Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm.

We have employed the technique of gene fusion to fuse the LacZ gene encoding the cytoplasmic enzyme beta-galactosidase with the malE gene encoding the periplasmic maltose binding protein (MBP). Strains were obtained which synthesize malE-lacZ hybrid proteins of various sizes. These proteins have, at their amino terminus, a portion of the MBP and at their carboxyl terminus, enzymatically active beta-galactosidase. When the hybrid protein includes only a small, amino-terminal portion of the MBP, the hybrid protein residues in the cytoplasm. When the hybrid protein contains enough of the MBP to include an intact MBP signal sequence, a significant portion of the hybrid protein is found in the cytoplasmic membrane, suggesting that secretion of the hybrid protein has been initiated. However, in no case is the hybrid protein secreted into the periplasm, even when the hybrid protein includes almost the entire MBP. In the latter case, the synthesis and attempted export of the hybrid protein interferes with the export of at least certain normal envelope proteins, which accumulate in the cell in their precursor forms, and the cell dies. These results suggest that a number of envelope proteins may be exported at a common site, and that there are only a limited number of such sites. Also, these results indicate that it is not sufficient to simply attach an amino-terminal signal sequence to a polypeptide to assure its export.     

Reconstitution of maltose transport in Escherichia coli: conditions affecting import of maltose-binding protein into the periplasm of calcium-treated cells.

The reconstitution of active transport by the Ca2+ -induced import of exogenous binding protein was studied in detail in whole cells of a malE deletion mutant lacking the periplasmic maltose-binding protein. A linear increase in reconstitution efficiency was observed by increasing the Ca2+ - concentration in the reconstitution mixture up to 400 mM. A sharp pH optimum around pH 7.5 was measured for reconstitution. Reconstitution efficiency was highest at 0 degree C and decreased sharply with increasing temperature. The time necessary for optimal reconstitution at 0 degree C and 250 mM Ca2+ was about 1 min. The competence for reconstitution was highest in exponentially growing cultures with cell densities up to 1 X 10(9)/ml and declined when the cells entered the stationary-growth phase. The apparent Km for maltose uptake was the same as that of wild-type cells (1 to 2 microM). Vmax at saturating maltose-binding protein concentration was 125 pmol per min per 7.5 X 10(7) cells (30% of the wild-type activity). The concentration of maltose-binding protein required for half-maximal reconstitution was about 1 mM. The reconstitution procedure appears to be generally applicable. Thus, galactose transport in Escherichia coli could also be reconstituted by its respective binding protein. Maltose transport in E. coli was restored by maltose-binding protein isolated from Salmonella typhimurium. Finally, in S. typhimurium, histidine transport was reconstituted by the addition of shock fluid containing histidine-binding protein to a hisJ deletion mutant lacking histidine-binding protein. The method is fast and general enough to be used as a screening procedure to distinguish between transport mutants in which only the binding protein is affected and those in which additional transport components are affected.     

Export of the periplasmic maltose-binding protein ofEscherichia coli

The export of the maltose-binding protein (MBP), themalE gene product, to the periplasm ofEschericha coli cells has been extensively investigated. The isolation of strains synthesizing MalE-LacZ hybrid proteins led to a novel genetic selection for mutants that accumulate export-defective precursor MBP (preMBP) in the cytoplasm. The export defects were subsequently shown to result from alterations in the MBP signal peptide. Analysis of these and a variety of mutants obtained in other ways has provided considerable insight into the requirements for an optimally functional MBP signal peptide. This structure has been shown to have multiple roles in the export process, including promoting entry of preMBP into the export pathway and initiating MBP translocation across the cytoplasmic membrane. The latter has been shown to be a late event relative to synthesis and can occur entirely posttranslationally, even many minutes after the completion of synthesis. Translocation requires that the MBP polypeptide exist in an export-competent conformation that most likely represents an unfolded state that is not inhibitory to membrane transit. The signal peptide contributes to the export competence of preMBP by slowing the rate at which the attached mature moiety folds. In addition, preMBP folding is thought to be further retarded by the binding of a cytoplasmic protein, SecB, to the mature moiety of nascent preMBP. In cells lacking this antifolding factor, MBP export represents a race between delivery of newly synthesized, export-competent preMBP to the translocation machinery in the cytoplasmic membrane and folding of preMBP into an export-incompetent conformation. SecB is one of threeE. coli proteins classified as molecular chaperones by their ability to stabilize precursor proteins for membrane translocation.     

