Lipid fluidity-dependent biosynthesis and assembly of the outer membrane proteins of E. coli.

The reduction of the membrane lipids of E. coli to a nonfluid state resulted in the accumulation in the cell envelope of a high molecular weight precursor of the protoIG protein, a major outer membrane protein. The protoIG protein was as sensitive to trypsin as the mature toIG protein assembled in the outer membrane. In contrast to the toIG protein, however, the accumulated protoIG protein was easily released from the envelope fraction by both sodium lauryl sarcosinate extraction and sonication. This indicated that the precursor protein was loosely associated with the cell membrane. When a fluid lipid state was restored, the protoIG protein was processed to the mature form which was then correctly assembled in the outer membrane. These results suggest that the protoIG protein produced under nonfluid lipid conditions was properly translocated across the cytoplasmic membrane, but could not be assembled in the outer membrane due either to the reversible inhibition of the processing of the ProtoIG to the toIG protein or to the lack of interaction with a specific outer membrane component(s). Reduced lipid fluidity also caused various alterations in the biosynthesis and assembly of other membrane proteins. In addition to the toIG protein, a large number of new proteins were accumulated in the membrane. Alternatively, the matrix protein as well as the promatrix protein were not detected in the cell envelope. On the other hand, the lipoprotein was normally produced, processed, modified and assembled in the outer membrane. These results indicate that the outer membrane proteins are synthesized and assembled according to several different mechanisms, on which the physical state of the membrane has various effects.     

Release of a special fraction of the outer membrane from both growing and phage T4-infected Escherichia coli B.

Growing Escherichia coli release envelope material into the medium. Upon infection with T4 phage increased amounts of this material are released and at a greater rate. In order to determine whether both inner and outer membranes are present in this material, and whether the material released by growing cells differs from that released by infected cells, we have examined the protein composition of envelope released by growing and T4-infected E. coli B. Our results show: (a) the protein composition of envelope released from growing or infected cells is similar, (b) the proteins present are representative of the outer membrane, (c) the major outer membrane protein of E. coli B, protein II, is deficient in the released material. We therefore conclude that the envelope material released from growing or infected E. coli represents a special fraction of the outer membrane. This finding is discussed in relation to outer membrane structure and function. In addition, data are presented on the differing outer membrane protein composition of substrains of E. coli B obtained from different laboratories.     

Effects of secA mutations on the synthesis and secretion of proteins in Escherichia coli. Evidence for a major export system for cell envelope proteins

We have followed the synthesis and secretion of a number of periplasmic and outer membrane proteins in three strains of Escherichia coli, a secA amber mutant, a secA temperature-sensitive mutant, and a strain that blocks protein secretion due to a high level of expression of an export-defective hybrid protein between maltose-binding protein and beta-galactosidase (MalE-LacZ). Our results show that after several hours under nonpermissive conditions the specificity and extent of the export blocks in the secA temperature-sensitive mutant and the strain producing the MalE-LacZ hybrid protein are identical, affecting at least four major outer membrane proteins and most but not all periplasmic proteins. The secA gene product, therefore, appears to be an essential component of the major export pathway in E. coli which is used by many envelope proteins independent of whether they are cotranslationally or post-translationally secreted. In contrast, the synthesis of only a subset of these envelope proteins is reduced in the secA amber mutant after shift to the nonpermissive condition. These results indicate that the SecA protein serves roles both in the synthesis and the secretion of certain cell envelope proteins.     

Characterization of the Salmonella typhimurium proteome by semi-automated two dimensional HPLC-mass spectrometry: detection of proteins implicated in multiple antibiotic resistance

The proteome of Salmonella enterica serovar Typhimurium was characterized by 2-dimensional HPLC mass spectrometry to provide a platform for subsequent proteomic investigations of low level multiple antibiotic resistance (MAR). Bacteria (2.15 +/- 0.23 x 10(10) cfu; mean +/- s.d.) were harvested from liquid culture and proteins differentially fractionated, on the basis of solubility, into preparations representative of the cytosol, cell envelope and outer membrane proteins (OMPs). These preparations were digested by treatment with trypsin and peptides separated into fractions (n = 20) by strong cation exchange chromatography (SCX). Tryptic peptides in each SCX fraction were further separated by reversed-phase chromatography and detected by mass spectrometry. Peptides were assigned to proteins and consensus rank listings compiled using SEQUEST. A total of 816 +/- 11 individual proteins were identified which included 371 +/- 33, 565 +/- 15 and 262 +/- 5 from the cytosolic, cell envelope and OMP preparations, respectively. A significant correlation was observed (r2 = 0.62 +/- 0.10; P < 0.0001) between consensus rank position for duplicate cell preparations and an average of 74 +/- 5% of proteins were common to both replicates. A total of 34 outer membrane proteins were detected, 20 of these from the OMP preparation. A range of proteins (n = 20) previously associated with the mar locus in E. coli were also found including the key MAR effectors AcrA, TolC and OmpF.     

