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B Witholt

Publications and source records attributed to B Witholt.

At least 109 records · Page 6Linked to original sources
PubMed1986

Induction kinetics and cell surface distribution of Escherichia coli lipoprotein under lac promoter control.

The induction kinetics and surface accessibility of the outer membrane lipoprotein were studied in an Escherichia coli strain with the lpp gene under control of the lac promoter. Free lipoprotein appeared rapidly after induction with isopropyl-beta-D-thiogalactopyranoside and reached a steady-state level after 30 min. The newly induced lipoprotein was slowly bound to the peptidoglycan layer. Immunological methods were developed to detect lipoprotein accessible at the cell surface after various pretreatments as well as peptidoglycan-bound lipoprotein at the surface of isolated peptidoglycan sacculi with specific antibodies in combination with 125I-protein A. With these methods an increase in lipoprotein molecules at the cell surface and bound to the peptidoglycan sacculus could be detected following induction. The topology of newly synthesized lipoprotein was examined in thin sections as well as at the cell surface and the surface of the peptidoglycan sacculus with immunoelectron microscopy. Ultrathin cell sections, whole cells, and isolated peptidoglycan sacculi showed lipoprotein distributed homogeneously over the entire surface.

Bacterial Outer Membrane Proteins↗
PubMed1985

Heat-labile enterotoxin in Escherichia coli. Kinetics of association of subunits into periplasmic holotoxin.

We have investigated the assembly of the heat-labile enterotoxin (LT) subunits after their processing and segregation into the periplasmic space as mature LT A and LT B polypeptides. LT B starts associating into oligomers during or immediately after translocation through the cytoplasmic membrane. Binding to LT A occurs immediately after oligomerization. Over 80% of the LT B subunits have oligomerized, and over 50% have associated with LT A into holotoxin within 1 min after synthesis. The fate of newly synthesized LT A is totally different. There is an extensive overproduction of LT A relative to LT B and after membrane translocation it becomes part of a periplasmic pool of free LT A. It is then bound by LT B oligomers or degraded at such a rate that the free periplasmic LT A disappears from the pool with a half-time of 20-25 min. About half of the LT A is incorporated into holotoxin, while the other half is degraded. We conclude that LT subunits are translocated and processed in a ratio of about 2 A to 5 B. Since free LT A is either degraded slowly or bound to newly synthesized LT B oligomers, the net result is a steady state of 1.4 to 1.7 A subunits to 5 B subunits in the periplasm. About 60% of this LT A is bound by LT B to form periplasmic holotoxin with a subunit ratio of about 1 A to 5 B. The remaining 40% of periplasmic LT A occurs free.

Bacterial Toxins↗
PubMed1985

Heat-labile enterotoxin of Escherichia coli. Characterization of different crystal forms.

Heat-labile enterotoxin (LT) was obtained in large quantities (several-gram amounts) and great purity from Escherichia coli C600 carrying the LT-coding multicopy plasmid EWD299. By growing this strain on a medium that allows high cell densities in the early stationary phase, we increased the net LT production per milliliter by a factor of 200, compared to natural porcine enterotoxigenic E. coli. Adsorption and redesorption on Controlled-Pore Glass usually resulted in a 50-100-fold purification of LT in one simple step, which was followed by established purification procedures. LT shows a natural tendency to form large crystals, which, however, are disordered. After numerous trials, conditions were found which virtually eliminated the disorder. Much better crystals were obtained by employing CdCl2 and KF as coprecipitating agents. CdCl2 yielded crystals which did not contain A subunits as judged by electrophoresis of dissolved crystals. Adding KF results in beautifully shaped crystals which diffracted beyond 2 A and are suitable for a high resolution structure determination.

Bacterial Toxins↗
PubMed1985

Developments in biotechnology of relevance to drinking water preparation.

This paper discusses strategies to increase the feasibility of microorganisms for the removal of toxic xenobiotics from waste water and drinking water. Based on the principles of adaptational mutations and genetic exchange of catabolic activities, it becomes possible to select and engineer microorganisms that are suitable for the degradation of recalcitrant compounds. The detailed biochemical knowledge that is required for this is now rapidly evolving, and especially for the degradation of chlorinated organics several detoxifying dehalogenation mechanisms have been studied in detail. The feasibility of specialized bacteria for waste and water treatment will be dependent on the possibility to obtain stable performance and maintenance in treatment systems.

