A mutation effecting gluconate catabolism in Escherichia coli: The locus for the main high affinity transport / Una mutación que afecta el catabolismo del gluconato en Escherichia coli: el locus para el transporte principal de alta afinidad

The bioH-malA region of the E.coli chromosome (min 75.5) includes the gntT gene which encodes a high affinity transport for gluconate. Other gnt loci have not been characterized in this region; nevertheless, because lesion in it affect severely the utilization of gluconate, it has been suggested as being more complex. This region was investigated with respect to gluconate catabolism through the characterization of suitable E.coli strains lysogenized with a specialized transducing phage carrying the bioH-malA region of the bacterial chromosome (cI857st68h80d2bioH-malA). It was found that the region transduced by this phage while includes the gntT gene lacks other gnt loci that might code additional activities for transport of gluconate or its phosphorylation. Moreover, the pleiotropic lesion gntM2, previously mapped into this region and suggested as altering gntT or a presumptive regulator gene that might be involved in this catabolism, resulted recessive in lysogens (partial diploids) containing the defective prophage. The results obtained supported the idea that gntM2 is an allele of gntT; consequently those results suggested the precise position of this gene on the cromosomic map and the central role that its product might have in the initial incorporation of gluconate in E.coli.

Penicillin-binding protein 1B of Escherichia coli exists in dimeric forms.

A high-molecular-weight band has been detected in Western immunoblots of nonboiled Escherichia coli samples incubated with polyclonal antiserum against penicillin-binding protein 1B (PBP 1B). This band was shown to be a dimer of PBP 1B. The dimer was more strongly associated with the envelope than the monomer, and it was still able to bind penicillin G. Analysis of the binding of fusion proteins of PBP 1B and beta-lactamase showed that the part of PBP 1B necessary for complex formation lies in the amino-terminal half of the protein.

Related RNA polymerase-binding regions in human RAP30/74 and Escherichia coli sigma 70.

RAP30/74 is a heteromeric general transcription initiation factor that binds to mammalian RNA polymerase II. The RAP30 subunit contains a region that is similar in amino acid sequence to the RNA polymerase-binding domain of the Escherichia coli transcription initiation factor sigma 70 (sigma 70). Mammalian RNA polymerase II specifically protected a serine residue in the sigma 70-related region of RAP30 from phosphorylation in vitro. In addition, human RAP30/74 bound to Escherichia coli RNA polymerase and was displaced by sigma 70. These results suggest that RAP30 and sigma 70 have functionally related RNA polymerase-binding regions.

Crystal structure of Escherichia coli CheY refined at 1.7-A resolution.

The three-dimensional structure of wild-type CheY from Escherichia coli has been refined by stereochemically restrained least squares minimization to a crystallographic R-factor of 15.1% at 1.7-A resolution. The structure contains 1165 atoms, including all atoms of the protein, 147 water molecules, and three sulfate ions. The final model has root mean square deviations of 0.018 and 0.049 A from idealized bond lengths and angle distances, respectively. Seven amino acid side chains have been modeled in dual conformations. CheY folds as a compact (beta/alpha)5 globular protein, with the phosphorylation region contained in a cavity on one face of the molecule. This active site area is bordered by the carboxyl termini of the three central beta-strands, by alpha 1, and by the loop connecting beta 5 to alpha 5. The Lys-109 side chain of this loop extends into the active site by virtue of its cis peptide bond conformation preceding Pro-110. The epsilon-amino group of Lys-109 is in close bonding contact with the carboxyl group of Asp-57, the residue that is phosphorylated in the activation process of CheY. The details of the hydrogen bonding network in the phosphorylation region indicate that structural rearrangements must accompany the phosphorylation of Asp-57.

Structural dynamics and functional domains of the fur protein.

Proteolytic enzymes were used to detect metal-induced conformational changes in the ferric uptake regulation (Fur) protein of Escherichia coli K12. Metal binding results in enhanced cleavage of the N-terminal region of Fur by trypsin and chymotrypsin. Activation of both trypsinolysis sensitivity and DNA binding have similar metal ion specificity and concentration dependencies, suggesting that the conformational change detected is required for operator DNA binding. Isolation and characterization of biochemically generated fragments of Fur as well as other data indicate that the N-terminal region is necessary for the interaction of the repressor with DNA and that a C-terminal domain is sufficient for binding to metal ions.

Affinity-chromatographic purification of sixteen cysteine-substituted maltoporin variants: thiol reactivity and cross-linking in an outer membrane protein of Escherichia coli.