Proteomic analysis of extracellular proteins from Escherichia coli W3110

While numerous proteomic analyses have been carried out on Escherichia coli, the vast majority have focused on expression of intracellular proteins. Yet, recent literature reports imply that even in laboratory strains, significant proteins may be found outside the cell. Here, we identify extracellular proteins associated with nonpathogenic E. coli strain W3110. Two-dimensional gel electrophoresis (2DE) revealed approximately 66 prominent protein spots during exponential growth (4 and 8 h shake flask culture) in minimal medium. The absence of detectable nucleic acids in the culture supernatant implies these proteins did not result from cell lysis. MALDI-TOF MS was used to identify 44 proteins, most of which have been previously identified as either outer membrane or extracellular proteins. In addition, 2DE protease zymogram analysis was carried out which facilitated identification of three extracellular proteases, one of which was not observed during standard 2DE. Our results are consistent with previous findings which imply outer membrane proteins are shed during growth.     

Comparative analysis of envelope proteomes in Escherichia coli B and K-12 strains

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane beta-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.     

Helical disposition of proteins and lipopolysaccharide in the outer membrane of Escherichia coli

In bacteria, several physiological processes once thought to be the products of uniformly dispersed reactions are now known to be highly asymmetric, with some exhibiting interesting geometric localizations. In particular, the cell envelope of Escherichia coli displays a form of subcellular differentiation in which peptidoglycan and outer membrane proteins at the cell poles remain stable for generations while material in the lateral walls is diluted by growth and turnover. To determine if material in the side walls was organized in any way, we labeled outer membrane proteins with succinimidyl ester-linked fluorescent dyes and then grew the stained cells in the absence of dye. Labeled proteins were not evenly dispersed in the envelope but instead appeared as helical ribbons that wrapped around the outside of the cell. By staining the O8 surface antigen of E. coli 2443 with a fluorescent derivative of concanavalin A, we observed a similar helical organization for the lipopolysaccharide (LPS) component of the outer membrane. Fluorescence recovery after photobleaching indicated that some of the outer membrane proteins remained freely diffusible in the side walls and could also diffuse into polar domains. On the other hand, the LPS O antigen was virtually immobile. Thus, the outer membrane of E. coli has a defined in vivo organization in which a subfraction of proteins and LPS are embedded in stable domains at the poles and along one or more helical ribbons that span the length of this gram-negative rod.     

Effect of mutation in phoB and phoM genes for the positive control of alkaline phosphatase biosynthesis on Escherichia coli envelope proteins

The suppression of some envelope proteins, localized in both the periplasm and the outer and inner membranes was shown in phoB and phoM phoR mutants of E. coli. Among these proteins are the proteins of the phosphate regulon and also those not pertaining them. As a result of phoB and phoM phoR mutations, the cytoplasmic membrane was found to be lacking in minor protein of 28,000 Mr, which belongs to the phosphate regulon. Besides, the phoM phoR mutation leads to the loss of protein of 55,000 Mr of the outer membranes, whereas phoB mutation causes loss of protein 37 000 Mr, identified as outer membrane protein OmpT. A damage in the phoB mutant of the protein proteolytic modification, probably determining the suppression of the biosynthesis of E. coli envelope secreted proteins is suggested.     

Export of hybrid proteins FhuA''-''LacZ and FhuA''-''PhoA to the cell envelope of Escherichia coli K-12.

