Molecular analysis of asmA, a locus identified as the suppressor of OmpF assembly mutants of Escherichia coli K-12

We present the molecular characterization of the asmA gene, whose product is involved in the assembly of outer membrane proteins in Escherichia coli K-12. The asmA locus was initially identified as a site for suppressor mutations of an assembly defective OmpF315. Our data suggest that these suppressor mutations either completely abolish or reduce asmA expression and can be complemented in trans by piasmid clones carrying asmA sequences. The recessive nature of asmA suppressor mutations suggests that the functional AsmA protein participates in Inhibiting the assembly of OmpF315 and other mutant OmpFs. As the assembly of wild-type and parental OmpF proteins was not affected by asmA mutations, AsmA must provide an environment refractory only to the assembly of mutant OmpF proteins. However, we cannot completely rule out the possibility that AsmA plays a minor role in the assembly of wild-type and parental OmpF in wild-type cells. The presence of a putative signal sequence within the amino-terminal sequence of AsmA suggests that it is either a periplasmic or an outer membrane protein. This predicted location of AsmA is compatible with its role in the assembly of outer membrane proteins.     

Examination of AsmA and its effect on the assembly of Escherichia coli outer membrane proteins

asmA mutations were isolated as extragenic suppressors of an OmpF assembly mutant, OmpF315. This suppressor locus produced a protein that was present in extremely low levels and could only be visualized by Western blotting in cells where AsmA expression was induced from a plasmid. Detailed fractionation analyses showed that AsmA localized with the inner membrane. Curiously, however, the mutant OmpF assembly step influenced by AsmA occurred in the outer membrane, perhaps indicating an indirect involvement of AsmA in the assembly of outer membrane proteins. Biochemical examination of the outer membrane showed that asmA null mutations reduce lipo-polysaccharide (LPS) levels, thereby lowering the ratios of glycerolphospholipids to LPS and envelope proteins to LPS in the outer membrane. Despite these quantitative alterations, no apparent structural changes in LPS or major phospholipids were noted. Reduced LPS levels in asmA mutants indicate a possible role of AsmA in LPS biogenesis. Data presented in this study suggest that asmA-mediated OmpF assembly suppression may have been achieved by altering the outer membrane fluidity, thus making it more amenable for the assembly of mutant proteins.     

Expression of chimeric ras protein with OmpF signal peptide in Escherichia coli: localization of OmpF fusion protein in the inner membrane

Summary The ras gene was fused with the DNA sequence of OmpF signal peptide or with the DNA sequence of OmpF signal peptide plus the amino terminal portion of the OmpF gene. They were placed in plasmids together with the bacteriophage P _L promoter. These plasmids were introduced into Escherichia coli strain K-12 and the OmpF signal peptide fusion proteins were expressed. These fusion proteins were idetified as 29.0 and 30.0 kDa proteins. However, processed products of these proteins were not found in the The fusion proteins were localized mostly in the cytoplasm and the inner membrane, but none of them was secreted into the periplasmic space. On the other hand, the ras protein alone was found in the cytoplasm and not in the inner membrane. Viable counts of E. coli harbouring these plasmids decreased when these fused proteins were induced. Induction of the ras protein alone did not harm cells. These observations suggest that insertion of the heterologous proteins into the inner membrane may cause the bactericidal effect. Offprint requests to: A. Kaji     

Role for Skp in LptD Assembly in Escherichia coli

The periplasmic chaperone Skp has long been implicated in the assembly of outer membrane proteins (OMPs) in Escherichia coli. It has been shown to interact with unfolded OMPs, and the simultaneous loss of Skp and the main periplasmic chaperone in E. coli, SurA, results in synthetic lethality. However, a Δskp mutant displays only minor OMP assembly defects, and no OMPs have been shown to require Skp for their assembly. Here, we report a role for Skp in the assembly of the essential OMP LptD. This role may be compensated for by other OMP assembly proteins; in the absence of both Skp and FkpA or Skp and BamB, LptD assembly is impaired. Overexpression of SurA does not restore LptD levels in a Δskp ΔfkpA double mutant, nor does the overexpression of Skp or FkpA restore LptD levels in the ΔsurA mutant, suggesting that Skp acts in concert with SurA to efficiently assemble LptD in E. coli. Other OMPs, including LamB, are less affected in the Δskp ΔfkpA and Δskp bamB::kan double mutants, suggesting that Skp is specifically necessary for the assembly of certain OMPs. Analysis of an OMP with a domain structure similar to that of LptD, FhuA, suggests that common structural features may determine which OMPs require Skp for their assembly.     

