Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli.

We have directly measured the stoichiometry of maltodextrin-binding sites in LamB. Scatchard plots and computer fitting of flow dialysis (rate-of-dialysis) experiments clearly establish three independent binding sites per LamB trimer, with a dissociation constant of approximately 60 microM for maltoheptaose. The current model for LamB's function as a specific pore is discussed with respect to the symmetry in LamB's kinetic properties and the implications of our results.     

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.     

Antibodies against synthetic peptides and the topology of LamB, an outer membrane protein from Escherichia coli K12

LamB, an outer membrane protein from Escherichia coli K12, is involved in the transport of maltose and maltodextrins across the outer membrane and constitutes a receptor for a number of bacteriophages. A recent folding model proposes that LamB spans the outer membrane through a number of transmembranous segments separated by regions exposed either to the cell exterior or to the periplasm. This model is essentially based on predictions of structure and genetic arguments relying on the hypothesis that the mutations studied did not alter the folding of the protein. In order to obtain direct evidence with the unaltered protein, we elicited polyclonal antibodies against synthetic peptides corresponding to several LamB sequences. We chose four regions. Three of them [aa 147-161 (peptide 2), aa 371-385 (peptide 3), and aa 399-413 (peptide 4)] are predicted to face the outside of the cell, and the fourth (aa 19-33 (peptide 1)] is predicted to be periplasmic. By immunoblotting against extracts of various mutants, these antibodies were shown to be specific for LamB and targeted to the selected regions. In some cases, the recognition sites for antibodies were narrowed down to parts of a region. In vivo, on intact cells, anti-peptides 2, 3, and 4 reacted with LamB in an ELISA; this confirmed that regions of peptide 2 and 3 are located, at least in part, at the cell exterior and provided the first proof for a similar, situation of the region of peptide 4. Under the same conditions, anti-peptide 1 did not react with LamB.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Identification and characterization of the TolC protein, an outer membrane protein from Escherichia coli

We used the cloned tolC gene to identify, locate, and purify its gene product. Strains carrying pPR13 or pPR42 overproduced a cell envelope protein (molecular weight, 52,000). A protein of the same molecular weight was identified in radioactively labeled minicells carrying pPR13; this protein was absent in pPR11-carrying minicells. This protein was the tolC gene product, since pPR11 differed from pPR13 in having a Tn10 insertion in the tolC gene. The protein seen in cell envelopes of whole cells (TolC protein) was found to exist in an aggregated state in the outer membrane; under conditions in which OmpC and OmpF were peptidoglycan associated, TolC protein was not likewise associated. Using these properties, we purified the TolC protein and determined the sequence of twelve amino acids from the amino-terminal end. The location of the TolC protein in the outer membrane was consistent with the proposed function for the tolC gene product as a processing protein in the outer membrane.     

Role of a disulfide bond in the thermal stability of the LamB protein trimer in Escherichia coli outer membrane

In order to understand the unusual heat resistance of LamB protein (the outer membrane component of the maltose transport system in Escherichia coli and its receptor for bacteriophage lambda), we investigated the role of its 2 cysteinyl residues. Our studies show that Cys22 and Cys38 form an intrasubunit disulfide bond which contributes to the heat stability of the LamB protein trimer. Physical evidence for the disulfide was obtained by using site-directed mutagenesis to convert Asn36 to Met, which allowed cyanogen bromide cleavage between the 2 cysteines. Upon reduction one of the N36M fragments migrated as two pieces, resolved by two-dimensional polyacrylamide gel electrophoresis. Other mutagenized LamB proteins, in which 1 or both Cys residues were converted to Ser, exhibited a sharp loss of thermal stability. In contrast to wild-type LamB protein trimer, which does not dissociate to monomers even after 60 min at 100 degrees C, only 10-15% of the mutant LamB proteins remain trimeric after boiling 10 min. The disulfide bond in LamB protein is not required for its transport function, since both mutagenized LamB protein and N-ethylmaleimide-labeled LamB protein exhibit normal uptake of sugars in proteoliposomes. Finally, the disulfide bond must not be between subunits of the LamB trimer since reversible dissociation of trimer is achieved by low pH or denaturants in the absence of reducing agent.     

Derepression of LamB protein facilitates outer membrane permeation of carbohydrates into Escherichia coli under conditions of nutrient stress.

