Ferric citrate transport in Escherichia coli requires outer membrane receptor protein fecA.

Mutants of Escherichia coli K-12 AB2847 and of E. coli K-12 AN92 were isolated which were unable to grow on ferric citrate as the sole iron source. Of 22 mutants, 6 lacked an outer membrane protein, designated FecA protein, which was expressed by growing cells in the presence of 1 mM citrate. Outer membranes showed an enhanced binding of radioactive iron, supplied as a citrate complex, depending on the amount of FecA protein. The FecA protein was the most resistant of the proteins involved in ferric irion iron translocation across the outer membrane (FhuA = TonA, FepA, Cir, or 83K proteins) to the action of pronase P. It is also shown that previously isolated fec mutants (G. C. Woodrow et al., J. Bacteriol. 133:1524-1526, 1978) which are cotransducible with argF all lack the FecA protein. They were termed fecA to distinguish them from the other ferric citrate transport mutants, now designated fecB, which mapped in the same gene region at 7 min but were not cotransducible with ArgF. E. coli W83-24 and Salmonella typhimurium, which are devoid of a citrate-dependent iron transport system, lacked the FecA protein. It is proposed that the FecA protein participates in the transport of ferric citrate.     

Role of a major outer membrane protein in Escherichia coli.

Mutants of Escherichia coli B/r lacking a major outer membrane protein, protein b, were obtained by selecting for resistance to copper. These mutants showed a decreased ability to utilize a variety of metabolites when the metabolites were present at low concentrations. Also, mutants of E. coli K-12 lacking proteins b and c from the outer membrane were shown to have an identical defect in the uptake of various metabolites. These results are discussed with regard to their implications as to the role of these proteins in permeability of the outer membrane,     

Escherichia coli outer membrane protein K is a porin.

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

Insertion of a minor protein into the outer membrane of Escherichia coli during inhibition of lipid synthesis.

The antibiotic cerulenin, a specific inhibitor of fatty acid synthetase systems, was used to demonstrate that a minor protein component of the outer membrane of Escherichia coli, which serves as the receptor for the phage lambda, can be synthesized and inserted into the outer membrane during inhibition of lipid synthesis.     

Insertion mutagenesis on a cell-surface-exposed region of outer membrane protein PhoE of Escherichia coli K-12

Amino acid residue arginine-158 of the outer membrane protein PhoE of Escherichia coli K-12 has been shown to be cell-surface-exposed [Korteland et al. (1985) Eur. J. Biochem. 152, 691-697]. To study the effects of small insertions in this region of the protein on its biogenesis and characteristics, a unique restriction site was created by site-directed mutagenesis in a plasmid carrying the phoE gene and oligonucleotides of 12-74 bp were inserted. The insertions did not interfere with incorporation into the outer membrane since (a) several monoclonal antibodies, directed against the cell-surface-exposed part of PhoE protein, bound to whole cells producing the altered proteins and (b) the proteins formed functional pores for the uptake of beta-lactam antibiotics. The binding of one monoclonal antibody and of the PhoE-specific phages TC45 and TC45hrN3 was disturbed by the insertions, showing that this region of the protein is immunogenic and is involved in the binding of both of these phages. The functioning of the mutant pores was characterized both in vivo by studying the uptake of beta-lactam antibiotics and in vitro after the reconstitution of the proteins in black lipid films. The pore characteristics changed depending on the nature of the inserted amino acids. Addition of a negatively charged amino acid resulted in decreased anion-selectivity, whereas insertion of a positive charge and deletion of a negative charge had only a small influence.     

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.     

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

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

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

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

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

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

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

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

Molecular characterization of a heat-modifiable protein from the outer membrane of Escherichia coli.

Discrete fractions of nonhistone chromosomal proteins (NHCP) were obtained from rabbit liver chromatin by their dissociation in 5 m urea with increasing concentrations of NaCl. Three fractions were obtained: M₀, M₁, and M₃. We found that M₀ can modify the conformation of DNA/histone complexes as depicted from their induced increase in the ellipticity of DNA/histone from 5100 to 6900 degree-cm²/dmol. This effect was found to be reversible, while M₁ and M₃ effects, if any, were not measurable. These results suggest that M₀ primarily interacts with the chromatin subunit.     

Optical properties of an outer membrane lipoprotein from Escherichia coli.

The infrared spectrum of a structural lipoprotein from the Escherichia coli outer membrane indicated the lipoprotein had an alpha-helical conformation but no sign for the existence of beta-structures. From circular dichroism spectra of the lipoprotein, the alpha-helical content of the protein was found to be as high as 88% in 0.01-0.03% sodium dodecyl sulfate in the presence of 10(-5) M Mg2+ at pH 7.1 and 23 degrees C. When sodium dodecyl sulfate concentration increased higher than 0.1%, the alpha-helical content of the lipoprotein decreased to about 57%. Divalent cations, such as Mg2+ and Mn2+, were found to increase the helical content of the lipoprotein. The high alpha-helical content of the lipoprotein was observed in a wide range of temperatures (23 to 55 degrees C). The significance of the high alpha-helical content of the lipoprotein is discussed in light of the three-dimensional molecular models of the lipoprotein proposed previously.     

Evidence for a coupling of synthesis and export of an outer membrane protein in Escherichia coli

We describe a lesion, lamB701-708, affecting the hydrophilic portion of the lambda receptor signal sequence. The C to A transversion of the sixth codon of the signal sequence changes a positively charged arginine to a neutral serine. The phenotype conferred by this alteration is unique among previously described signal sequence mutations. The results suggest an essential role for the charged amino acids of the hydrophilic segment in the initial interaction between a nascent secreted protein and a membrane export site. The results further suggest that synthesis of lambda receptor is coupled to its export.     