Export and purification of a cytoplasmic dimeric protein by fusion to the maltose-binding protein of Escherichia coli

A hybrid between the maltose-binding protein (MalE) of Escherichia coli and the gene 5 protein (G5P) of phage M13 was constructed at the genetic level. MalE is a monomeric and periplasmic protein while G5P is dimeric and cytoplasmic. The hybrid (MalE-G5P) was synthesized in large amounts from a multicopy plasmid and efficiently exported into the periplasmic space of E. coli. The export was dependent on the integrity of the signal peptide. MalE-G5P was purified from a periplasmic extract by affinity chromatography on cross-linked amylose, with a yield larger than 50,000 molecules/E. coli cell. The hybrid specifically bound denatured but not double-stranded DNA cellulose, as native G5P. Sedimentation velocity and gel-filtration experiments showed that MalE-G5P exists as a dimer. Thus, it was possible to efficiently translocate through the membrane a normally cytoplasmic and dimeric protein, by fusion to MalE. Moreover, the passenger protein kept its activity, specificity and quaternary structure in the purified hybrid. MalE-G5P will enable the study of mutant G5P that no longer binds single-stranded DNA and therefore cannot be purified by DNA-cellulose chromatography.     

Thirty-three amino acids of the mature moiety of an unprocessed maltose-binding protein are sufficient for export in Escherichia coli.

Maltose-binding protein (MBP) is translocated across the cytoplasmic membrane of Escherichia coli; successful export depends on information in both the signal peptide and the mature moiety of the protein. To determine the shortest portion of the mature region that would maintain detectable entry of MBP into the export pathway, we took advantage of the properties of an MBP species with proline substituted in the +1 position relative to the cleavage site (MBP27-P). This protein efficiently crosses the cytoplasmic membrane but is not processed and acts as a competitive inhibitor of signal peptidase I (leader peptidase). Export of MBP27-P is measured by the inhibition of processing of other proteins, such as ribose-binding protein (RBP). A series of truncated derivatives of MBP27-P were tested for the ability to inhibit processing of RBP. An MBP27-P species with only 33 amino acids of the mature moiety inhibited processing of RBP, indicating that this truncated polypeptide was probably exported and interacted with signal peptidase I.     

Mutations that improve export of maltose-binding protein in SecB- cells of Escherichia coli.

It previously has been proposed that the Escherichia coli SecB protein promotes the export of the maltose-binding protein (MBP) from the cytoplasm by preventing the folding of the precursor MBP (preMBP) into a translocation-incompetent conformation. The export of wild-type MBP is only partially blocked in SecB- cells. In contrast, the export of MBP16-1, an MBP species with a defective signal peptide, is totally dependent on SecB; hence, SecB- cells that synthesize MBP16-1 are unable to utilize maltose as a sole carbon source. The selection of Mal+ revertants primarily yielded mutants with alterations in the MBP16-1 signal peptide that permitted SecB-independent MBP export to the periplasm to various extents. Although each of these alterations increased the overall hydrophobicity of the signal peptide, it was not possible to strictly equate changes in hydrophobicity with the degree of SecB-independent export. Somewhat unexpectedly, two mutants were obtained in which MBP export in SecB- cells was markedly superior to that of the wild-type MBP. Although wild-type MBP is not cotranslationally translocated in SecB- cells, the two mutant proteins designated MBP172 and MBP173 exhibited significant cotranslational export in the absence of SecB. Thus, the role of SecB was partially supplanted by a signal peptide that promoted more rapid movement of MBP through the export pathway. When preMBP included the MBP172 signal peptide as well as an alteration in the mature moiety that slows folding, the SecB requirement for maximal MBP export efficiency was almost totally eliminated. These results provide additional strong support for the proposed antifolding role of SecB in MBP export.     

Interaction of the maltose-binding protein with membrane vesicles of Escherichia coli.

The interaction of the radioactively labeled purified maltose-binding protein of Escherichia coli with membrane vesicles was studied. The maltose-binding protein bound specifically to the vesicles, in the presence of maltose, on few sites. Under conditions in which a potential was imposed across the membrane, the specific binding was (i) increased, (ii) dependent on maltose, and (iii) abolished in a mutant defective in the tar gene product, one of the methyl-accepting chemotaxis proteins. At least 1,300 binding sites were present in the membrane fraction of logarithmically growing cells.     

Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli

The twin-arginine translocation (Tat) system targets cofactor-containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif. In this study, we have analysed the mechanism and capabilities of the E. coli Tat system using green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild-type cells corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state. These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.     

Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations.

