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.     

Role of SecA and SecY in protein export as revealed by studies of TonA assembly into the outer membrane of Escherichia coli

The growth of secAts or secYts mutants at the restrictive temperature has been shown to inhibit the export of many outer membrane proteins. We report here that in two secAts strains the rate of incorporation of newly synthesized protein into both inner and outer membrane fractions decreased by about 70% at the restrictive temperature. The export of the outer membrane protein TonA was used as a model system in which to study the effects of SecA or SecY inactivation. pre-TonA that accumulated at the restrictive temperature was found to co-sediment with the outer membrane fraction. However, the precursor was sensitive to protease and did not float up a sucrose gradient with the membrane fractions. It was therefore concluded that pre-TonA was not integrated into the outer membrane fraction but probably accumulated in the cytoplasm. Studies on the rate of processing of pre-TonA, pulse-labelled at the restrictive temperature then chased at the permissive temperature, revealed differences between secA and secY mutants. In the secAts mutant the great majority of cytoplasmic pre-TonA was not apparently processed to the mature form, whereas in the secYts mutant significant amounts of precursors were rapidly chased into mature TonA, which appeared in the outer membrane. These results suggest that SecA and SecY may act sequentially in the export of proteins to the outer membrane. In particular these data indicate that SecA is required to maintain pre-TonA in a translocationally competent form prior to interaction with the SecY export site.     

Effects of two sec genes on protein assembly into the plasma membrane of Escherichia coli

We have examined the effects of thermosensitive mutations in secA and secY (prlA) genes on the export of proteins to the three layers of the Escherichia coli cell surface. After several hours at the nonpermissive temperature, the export of two major outer membrane proteins, lipoprotein and OmpA, is delayed, then essentially blocked, in either a secA or secY strain. These mutations also have a strong effect on the export of several proteins, such as maltose binding protein, to the periplasm, though the export of many periplasmic proteins is not affected. secA and secY block the assembly of leader peptidase, which is made without a leader sequence, into the inner membrane. However, the membrane assembly of M13 coat protein (an inner membrane protein made with an amino-terminal leader sequence) is not affected. Thus, the requirement for sec function for export does not correlate with the presence or absence of leader peptide or with a particular subcellular compartment, but rather is specific to each particular protein.     

Involvement of Neisseria meningitidis Lipoprotein GNA2091 in the Assembly of a Subset of Outer Membrane Proteins

GNA2091 of Neisseria meningitidis is a lipoprotein of unknown function that is included in the novel 4CMenB vaccine. Here, we investigated the biological function and the subcellular localization of the protein. We demonstrate that GNA2091 functions in the assembly of outer membrane proteins (OMPs) because its absence resulted in the accumulation of misassembled OMPs. Cell fractionation and protease accessibility experiments showed that the protein is localized at the periplasmic side of the outer membrane. Pulldown experiments revealed that it is not stably associated with the β-barrel assembly machinery, the previously identified complex for OMP assembly. Thus, GNA2091 constitutes a novel outer membrane-based lipoprotein required for OMP assembly. Furthermore, its location at the inner side of the outer membrane indicates that protective immunity elicited by this antigen cannot be due to bactericidal or opsonic activity of antibodies.     

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.     

Assembly of outer membrane lipoprotein in an Escherichia coli mutant with a single amino acid replacement within the signal sequence of prolipoprotein.

We have compared the rate of assembly of outer membrane proteins including the lipoprotein in a pair of isogenic mlpA+ (lpp+) and mlpA (lpp) strains by pulse-chase experiments. The rate of assembly of the mutant prolipoprotein into the outer membrane was slightly slower than that of the wild-type lipoprotein. The rate of assembly of protein I and protein H-2 was similar in the wild type and the mutant, whereas the rate of assembly of protein II into the outer membrane was slightly reduced in the mutant strain. The organization of outer membrane was slightly reduced in the mutant strain. The organization of outer membrane proteins in the mutant cells appeared not to be grossly altered, based on the apparent resistance (or susceptibility) of these proteins toward trypsin treatment and their resistance to solubilization by Sarkosyl. Like the wild-type lipoprotein, the mutant prolipoprotein in the outer membrane was resistant to trypsin. On the other hand, the prolipoprotein in the cytoplasmic membrane fraction of the mutant cell envelope was susceptible to trypsin digestion. We conclude from these data that proteolytic cleavage of prolipoprotein is not essential for the translocation and proper assembly of lipoprotein into outer membrane.     

