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Biomedical subjects

W Boos

Publications and source records attributed to W Boos.

At least 127 records · Page 7Linked to original sources
PubMed1982

Mapping of two ugp genes coding for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli.

Two genes, ugpA and ugpB, coding for a binding protein-dependent sn-glycerol-3-phosphate transport system, were mapped at 75.3 min on the Escherichia coli chromosome. A Tn10 insertion in ugpA resulted in loss of transport activity but still allowed the synthesis of the sn-glycerol-3-phosphate-binding protein. This Tn10 insertion was found to be linked by P1 transduction to pit, aroB, malA, asd, and livH with 2.5, 2.8, 25, 63.5, and 83% cotransduction frequency. An insertion of Mud (Ampr lac) in ugpB resulted in the loss of the binding protein. ugpB is closely linked to ugpA. It is either the structural gene for the binding protein or located proximal to it. The analysis of the crosses allowed the ordering of the markers in the clockwise direction as follows: aroB, malA, asd, ugpA, ugpB, livH, pit.

ATP-Binding Cassette Transporters↗
PubMed1982

Identification of the glpT-encoded sn-glycerol-3-phosphate permease of Escherichia coli, an oligomeric integral membrane protein.

A collection of hybrid plasmids carrying either the wild-type or mutated glpT gene was generated in vitro and used to characterize the glpT-dependent active transport system for sn-glycerol-3-phosphate in Escherichia coli K-12. Restriction endonuclease analysis and recloning of DNA fragments localized glpT to a 3-kilobase pair PstI-HpaI segment of DNA. Comparison of DNA carrying glpT-lacZ fusions with DNA carrying intact glpT allowed determination of the direction of transcription. Through characterization of the proteins synthesized by strains harboring hybrid plasmids carrying amber, missense, or deletion mutations in glpT, it was shown that glpT is a promoter-proximal gene in an operon consisting of at least two genes. The gene product of glpT, the sn-glycerol-3-phosphate permease, was found associated with the inner membrane. It could be solubilized by treatment with sodium dodecyl sulfate at 50 degrees C. Its molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was dependent upon sample treatment before electrophoresis. The apparent molecular weight was 44,000 when membrane fractions were heated to 50 degrees C; subsequent treatment at 95 degrees C modified the protein such that it migrated faster (apparent molecular weight = 33,000). Several missense mutations in glpT were negatively dominant over wild-type glpT, indicating that the active form of the permease is multimeric. A gene (named glpQ) promoter distal to glpT codes for a periplasmic protein. This protein had previously been named GLPT protein to indicate its relationship to the glpT gene. The present report demonstrates that it is not the gene product of glpT and is not required for active transport of sn-glycerol-3-phosphate.

Cell Membrane↗
PubMed1982

sn-Glycerol-3-phosphate transport in Escherichia coli and Salmonella typhimurium.

The gene necessary for the synthesis of the active transport system of sn-glycerol-3-phosphate (G3P) is located at 48 min on the Escherichia coli linkage map. Complementation analysis revealed that there is only one gene necessary for G3P transport. The gene was cloned into the multicopy plasmid pBR322. Strains harboring the hybrid plasmid synthesized large amounts of a protein of 33,000 molecular weight that was found in the cytoplasmic membrane. This protein was identified as the G3P permease. In addition, the periplasm of the hybrid plasmid carrying strain contained large amounts of a soluble protein, identical with the previously recognized GLPT-protein of 40,000 molecular weight. The analysis of amber mutants isolated on the hybrid plasmid showed that the gene for the G3P permease is the first gene in an operon that codes for two genes; the distal gene being the structural gene for the periplasmic GLPT-protein. The corresponding gene region from Salmonella typhimurium has been cloned from an EcoRI libary in lambda gt7. The EcoRI fragment containing the gene necessary for G3P transport was subcloned into the multicopy plasmid pACYC184. The hybrid plasmid directed the synthesis of the G3P permease that behaved identically to the protein from Escherichia coli. However, the gene for the GLPT-protein was not intact but truncated by the EcoRI restriction site. The synthesis of the remaining polypeptide of 30,000 prevented the proper assembly of other transport related binding proteins such as the ribose- and galactose-binding protein.