Adaptation, Physiological↗
PubMed1985

Degradation of halogenated aliphatic compounds by Xanthobacter autotrophicus GJ10.

A bacterium that is able to utilize a number of halogenated short-chain hydrocarbons and halogenated carboxylic acids as sole carbon source for growth was identified as a strain of Xanthobacter autotrophicus. The organism constitutively produces two different dehalogenases. One enzyme is specific for halogenated alkanes, whereas the other, which is more heat stable and has a higher pH optimum, is specific for halogenated carboxylic acids. Haloalkanes were hydrolyzed in cell extracts to produce alcohols and halide ions, and a route for the metabolism of 1,2-dichlorethane is proposed. Both dehalogenases show a broad substrate specificity, allowing the degradation of bromine- and chlorine-substituted organic compounds. The results show that X. autotrophicus may play a role in the degradation of organochlorine compounds and that hydrolytic dehalogenases may be involved in the microbial metabolism of short-chain halogenated hydrocarbons in microorganisms.

Biodegradation, Environmental↗
PubMed1985

Purification and characterization of hydrolytic haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.

A new enzyme, haloalkane dehalogenase, was isolated from the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10. The purified enzyme catalyzed the hydrolytic dehalogenation of n-halogenated C1 to C4 alkanes, including chlorinated, brominated, and iodinated compounds. The highest activity was found with 1,2-dichloroethane, 1,3-dichloropropane, and 1,2-dibromoethane. The enzyme followed Michaelis-Menten kinetics, and the Km for 1,2-dichloroethane was 1.1 mM. Maximum activity was found at pH 8.2 and 37 degrees C. Thiol reagents such as p-chloromercuribenzoate and iodoacetamide rapidly inhibited the enzyme. The protein consists of a single polypeptide chain of a molecular weight of 36,000, and its amino acid composition and N-terminal sequence are given.

Amino Acid Sequence↗
PubMed1984

Kinetics of synthesis, processing, and membrane transport of heat-labile enterotoxin, a periplasmic protein in Escherichia coli.

We report the detection in vivo of precursors to the A and the B subunits of the heat-labile enterotoxin (LT) in Escherichia coli. Both pre-LT A (Mr = 29,500) and pre-LT B (Mr = 13,500) are present in the spheroplast fraction of the bacteria after separation of the cells in spheroplasts and periplasm. Two smaller LT A related polypeptides (17 and 23 kDa) were also detected in the spheroplast fraction. Both were degraded with a half-time of about 40 s. Mature subunits (Mr = 27,500 for LT A, and 11,500 for LT B) are released from the spheroplasts soon after processing and occur freely in the periplasm not associated with the cytoplasmic or the outer membranes. Processing occurs mainly post-translationally for both the A and the B subunits. However, they show different kinetics of processing and subsequent segregation into the periplasm. Whereas pre-LT B is processed and released within seconds after chain termination, pre-LT A is processed and released more slowly, and a subfraction of mature LT A may reside in the cytoplasmic membrane for several minutes.

Bacterial Toxins↗
PubMed1984

Assembly pathway of newly synthesized LamB protein an outer membrane protein of Escherichia coli K-12.

The assembly of newly induced LamB protein (phage lambda receptor) was investigated in an operon fusion strain of Escherichia coli, in which the lamB gene is expressed under lac promoter control. The induction kinetics both for total cellular and for cell surface-exposed LamB protein were studied by immunochemical detection methods, using two distinct antisera directed against detergent-solubilized LamB trimers and completely denatured LamB monomers, respectively. Anti-trimer antibodies recognized both monomers and trimers, whereas anti-monomer antibodies only reacted with monomers. Provided appropriate solubilization conditions were used, both antisera were able to immunoprecipitate intracellular mature LamB protein quantitatively. Following induction, the first LamB antigenic determinants were detected after 60 to 80 seconds; detection of the newly synthesized protein by anti-monomer antibodies slightly preceded that by anti-trimer antibodies, a finding that could be partly explained by the observation that anti-monomer antibodies recognized a larger fraction of nascent LamB than did anti-trimer antibodies. Exposure of antigenic determinants at the cell surface was delayed for 30 to 50 seconds with respect to their synthesis. Therefore, either translocation or conformational changes must be rate-limiting in the series of processes that eventually convert the newly synthesized protein into its mature outer membrane state. LamB protein was found to occur in at least three clearly distinguishable states. State I is the LamB monomer, state II corresponds to a metastable trimer that dissociates in sodium dodecyl sulphate above 60 degrees C, and state III is the state LamB trimer that dissociates in sodium dodecyl sulphate only at temperatures above 90 degrees C. The chase kinetics of these states showed that conversion of newly synthesized LamB monomers to stable LamB trimers occurred in two stages: state I monomers were chased into metastable state II trimers rapidly (t 1/2 = 20 s), whereas stabilization of state II trimers to state III trimers was a relatively slow (t 1/2 = 5.7 min) process. Based on our results, a timing sequence in the assembly of outer membrane LamB protein is proposed.