Carboxy-terminal phenylalanine is essential for the correct assembly of a bacterial outer membrane protein

Bacterial outer membrane proteins are supposed to span the membrane repeatedly, mostly in the form of amphipathic beta-sheets. The last ten C-terminal amino acid residues of PhoE protein are supposed to form such a membrane-spanning segment. Deletion of this segment completely prevents incorporation into the outer membrane. Comparison of the last ten amino acid residues of other outer membrane proteins from different Gram-negative bacteria revealed the presence of a potential amphipathic beta-sheet with hydrophobic residues at positions 1 (Phe), 3 (preferentially Tyr), 5, 7 and 9 from the C terminus, in the vast majority of these proteins. Since such sequences were not detected at the C termini of periplasmic proteins, it appears to be possible to discriminate between the majority of outer membrane proteins and periplasmic proteins on the basis of sequence data. The highly conserved phenylalanine at the C termini of outer membrane proteins suggests an important function for this amino acid in assembly into the outer membrane. Site-directed mutagenesis was applied to study the role of the C-terminal Phe in PhoE protein assembly. All mutant proteins were correctly incorporated into the outer membrane to some extent, but the efficiency of the process was severely affected. It appears that both the hydrophobicity and the aromatic nature of Phe are of importance.     

Synthesis of OmpA protein of Escherichia coli K12 in Bacillus subtilis

We have inserted a C-terminally truncated gene of the major outer membrane protein OmpA of Escherichia coli downstream from the promoter and signal sequence of the secretory alpha-amylase of Bacillus amyloliquefaciens in a secretion vector of Bacillus subtilis. B. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as OmpA. All the protein was present in the particulate fraction. The size of the protein compared to the peptide synthesized in vitro from the same template indicated that the alpha-amylase derived signal peptide was not removed; this was verified by N-terminal amino acid sequence determination. The lack of cleavage suggests that there was little or no translocation of OmpA protein across the cytoplasmic membrane. This is an unexpected difference compared with periplasmic proteins, which were both secreted and processed when fused to the same signal peptide. A requirement of a specific component for the export of outer membrane proteins is suggested.     

Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12

The 325-residue outer membrane protein OmpA of Escherichia coli has been proposed to consist of a membrane-embedded moiety (residues 1 to about 170) and a C-terminal periplasmic region. The former is thought to comprise eight transmembrane segments in the form of antiparallel β-strands, forming an amphiphilic β connected by exposed turns. Several questions concerning this model were addressed. Thus no experimental evidence had been presented for the turns at the inner leaflet of the membrane and it was not known whether or not the periplasmic part of the polypeptide plays a role in the process of membrane incorporation. Oligonucleotides encoding trypsin cleavage sites were inserted at the predicted turn sites of the ompA gene and it was shown that the encoded proteins indeed become accessible to trypsin at the modified sites. Together with previous results, these data also show that the turns on both sides of the membrane do not possess specifically topogenic information. In two cases one of the two expected tryptic fragments was lost and could be detected at low concentration in only one case. Therefore, bilateral proteolytic digestion of outer membranes can cause loss of β-strands and does not necessarily produce a reliable picture of protein topology. When ompA genes were constructed coding for proteins ending at residue 228 or 274, the membrane assembly of these proteins was shown to be partially defective with about 20% of the proteins not being assembled. No such defect was observed when, following the introduction of a premature stop codon, a truncated protein was produced ending with residue 171. It is concluded that (1) the proposed β-barrel structure is essentially correct and (2) the periplasmic part of OmpA does not play an active role in, but can, when present in mutant form, interfere with membrane assembly.     

In vivo membrane assembly of split variants of the E.coli outer membrane protein OmpA.

The two-domain, 325 residue outer membrane protein OmpA of Escherichia coli is a well-established model for the study of membrane assembly. The N-terminal domain, consisting of approximately 170 amino acid residues, is embedded in the membrane, presumably in the form of a beta-barrel consisting of eight antiparallel transmembrane beta-strands. A set of 16 gene variants carrying deletions in the membrane-embedded domain of OmpA was constructed. When pairs of these mutant genes were co-expressed in E.coli, it was found that a functional OmpA protein could be assembled efficiently from two complementary protein fragments. Assembly was found when the polypeptide chain was split at the second or third periplasmic turn. All four protein termini were located in the periplasmic space. Interestingly, duplication of transmembrane strands five and six led to a variant with an unusual topology: the N-terminus of one fragment and the C-terminus of the other fragment were exposed at the cell surface. This is the first demonstration of correct membrane assembly of split beta-structured membrane proteins. These findings are important for a better understanding of their folding/assembly pathway and may have implications for the development of artificial outer membrane proteins and for the cell surface display of heterologous peptides or proteins.     

