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.     

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 Pi through the outer membrane, such as a pore protein deficient strain behaves as a Km mutant. Comparison of the rates of permeation of Pi, 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 Pi 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 Pi and Pi-containing solutes are discussed.     

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.     

Mutants (ompA) affecting a major outer membrane protein of Escherichia coli K12.

Seventy independent mutants have been analyzed affecting a major protein, polypeptide II, of the outer cell envelope membrane from Escherichia coli K12. They were classified as nonsense mutants of the amber type (20%), mutants most likely of the missense type possessing the protein at normal concentrations (9%), and mutants either missing the protein or harboring it at much reduced concentrations for unknown reasons (71%). Forty of the mutants were analyzed genetically and all were found to map at or near ompA, the structural gene for protein II. Two-dimensional electrophoretic analyses of envelopes from such mutants revealed an unusual heterogeneity of the protein which on such patterns appeared as at least 12 well separated spots, and the majority of these is due to artifacts of the method but apparently specific for this protein. In no case was a polypeptide fragment found in envelopes from the nonsense mutants. The results are discussed regarding two different phages which use the protein as a receptor and concerning the biosynthetic incorporation of the protein into the outer membrane.     

Defective growth functions in mutants of Escherichia coli K12 lacking a major outer membrane protein.

Various properties of mutants of Escherichia coli K12 lacking specific outer membrane proteins have been studied. ompA mutants are shown to grow less well than their parent strains under a variety of growth conditions, and after completion of growth to enter a decline phase in which viability is lost and the cells become heavily piliated and clump. They are defective in the uptake of amino acids, whereas the uptakes of the larger transport substrates ferrienterochelin and cyanocobalamin (vitamin B12) are normal. These ompA mutants also grow poorly at 42 °C. The implications of these results are discussed in terms of the function of the ompA. gene product. No growth or uptake defects were observed for ompB or tsx mutants.     

Increased efficiency of the outer membrane PhoE protein pore in Escherichia coli K-12 mutants with heptose-deficient lipopolysaccharide

The pore properties of PhoE protein channels in the outer membrane of a lipoprotein-deficient mutant and in a mutant with heptose-deficient lipopolysaccharide were investigated. The absence of lipoprotein neither affects the rate of permeation of glucose 6-phosphate or of the beta-lactam antibiotic cephsulodin through the PhoE pore nor the inhibition of cephsulodin permeation by polyphosphate. In contrast, heptose deficiency results in a 6- to 8-fold increase in the rates of permeation of glucose 6-phosphate and cephsulodin. Possible explanations for these data are discussed. It is argued that the lipopolysaccharide structure synthesized under phosphate limitation may be similar to that of the heptoseless mutant and hence that not only the structure of the PhoE protein pore but also the structure of the lipopolysaccharide may promote the uptake of Pi and Pi-containing solutes under phosphate limitation.     

Freeze etch morphology of outer membrane mutants of Escherichia coli K12

Freeze etching showed that the loss of each of the major outer membrane proteins b, c or d in mutants of Escherichia coli K12 does not influence the morphology of fracture faces of the outer membrane.Mutants that possess a heptose-deficient lipopolysaccharide and which in addition are deficient in one or more major outer membrane proteins exhibit a reduction in the number of intramembranous particles of the outer membrane.Moreover it was shown that lipid phase transitions induce a lateral lipid protein separation in the outer membrane, similar to that found in the cytoplasmic membrane.     

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.     