Secretion into the culture medium of a foreign gene product from Escherichia coli: use of the ompF gene for secretion of human beta-endorphin.

The ompF gene codes for a major outer membrane protein of Escherichia coli. A plasmid was constructed in which the structural gene for human beta-endorphin is preceded by the upstream region of the ompF gene consisting of the promoter region and the coding regions for the signal peptide and the N terminus of the OmpF protein. When the plasmid was introduced into E. coli N99, and OmpF-beta-endorphin fused peptide was synthesized and secreted into the culture medium through both the cytoplasmic and outer membranes. The OmpF signal peptide was cleaved correctly during the secretion, indicating that the export of the fused protein across the cytoplasmic membrane was dependent on the signal peptide. The secretion into the culture medium was apparently selective. Neither beta-lactamase nor alkaline phosphatase (both are periplasmic proteins) appeared in the culture medium in significant amounts. The mode of passage of the fused peptide across the outer membrane is discussed.     

Genetic and biochemical characterization of anEscherichia coli K-12 mutant with an altered outer membrane protein

The properties of anEscherichia coli K-12 mutant are described which seemingly produces a “new” major outer membrane protein with an apparent molecular weight of 40000. This 40K protein was purified and its cyanogen bromide (CNBr) fragments were compared with those of several known major outer membrane proteins. A similarity was found between the CNBr fragments of the 40K protein and those of the OmpF protein (molecular weight 37000). In addition, the 40K protein was found to be regulated exactly like the OmpF protein, and the mutation which causes the production of the 40K protein has been localized in (or very close to) theompF gene. It is concluded that the 40K protein is a mutant form of the OmpF protein. The results provide additional evidence that theompF gene at minute 21 is the structural gene for the OmpF protein.     

An inner membrane protein N-terminal signal sequence is able to promote efficient localisation of an outer membrane protein in Escherichia coli

To test the importance of N-terminal pre-sequences in translocation of different classes of membrane proteins, we exchanged the normal signal sequence of an Escherichia coli outer membrane protein, OmpF, for the pre-sequence of the inner membrane protein, DacA. The DacA-OmpF hybrid was efficiently assembled into the outer membrane in a functionally active form. Thus the pre-sequence of DacA, despite its relatively low hydrophobicity compared with that of OmpF, contains all the essential information necessary to initiate the translocation of OmpF to the outer membrane. Since processing of DacA was also shown to be dependent upon SecA we conclude that the initiation of translocation of this inner membrane polypeptide across the envelope occurs by the same mechanism as outer membrane and periplasmic proteins. The N-terminal 11 amino acids of mature OmpF, which in the hybrid are replaced by the N-terminal nine amino acids of DacA, carry no essential assembly signals since the hybrid protein is apparently assembled with equal efficiency to OmpF.     

Lethal mutations in the structural gene of an outer membrane protein (OmpA) of Escherichia coli K12

Summary The gene ompA encodes a major outer membrane protein of Escherichia coli. Localized mutagenesis of the part of the gene corresponding to the 21-residue signal sequence and the first 45 residues of the protein resulted in alterations which caused cell lysis when expressed. DNA sequence analyses revealed that in one mutant type the last CO₂H-terminal residue of the signal sequence, alanine, was replaced by valine. The proteolytic removal of the signal peptide was much delayed and most of the unprocessed precursor protein was fractioned with the outer membrane. However, this precursor was completely soluble in sodium lauryl sarcosinate which does not solubilize the OmpA protein or fragments thereof present in the outer membrane. Synthesis of the mutant protein did not inhibit processing of the OmpA or OmpF proteins. In the other mutant type, multiple mutational alterations had occurred leading to four amino acid substitutions in the signal sequence and two affecting the first two residues of the mature protein. A reduced rate of processing could not be clearly demonstrated. Membrane fractionation suggested that small amounts of this precursor were associated with the plasma membrane but synthesis of this mutant protein also did not inhibit processing of the wild-type OmpA or OmpF proteins. Several lines of evidence left no doubt that the mature, mutant protein is stably incorporated into the outer membrane. It is suggested that the presence, in the outer membrane, of the mutant precursor protein in the former case, or of the mutant protein in the latter case perturbs the membrane architecture enough to cause cell death.     