Nine amino acid residues at the NH2-terminal of lipoprotein are sufficient for its modification, processing, and localization in the outer membrane of Escherichia coli

We have examined the structural requirements at the NH2-terminal region of the lipoprotein for its assembly in the outer membrane of Escherichia coli by constructing a hybrid protein consisting of an NH2-terminal portion of the prolipoprotein, consisting of the signal peptide and 9 amino acid residues of lipoprotein, and the entire beta-lactamase sequence. The results from this study indicate that the hybrid protein is modified with glyceride, processed in a globomycin-sensitive step, and localized in the outer membrane. The translocation of the hybrid protein across the cytoplasmic membrane occurs post-translationally and is inhibited by carbonyl cyanide m-chlorophenylhydrazone. Our results, therefore, indicate that the signal peptide and 9 amino acid residues of prolipoprotein are sufficient for its modification, processing, and localization in the outer membrane.     

Localization of the outer membrane subunit OprM of resistance-nodulation-cell division family multicomponent efflux pump in Pseudomonas aeruginosa

The outer membrane subunit OprM of the multicomponent efflux pump of Pseudomonas aeruginosa has been assumed to form a transmembrane xenobiotic exit channel across the outer membrane. We challenged this hypothesis to clarify the underlying ambiguity by manipulating the amino-terminal signal sequence of the OprM protein of the MexAB-OprM efflux pump in P. aeruginosa. [(3)H]Palmitate uptake experiments revealed that OprM is a lipoprotein. The following lines of evidence unequivocally established that the OprM protein functioned at the periplasmic space. (i) The OprM protein, in which a signal sequence including Cys-18 was replaced with that of periplasmic azurin, appeared in the periplasmic space but not in the outer membrane fraction, and the protein fully functioned as the pump subunit. (ii) The hybrid OprM containing the N-terminal transmembrane segment of the inner membrane protein, MexF, appeared exclusively in the inner membrane fraction. The hybrid protein containing 186 or 331 amino acid residues of MexF was fully active for the antibiotic extrusion, but a 42-residue protein was totally inactive. (iii) The mutant OprM, in which the N-terminal cysteine residue was replaced with another amino acid, appeared unmodified with fatty acid and was fractionated in both the periplasmic space and the inner membrane fraction but not in the outer membrane fraction. The Cys-18-modified OprM functioned for the antibiotic extrusion indistinguishably from that in the wild-type strain. We concluded, based on these results, that the OprM protein was anchored in the outer membrane via fatty acid(s) attached to the N-terminal cysteine residue and that the entire polypeptide moiety was exposed to the periplasmic space.     

Organization of K88ac-encoded polypeptides in the Escherichia coli cell envelope: use of minicells and outer membrane protein mutants for studying assembly of pili

Escherichia coli K-12 minicells, harboring recombinant plasmids encoding polypeptides involved in the expression of K88ac adhesion pili on the bacterial cell surface, were labeled with [35S]methionine and fractionated by a variety of techniques. A 70,000-dalton polypeptide, the product of the K88ac adhesion cistron adhA, was primarily located in the outer membrane of minicells, although it was less clearly associated with this membrane than the classical outer membrane proteins OmpA and matrix protein. Two polypeptides of molecular weights 26,000 and 17,000 (the products of adhB and adhC, respectively) were located in significant amounts in the periplasmic space. The 29,000-dalton polypeptide was shown to be processed in E. coli minicells. The 23.500-dalton K88ac pilus subunit (the product of adhD) was detected in both inner and outer membrane fractions. E. coli mutants defective in the synthesis of murein lipoprotein or the major outer membrane polypeptide OmpA were found to express normal amounts of K88ac antigen on the cell surface, whereas expression of the K88ac antigen was greatly reduced in perA mutants. The possible functions of the adh cistron products are discussed.     

Effect of OmpA signal peptide mutations on OmpA secretion, synthesis, and assembly.