Wild-type and 16 variant maltoporins with site-directed cysteine substitutions at 14 sites were purified by a novel one-step affinity-chromatographic procedure. The trimer stability of purified proteins with C22S, C38S and G103C substitutions was reduced compared to wild-type maltoporin. Quantitative labelling with N-ethyl[14C]maleimide, cross-linking with bifunctional bismaleimides and disulphide formation was used to test the reactivity of cysteines in the folded protein. The maleimide reactivity of the residues was in the order: 152 approximately equal to 153 greater than 265 greater than 30 approximately equal to 103 approximately equal to 120 approximately equal to 154 approximately equal to 382 greater than 57 approximately equal to 146, with the other sites (22, 38, 97, 184) poorly labelled. Only cysteines at 152 or 153 permitted the formation of inter-subunit disulphide bonds suggesting these residues are located within 0.5-0.9 nm of each other in homotrimers of maltoporin. S152C and S153C as well as S154C permitted the formation of inter-subunit cross-links using bifunctional bismaleimides. The cross-linkability and the high reactivity to N-ethylmaleimide of the 150 region was consistent with the current model of the structure of maltoporin in the outer membrane; the reactivity of the other sites is also discussed within the context of this model.

Polyamines as constituents of the outer membranes of Escherichia coli and Salmonella typhimurium.

Extraction of whole cells of Salmonella typhimurium and Escherichia coli with 1 M NaCl released 8 to 13% of their total cellular polyamines (putrescine, cadaverine, and spermidine). This extraction did not cause significant cell lysis, release of outer membrane (OM) constituents, or leakage of periplasmic beta-lactamase. The extraction released nearly equal amounts of polyamines from mdo (membrane-derived oligosaccharide) mutants and wild type. These findings suggest that the released polyamines are apparently bound to the cell envelope. NaCl (1 M) was as effective as trichloroacetic acid in releasing polyamines from isolated OM and lipopolysaccharide (LPS). Isolated OM contained four times more polyamines than the cytoplasmic membrane. The increased binding to the OM is apparently due to the association of polyamines with the polyanionic LPS. Nearly identical amounts of polyamines were found in the OM and LPS preparations (as quantified per milligram of LPS). These amounts are equal to those released from the intact cells by 1 M NaCl (quantitation as above). However, redistribution of polyamines took place after cell disruption, because the relative proportions of different polyamines varied in the OM and LPS preparations. These results indicate that polyamines released from intact cells during 1 M NaCl extraction are preferentially derived from the OM.

Assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin.

As a necessary first step in the use of heteronuclear correlated spectra to obtain high resolution solution structures of the protein, assignment of the 15N NMR spectra of reduced and oxidized Escherichia coli thioredoxin (Mr 12,000) uniformly labeled with 15N has been performed. The 15N chemical shifts of backbone amide nitrogen atoms have been determined for both oxidation states of thioredoxin using 15N-1H correlated and two-dimensional heteronuclear single-quantum coherence (HSQC) TOCSY and NOESY spectra. The backbone assignments are complete, except for the proline imide nitrogen resonances and include Gly33, whose amide proton resonance is difficult to observe in homonuclear 1H spectra. The differences in the 15N chemical shift between oxidized and reduced thioredoxin, which occur mainly in the vicinity of the two active site cysteines, including residues distant in the amino acid sequence which form a hydrophobic surface close to the active site, are consistent with the differences observed for proton chemical shifts in earlier work on thioredoxin.

Alteration in the electrophoretic mobility of OmpC due to variations in the ammonium persulfate concentration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Studies on Salmonella typhi and Salmonella typhimurium outer membrane proteins have shown that the relative position of OmpC porin in sodium dodecyl sulfate.polyacrylamide gel electrophoresis undergoes an important shift when the concentration of ammonium persulfate in the running gel is increased from 6 to 12 mM. The apparent molecular mass at these concentrations was estimated to be 34 and 40 kDa, respectively. Under similar electrophoretic conditions the apparent molecular mass estimated for OmpF was 37.6 and 38.2 kDa. Therefore, OmpC moves from a leading position to a position behind OmpF. For Escherichia coli OmpC the shift observed is less pronounced than that occurring in Salmonellae.

Salt dependent changes in structure and dynamics of circular single stranded DNA of filamentous phages of Escherichia coli.