Biological Transport↗
PubMed1982

Aspects of maltose transport in Escherichia coli: established facts and educated guesses.

The transport system can translocate maltose and maltodextrins (up to 7 glucose moieties) in chemically unmodified form against concentration gradients that can reach 1:10(5). To overcome the problems of diffusion of substrate across the outer membrane at substrate concentrations below 0.1 mM, the receptor for phage lambda has to be present in the outer membrane. The facilitated diffusion via the lambda receptor is accomplished by its interaction with maltose-binding protein. The maltose-binding protein-substrate complex, but not free substrates is recognized at the cytoplasmic membrane. This implies that the maltose-binding protein is essential for substrate translocation and that the cytoplasmic membrane has no second maltodextrin recognition site. Substrate translocation through the cytoplasmic membrane is tightly coupled to energy consumption and unidirectionally inward. The energy source used for substrate accumulation is not the electrochemical potential of protons, but phosphate-bound energy, likely to be ATP. In mutants that are blocked in maltose metabolism, exit of maltose occurs in chemically modified form as acetylmaltose. It is energy-dependent, blocked by uncouplers of the proton-conductive type and is stimulated by energy sources. It is not mediated by any malB-dependent function. The membrane-bound components of the system may establish a non-specific pore that is triggered by its interaction with the maltose-binding protein bound to its substrate. After translocation of one molecule substrate the pore has to be re-energized, possibly by ATP hydrolysis.

ATP-Binding Cassette Transporters↗
PubMed1982

Maltose transacetylase of Escherichia coli: a preliminary report.

Crude extracts of Escherichia coli K12 contain an enzyme that is able to transfer the acetyl group of acetyl-co-enzyme A to maltose (maltose transacetylase). Half maximal acetylation occurs at about 20 microM acetyl-coenzyme A. Half maximal concentration of maltose has not been determined precisely, but it is clear that it exceeds 10 mM. The appearance of maltose transacetylase is not induced by growth on maltose. Mutants carrying a defect in the malT gene, the positive regulator for the well known malA and malB regions, exhibit elevated levels of maltose transacetylase. The gene coding for the enzyme is not known that mutant analysis demonstrated that it is not part of malA, malB or malT. In addition, it is clear that the enzyme is not identical with thiogalactoside transacetylase, the gene product of the lacA gene. Besides maltose, maltodextrins and thiomaltose are substrates for maltose transacetylase.

Acetyl Coenzyme A↗
PubMed1981

Mapping of mglB, the structural gene of the galactose-binding protein of Escherichia coli.

The tetracycline resistance transposon Tn10 was inserted into the E. coli chromosome near mglB550, a structural gene for the galactose-binding protein. P1 transductions established the position of these Tn10 insertions (zee-700, 701, 702::Tn10) close to the genes ptsF, fpk, cdd, mglB550, his, and gatA with 85%-95%, 85%, 36%, 20%-40%, 12%-15%, and 0.5% cotransduction frequency. Three factor crosses revealed the relative sequence of the genes as: mglB550, zee-700::Tn10, ptsF, fpk, cdd, his, gatA was found to be 1.3% cotransducible with mglB550. Two Tn10 insertions near gatA were isolated and characterized. One, zef-704::Tn10, was 3% cotransducible with fpk, 8% with mglB550, and 42% with gatA. The other, zef-703::Tn10, was 98% cotransducible with gatA but not with mglB550 or fpk. Neither of these two Tn10 insertions was cotransducible with cdd. Four factor crosses revealed the sequence gatA, zef-704::Tn10, mglB550, fpk. Neither zee-700::Tn10 nor zef-703::Tn10 showed an (0/300) cotransduction with either glpT or gyrA. The clockwise order of genes is then: his, cdd, fpk, ptsF, zee-700::Tn10, mglB550, zef-704::Tn10, gatA. With a fix-point for his at 44 min, fpk would be placed at 45 min and mglB550 at 45.5 min. During the course of this work we noticed that the cotransduction frequency between Tn10 insertions and nearby markers tended to increase when new P1 lysates were prepared from freshly reisolated strains. This may indicate loss of nonessential genes adjacent to Tn10 insertions. Using insertion zee-703::Tn10, we isolated deletions extending into an mgl gene other than mglB. Crosses between such a deletion mutant and an mglB550 mutant were done. The analysis of the periplasmic proteins of these as well as other transductants or recombinants involving the mglB550 or the mglB551 gene revealed the existence of strains synthesizing both the wild-type as well as the corresponding mutant protein. Strains containing both proteins exhibit either wild-type or mutant phenotype. These strains appeared unstable. Upon reisolation from purified stock cultures kept in glycerol at -20 degrees C, colonies could be isolated that carried only mutant or wild-type protein.

