation of proAmy protein.

The SecA protein of Streptomyces lividans was purified to near electrophoretic homogeneity by means of FPLC from an overproducing strain harbouring plasmid pULA400, in which the secA gene (Blanco, J., Coque, J. J. R. & Martín, J. F. (1996) Gene (Amst.) 176, 61-65) was expressed from the strong promoter of the Streptomyces griseus saf gene. The native form of SecA was shown to be a dimer (Mr 209 kDa) by gel filtration. It crossreacted with antibodies raised against Escherichia coli or Bacillus subtilis SecA proteins. Purified S. lividans SecA showed a low endogenous ATPase activity that was stimulated by addition of a S. lividans lipid fraction. SecA contains a high-affinity and a low-affinity nucleotide-binding site (NBS). [Alpha-32P]ATP could be crosslinked by ultraviolet radiation at the high-affinity site. The intrinsic tryptophan fluorescence of SecA decreased on addition of increasing concentrations of ADP and reached a saturation level at about 1 microM (the range of saturation at the NBS I). The calculated Kd of the high-affinity binding site for ADP was 150 nM. Millimolar concentrations of ATP or ADP did not render the S. lividans SecA protein resistant to V8 protease degradation, in contrast to what occurs with the E. coli and B. subtilis SecA proteins. SecA was found to bind to urea-washed S. lividans membrane vesicles with high-affinity, i.e. 10 nM. SecA-dependent binding of E. coli SecB to membrane vesicles was observed when E. coli SecA was used, but not with the S. lividans SecA, suggesting that this interaction may be specific for the Gram-negative bacteria. An in vitro translocation system has been developed using inverted membrane vesicles of S. lividans. SecA supported in vitro translocation of proAmy into S. lividans membrane vesicles in an ATP-dependent manner.

Adenosine Diphosphate↗
PubMed1998

Translocase-bound SecA is largely shielded from the phospholipid acyl chains.

Protein translocation in Escherichia coli is mediated by the SecA ATPase bound to the SecYEG membrane protein complex. SecA translocation ATPase activity as well as protein translocation is dependent on the presence of negatively charged lipids. By using a phospholipid with an acyl chain linked photoactivatable group, the lipid accessibility of SecA bound at the translocase was explored. SecA bound to lipid vesicles containing negatively charged lipids was found to be readily accessible for labeling by the photoactivatable phospholipid. The presence of an excess amount of SecYEG complex resulted in a remarkable reduction in the amount of lipid-accessible SecA irrespective of the nucleotide-bound form of SecA. These data demonstrate that the SecYEG-bound SecA is largely shielded from the phospholipid acyl chains and suggest the presence of two distinct pools of membrane-bound SecA that differ in the degree of lipid association.

Adenosine Triphosphatases↗
PubMed1998

Translocation of the precursor of alpha-amylase into Bacillus subtilis membrane vesicles.

Bacilli vigorously secrete proteins into the extracellular environment, and are therefore used in industry for the bulk production of enzymes such as proteinases and amylases. Studies on the mechanism of protein translocation in these Gram-positive bacteria have been hampered by the lack of an in vitro system. To establish such a system for Bacillus subtilis, everted membranes were isolated from a strain deficient in the alkaline and neutral protease. Translocation-competent membrane vesicles were obtained only when a broad range proteinase-inhibitor cocktail was used during membrane isolation. This method efficiently prevented proteolysis of SecY, one of the core integral membrane components of the preprotein translocase. Translocation of the urea-denatured precursor of the Bacillus licheniformis alpha-amylase, preAmyL, and B. subtilis alkaline phosphatase, prePhoB, into the B. subtilis membrane vesicles require the B. subtilis SecA protein and are driven by ATP hydrolysis and the proton-motive force. These studies establish an authentic in vitro translocation system for protein secretion in B. subtilis.

Adenosine Triphosphate↗
PubMed1998

PrlA4 prevents the rejection of signal sequence defective preproteins by stabilizing the SecA-SecY interaction during the initiation of translocation.

In Escherichia coli, precursor proteins are translocated across the cytoplasmic membrane by translocase. This multisubunit enzyme consists of a preprotein-binding and ATPase domain, SecA, and the SecYEG complex as the integral membrane domain. PrlA4 is a mutant of SecY that enables the translocation of preproteins with a defective, or missing, signal sequence. Inner membranes of the prlA4 strain efficiently translocate Delta8proOmpA, a proOmpA derivative with a non-functional signal sequence. Owing to the signal sequence mutation, Delta8proOmpA binds to the translocase with a lowered affinity and the recognition is not restored by the prlA4 SecY. At the ATP-dependent initiation of translocation, the binding affinity of SecA for SecYEG is lowered causing the premature loss of bound preproteins from the translocase. The prlA4 membranes, however, bind SecA with a much higher affinity than the wild-type, and during initiation, the SecA and preprotein remain bound at the translocation site allowing an improved efficiency of translocation. It is concluded that the prlA4 strain prevents the rejection of defective preproteins from the export pathway by stabilizing SecA at the SecYEG complex.