In previous investigations, we have examined the effect of OmpA signal peptide mutations on the secretion of the two heterologous proteins TEM beta-lactamase and nuclease A. During these studies, we observed that a given signal peptide mutation could affect differentially the processing of precursor OmpA-nuclease or precursor OmpA-lactamase. This observation led us to further investigate the influence of the mature region of a precursor protein on protein export. Preexisting OmpA signal peptide mutations of known secretion phenotype when directing heterologous protein export (nuclease A or beta-lactamase) were fused to the homologous mature OmpA protein. Four signal peptide mutations that have previously been shown to prevent export of nuclease A and beta-lactamase were found to support OmpA protein export, albeit at reduced rates. This remarkable retention of export activity by severely defective precursor OmpA signal peptide mutants may be due to the ability of mature OmpA to interact with the cytoplasmic membrane. In addition, these same signal peptide mutations can affect the level of OmpA synthesis as well as its proper assembly in the outer membrane of Escherichia coli. Two signal peptide mutations dramatically stimulate the rate of precursor OmpA synthesis three- to fivefold above the level observed when a wild-type signal peptide is directing export. The complete removal of the OmpA signal peptide does not result in increased OmpA synthesis. This finding suggests that the signal peptide mutations function positively to stimulate OmpA synthesis, rather than bypass a down-regulatory mechanism effected by a wild-type signal peptide. Overproduction of wild-type precursor OmpA or precursors containing signal peptide mutations which lead to relatively minor kinetic processing defects results in accumulation of an improperly assembled OmpA species (imp-OmpA). In contrast, signal peptide mutations which cause relatively severe processing defects accumulate no or only small quantities of imp-OmpA. All mutations result in equivalent levels of properly assembled OmpA. Thus, a strong correlation between imp-OmpA accumulation and cell toxicity was observed. A mutation in the mature region of OmpA which prevents the proper outer membrane assembly of OmpA was suppressed when export was directed by a severely defective signal peptide. These findings suggest that signal peptide mutations indirectly influence OmpA assembly in the outer membrane by altering both the level and rate of OmpA secretion across the cytoplasmic membrane.     

Trimeric structure and localization of the major lipoprotein in the cell surface of Escherichia coli

A hybrid gene consisting of the ompF promoter, the coding regions for the signal peptide, and the Ala-Glu residue of the OmpF NH2 terminus and the coding region for the major outer membrane lipoprotein devoid of the NH2-terminal cysteine residue was constructed. Escherichia coli carrying the cloned gene produced the predicted hybrid protein that is the same as the major lipoprotein except that the diacyl glycerylcysteine residue at the NH2 terminus is replaced by the Ala-Glu residue. The hybrid protein was localized in the periplasmic space as a trimer with a noncovalent interaction in addition to the previously known covalent interaction with the peptidoglycan. These results strongly indicate that the major lipoprotein exists as a trimer in the periplasmic space with covalent and noncovalent interactions with the peptidoglycan layer through the protein domain on one side and with the hydrophobic interaction with the outer membrane through the lipid domain on the other side. The trimeric structure of the lipoprotein was directly demonstrated by the chemical cross-linking of the native lipoprotein with both cleavable and uncleavable reagents. The cross-linking study also revealed interaction between the lipoprotein and the OmpA protein, a major outer membrane protein.     

The dynamics of assembly of a cytoplasmic membrane protein in Escherichia coli.

The topology of integral cytoplasmic membrane proteins can be analyzed using alkaline phosphatase fusions by determining which constructs have low and which have high specific activity. We show that in all cases the enzymatic activity is due to the fraction of the alkaline phosphatase moiety of the fusion protein localized to the periplasm. We present evidence that these fusions can also be used to analyze the process of assembly of cytoplasmic proteins into the membrane. The rate of acquisition of protease resistance of the alkaline phosphatase moiety of such hybrid proteins is compared for fusions to periplasmic and cytoplasmic domains. We show that this process, which is assumed to be representative of export of alkaline phosphatase, is significantly slower for fusions to cytoplasmic and certain periplasmic domains than for most periplasmic domains. These results are discussed in the context of the normal assembly of integral membrane proteins.     