It is believed that one or more basic residues at the extreme amino terminus of precursor proteins and the lack of a net positive charge immediately following the signal peptide act as topological determinants that promote the insertion of the signal peptide hydrophobic core into the cytoplasmic membrane of Escherichia coli cells with the correct orientation required to initiate the protein export process. The export efficiency of precursor maltose-binding protein (pre-MBP) was found to decrease progressively as the net charge in the early mature region was increased systematically from 0 to +4. This inhibitory effect could be further exacerbated by reducing the net charge in the signal peptide to below 0. One such MBP species, designated MBP-3/+3 and having a net charge of -3 in the signal peptide and +3 in the early mature region, was totally export defective. Revertants in which MBP-3/+3 export was restored were found to harbor mutations in the prlA (secY) gene, encoding a key component of the E. coli protein export machinery. One such mutation, prlA666, was extensively characterized and shown to be a particularly strong suppressor of a variety of MBP export defects. Export of MBP-3/+3 and other MBP species with charge alterations in the early mature region also was substantially improved in E. coli cells harboring certain other prlA mutations originally selected as extragenic suppressors of signal sequence mutations altering the hydrophobic core of the LamB or MBP signal peptide. In addition, the enzymatic activity of alkaline phosphatase (PhoA) fused to a predicted cytoplasmic domain of an integral membrane protein (UhpT) increased significantly in cells harboring prlA666. These results suggest a role for PrlA/SecY in determining the orientation of signal peptides and possibly other membrane-spanning protein domains in the cytoplasmic membrane.     

Two-dimensional crystals of Escherichia coli maltoporin and their interaction with the maltose-binding protein.

We have reconstituted Escherichia coli maltoporin into phospholipid membranes at low lipid-to-protein ratios to produce two-dimensional crystals of this membrane protein. Electron microscopy of negatively stained membranes showed three different types of arrays, two of them hexagonal and the third rectangular, all diffracting to approximately (2 nm)-1. Furthermore, we have core-constituted maltoporin with the maltose-binding protein from E. coli, a soluble periplasmic protein that has been proposed to interact with maltoporin. One of the hexagonal arrays was found to bind maltose-binding protein molecules in a regular way, while the maltose-binding protein binding sites were not accessible in the other crystal forms. Difference maps from averaged decorated arrays and undecorated controls showed three symmetry-related maltose-binding protein binding sites per maltoporin trimer, of which not more than one is likely to be occupied at a given time. Using multivariate statistical analysis to select similar unit cells of the decorated maltoporin array, we have obtained a map showing the rough outline of a maltose-binding protein molecule interacting with the pore formed by a maltoporin trimer.     

Factors influencing the in vitro translocation of the Escherichia coli maltose-binding protein

An in vitro system has been utilized to study the translocation of newly synthesized Escherichia coli maltose-binding protein (MBP) into inverted membrane vesicles. Approximately 40% of precursor MBP (pMBP) synthesized with a wild-type signal peptide was imported into vesicles. However, MBP species with even minor alterations in the signal peptide hydrophobic core were imported into vesicles with an efficiency much lower than predicted from in vivo studies. Posttranslational import of wild-type pMBP into vesicles could be demonstrated if membranes were added after the termination of protein synthesis. However, if vesicles were present throughout the synthesis reaction, most pMBP import occurred either cotranslationally or very soon after completion of synthesis. The wild-type pMBP rapidly became incompetent for posttranslational translocation upon continued incubation in the absence of membranes, whereas pMBP species with altered folding properties remained competent for significantly longer periods. The rate of in vitro pMBP folding was affected by the nature of the signal peptide. The evidence suggests that one or more soluble factors may interact with the newly synthesized pMBP to help maintain it in a translocation-competent state and to promote its entrance into the export pathway.     

Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein.

Attempts to reconstitute periplasmic binding protein-dependent transport activity in membrane vesicles have often resulted in systems with poor and rather inconsistent activity, possibly because of the need to add a large excess of purified binding protein to the vesicles. We circumvented this difficulty by using a mutant which produces a precursor maltose-binding protein that is translocated across the cytoplasmic membrane but is not cleaved by the signal peptidase (J. D. Fikes and P. J. Bassford, Jr., J. Bacteriol. 169:2352-2359, 1987). The protein remains tethered to the cytoplasmic membrane, presumably through the hydrophobic signal sequence, and we show here that the spheroplasts and membrane vesicles prepared from this mutant catalyze active maltose transport without the addition of purified maltose-binding protein. In vesicles, the transport requires electron donors, such as ascorbate and phenazine methosulfate or D-lactate. However, inhibition by dicyclohexylcarbodiimide and stimulation of transport by the inculsion of ADP or ATP in the intravesicular space suggest that ATP (or compounds derived from it) is involved in the energization of the transport. The transport activity of intact cells can be recovered without much inactivation in the vesicles, and their high activity and ease of preparation will be useful in studies of the mechanism of the binding protein-dependent transport process.     