We have analyzed the static and dynamic behaviour of the circular single stranded DNA of the filamentous Escherichia coli phages F1 and M13mp8 in solution as a function of salt concentration using static and dynamic light scattering and sedimentation analysis in the analytical ultracentrifuge. We show by static light scattering that native and denatured single stranded DNA behave like a randomly coiled macromolecule at all salt concentrations used. The size of the native single stranded DNA is governed by the formation of secondary structures. While the radius of gyration decreases with increasing salt concentration the translational diffusion of the center-of-mass of native single stranded DNA and the sedimentation coefficient increase with increasing salt concentration in a biphasic manner. Below 100 mM monovalent cation concentration there is a strong dependence of the hydrodynamic parameters upon salt which is reduced approx. 3-fold at higher salt concentrations. We attribute the compaction of single stranded DNA by salt to electrostatic shielding and, in case of native single stranded DNA, secondary structure formation. Internal motions of the native single stranded DNA are observable at all salt concentrations and can be interpreted with a model of segmental diffusion of the elements of the polymer chain. The observed segmental diffusion coefficient of the native single stranded polynucleotide increases with increasing salt under the conditions investigated.

The phosphate pool in Escherichia coli THU.

The phosphate pool of Escherichia coli was determined as a fraction of the total cell phosphate. This relative pool size was found to be essentially independent of cell age.

Tritium NMR spectroscopy of ligand binding to maltose-binding protein.

Tritium-labeled alpha- and beta-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR spectroscopy of the labeled sugars showed large upfield chemical shift changes upon binding and strong anomeric specificity. At 10 degrees C, MBP bound alpha-maltose with 2.7 +/- 0.5-fold higher affinity than beta-maltose, and, for longer maltodextrins, the ratio of affinities (KD beta/KD alpha) was even larger (between 10 and 30). The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound alpha-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound beta-maltotriose resonances in rapid exchange. We interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the beta-maltodextrin is bound by its reducing end, and, in the other complex, the beta-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins.

Molecular modelling of bacterial deep rough mutant lipopolysaccharide of Escherichia coli.

Molecular modelling techniques have been applied to compute the conformation accessible to bacterial deep rough lipopolysaccharide of Escherichia coli (Re-LPS). Analyses of the results showed that the models typically exhibit a tilt of the diglucosamine backbone with respect to the membrane normal of 53 +/- 7 degrees while both the glucosamine ring planes are oriented approximately parallel to the membrane normal. Different models were found to show compact and elongated types of acyl chain arrangements, both producing anisotropic lateral dimensions of the models of 1.0-1.1 nm and 1.7-2.0 nm for the shorter and the longer side, respectively. The conformationally allowed range of the isolated dOclA(alpha-2-4)dOclA disaccharide (dOclA = 3-deoxy-D-mannooctulosonic acid) was found to be extremely limited. It appeared that the dOclA disaccharide (dOclA)2 is centred at the top of the Re-LPS molecule preferring two orientations stabilized by hydrogen bonds involving only one phosphate group of the lipid A moiety at a time. The effect of charges on the Re-LPS conformations has been studied in separate calculations. From these calculations it was obvious that charges have no significant effects on the conformations of the isolated lipid A and (dOclA)2 moieties. However, it was found that the orientation of (dOclA)2 with respect to the lipid A part is highly sensitive to charges, i.e. in the charged models the proximity of phosphate and carboxyl groups is prevented by strong electrostatic repulsion between these negatively charged groups. In order to rationalize the acyl chain packing of the models, a simple geometrical model which correlates the tilt of the diglucosamine backbone with the energically favoured close packing of the acyl chains is proposed. Furthermore, the possibility of a chelate-like complexation of divalent cations and its contribution to head group mobility is discussed.

Escherichia coli DNA-binding protein H-NS is localized in the nucleoid.

Electron microscope localization of the 15.4-kDa DNA-binding protein H-NS was carried out in Escherichia coli cells subjected to cryosubstitution followed by immuno-labelling with the protein A/gold technique. Three types of E. coli cells were used: (1) "normal" cells growing exponentially at 37 degrees C; (2) "cold-shocked" cells two hours after the shift from 37 degrees C to 10 degrees C; and (3) cells in which an expression vector had been induced to overproduce H-NS. The results clearly indicate that in all 3 cases, the vast majority of the molecules reacting with anti-H-NS antibodies are localized within the nucleoid and at the border between the nucleoid and the ribosome-rich cytoplasm, which supports the premise that H-NS is implicated in the condensation of the nucleoid.