The level of LamB protein in the outer membrane of Escherichia coli was derepressed in the absence of a known inducer (maltodextrins) under carbohydrate-limiting conditions in chemostats. LamB protein contributed to the ability of the bacteria to remove sugar from glucose-limited chemostats, and well-characterized lamB mutants with reduced stability constants for glucose were less growth competitive under glucose limitation than those with wild-type affinity. In turn, wild-type bacteria were less growth competitive than lamB mutants with enhanced sugar affinity. In contrast to an earlier report, we found that LamB- bacteria were less able to compete in carbohydrate-limited chemostats (with glucose, lactose, arabinose, or glycerol as the carbon and energy sources) when mixed with LamB+ bacteria. The transport Km for [14C]glucose was affected by the presence or affinity of LamB, but only in chemostat-grown bacteria, with their elevated LamB levels. The pattern of expression of LamB and the advantage it confers for growth on low concentrations of carbohydrates are consistent with a wider role in sugar permeation than simply maltosaccharide transport, and hence the well-known maltoporin activity of LamB is but one facet of its role as the general glycoporin of E. coli. A corollary of these findings is that OmpF/OmpC porins, present at high levels in carbon-limited bacteria, do not provide sufficient permeability to sugars or even glycerol to support high growth rates at low concentrations. Hence, the sugar-binding site of LamB protein is an important contributor to the permeability of the outer membrane to carbohydrates in habitats with low extracellular nutrient concentrations.     

Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein.

Only one of the characterized components of the main terminal branch of the general secretory pathway (GSP) in Gram-negative bacteria, GspD, is an integral outer membrane protein that could conceivably form a channel to permit protein transport across this membrane. PulD, a member of the GspD protein family required for pullulanase secretion by Klebsiella oxytoca, is shown here to form outer membrane-associated complexes which are not readily dissociated by SDS treatment. The outer membrane association of PulD is absolutely dependent on another component of the GSP, the outer membrane-anchored lipoprotein PulS. Furthermore, the absence of PulS resulted in limited proteolysis of PulD and caused induction of the so-called phage shock response, as measured by increased expression of the pspA gene. We propose that PulS may be the first member of a new family of periplasmic chaperones that are specifically required for the insertion of a group of outer membrane proteins into this membrane. PulS is only the second component of the main terminal branch of the GSP for which a precise function can be proposed.     

Folding studies of purified LamB protein, the maltoporin from the Escherichia coli outer membrane: trimer dissociation can be separated from unfolding

The folding mechanisms for β-barrel membrane proteins present unique challenges because acquisition of both secondary and tertiary structure is coupled with insertion into the bilayer. For the porins in Escherichia coli outer membrane, the assembly pathway also includes association into homotrimers. We study the folding pathway for purified LamB protein in detergent and observe extreme hysteresis in unfolding and refolding, as indicated by the shift in intrinsic fluorescence. The strong hysteresis is not seen in unfolding and refolding a mutant LamB protein lacking the disulfide bond, as it unfolds at much lower denaturant concentrations than wild type LamB protein. The disulfide bond is proposed to stabilize the structure of LamB protein by clasping together the two sides of Loop 1 as it lines the inner cavity of the barrel. In addition we find that low pH promotes dissociation of the LamB trimer to folded monomers, which run at about one third the size of the native trimer during SDS PAGE and are much more resistant to trypsin than the unfolded protein. We postulate the loss at low pH of two salt bridges between Loop 2 of the neighboring subunit and the inner wall of the monomer barrel destabilizes the quaternary structure.     

Linker mutagenesis in the gene of an outer membrane protein of Escherichia coli, lamB

In order to identify sequences involved in the localization of LamB, an outer membrane protein from E coli K12, mutagenesis by linker insertion has been performed on a lamB gene copy carried on a plasmid devised for this purpose. An analysis of the first set of 16 clones constructed by this technique shows that, in these clones, the lamB protein is altered either by frameshift mutations leading to abnormal COOH terminal (usually premature termination) or by in-phase deletions or small insertions. Except for two in-phase linker insertions, which only slightly changed the behavior of the protein, the modified proteins are either toxic to cell growth or unstable. In all cases examined so far, the modified proteins were in the outer membrane. We suggest that toxicity is due to incorrect folding, which leads to disruption of the outer membrane. The nature of the genetic alterations leads to the hypothesis that the first 183 amino acids of the LamB mature protein contain, together with the signal sequence, all the instructions needed for proper localization.     