Calcium-Binding Proteins↗
PubMed1981

Ultrastructural localization of the maltose-binding protein within the cell envelope of Escherichia coli.

Logarithmically growing cells of Escherichia coli were fixed with glutaraldehyde and incubated with antimaltose-binding protein Fab coupled to horseradish peroxide (molecular weight of the complex 80,000). The position of this complex within the cell envelope was determined by reacting with diaminobenzidine-H2O2, staining with osmium tetroxide and processing for thin section electron microscopy. The following observations were made: (i) induction of the maltose-binding protein resulted in swelling and staining of the outer membrane; (ii) the swelling and staining was more prominent in short cells, less prominent or absent in long cells; (iii) rare examples exhibited granular staining in the space between the plasma membrane and the peptidoglycan layer. These stainings were observable mainly in pole caps; (iv) a mutant lacking the receptor for phage lambda showed altered staining pattern. Treatment of glutaraldehyde-fixed cells with EDTA-lysozyme prevented the specific labelling of the maltose-binding protein.

ATP-Binding Cassette Transporters↗
PubMed1981

Reconstitution of maltose transport in malB mutants of Escherichia coli through calcium-induced disruptions of the outer membrane.

The barrier function of the Escherichia coli outer membrane against low concentrations of maltose in strains missing the lambda receptor was partially overcome by treating the cells for 3 h with 25 mM Ca2+. Kinetic analysis of maltose-transport revealed a Ca2+-induced shift of the apparent Km of the system from about 100 microM in cells pretreated with Tris to about 15 microM in cells pretreated with Tris plus Ca2+. In contrast to maltose transport in untreated cells, that of Ca2+-treated lamB cells was inhibited by molecules with a high molecular weight, such as amylopectin (molecular weight, 20,000), and anti-maltose-binding protein antibodies. In addition, lysozyme was shown to attack Ca2+-treated cells in contrast to untreated cells. The Ca2+-induced permeability increase of the outer membrane allowed reconstitution of maltose transport in a mutant missing the maltose-binding protein with osmotic shock fluid containing the maltose-binding protein. Even though Ca2+-treatment allowed the entry of large molecules, the release of the periplasmic maltose-binding protein or alkaline phosphatase was negligible.

ATP-Binding Cassette Transporters↗
PubMed1981

Formation and excretion of acetylmaltose after accumulation of maltose in Escherichia coli.