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.     

SurA assists the folding of Escherichia coli outer membrane proteins.

Many proteins require enzymatic assistance in order to achieve a functional conformation. One rate-limiting step in protein folding is the cis-trans isomerization of prolyl residues, a reaction catalyzed by prolyl isomerases. SurA, a periplasmic protein of Escherichia coli, has sequence similarity with the prolyl isomerase parvulin. We tested whether SurA was involved in folding periplasmic and outer membrane proteins by using trypsin sensitivity as an assay for protein conformation. We determined that the efficient folding of three outer membrane proteins (OmpA, OmpF, and LamB) requires SurA in vivo, while the folding of four periplasmic proteins was independent of SurA. We conclude that SurA assists in the folding of certain secreted proteins.     

Overexpression of protease-deficient DegP(S210A) rescues the lethal phenotype of Escherichia coli OmpF assembly mutants in a degP background

Replacement of OmpF's conserved carboxy-terminal phenylalanine with dissimilar amino acids severely impaired its assembly into stable trimers. In some instances, interactions of mutant proteins with the outer membrane were also affected, as judged by their hypersensitivity phenotype. Synthesis of all mutant OmpF proteins elevated the expression of periplasmic protease DegP, and synthesis of most of them made its presence obligatory for cell viability. These results showed a critical role for DegP in the event of aberrant outer membrane protein assembly. The lethal phenotype of mutant OmpF proteins in a degP null background was eliminated when a protease-deficient DegP(S210A) protein was overproduced. Our data showed that this rescue from lethality and a subsequent increase in mutant protein levels in the envelope did not lead to the proper assembly of the mutant proteins in the outer membrane. Rather, a detergent-soluble and thermolabile OmpF species resembling monomers accumulated in the mutants, and to a lesser extent in the parental strain, when DegP(S210A) was overproduced. Interestingly, this also led to the localization of a significant amount of mutant polypeptides to the inner membrane, where DegP(S210A) also fractionated. These results suggested that the DegP(S210A)-mediated rescue from toxicity involved preferential sequestration of misfolded OmpF monomers from the normal assembly pathway.     

Dissection of β‐barrel outer membrane protein assembly pathways through characterizing BamA POTRA 1 mutants of Escherichia coli

BamA of Escherichia coli is an essential component of the hetero‐oligomeric machinery that mediates β‐barrel outer membrane protein (OMP) assembly. The C‐ and N‐termini of BamA fold into trans‐membrane β‐barrel and five soluble POTRA domains respectively. Detailed characterization of BamA POTRA 1 missense and deletion mutants revealed two competing OMP assembly pathways, one of which is followed by the archetypal trimeric β‐barrel OMPs, OmpF and LamB, and is dependent on POTRA 1. Interestingly, our data suggest that BamA also requires its POTRA 1 domain for proper assembly. The second pathway is independent of POTRA 1 and is exemplified by TolC. Site‐specific cross‐linking analysis revealed that the POTRA 1 domain of BamA interacts with SurA, a periplasmic chaperone required for the assembly of OmpF and LamB, but not that of TolC and BamA. The data suggest that SurA and BamA POTRA 1 domain function in concert to assist folding and assembly of most β‐barrel OMPs except for TolC, which folds into a unique soluble α‐helical barrel and an OM‐anchored β‐barrel. The two assembly pathways finally merge at some step beyond POTRA 1 but presumably before membrane insertion, which is thought to be catalysed by the trans‐membrane β‐barrel domain of BamA.     