Reconstitution of maltose chemotaxis in Escherichia coli by addition of maltose-binding protein to calcium-treated cells of maltose regulon mutants.

Maltose chemotaxis was reconstituted in delta malE cells lacking maltose-binding protein (MBP). Purified MBP was introduced into intact cells during incubation with 250 mM CaCl2 in Tris-hydrochloride buffer at 0 degrees C. After removal of extracellular CaCl2 and MBP, chemotaxis was measured with tethered bacteria in a flow chamber or with free-swimming cells in a capillary assay. About 20% of tethered cells responded to 10(-4) M maltose; the mean response times were about half those of CaCl2-treated wild-type cells (100 s as opposed to 190 s). In capillary tests, the maltose response of reconstituted cells was between 15 and 40% of the aspartate response, about the same percentage as in wild-type cells. The best reconstitution was seen with 0.5 to 1 mM MBP in the reconstitution mixture, which is similar to the periplasmic MBP concentration estimated for maltose-induced wild-type cells. Strains containing large deletions of the malB region and malT mutants lacking the positive regulator gene of the mal regulon also could be reconstituted for maltose chemotaxis, showing that no product of the mal regulon other than MBP is essential for maltose chemotaxis.     

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.Non Standard Abbreviations GLPT periplasmic protein, related to transport of glycerolphosphate in Escherichia coli (Silhavy et al., 1976b)     

Recovery of a foreign protein from the periplasm of Escherichia coli by chemical permeabilization.

We have applied the technique of protein release by chemical permeabilization to recover a foreign protein in active form from the periplasm of a recombinant strain of Escherichia coli. The two agents used in our chemical permeabilization scheme, guanidine hydrochloride and Triton X-100, have different modes of action, allowing selectivity in protein release based on intracellular location under different treatment conditions. Specifically, treatment of E. coli C600-1 cells by guanidine alone resulted in 40-fold purification of recombinant beta-lactamase, which is periplasmically expressed in this host. Achieving such high purification in the cell disruption stage could alleviate some of the problems associated with recovery of intracellular products, such as low expression or the need to solubilize cytoplasmic inclusion bodies. Recovery of periplasmic proteins by chemical permeabilization is simpler than by osmotic shock and is less expensive than using enzymatic digestion.     

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 F_ab coupled to horseradish peroxidase (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 showed altered staining pattern. Treatment of glutaraldehyde-fixed cells with EDTA-lysozyme prevented the specific labelling of the maltose-binding protein.Lists of Non Common Abbreviations MBP maltose-binding protein - MBP-F_ab)-HRPO F_ab fragments against maltose-binding, protein coupled to horseradish peroxidase - IgG immunoglobulin - PBS pnosphate buffered saline     

Electrochemical potential releases a membrane-bound secretion intermediate of maltose-binding protein in Escherichia coli.

A secretionary intermediate of the Escherichia coli maltose-binding protein accumulated in the inner membrane when the membrane electrochemical potential was reduced and the cytosolic ATP concentration was normal. The intermediate was mature in size, but maintained a conformation similar to the cytosolic precursor form, and not the mature periplasmic protein, as measured by differences in susceptibility to proteinase K in vitro. The intermediate was located on the periplasmic side of the inner membrane. Restoration of the membrane electrochemical potential resulted in the movement of the intermediate from the inner membrane to the periplasm. In other experiments in which the ATP concentration was reduced by 96% and the electrochemical potential remained normal, no intermediate accumulated. Thus, the final step in the export of maltose-binding protein requires the electrochemical potential of the inner membrane and does not require ATP.     

Periplasmic maltose-binding protein confers specificity on the outer membrane maltose pore of Escherichia coli.

ompB mutants of Escherichia coli K-12 are markedly deficient in porin in their outer membrane. This results in a decreased rate of uptake for many substrates: the maltose pore (lambda receptor) can in some circumstances, in the absence of the periplasmic maltose-binding protein, compensate for the consequent defects in permeability to lactose, mannitol, glycylglycyl-L-valine, and tri-L-ornithine. It is postulated that the maltose-binding protein associates with the maltose pore and confers on it the specificity for maltose, and that the absence of the maltose-binding protein leaves the pore open and results in enhanced transmembrane diffusion of molecules other than maltose. This paper presents evidence to support this hypothesis.