The fhuA gene of Escherichia coli K-12 encodes an outer membrane protein that acts as the ferrichrome-iron(III) receptor. To determine the export signals and sorting information within FhuA, gene fusions of fhuA'-'lacZ and fhuA'-'phoA were constructed. Although a FhuA'-'LacZ hybrid protein was detected in the Triton X-100-insoluble fraction of the cell envelope, direct immunoelectron microscopic observation showed that this protein remained in the cytoplasm. FhuA'-'PhoA hybrid proteins were all exported across the cytoplasmic membrane. Those hybrids containing up to 88 amino acids of FhuA (FhuA88) fused to PhoA were released along with other periplasmic proteins. Hybrids containing 180 or more amino acids of FhuA (FhuA180) fused to PhoA were associated with the outer membrane. It is proposed that some information inherent in the sequences between FhuA88 and FhuA180 confers stable association with the outer membrane.     

Components of the RP4 conjugative transfer apparatus form an envelope structure bridging inner and outer membranes of donor cells: implications for related macromolecule transport systems

During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell. A membrane-spanning transfer apparatus encoded by conjugative plasmids has been proposed to facilitate protein and DNA transport. For the IncPalpha plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins. We localized essential RP4 transfer functions to Escherichia coli cell fractions by immunological detection with specific polyclonal antisera. Each of the gene products of the RP4 mating pair formation (Mpf) system, specified by the Tra2 core region and by traF of the Tra1 region, was found in the outer membrane fraction with one exception, the TrbB protein, which behaved like a soluble protein. The membrane preparation from Mpf-containing cells had an additional membrane fraction whose density was intermediate between those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the two E. coli membranes. The Tra1 region is known to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction. This indicates that the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the E. coli cell envelope. The Tra1 protein TraG was predominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.     

Interaction between two major outer membrane proteins of Escherichia coli: the matrix protein and the lipoprotein.

The affinity to the matrix protein, one of the major outer membrane proteins of Escherichia coli, for the peptidoglycan was examined of extracting the cell envelope complex at 55 degrees C and 2% sodium dodecyl sulfate containing different amounts of NaCl. It was found that the matrix protein was extracted from the peptidoglycan of a mutant strain (lpo) that lacks another major membrane protein, the lipoprotein, at a lower NaCl concentration than was the matrix protein of the wild-type cell (lpo+). When the envelope fraction of the wild-type strain was treated with trypsin, which is known to cleave the bound-form lipoprotein from the peptidoglycan, the affinity of the matrix protein for the peptidoglycan decreased to the same level as that of the affinity of the matrix protein for the peptidoglycan of the mutant strain. It was further shown that the free-form lipoprotein was also retained in the matrix protein-peptidoglycan complex, although the extent of retention of the free form of the lipoprotein was less than that of the matrix protein. These results indicate that both the free and the bound forms of the lipoprotein are closely associated with the matrix protein and that the bound form of the lipoprotein plays and important role in the association between the matrix protein and the peptidoglycan.     

Bacterial proteins with cleaved or uncleaved signal peptides of the general secretory pathway

Correct protein compartmentalization is a key step for molecular function and cell viability, and this is especially true for membrane and externalized proteins of bacteria. Recent proteomic reports of Bacillus subtilis have shown that many proteins with Sec-like signal peptides and absence of a transmembrane helix domain are still observed in membrane-enriched fractions, but further evidence about signal peptide cleavage or soluble protein contamination is still needed. Here we report a proteomic screening of identified peptides in culture filtrate, membrane fraction and whole cell lysate of Mycobacterium tuberculosis. We were able to detect peptide sequencing evidence that shows that the predicted signal peptide was kept uncleaved for several types of proteins such as mammalian cell entry (Mce) proteins and PE or PE-PGRS proteins. Label-free quantitation of all proteins identified in each fraction showed that the majority of these proteins with uncleaved signal peptides are, indeed, enriched in the Triton X-114 lipid phase. Some of these proteins are likely to be located in the inner membrane while others may be outer membrane proteins.     

Specific location of penicillin-binding proteins within the cell envelope of Escherichia coli.