malB(+)malQ strains accumulate maltose via the maltose-binding-protein-dependent transport system but are unable to metabolize it. Nevertheless, some of the maltose is modified after entering the cell. This newly formed compound exhibited a higher R(f) value than did maltose upon thin-layer and paper chromatography with the usual sugar-separating solvents. Treatment of this compound with acid and alkali reformed maltose. The identity of this compound with acetylmaltose was derived from mass spectrometry. Nuclear magnetic resonance spectra of the compound confirmed the presence of the acetyl group but did not allow its precise location on the maltose moiety. However, linkage to the 1-position of maltose could be excluded. Analysis of the mass spectra indicated that the nonreducing end of maltose was acetylated. Other substrates of the maltose transport system, such as maltotetraose, maltopentaose, and maltohexaose, were also modified after accumulation into the cell. Several products were formed; the heterogeneity of these products was probably caused by different degrees of acetylation. The enzymatic activity responsible for maltose and maltodextrin acetylation is unknown. However, it is clear that the lacA-dependent thiogalactoside transacetylase was not necessary for the acetylation of maltose. Strains that accumulate maltose via a bypass of the normal malB-dependent transport system also acetylated maltose even in the absence of any malB gene products. Thus, the acetylating activity was not connected to the malB system. Acetylmaltose as well as acetylated maltodextrins was excreted into the medium. Acetylmaltose is not a substrate of the maltose transport system. Thus, maltose acetylation may be an effective detoxification mechanism.

ATP-Binding Cassette Transporters↗
PubMed1980

The role of the Escherichia coli lambda receptor in the transport of maltose and maltodextrins.

The lambda receptor is a peptidoglycan-associated integral protein that spans the outer membrane. Beside its function in phage lambda adsorption it participates in transport. The latter function can be summarized as follows: 1) Receptor allows the nonspecific permeation of small molecules other than maltose and maltodextrins (in close analogy to a molecular sieve). Here the only criterion for selectivity is size and it has the properties of an unspecific pore. In this respect, it is similar to the outer membrane proteins Ia, Ib, and Ic, the porins. 2) It is a binding protein for maltodextrins. Binding affinity is low but increases by a factor of 500 as the chain length of the maltodextrins increases. In contrast, the affinity of the periplasmic maltose-binding protein for maltose and maltodextrins is similarly high (in the microM range). 3) In the in vitro system of liposomes, the lambda receptor facilitates specifically the diffusion of maltodextrins that exceed the size limit given by its porin function. This clearly demonstrates that the lambda receptor alone is able to specifically overcome the permeability barrier of the outer membrane for maltodextrins. 4) From the genetic and kinetic analysis of maltose and maltodextrin transport, it can be concluded that the lambda receptor interacts with the periplasmic maltose-binding protein. 5) Electron microscopic studies indicate a location for the maltose-binding protein in the outer cell envelope. This location is dependent on the presence of the lambda receptor.

ATP-Binding Cassette Transporters↗
PubMed1980

Co-regulation in Escherichia coli of a novel transport system for sn-glycerol-3-phosphate and outer membrane protein Ic (e, E) with alkaline phosphatase and phosphate-binding protein.

Mutants constitutive for the novel outer membrane protein Ic (e or E) contained a recently discovered binding protein for sn-glycerol-3-phosphate. The corresponding parental strains missing the outer membrane protein Ic (e, E) were negative or strongly reduced in the synthesis of the binding protein. In addition, strains that were previously isolated as mutants constitutive for the sn-glycerol-3-phosphate transport system (ugp(+) mutants) and that produced the novel periplasmic proteins GP1 to GP4 also synthesized a new outer membrane protein with the same electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as protein Ic. Screening of different ugp(+) mutants revealed the existence of three types in respect to the four novel periplasmic proteins GP1, -2, -3, and -4: (i) one containing all four proteins; (ii) one containing only proteins GP1, -2, and -3; (iii) one containing only proteins GP1, -2, and -4. In confirmation of the data presented in the accompanying paper by Tommassen and Lugtenberg (J. Bacteriol. 143:151-157, 1980), we found that purified GP1 is identical to alkaline phosphatase, whereas purified GP3 has binding activity of inorganic phosphate and is identical to the phosphate-binding protein. Moreover, growth conditions that lead in a wild-type strain to the derepression of alkaline phosphatase synthesis also derepressed the synthesis of the sn-glycerol-3-phosphate-binding protein as well as the corresponding transport system. Thus, the new sn-glycerol-3-phosphate transport system is part of the alkaline phosphatase regulatory system.

Alkaline Phosphatase↗
PubMed1979

Purification and properties of the sn-glycerol 3-phosphate-binding protein of Escherichia coli.