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

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

Role of the Arg158 residue of the outer membrane PhoE pore protein of Escherichia coli K 12 in bacteriophage TC45 recognition and in channel characteristics

In order to study the structure-function relationship of the PhoE protein pore we have isolated five independent, TC45-resistant, phoE mutants all of which appeared to produce normal amounts of an electrophoretically altered PhoE protein, designated as PhoE* protein. Nucleotide sequence analysis of the DNA fragments carrying the mutations showed that the mutations all correspond to a G.C to A.T transition at the same place within the phoE gene resulting in a deduced change of amino acid residue arginine 158 into histidine. This result shows that the arginine 158 residue plays an important role in the interaction of the PhoE protein pore with phage TC45. Moreover, studies on the channel properties of the PhoE* protein showed that the PhoE channel has lost part of its preference for negatively charged solutes, as a result of the arginine to histidine change. The results are discussed in terms of the structure-function relationship of PhoE protein as well as in terms of the topological organization of the protein channel in the outer membrane.     

Role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein

To investigate the role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein, deletions were generated in two presumed cell surface-exposed regions of the protein. Intact cells expressing these mutant proteins were recognized by PhoE-specific monoclonal antibodies, which recognize conformational epitopes on the cell surface-exposed parts of the protein and/or were sensitive to a PhoE-specific phage. This shows that the polypeptides were normally incorporated into the outer membrane. When the deletions extended four amino acid residues into the seventh presumed membrane-spanning segment, the polypeptides accumulated in the periplasm. In conclusion, exposed regions of PhoE protein apparently do not play an essential role in outer membrane localization, which is consistent with the observation that these regions are hypervariable when PhoE is compared to the related proteins OmpF and OmpC. In contrast, the membrane-spanning segments are essential for the assembly process.     

Protein K: a new major outer membrane protein found in encapsulated Escherichia coli.

The protein composition of purified outer membranes of 47 Escherichia coli strains was examined by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. Of 33 encapsulated strains, all contained an outer membrane protein distinguishable from previously reported proteins. The 14 non-encapsulated strains with one exception lacked this protein. Because of its apparent association with encapsulation (K antigen) we have named it K protein. The protein was purified nearly to homogeneity by chromatography in the presence of detergents, and its composition was determined. Its amino acid composition does not differ significantly from that reported for protein I, another E. coli major outer membrane protein. Furthermore, the N-terminal amino acid sequence of protein K indicates that it is related to protein I.     

Analysis of structure-function relationships in Escherichia coli K12 outer membrane porins with the aid of ompC-phoE and phoE-ompC hybrid genes

Summary To study structure-function relationships in the outer membrane pore proteins OmpC and PhoE of Escherichia coli K12, we have constructed a series of phoE-ompC hybrid genes in which DNA encoding part of one protein is replaced by the homologous part of the other gene. The hybrid gene products were incorporated normally into the outer membrane, allowing their functional characterization. Combined with previous studies, the present results permit the identification of regions involved in determining functions and properties in which the native PhoE and OmpC proteins differ, such as pore characteristics, receptor activity for phages and binding of monoclonal antibodies. Most of these properties were found to be determined by multiple regions clearly separated in the primary structure. The combined phage and antibody binding data have demonstrated that at least five distinct regions in PhoE and OmpC are exposed at the cell surface. The locations of these regions are in agreement with a previously proposed model for porin topology.     

Cloned DNA fragment specifying major outer membrane protein a in Escherichia coli K-12.

Plasmid pMC44 is a recombinant plasmid that contains a 2-megadalton EcoRI fragment of Escherichia coli K-12 DNA joined to the cloning vehicle, pSC101. The polypeptides specified by plasmid pMC44 were identified and compared with those specified by pSC101 to determine those that are unique to pMC44. Three polypeptides specified by plasmid pMC44 were localized in the cell envelope fraction of minicells: a Sarkosyl-insoluble outer membrane polypeptide (designated M2), specified by the cloned 2-megadalton DNA fragment, and two Sarkosyl-soluble membrane polypeptides specified by the cloning plasmid pSC101. Bacteria containing plasmid pMC44 synthesized quantities of M2 approximately equal to the most abundant E. coli K-12 outer membrane protein. Evidence is presented that outer membrane polypeptide M2, specified by the recombinant plasmid pMC44, is the normal E. coli outer membrane protein designated protein a by Lugtenberg and 3b by Schnaitman.