Bacterial Outer Membrane Proteins↗
PubMed1984

High-sensitivity detection of newly induced LamB protein on the Escherichia coli cell surface.

The kinetics of the appearance at the cell surface of the outer membrane LamB protein after induction were determined by using specific antibodies and radioiodinated protein A as a probe. This was done in two different induction systems. First, LamB protein was induced in a wild-type strain by the simultaneous addition of cyclic AMP and maltose. Second, an operon fusion strain in which the lamB gene is expressed under lac promoter control was used; in this system, LamB protein can be induced by isopropyl-beta-D-thiogalactopyranoside. When uninduced cells were grown in glucose minimal medium, background expression of the lamB gene was found to be ca. 10-fold lower in lac-lamB cells than in wild-type cells. The level of LamB protein present in uninduced wild-type cells could, however, be reduced by supplementing the growth medium with Casamino Acids. After induction, the LamB protein appeared at the cell surface of both strains within a few minutes, and then the LamB level per cell increased linearly. The time lag in cell surface exposure of LamB protein differed slightly under both induction conditions: the LamB protein appeared at the surface of lac-lamB cells within 3 min of induction, whereas in wild-type cells it could not be detected earlier than after 4 to 5 min of induction.

Bacterial Outer Membrane Proteins↗
PubMed1984

Topography of the insertion of LamB protein into the outer membrane of Escherichia coli wild-type and lac-lamB cells.

The appearance of newly induced LamB protein at the cell surface of Escherichia coli was followed topographically by immuno-electron microscopy. LamB protein was induced in E. coli wild-type or lac-lamB cells for a short period of time (4 to 6 min), such that the overall level of LamB protein in induced cells was at least twofold higher than that in uninduced cells. Antibodies bound to LamB protein exposed at the cell surface were labeled with a protein A-gold probe, and the probe distribution in briefly induced cells was compared to that in uninduced cells. Analysis of large numbers of cells showed that newly inserted LamB protein appeared homogeneously over the entire cell surface, both in wild-type cells and in lac-lamB cells. A peak of insertion which was observed at the division site of the cell was also observed in the absence of induction and in control experiments in which a nonspecific probe was used. It is concluded therefore that insertion of LamB protein into the cell envelope of E. coli occurs at multiple sites over the entire cell surface. The average amount of LamB protein which appeared at the cell surface after induction was determined for various cell size classes. It was found that cells of various size classes all synthesized LamB protein after induction, indicating that synthesis of the protein was not restricted to cells in a particular stage of the cell cycle. However, the rate of LamB synthesis was found to vary during the cell cycle: this rate was constant regardless of cell size in nondividing cells, whereas it increased in dividing cells. It is concluded that the accumulation of newly induced LamB protein follows a linear pattern.

Bacterial Outer Membrane Proteins↗
PubMed1983

An in vitro system to study interactions between bacteria and epithelial cells at the molecular level.

This paper describes an experimental system to study interactions between porcine enterotoxigenic Escherichia coli (ETEC) and porcine intestinal epithelial cells in vitro at the molecular level. Radiolabelled bacteria or bacterial membrane fractions were incubated with brush borders prepared from purified epithelial cells, which were then washed repeatedly. The bacterial components removed by washing or retained by the brush borders were analysed to determine their composition and source. For this it was necessary to develop a minimal medium in which attachment factors of porcine ETEC could be radiolabelled. Furthermore, an improved method for the isolation of porcine intestinal epithelial cells was developed, since other procedures did not yield sufficiently pure preparations. The resulting method was rapid and yielded large quantities of viable epithelial cells, free from crypt cells and contaminating intestinal contents. Finally, we adapted existing procedures to isolate brush borders from these epithelial cells with special emphasis on the removal of nuclear and cytosolic material and on the isolation of morphologically intact brush borders. Using this system, mixtures of bacterial cytoplasmic and outer membranes were incubated with brush borders. Cytoplasmic membranes were easily removed by washing, while the outer membranes were not.