This communication deals with the location of penicillin-binding proteins in the cell envelope of Escherichia coli. For this purpose, bacterial cells have been broken by various procedures and their envelopes have been fractioned. To do so, inner (cytoplasmic) and outer membranes were separated by isopycnic centrifugation in sucrose gradients. Some separation methods (Osborn et al., J. Biol. Chem. 247:3962-3972, 1972; J. Smit, Y. Kamio, and H. Nikaido, J. Bacteriol. 124:942-958, 1975) revealed that penicillin-binding proteins are not exclusively located in the inner membrane. They are also found in the outer membrane (A. Rodríguez-Tébar, J. A. Barbas, and D. Vásquez, J. Bacteriol. 161:243-248, 1985). Under the milder conditions for cell rupture used in this work, an intermembrane fraction, sedimenting between the inner and outer membrane, can be recovered from the gradients. This fraction has a high content of both penicillin-binding proteins and phospholipase B activity and may correspond to the intermembrane adhesion sites (M. H. Bayer, G. P. Costello, and M. E. Bayer, J. Bacteriol. 149:758-769, 1982). We postulate that this intermembrane fraction is a labile structure that contains a high amount of all penicillin-binding proteins which are usually found in both the inner and outer membranes when the adhesion sites are destroyed by the cell breakage and fractionation procedures.     

In vivo labeling of Escherichia coli cell envelope proteins with N-hydroxysuccinimide esters of biotin.

The primary amine coupling reagents succinimidyl-6-biotinamido-hexanoate (NHS-A-biotin) and sulfosuccinimidyl-6-biotinamido-hexanoate (NHS-LC-biotin) were tested for their ability to selectively label Escherichia coli cell envelope proteins in vivo. Probe localization was determined by examining membrane, periplasmic, and cytosolic protein fractions. Both hydrophobic NHS-A-biotin and hydrophilic NHS-LC-biotin were shown to preferentially label outer membrane, periplasmic, and inner membrane proteins. NHS-A- and NHS-LC-biotin were also shown to label a specific inner membrane marker protein (Tet-LacZ). Both probes, however, failed to label a cytosolic marker (the omega fragment of beta-galactosidase). The labeling procedure was also used to label E. coli cells grown in low-salt Luria broth medium supplemented with 0, 10, and 20% sucrose. Outer membrane protein A (OmpA) and OmpC were labeled by both NHS-A- and NHS-LC-biotin at all three sucrose concentrations. In contrast, OmpF was labeled by NHS-A-biotin but not by NHS-LC-biotin in media containing 0 and 10% sucrose. OmpF was not labeled by either NHS-A- or NHS-LC-biotin in E. coli cells grown in medium containing 20% sucrose. Coomassie-stained gels, however, revealed similar quantities of OmpF in E. coli cells grown at all three sucrose concentrations. These data indicate that there was a change in outer membrane structure due to increased osmolarity, which limits accessibility of NHS-A-biotin to OmpF.     

Replication of an RK2 miniplasmid derivative in vitro by a DNA/membrane complex extracted from Escherichia coli: involvement of the dnaA but not dnaK host proteins and association of these and plasmid-encoded proteins with the inner membrane

A DNA/membrane complex extracted from a miniplasmid derivative of the broad host range plasmid RK2 cultured in Escherichia coli capable of synthesizing new plasmid supercoiled DNA in vitro was treated with antibodies that were made against or reacted with the dnaA and dnaK host-encoded proteins, respectively. Anti-dnaA protein antibody inhibited total plasmid DNA synthesis significantly and the synthesis of supercoil plasmid DNA almost completely. In contrast, anti-dnaK protein antibody and nonimmune serum had little or no effect on total plasmid DNA synthesis. Both proteins were found to be present in the inner but not outer membrane fraction of E. coli. A variety of miniplasmid-encoded proteins which had previously been found in the DNA/membrane complex have also been localized to the inner but not outer membrane fraction. These include an essential initiation protein of 32 kDa (and an overlapping protein of 43 kDa coded for by the same gene), as well as a 30-kDa protein that may be linked to incompatibility functions. Various extraction methods were used to distinguish between the associated and the integral nature of the plasmid-encoded proteins. The results demonstrated that the essential replication proteins (32 and 43 kDa) as well as the 30-kDa protein was tightly bound to the inner membrane. Computer analysis of the amino acid sequence of the 32 (and 43)-kDa protein revealed a hydrophobic region that is only half that normally required to span the membrane. Other interactions are discussed with respect to attaching this protein to the membrane.     

