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.     

Cell envelope and shape of Escherichia coli: multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins.

Starting with an Escherichia coli strain missing the outer membrane lipoprotein, multiple mutants were constructed than in addition to this defect miss the outer membrane proteins II, Ia and Ib, or Ia, Ib, and II. In contrast to all single mutants or strains missing the lipoprotein and polypeptides Ia and Ib, drastic influences on the integrity of the outer membrane and cell morphology were observed in mutants without lipoprotein and protein II. Such strains exhibited spherical morphology. They required increased concentrations of electrolytes for optimal growth, and Mg2+ or Ca2+ were the most efficient. These mutants were sensitive to hydrophobic antibiotics and detergents. Electron microscopy revealed abundant blebbing of the outer membrane, and it could clearly be seen that the murein layer was no longer associated with the outer membrane.     

Pleiotropic transport mutants of Escherichia coli lack porin, a major outer membrane protein.

Summary Four pleiotropic transport mutants of Escherichia coli B/r with decreased affinity for the uptake of most nutrients were found to lack a major outer membrane protein of 36,500 daltons (porin) previously shown to produce transmembrane diffusion channels in in vitro reconstitution experiments. Consequent decrease in outer membrane permeability was confirmed by measuring the transmembrane diffusion rate of 6-aminopenicillanic acid. Quantitative considerations on the porin-dependent permeability of the outer membrane show that (a) there may be very large differences in the actual rates of penetration, even among the permeable substances and (b) the numbers of porin molecules present in wild type cells is several orders of magnitude higher than that necessary for the uptake of rapidly diffusing substrates such as glocose from ordinary culture media. The absence of porin and the pleiotropic transport defect were always contransduced, and the mutation was mapped at 73.7 min between aroB and malT by P1 transduction. When revertants able to grow on low concentrations of lactose were selected, in addition to true revertants suppressor strains with increased amounts of non-porin membrane proteins were isolated.This paper corresponds to paper XVI of the series dealing with the bacterial outer membrane from the laboratory of H.N. The preceding paper in the series is Nikaido, Bavoil, and Hirota, J. Bacteriol., in press     

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.     

Concentration of a major outer membrane protein at the cell poles in Escherichia coli

Autoradiography of cell envelope ghosts obtained from a strain of Escherichia coli which lacks two major outer membrane proteins has been used to demonstrate the polar concentration of another major outer membrane protein, ompA protein. The beta-lactam antibiotic cephalexin prevents the insertion of newly synthesized ompA protein into the poles but removal of the antibiotic allows the randomly dispersed protein to migrate to the polar and possibly the septal areas of the cell. Labelling of whole cells with bacteriophage K3 has confirmed a polar concentration of ompA protein.     

Conjugation-deficient mutants of Escherichia coli distinguish classes of functions of the outer membrane OmpA protein

Summary Sixty-two E. coli mutants, selected as being deficient as recipients in F factor conjugation, are altered either in the amount or function of the outer membrane OmpA protein or in lipopolysaccharide structure. These two components may function together in conjugation, since the residual conjugation activity of a mutant lacking OmpA protein was unaffected by the additional presence of a lipopolysaccharide defect. Sixty of the strains carried mutations mapping to ompA, and these could be divided into classes depending on the amount of OmpA protein in their membranes. Representatives of these classes of mutant alleles failed to complement in diploids, indicating that they all affect the ompA structural gene and nearby sequences needed for its expression. The properties of these classes distinguish three groups of OmpA protein functions: 1) the structural function in the outer membrane in providing resistance to chelating agents and the hydrophobic antibiotic novobiocin, 2) the receptor functions in phage Tull^* and K3 infection, and 3) the functions of binding cells together during conjugation, facilitating the uptake of receptorbound colicin K or L, and allowing phage Ox2 to infect. Different cellular amounts or sites in OmpA protein are thus required for these three groups of functions.     

Escherichia coli K-12 tolF mutants: alterations in protein composition of the outer membrane.