A binding protein for sn-glycerol 3-phosphate was isolated from the cell envelope of Escherichia coli by the cold osmotic shock procedure. The protein was purified to homogeneity. It has a molecular weight of 45,000 and binds sn-glycerol 3-phosphate with a KD of 0.2 microM. The protein is monomeric and has L-leucine as NH2-terminal amino acid. The intrinsic fluorescence of the protein is altered upon binding of substrate. At an excitation of 285 nm, the emission maximum at 340 nm is quenched and shifted to 330 nm. Binding of sn-glycerol 3-phosphate is reversible and no chemical alteration occurs with the substrate. The appearance of the binding protein in the periplasm is the result of a mutation that renders the cells constitutive for sn-glycerol 3-phosphate transport. Simultaneously, two other proteins appear in the periplasm. These proteins were also purified. They do not bind sn-glycerol 3-phosphate and do not cross-react with antibodies against the pure binding protein.

Binding, Competitive↗
PubMed1979

Receptor for bacteriophage lambda of Escherichia coli forms larger pores in black lipid membranes than the matrix protein (porin).

The receptor for phage lambda in Escherichia coli was isolated by cholate extraction and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands corresponding to the monomer and the dimer were eluted from the gel and tested for their activity to inactivate phage lambda and to form pores in black lipid membranes. It was found that only the dimer inactivated phage lambda, whereas both the monomer and the dimer were active in forming pores. The pore characteristics were similar to those exhibited by the matrix protein (porin) (R. Benz, K. Janko, W. Boos, and P. Läuger, Biochim. Biophys. Acta 511:305--319, 1978). In comparison, the lambda receptor showed a somewhat higher degree of cation specificity, and its pore size was larger. Assuming that the thickness of the outer membrane is 7.5 nm and that the pore is an ideal hydrophilic channel, the pore diameter in vivo was estimated to be 1.6 nm for the lambda receptor and 1.2 nm for the matrix protein.

Bacterial Proteins↗
PubMed1978

Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli.

One of the major proteins of the outer membrane of Escherichia coli, the matrix protein (porin), has been isolated by detergent solubilisation. When the protein is added in concentrations of the order 10 ng/cm3 to the outer phases of a planar lipid bilayer membrane, the membrane conductance increases by many orders of magnitude. At lower protein concentrations the conductance increases in a stepwise fashion, the single conductance increment being about 2 nS (1 nS = 10(-9) siemens = 10(-9) omega -1) in 1 MKCl. The conductance pathway has an ohmic current vs. voltage character and a poor selectivity for chloride and the alkali ions. These findings are consistent with the assumption that the protein forms large aqueous channels in the membrane. From the average value of the single-channel conductance a channel diameter of about 0.9 nm is estimated. This channel size is consistent with the sugar permeability which has been reported for lipid vesicles reconstituted in the presence of the protein.

Cations, Monovalent↗
PubMed1978

Pole cap formation in Escherichia coli following induction of the maltose-binding protein.

After induction with maltose, 30--40% of the total protein in the osmotic shock fluid consist of maltose-binding protein while the induction ratio (maltose versus glycerol grown cells) for the amount of binding protein synthesized as well as for maltose transport is in the order of 10. Induction of maltose transport does not occur during all times of the cell cycle, but only shortly before cell division. Electronmicroscopic analysis of cells grown logarithmically on glycerol or maltose revealed in the latter the formation of large pole caps. These pole caps arise from an enlargement of the periplasmic space. Small cells contain one pole cap, large cells contain two. Pulse label studies with strain BUG-6, a mutant that is temperature sensitive for cell division reveal the following: Growth at the non-permissive temperature prevents maltose-binding protein synthesis and formation of new transport capacity. After shifting to the permissive temperature the cells regain both functions. Simultaneously, the newly formed cells exhibit pole caps. We conclude that the induction of maltose-binding protein is responsible for the formation of pole caps. In addition, beside the presence of inducer, cell cycle events occuring during division are necessary for the synthesis of maltose-binding protein.

Biological Transport, Active↗