PhoE protein pore of the outer membrane of Escherichia coli K12 is a particularly efficient channel for organic and inorganic phosphate

This study was undertaken to investigate the proposed in vivo pore function of PhoE protein, an Escherichia coli K12 outer membrane protein induced by growth under phosphate limitation, and to compare it with those of the constitutive pore proteins OmpF and OmpC. Appropriate mutant strains were constructed containing only one of the proteins PhoE, OmpF or OmpC, or none of these proteins at all. By measuring rates of nutrient uptake at low solute concentrations, the proposed pore function of PhoE protein was confirmed as the presence of the protein facilitates the diffusion of P_i through the outer membrane, such that a pore protein deficient strain behaves as a K_m mutant. Comparison of the rates of permeation of P_i, glycerol 3-phosphate and glucose 6-phosphate through pores formed by PhoE, OmpF and OmpC proteins shows that PhoE protein is the most effective pore in facilitating the diffusion of P_i and phosphorus-containing compounds. The three types of pores were about equally effective in facilitating the permeation of glucose and arsenate. Possible reasons for the preference for P_i and P_i-containing solutes are discussed.     

Transport of iron across the outer membrane

Summary The TonB protein is involved in energy-coupled receptor-dependent transport processes across the outer membrane. The TonB protein is anchored in the cytoplasmic membrane but exposed to the periplasmic space. To fulfill its function, it has to couple the energy-providing metabolism in the cytoplasmic membrane with regulation of outer membrane receptor activity. Ferrichrome and albomycin transport, uptake of colicin M, and infection by the phages T1 and80 occur via the same receptor, the FhuA protein in the outer membrane. Therefore, this receptor is particularly suitable for the study of energy-coupled TonB-dependent transport across the outer membrane. Ferrichrome, albomycin and colicin M bind to the FhuA receptor but are not released into the periplasmic space of unenergized cells, ortonB mutants. In vivo interaction between FhuA and TonB is suggested by the restoration of activity of inactive FhuA proteins, bearing amino acid replacements in the TonB box, by TonB derivatives with single amino acid substitutions. Point mutations in thefhuA gene are suppressed by point mutations in thetonB gene. In addition, naturally occurring degradation of the TonB protein and its derivatives is preferentially prevented in vivo by FhuA and FhuA derivatives where functional interaction takes place. It is proposed that in the energized state, TonB induces a conformation in FhuA which leads to the release of the FhuA-bound compounds into the periplasmic space. Activation of FhuA by TonB depends on the ExbBD proteins in the cytoplasmic membrane. They can be partially replaced by the TolQR proteins which show strong sequence similarity to the ExbBD proteins. A physical interaction of these proteins with the TonB protein is suggested by TonB stabilization through ExbB and TolQR. We propose a permanent or reversible complex in the cytoplasmic membrane composed of the TonB protein and the ExbBD/TolQR proteins through which TonB is energized.     

Gene encoding a hybrid OmpF-PhoE pore protein in the outer membrane of Escherichia coli K12

Summary To study the structure-function relationship of outer membrane pore proteins of E. coli K12, a hybrid gene was constructed in which the DNA encoding amino acid residues 2–73 of the mature PhoE protein is replaced by the homologous part of the related ompF gene. The product of this gene is incorporated normally into the outer membrane. It was characterized with respect to its pore activity and its phage receptor and colicin receptor properties. It is concluded (i) that the preference of the PhoE protein pore for negatively charged solutes is partly determined by the amino terminal 73 amino acids, (ii) that part of the receptor site of PhoE protein for phage TC45 is located in this part of the protein, (iii) that colicin N uses OmpF protein as (part of) its receptor, (iv) that the specificity of OmpF protein as a colicin N receptor is completely located within the 80 amino terminal amino acid residues, whereas the specificity of this protein as a colicin A receptor is completely located within the 260 carboxy terminal amino acid residues, and (v) that the amino terminal 73 amino acid residues of PhoE protein span the membrane at least once.     