Outer membrane materials prepared from three independently isolated spontaneous Escherichia coli tolF mutants contained no detectable protein Ia. The loss of this protein was nearly completely compensated for by an increase in other major outer membrane proteins, Ib and II. Thus, the major outer membrane proteins accounted for 40% of the total cell envelope protein in both tol+ and tolF strains. No changes were found in the levels of inner membrane proteins prepared from tolF strains when compared with similar preparations from the tol+ strain. Phage-resistant mutants were selected starting with a tolF strain by using either phage TuIb or phage PA2. These phage-resistant tolF strains contained neither protein Ia nor protein Ib. The mutation leading to the loss of protein Ib in these strains is independent of the tolF mutation and is located near malP on the E. coli genetic map.     

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.     

Normal iron-enterochelin uptake in mutants lacking the colicin I outer membrane receptor protein of Escherichia coli.

The outer membranes of two independent colicin Ia-resistant mutants of Escherichia coli K-12 lack the colicin Ia receptor protein. Such mutants exhibit normal capacity for enterochelin (enterobactin)-mediated iron uptake. It is concluded that the colicin Ia receptor is not involved in iron-enterochelin uptake.     

Synthesis and export of the outer membrane lipoprotein in Escherichia coli mutants defective in generalized protein export.

Export of the outer membrane lipoprotein in Escherichia coli was examined in conditionally lethal mutants that were defective in protein export in general, including secA, secB, secC, and secD. Lipoprotein export was affected in a secA(Ts) mutant of E. coli at the nonpermissive temperature; it was also affected in a secA(Am) mutant of E. coli at the permissive temperature, but not at the nonpermissive temperature. The export of lipoprotein occurred normally in E. coli carrying a null secB::Tn5 mutation; on the other hand, the export of an OmpF::Lpp hybrid protein, consisting of the signal sequence plus 11 amino acid residues of mature OmpF and mature lipoprotein, was affected by the secB mutation. The synthesis of lipoprotein was reduced in the secC mutant at the nonpermissive temperature, as was the case for synthesis of the maltose-binding protein, while the synthesis of OmpA was not affected. Lipoprotein export was found to be slightly affected in secD(Cs) mutants at the nonpermissive temperature. These results taken together indicate that the export of lipoprotein shares the common requirements for functional SecA and SecD proteins with other exported proteins, but does not require a functional SecB protein. SecC protein (ribosomal protein S15) is required for the optimal synthesis of lipoprotein.     

Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein.

Only one of the characterized components of the main terminal branch of the general secretory pathway (GSP) in Gram-negative bacteria, GspD, is an integral outer membrane protein that could conceivably form a channel to permit protein transport across this membrane. PulD, a member of the GspD protein family required for pullulanase secretion by Klebsiella oxytoca, is shown here to form outer membrane-associated complexes which are not readily dissociated by SDS treatment. The outer membrane association of PulD is absolutely dependent on another component of the GSP, the outer membrane-anchored lipoprotein PulS. Furthermore, the absence of PulS resulted in limited proteolysis of PulD and caused induction of the so-called phage shock response, as measured by increased expression of the pspA gene. We propose that PulS may be the first member of a new family of periplasmic chaperones that are specifically required for the insertion of a group of outer membrane proteins into this membrane. PulS is only the second component of the main terminal branch of the GSP for which a precise function can be proposed.     

Action of a major outer cell envelope membrane protein in conjugation of Escherichia coli K-12.

Protein II, a major outer cell envelope membrane protein, was found together with lipopolysaccharide to stoichiometrically inhibit conjugation in Escherichia coli K12.     

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.     

Pore-forming activity of the Tsx protein from the outer membrane of Escherichia coli. Demonstration of a nucleoside-specific binding site

The Tsx protein from the outer membrane of Escherichia coli is known to be involved in the permeation of nucleosides across the outer membrane under limiting substrate conditions. We purified Tsx from an E. coli strain that overproduces Tsx. The purified protein was still functional since it could neutralize the Tsx-specific bacteriophage T6 in vitro. When the purified Tsx was reconstituted into a lipid bilayer, there was a large increase of the membrane conductance, indicating pore-forming activity of Tsx in vitro. This increase could be strongly blocked with adenosine and to a much lesser extent with cytidine. Titration of the pore conductance with adenosine or cytidine suggested the presence of a binding site for nucleosides in the Tsx pore, with a Ks of 6 X 10(-4) and 2 X 10(-2) M for adenosine and cytidine, respectively. We propose that the Tsx protein functions in vivo as a pore that specifically facilitates the permeation of nucleosides across the outer membrane due to its binding site for nucleosides.     

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.