Temperature-sensitive processing of outer membrane lipoprotein in an Escherichia coli mutant.

A mutant of Escherichia coli that accumulated prolipoprotein, a secretory precursor of the outer membrane lipoprotein, was isolated. The prolipoprotein accumulated in this mutant was modified by glyceride, but the in vitro cleavage of the signal peptide of the accumulated prolipoprotein was found to be temperature sensitive. The mutation appears to be located outside the gene for the lipoprotein, thus suggesting that the gene for the signal peptidase for the prolipoprotein was mutated.     

The assembly pathway of outer membrane protein PhoE of Escherichia coli.

The assembly of the wild-type and several mutant forms of the trimeric outer membrane porin PhoE of Escherichia coli was investigated in vitro and in vivo. In in vivo pulse-chase experiments, approximately half of the wild-type PhoE molecules assembled within the 30-s pulse in the native conformation in the cell envelope. The other half of the molecules followed slower kinetics, and three intermediates in this multistep assembly process were detected: a soluble trypsin-sensitive monomer, a trypsin-sensitive monomeric form that was loosely associated with the cell envelope and a metastable trimer, which was integrated into the membranes and converted to the stable trimeric configuration within minutes. The metastable trimers disassembled during sample preparation for standard SDS/PAGE into folded monomers. In vitro, the isolated PhoE protein could efficiently be folded in the presence of N,N-dimethyldodecylamine-N-oxide (LDAO). A mutant PhoE protein, DeltaF330, which lacks the C-terminal phenylalanine residue, mainly followed the slower kinetic pathway observed in vivo, resulting in increased amounts of the various assembly intermediates. It appears that the DeltaF330 mutant protein is intrinsically able to fold, because it was able to fold in vitro with LDAO with similar efficiencies as the wild-type protein. Therefore, we propose that the conserved C-terminal Phe is (part of) a sorting signal, directing the protein efficiently to the outer membrane. Furthermore, we analysed a mutant protein with a hydrophilic residue introduced at the hydrophobic side of one of the membrane-spanning amphipathic beta strands. The assembly of this mutant protein was not affected in vivo or in vitro in the presence of LDAO. However, it was not able to form folded monomers in a previously established in vitro folding system, which requires the presence of lipopolysaccharides and Triton. Hence, a folded monomer might not be a true assembly intermediate of PhoE in vivo.     

Arrangement of protein I in Escherichia coli outer membrane: cross-linking study.

The arrangement of protein I in the outer membrane of Escherichia coli was investigated by cross-linking whole cells, isolated cell wall, protein-peptidoglycan complexes, and protein I released from peptidoglycan with NaCl. Both cleavable azide cross-linkers and imidoester reagents were used. The data presented suggest that protein I exists in the outer membrane as a trimer.     

Restoration of membrane incorporation of an Escherichia coli outer membrane protein (OmpA) defective in membrane insertion

The mechanism of sorting, to the outer membrane, of the 325-residue Escherichia coli protein OmpA has been investigated. It is thought to traverse the membrane eight times in antiparallel beta-strands, forming an amphiphilic beta-barrel which encompasses residues 1 to about 170; the COOH-terminal moiety is periplasmic. A mutant, carrying the substitutions Leu164----Pro and Val166----Asp within the last beta-strand (residues 160-170), has been described which was unable to assemble in the membrane (Klose, M., MacIntyre, S., Schwarz, H., and Henning, U. (1988) J. Biol. Chem. 263, 13297-13302). Linkers were inserted between the codons for residues 164 and 165 of the mutant protein. Of 13 different genes recovered, five encoded proteins which had regained the ability to assemble in the membrane. The properties of the mutant proteins, together with a structure prediction method, indicate the following rules for the final beta-strand to be compatible with, or possibly initiate, membrane insertion: (i) it must be amphiphilic or hydrophobic while its primary structure as such is fairly unimportant, (ii) it must extend over at least 9 residues, and (iii) it must not contain a proline residue around its center. One of the genes recovered coded for OmpA up to residue 164 and then followed by 10 linker-encoded residues. This 174-residue polypeptide was assembled in the membrane but did not, in contrast to all other proteins, expose sites sensitive to trypsin at the inner face of the membrane. This behavior agrees perfectly well with the OmpA model.     

Ultrastructure of a periodic protein layer in the outer membrane of Escherichia coli

Matrix protein (36,500 daltons), one of the major polypeptides of the Escherichia coli cell envelope, is arranged in a periodic monolayer which covers the outer surface of the peptidoglycan. Although its association with the peptidoglycan layer is probably tight, the periodic structure of the peptidoglycan. Although its association with the peptidoglycan later is probably tight, the periodic structure is maintained in the absence of peptidoglycan, and is therefore based on strong protein-protein interactions. A detailed analysis of the ultrastructure of the matrix protein array by electron microscopy and image processing of specimens prepared by negative staining or by freeze-drying and shadowing shows that the molecules are arranged according to three fold symmetry on a hexagonal lattice whose repeat is 7.7 nm. The most pronounced feature of the unit cell, which probably contains three molecules of matrix protein, is a triplet of indentations, each approx. 2 nm in diameter, with a center-to-center spacing of 3nm. They are readily penetrated by stain and may represent channels which span the protein monolayer.