Adenosine Triphosphatases↗
PubMed1998

Interaction between SecA and SecYEG in micellar solution and formation of the membrane-inserted state.

Preprotein translocation in Escherichia coli is mediated by the translocase with SecA as peripheral ATPase and SecY, SecE, and SecG as membrane domain. To facilitate large-scale purification of the SecYEG heterotrimer, SecY was fused at its amino terminus with a hexahistidine tag and co-overexpressed with SecE and SecG. The presence of the His tag allowed purification of homogeneously pure SecYEG complex by a single anion-exchange chromatographic step starting from octyl glucoside-solubilized inner membranes. Endogenous levels of SecD and SecF copurified with the SecYEG protein. Purified SecYEG complex retained a nativelike, alpha-helical conformation in octyl glucoside and in micellar solution binds SecA with high affinity. In the presence of the nonhydrolyzable nucleotide analogue adenosine 5'-(beta, gamma-imidotriphosphate), octyl glucoside-solubilized SecYEG is nearly as effective as the reconstituted enzyme in inducing the formation of a proteinase K-protected 30 kDa fragment of 125I-labeled SecA, while SecYEG is proteolyzed to fragments smaller than 6 kDa. These data demonstrate that the 30-kDa SecA fragment is not protected by the lipid phase nor by SecYEG but rather indicate that it represents a SecYEG- and nucleotide-induced stable conformational state of a SecA domain.

Adenosine Triphosphatases↗
PubMed1998

Bioenergetics and cytoplasmic membrane stability of the extremely acidophilic, thermophilic archaeon Picrophilus oshimae.

Picrophilus oshimae is an extremely acidophilic, thermophilic archaeon that grows optimally at 60 degrees C and at pH 0.7. It is an obligatory acidophile that does not grow at pH values above 4.0. The proton motive force in respiring cells is composed of a large transmembrane pH gradient, inside less acid, and a reversed transmembrane electrical potential, inside positive. Cells maintain an intracellular pH at around 4.6 at extracellular pH values ranging from 0.8 to 4.0. Above pH 4.0 cells lyse rapidly and lose their viability. Liposomes prepared from lipids derived from P. oshimae have an extremely low proton permeability at acidic pH. However, at neutral pH, the lipids are unable to assemble into regular liposomal structures. These observations suggest that the loss of viability and cell integrity above pH 4.0 is due to an impairment of the barrier function of the cytoplasmic membrane.

Cell Membrane↗
PubMed1998

The essence of being extremophilic: the role of the unique archaeal membrane lipids.

In extreme environments, mainly Archaea are encountered. The archaeal cytoplasmic membrane contains unique ether lipids that cannot easily be degraded, are temperature- and mechanically resistant, and highly salt tolerant. Moreover, thermophilic and extreme acidophilic Archaea possess membrane-spanning tetraether lipids that form a rigid monolayer membrane which is nearly impermeable to ions and protons. These properties make the archaeal lipid membranes more suitable for life and survival in extreme environments than the ester-type bilayer lipids of Bacteria or Eukarya.

Archaea↗
PubMed1998

The Sec system.

Proteins designated to be secreted by Escherichia coli are synthesized with an amino-terminal signal peptide and associate as nascent chains with the export-specific chaperone SecB. Translocation occurs at a multisubunit membrane-bound enzyme termed translocase, which consists of a peripheral preprotein-binding site and an ATPase domain termed SecA, a core heterotrimeric integral membrane protein complex with SecY, SecE and SecG as subunits, and an accessory integral membrane protein complex containing SecD and SecF. Major new insights have been gained into the cascade of preprotein targeting events and the enzymatic mechanism or preprotein translocation. It has become clear that preproteins are translocated in a stepwise fashion involving large nucleotide-induced conformational changes of the molecular motor SecA that propels the translocation reaction.

Adenosine Triphosphatases↗
PubMed1998

Preprotein transfer to the Escherichia coli translocase requires the co-operative binding of SecB and the signal sequence to SecA.

In Escherichia coli, precursor proteins are targeted to the membrane-bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxy-terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo, are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA-dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (delta8proOmpA) bearing a non-functional signal sequence. Delta8proOmpA is not translocated across wild-type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prIA4 strain. SecB reduces the translocation of delta8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB-SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase.

Adenosine Triphosphatases↗