A genetic approach for analyzing the pathway of LamB assembly into the outer membrane of Escherichia coli

Results presented in this study demonstrate that a mutation which inserts an additional tyrosine between the 2 tyrosines at residues 118 and 119 of mature LamB protein results in a temperature-dependent assembly defect. This defect leads to the accumulation of an intermediate at the restrictive temperature that is most likely an assembly-defective monomer. These monomers are rapidly degraded in the wild type (htrA+) strain, and the biphasic kinetics of this degradation indicate that the mutation affects the assembly process and not the final product, i.e. stable trimers. In addition, our data show that the temperature-dependent assembly defect in the mutant strain is reversible, and therefore the accumulated monomers represent a true assembly intermediate. Fractionation studies show that the monomers, which can be accumulated in htrA (degP) mutants at the restrictive temperature, are associated with the outer membrane, indicating that trimerization of LamB is not a prerequisite for localization.     

Mutations affecting antigenic determinants of an outer membrane protein of Escherichia coli.

The Escherichia coli LamB protein is located in the outer membrane. It is both a component of the maltose and maltodextrin transport system, and the receptor for phages lambda and K10. It is a trimer composed of three identical polypeptide chains, each containing 421 residues. Six independent mutants have been isolated, in which the LamB protein is altered in its interaction with one or more monoclonal antibodies specific for regions of the protein that are exposed at the cell surface. Some of the mutations also altered the binding site for phage lambda. All of the mutations were clustered in the same region of the lamB gene, corresponding to residues 333-394 in the polypeptide. This and previous results strongly suggest that a rather large segment of the LamB polypeptide, extending from residue 315 to 401, is exposed at the outer face of the outer membrane. This segment would bear the epitopes for the four available anti-LamB monoclonal antibodies that react with the cell surface, and part of the binding site for phage lambda.     

Apparent bacteriophage-binding region of an Escherichia coli K-12 outer membrane protein.

The 325-residue OmpA protein is one of the major outer membrane proteins of Escherichia coli. It serves as the receptor for several T-even-like phages and is required for the action of certain colicins and for the stabilization of mating aggregates in conjugation. We have isolated two mutant alleles of the cloned ompA gene which produce a protein that no longer functions as a phage receptor. Bacteria possessing the mutant proteins were unable to bind the phages, either reversibly or irreversibly. However, both proteins still functioned in conjugation, and one of them conferred colicin L sensitivity. DNA sequence analysis showed that the phage-resistant, colicin-sensitive phenotype exhibited by one mutant was due to the amino acid substitution Gly leads to Arg at position 70. The second mutant, which contained a tandem duplication, encodes a larger product with 8 additional amino acid residues, 7 of which are a repeat of the sequence between residues 57 and 63. In contrast to the wild-type OmpA protein, this derivative was partially digested by pronase when intact cells were treated with the enzyme. The protease removed 64 NH2-terminal residues, thereby indicating that this part of the protein is exposed to the outside. It is argued that the phage receptor site is most likely situated around residues 60 to 70 of the OmpA protein and that the alterations characterized have directly affected this site.     

The Hek outer membrane protein of Escherichia coli is an auto-aggregating adhesin and invasin

Escherichia coli is the principal gram-negative causative agent of sepsis and meningitis in neonates. The pathogenesis of meningitis due to E. coli K1 involves mucosal colonization, transcytosis of epithelial cells, survival in the blood stream and eventually invasion of the meninges. The latter two aspects have been well characterized at a molecular level in the last decade. Less is known about the early stages of pathogenesis, i.e. adhesion to and invasion of gastrointestinal cells. Here, the characterization of the Hek protein is reported, which is expressed by neonatal meningitic E. coli (NMEC) and is localized to the outer membrane. It is demonstrated that this protein can cause agglutination of red blood cells and can mediate autoaggregation. Escherichia coli expressing this protein can adhere to and invade epithelial cells. So far, this is the first outer membrane protein in NMEC to be directly implicated in epithelial cell invasion.     