The secretin-specific, chaperone-like protein of the general secretory pathway: separation of proteolytic protection and piloting functions

The chaperone-like protein of the main terminal branch of the general secretory pathway from Klebsiella oxytoca , the outer membrane lipoprotein PulS, protects the multimeric secretin PulD from degradation and promotes its correct localization to the outer membrane. To determine whether these are separable functions, or whether resistance to proteolysis results simply from correct localization of PulD, we replaced the lipoprotein-type signal peptide of PulS by the signal peptide of periplasmic maltose-binding protein. The resulting periplasmic PulS retained its ability to protect PulD, but not its ability to localize PulD to the outer membrane and to function in pullulanase secretion. Periplasmic PulS competed with wild-type PulS to prevent pullulanase secretion, presumably again by causing mislocalization of PulD. A hybrid protein comprising the mature part of PulS fused to the C-terminus of full-length maltose-binding protein (MalE–PulS) had similar properties to the periplasmic PulS protein. Moreover, MalE–PulS was shown to associate with PulD by amylose-affinity chromatography. The MalE–PulS hybrid was rendered completely functional (i.e. it restored pullulanase secretion in a pulS mutant) by replacing its signal peptide with a lipoprotein-type signal peptide. However, this fatty-acylated hybrid protein was only functional if it also carried a lipoprotein sorting signal that targeted it to the outer membrane. Thus, the two functions of PulS are separate and fully dissociable. Incorrect localization, rather than proteolysis, of PulD in the absence of PulS was shown to be the factor that causes high-level induction of the phage shock response. The Erwinia chrysanthemi PulS homologue, OutS, can substitute for PulS, and PulS can protect the secretin OutD from proteolysis in Escherichia coli , indicating the possible existence of a family of PulS-like chaperone proteins.     

The TolB protein interacts with the porins of Escherichia coli.

TolB is a periplasmic protein of the cell envelope Tol complex. It is partially membrane associated through an interaction with the outer membrane lipoprotein PAL (peptidoglycan-associated lipoprotein), which also belongs to the Tol system. The interaction of TolB with outer membrane porins of Escherichia coli was investigated with a purified TolB derivative harboring a six-histidine tag. TolB interacted with the trimeric porins OmpF, OmpC, PhoE, and LamB but not with their denatured monomeric forms or OmpA. These interactions took place both in the presence and in the absence of lipopolysaccharide. TolA, an inner membrane component of the Tol system, also interacts with the trimeric porins via its central periplasmic domain (R. Dérouiche, M. Gavioli, H. Bénédetti, A. Prilipov, C. Lazdunski, and R. Lloubès, EMBO J. 15:6408-6415, 1996). In the presence of the purified central domain of TolA (TolAIIHis), the TolB-porin complexes disappeared to form TolAIIHis-porin complexes. These results suggest that the interactions of TolA and TolB with porins might take place in vivo and might be concomitant events participating in porin assembly. They also suggest that the Tol system as a whole may be involved in porin assembly in the outer membrane.     

Effect of reduced membrane lipid fluidity on the biosynthesis of lipopolysaccharide of Escherichia coli

A low molecular weight precursor of lipopolysaccharide was accumulated under conditions in which the membrane lipids of a fatty acid auxotroph of Escherichia coli were reduced to a non-fluid state. The lipopolysaccharide precursor was detected, by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography, in membranes isolated from cells which were pulse-labeled with N-acetyl-[1-14C]glucosamine. The precursor could be chased into mature lipopolysaccharide by returning the membrane lipids to a normal fluid state. Conversion of the precursor to lipopolysaccharide was inhibited by the presence of potassium cyanide or sodium arsenate. The processing of several outer membrane protein precursors, including the promatrix proteins, was also inhibited under these conditions. Preliminary characterization of the lipopolysaccharide precursor was undertaken.     

Effect of uncoupler on assembly pathway for pigment-binding protein of bacterial photosynthetic membranes.

The uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) was used to investigate membrane protein assembly in the phototrophic bacterium Rhodobacter capsulatus. As found for Escherichia coli (T. Date, G. Zwizinsky, S. Ludmerer, and W. Wickner, Proc. Natl. Acad. Sci. 77:827-831, 1980) and mitochondrial proteins (N. Nelson and G. Schatz, Proc. Natl. Acad. Sci. USA 76:4365-4369, 1979), assembly across the bacterial photosynthetic membranes was sensitive to CCCP. At uncoupler concentrations which were sufficient to block the export of the periplasmic cytochrome c2 and an outer membrane protein, the integration of pigment-binding protein into the photosynthetic apparatus was abolished. The unassembled protein was detected on the inner surface of the intracytoplasmic membrane. After inactivation of CCCP, accumulated protein continued insertion into the membrane. The data suggest that after binding to the cytoplasmic face of the membrane, translocation of protein into a transmembrane orientation takes place, which is a prerequisite for the formation of a functional pigment-protein complex.