Adhesiveness↗
PubMed1983

Characterization of intracellular inclusions formed by Pseudomonas oleovorans during growth on octane.

The growth of Pseudomonas oleovorans on n-octane was characterized by the formation of intracellular structures. These inclusions were isolated and characterized. Morphologically, they resembled the poly-beta-hydroxybutyrate granules found in Bacillus cereus, as shown by freeze-fracture electron microscopy. The elemental analysis of isolated granules showed, however, that they do not contain poly-beta-hydroxybutyric acid. Instead, the analysis was consistent with a C8 polyester, which interpretation was supported by the fatty acid analysis of hydrolyzed granules. From the evidence presented here, we conclude that P. oleovorans forms poly-beta-hydroxyoctanoate granules when grown on n-octane.

Caprylates↗
PubMed1982

Attachment of lipoprotein to the murein of Escherichia coli.

The rate of incorporation of [3H]diaminopimelate into the lipoprotein attachment sites was compared to the rate of its incorporation into total acid-insoluble and dodecylsulphate-insoluble murein. In contrast to earlier findings, we could detect no significant difference in these rates of incorporation, suggesting that the lipoprotein molecules are not specifically excluded from the murein growth sites. The amount of lipoproteins bound to the murein increased when the cells entered the stationary phase of growth.

Bacterial Proteins↗
PubMed1981

Outer-membrane vesicles released by normally growing Escherichia coli contain very little lipoprotein.

The lipoprotein content of the outer-membrane medium vesicles, which are released from Escherichia coli during normal growth, was compared to the lipoprotein content of the corresponding cellular outer membranes. It was found that the medium vesicles contained only 35% free lipoprotein and almost none of the bound lipoprotein when compared with cellular outer membranes. Medium vesicles also had reduced amounts of protein II and a protein V (Mr = 16 000), while they contained large amounts of pore-forming proteins I and lamB. A mechanism is proposed in which outer membrane vesicles are formed when the outer membrane expands faster than the underlying peptidoglycan layer. The lack or enrichment of individual proteins in medium vesicles may be determined by their interactions with the peptidoglycan-bound lipoprotein complex.

Cell Membrane↗
PubMed1981

Identification of different forms of the murein-bound lipoprotein found in isolated outer membranes of Escherichia coli.

The identification of the free and murein-bound forms of the Escherichia coli lipoprotein on dodecylsulphate-polyacrylamide gels was systematically investigated by analyzing the low-molecular-weight proteins (Mr less than 20 000) of both cytoplasmic and outer membranes. The free form of the lipoprotein was identified on 15% polyacrylamide gels as the fastest migrating component (Mr = 7200-7500) of isolated outer membranes; it could be separated from a small cytoplasmic membrane protein (Mr = 6500) which was probably identical to the dicyclohexylcarbodiimide binding proteolipid of the membrane-bound ATPase. Lysozyme treatment of both outer membranes and murein sacculi failed to convert the murein-bound lipoprotein into a fragment of uniform size; instead the bound form appeared as a series of bands consisting of lipoprotein bound to one, two,...eight murein subunits. The composition of this ladder depended on the method used to isolate outer membranes. Beside these lipoprotein bands the outer membrane contained two other proteins, III and V; the relation of these proteins to previously described proteins is discussed.

Bacterial Proteins↗
PubMed1981

Conversion of free lipoprotein to the murein-bound form.

The time-course of the conversion of free lipoprotein to the murein-bound form was followed after pulsing growing cells of Escherichia coli for 2 min with [35S]methionine. The conversion of the free to the bound form was measured by two independent methods. First, the appearance of murein-bound lipoprotein was determined in isolated murein sacculi after extraction with hot 4% sodium dodecylsulphate. Second, the ratio of various forms of the bound lipoprotein to the free lipoprotein was determined in outer membranes. Both methods indicated that the half-maximal amount of bound lipoprotein was reached within one-sixth of a generation time.

Cell Membrane↗