Production of Escherichia coli LamB protein in Yersinia enterocolitica

Abstract Plasmid pBCP 68 carrying the lamB gene of Escherichia coli was introduced and expressed in Yersinia enterocolitica cells. The presence of LamB protein in the outer membrane of the wild-type strain of Y. enterocolitica coincided with the loss of the OmpC and OmpF porins. Western blot analysis showed that LamB in Y. enterocolitica cells co-migrated with authentic monomeric LamB, indicating that its signal peptide was recognized and cleaved by Y. enterocolitica and properly integrated into the outer membrane. The expression of LamB made Y. enterocolitica sensitive to phage λ.     

Immunological approach of assembly and topology of OmpF, an outer membrane protein of Escherichia coli.

Various monoclonal antibodies (MoF) directed against cell-surface-exposed epitopes of OmpF, one major outer membrane pore protein of Escherichia coli B and K-12, have been used to study the assembly and the topology of the protein. This paper firstly describes the characterization of the OmpF epitopes recognized by the various monoclonal antibodies. A comparison between OmpC, OmpF and PhoE porins with respect to their primary amino acid sequence and their cell-surface exposed regions allows us to propose a rough model including 2 antigenic sites. The second part is focused on the assembly of the OmpF protein in the outer membrane. Various forms, precursor, unassembled monomer, metastable oligomer (pre-trimer) and trimer are detected with immunological probes directed against OmpF during a kinetic analysis of the process. The requirement for a concomitant lipid synthesis during the trimerization has been demonstrated by investigating the presence of a specific native epitope. The role of lipopolysaccharide during the stabilization of the conformation is discussed with regard to the various steps of assembly.     

X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes

An integral membrane protein forming channels across Escherichia coli outer membranes, porin, has been crystallized using a polyethylene glycol or salt-generated two-phase system. Monodispersity and homogeneity of protein-detergent complexes were found to be prerequisites for reproducible formation of crystals amenable to X-ray structural analysis. By varying pH, detergent and buffer type, large crystals of three different habits can be obtained, two of which are discussed in this paper. The tetragonal form (space group P4(2); unit cell dimensions, a = b = 155 A, c = 172 A) is suitable for X-ray analysis. Low temperature induces a change of the space group to P4(2)22, with a single trimer in the asymmetric unit. This crystal form diffracts to a resolution beyond 2.9 A. The hexagonal crystal form (space group P6(3)22; unit cell dimensions, a = b = 93 A, c = 220 A) is limited in resolution to 4.5 A, but reveals a packing arrangement very similar to that in two-dimensional membrane-like crystalline arrays.     

Escherichia coli outer membrane protein K is a porin.

Protein K is an outer membrane protein found in pathogenic encapsulated strains of Escherichia coli. We present evidence here that protein K is structurally and functionally related to the E. coli K-12 porin proteins (OmpF, OmpC, and PhoE). Protein K was found to cross-react with antibody to OmpF protein and to share 8 out of 17 peptides in common with the OmpF protein. Strains that are OmpC porin- and OmpF porin- and contain protein K as their major outer membrane protein have increased rates of uptake of nutrients and a faster growth rate relative to the parental porin- strain. The protein K-containing strains are at least 1,000-fold more sensitive to colicins E2 and E3 than is the porin -deficient strain. These data suggest that protein K is a functional porin in E. coli. The porin function of protein K was also demonstrated in vitro, using black lipid membranes. Protein K increased the conductance in these membranes in discrete, uniform steps characteristic of channels with a size of about 2 nS.     

The penicillinase of Bacillus licheniformis is an outer membrane protein in Escherichia coli.

The cloned gene coding for Bacillus licheniformis penicillinase (penP) was introduced into Escherichia coli in a heat-inducible lambda Qam vector. After induction, significant amounts of penicillinase were synthesized in the new host. The cellular location of the penicillinase was found to be almost exclusively the outer membrane fraction of E. coli, and virtually no soluble penicillinase was found. According to sodium dodecyl sulfate-gel electrophoresis, the size of the penicillinase from E. coli was identical to that of the membrane-bound form of the B. licheniformis penicillinase. Gel filtration in the presence of Triton X-100 suggested that the penicillinase from E. coli had amphiphilic properties, as does B. licheniformis membrane penicillinase. These results show that the export of the penicillinase to the outer membrane of E. coli involves the cleavage of the signal peptide from the prepenicillinase, giving an outer membrane component indistinguishable from the membrane penicillinase of B. licheniformis.