Proline 21, a residue within the α-helical domain of ΦX174 lysis protein E, is required for its function in Escherichia coli

ΦX174 lysis protein E-mediated lysis of Escherichia coli is characterized by a protein E-specific fusion of the inner and outer membrane and formation of a transmembrane tunnel structure. In order to understand the fusion process, the topology of protein E within the envelope complex of E. coli was investigated. Proteinase K protection studies showed that, during the time course of protein E-mediated lysis process, more of the fusion protein E-FXa-streptavidin gradually became accessible to the protease at the cell surface. These observations postulate a conformational change in protein E during induction of the lysis process by movement of the C-terminal end of the protein throughout the envelope complex from the inner side to the outer side spanning the entire pore and fusing the inner and outer membranes at distinct areas. The initiation mechanism for such a conformational change could be the cis–trans isomerization of proline residues within α-helical membrane-spanning segments. Conversion of proline 21, presumed to be in the membrane-embedded α-helix of protein E, to alanine, glycine, serine and valine, respectively, resulted in lysis-negative E mutant proteins. Proteinase K accessibility studies using streptavidin as a reporter fused to the P21G mutant protein showed that the C-terminal part of the fusion protein is not translocated to the outer side of the membrane, suggesting that this proline residue is essential for the correct folding of protein E within the cell wall complex of E. coli . Oligomerization of protein P21G-StrpA was not disturbed.     

Insertion of newly synthesized proteins into the outer membrane of Escherichia coli.

The insertion of newly synthesized proteins into the outer membrane of Escherichia coli has been examined. The results show that there is no precurser pool of outer membrane proteins in the cytoplasmic membrane because first, the incorporation of a [35S]methionine pulse into outer membrane proteins completely parallels its incorporation into cytoplasmic membrane proteins, and second, under optimal isolation conditions, no outer membrane proteins are found in the cytoplasmic membrane, even when the membranes are analysed after being labeled for only 15 s. The [35S]methionine present in the outer membrane after a pulse of 15 s was found in protein fragments of varying sizes rather than in specific outer membrane proteins. This label could however be chased into specific proteins within 30--120 s, depending on the size of the protein, indicating that although unfinished protein fragments were present in the outer membrane, they were completed by subsequent chain elongation. Thus, outer membrane proteins are inserted into the outer membrane while still attached to ribosomes. Since ribosomes which are linked to the cell envelope by nascent polypeptide chains are stationary, the mRNA which is being translated by these ribosomes moves along the inner cell surface.     

Purification of protein A, an outer membrane component missing in Escherichia coli K-12 ompA mutants.

Outer membrane materials prepared from an Escherichia coli ompA (tolG) strain do not contain one of the major outer membrane proteins found in ompA+ strains. This protein has been purified in high yield from detergent-solubilized cell envelope material prepared from an ompA+ strain by preparative electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. The purified protein is homogeneous in three electrophoretic systems, contains 2 mol of reducing sugar/mol of peptide and has alanine as the N-terminal amino acid. The amino acid composition is nearly identical to outer membrane protein II or B purified by others from incompletely solubilized cell envelope material. Thus, the fraction of outer membrane protein II or B that is difficult to solubilize is identical with the more readily solubilized fraction.     

Fate of predator and prey proteins during growth of Bdellovibrio bacteriovorus on Escherichia coli and Pseudomonas syringae prey

A two-dimensional electrophoretic analysis of protein distribution followed by identification of selected proteins by mass spectrometry was performed on fresh bdellovibrio cultures containing attack phase cells of the predatory bacterium Bdellovibrio bacteriovorus strain 109J-1 and the remains of an Escherichia coli or a Pseudomonas syringae pv. tomato prey. Cleavage of the peptidoglycan-associated outer membrane proteins (OMPs) OmpA in E. coli and OprF in P. syringae occurred in both prey. The tryptic peptides obtained from the cleavage products of OmpA and OprF were all located within the 19-kDa pronase-resistant N-terminal parts of the corresponding proteins. The predator cell fraction was separated from the prey ghosts in fresh bdellovibrio cultures by centrifugation on a Percoll-sucrose cushion. Proteins from each fraction were separated by two-dimensional electrophoresis and identified by mass spectrometric analysis. As no prey OMP could be detected in the predator cell fraction, it was concluded that prey OMPs are not transferred to the predator, as had been suggested previously. However, a protein from the predator was found bound to ghost cell envelopes. This protein may correspond to a protein earlier suggested to be associated with the prey outer or cytoplasmic membranes. Along with recently described polypeptides from B. bacteriovorus strains 100 and 114, it forms a new family of putative outer membrane proteins.     

Insight from TonB hybrid proteins into the mechanism of iron transport through the outer membrane

We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB(+) bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepADelta3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.