Separation of ribosomal subunits on Trisacryl GF 2000.

The separation of rat liver and E. coli ribosomal subunits was attempted on Trisacryl GF 2000. Contrary to experiments with Sepharose 4B and Bio-Gel A-15 the 60S mammalian subunit did not bind to the resin at 4 degrees C but eluted within the column volume ahead of the 40S subunit. Puromycin, however, used to prepare the subunits, which on the agarose gels had eluted at the total column volume, exhibited anomalous retardation on the Trisacryl resin. Trisacryl therefore behaves as the more non-polar resin, and the binding of 60S subunits to agarose gels is a result of hydrophilic interaction.

Selective precipitation of proteins from guanidine hydrochloride-containing solutions with ethanol.

The solubility of guanidine hydrochloride in ethanol and conversely the low solubility of proteins have been used as the basis for a procedure for recovering proteins from guanidine-containing solutions by selective precipitation. Yields of greater than 94% were observed with as little as 28 ng protein and of 98% for larger quantities of protein up to 20 mg/ml. The precipitations were independent of molecular weight for proteins in the range of 6-100 kDa and could be run in as little as 5 min at room temperature. Unlike the conventional desalting methods for removing guanidine, ethanol precipitation is rapid, efficient, and can be applied simultaneously to a large number of samples. The approach should have a wide range of applications.

Physical, chemical and immunological properties of the bacterioferritins of Escherichia coli, Pseudomonas aeruginosa and Azotobacter vinelandii.

The 70-amino-acid-residue N-terminal sequence of the bacterioferritin (BFR) of Azotobacter vinelandii was determined and shown to be highly similar to the N-terminal sequences of the Escherichia coli and Nitrobacter winogradskyi bacterioferritins. Electrophoretic and immunological analyses further indicate that the bacterioferritins of E. coli, A. vinelandii and Pseudomonas aeruginosa are closely related. A novel, two-subunit assembly state that predominates over the 24-subunit form of BFR at low pH was demonstrated. The results indicate that the bacterioferritins form a family of proteins that are distinct from the ferritins of plants and animals.

A resuscitation/selection system for rapid determination of Salmonella in foods.

A resuscitation medium was developed consisting of a trypticase soy broth base supplemented with 0.5% yeast extract, 0.25% sodium pyruvate, 0.01% sodium thioglycollate, and 0.1% chicken fat. After a resuscitation period of 4 h, the medium was made selective by addition of either sodium thiosulfate, bile salts and iodine, or sodium selenite and L-cystine. The now selective medium was incubated for 16 h. The presence or absence of Salmonella was determined by the Salmonella-Tek antibody-based detection kit. The present system was compared with a method of the Bacteriological Analytical Manual (BAM) for naturally contaminated foods. Nineteen egg products were screened; 3/19 were positive using the BAM method, 9/19 were positive using the present system. Seventeen chicken samples were assayed; 10/17 were positive using the BAM method; 13/17 were positive using the present system. Of 8 pepper samples, 4/8 were positive using the BAM method; 6/8 were positive using the present system. Of 8 spice samples, 6/8 were positive using the BAM method, 7/8 were positive using the present system. Of 6 onion products sampled, 5/6 were positive using the BAM method; 6/6 were positive using the present system.

Purification and characterization of the membrane-associated components of the maltose transport system from Escherichia coli.

Maltose is transported across the cytoplasmic membrane of Escherichia coli by a binding protein-dependent transport system. The three membrane-associated components of the transport system, the MalK, MalF, and MalG proteins, have been solubilized from the membrane and maltose transport activity has been reconstituted in proteoliposome vesicles (Davidson, A. L., and Nikaido, H. (1990) J. Biol. Chem. 265, 4254-4260). A modification of the reconstitution technique is presented which permits reconstitution from the detergent dodecyl maltoside. Utilizing reconstitution of maltose transport as an assay, we have purified these proteins in the presence of n-dodecyl-beta-D-maltoside. The purified proteins catalyze both maltose transport activity and ATP hydrolysis. In all experiments, the MalF, MalG, and MalK proteins behaved as a multiprotein complex; all three proteins were immunoprecipitated using antibody prepared against MalF, and they copurified, eluting from a gel filtration column between markers of Mr 160,000 and 200,000. Each complex contains two MalK, one MalF, and one MalG proteins, providing two putative sites for ATP hydrolysis. Chemical cross-linking detected specific interactions between MalF and MalG and between MalF and MalK.