The ultimate localization of an outer membrane protein of Escherichia coli K-12 is not determined by the signal sequence

To study the role of the signal sequences in the biogenesis of outer membrane proteins, we have constructed two hybrid genes: a phoE-ompF hybrid gene, which encodes the signal sequence of outer membrane PhoE protein and the structural sequence of outer membrane OmpF protein, and a bla-phoE hybrid gene which encodes the signal sequence as well as 158 amino acids of the structural sequence of the periplasmic enzyme beta-lactamase and the complete structural sequence of PhoE protein. The products of these genes are normally transported to and assembled into the outer membrane These results show: (i) that signal sequences of exported proteins are export signals which function independently of the structural sequence, and (ii) that the information which determines the ultimate location of an outer membrane protein is located in the structural sequence of this protein, and not in the signal sequence.     

Export of altered forms of an Escherichia coli K-12 outer membrane protein (OmpA) can inhibit synthesis of unrelated outer membrane proteins

Expression of mutant ompA genes, encoding the 325 residue Escherichia coli outer membrane protein OmpA, caused an inhibition of synthesis of the structurally unrelated outer membrane porins OmpC and OmpF and of wild-type OmpA, but not of the periplasmic beta-lactamase. There was no accumulation of precursors of the target proteins and the inhibitory mechanism operated at the level of translation. So far only alterations around residue 45 of OmpA have been found to affect this phenomenon. Linkers were inserted between the codons for residues 45 and 46. A correlation between size and sequence of the resulting proteins and presence or absence of the inhibitory effect was not found, indicating that the added residues acted indirectly by altering the conformation of other parts of the mutant OmpA. To be effective, the altered polypeptides had to be channelled into the export pathway. Internal deletions in effector proteins, preventing incorporation into the membrane, abolished effector activity. The results suggest the existence of a periplasmic component that binds to OmpA prior to membrane assembly; impaired release of this factor from mutant OmpA proteins may trigger inhibition of translation. The factor could be a See B-type protein, keeping outer membrane proteins in a form compatible with membrane assembly.     

Construction of a series of ompC-ompF chimeric genes by in vivo homologous recombination in Escherichia coli and characterization of their translational products

Summary OmpC and OmpF are major outer membrane proteins and although they are homologous proteins, they function differently in several respects. As an approach to elucidate the submolecular structures that determine their differences, we have constructed a series of ompC-ompF chimeric genes by in vivo homologous recombination between these two genes, which are adjacent on a plasmid. The recombination sites in the chimeric genes were localized by means of restriction endonuclease analysis and nucleotide sequence determination. Most of the chimeric gene products were accumulated in the outer membrane. One of the chimeric gene products, with a fusion site in a central region between the OmpC and OmpF proteins, was normally expressed but not accumulated in the outer membrane. The trimeric structures of some of the chimeric gene products appeared to be extremely unstable in a SDS solution. From these results, domains contributing to the formation of specific structures in which the OmpC and OmpF proteins differ were identified. Bacterial cells possessing the chimeric gene products were also investigated as to their sensitivity to phages that require either OmpC or OmpF as a receptor component. With the aid of the chimeric gene products, the immunogenic determinants for three anti-OmpC monoclonal antibodies were found to be localized at different portions of the OmpC polypeptide: the N-terminal, central and C-terminal portions, respectively.     

Liprotides assist in folding of outer membrane proteins

Proteins and lipids can form complexes called liprotides, in which the partially denatured protein forms a shell encasing a lipid core. This effectively stabilizes a lipid micelle in an aqueous solvent and suggests that liprotides may provide a suitable vessel for membrane proteins. Accordingly we have investigated if liprotides consisting of α‐lactalbumin and oleate could aid folding of four different outer membrane proteins (OMPs) tOmpA, PagP, BamA, and OmpF. tOmpA was able to fold in the presence of the liprotide, and folding did not occur if only oleate or α‐lactalbumin were added. Although the liprotides did not fold the other three OMPs on its own, it was able to assist their folding in the presence of vesicles. Incubation with liprotides before folding into vesicles increased the folding yield of the outer membrane proteins to a level higher than using micelles of the non‐ionic surfactant DDM. Even though the liprotide was stable at both high urea concentrations and high pH, it failed to efficiently fold OmpA at high pH. Instead, optimal folding was seen at pH 8–9, suggesting that important changes in the liprotide occurred when increasing the pH. We conclude that an otherwise folding‐inactive fatty acid can be activated when presented by a liprotide and thereby work as an in vitro chaperone for outer membrane proteins.