Outer membrane of gram-negative bacteria. XVIII. Electron microscopic studies on porin insertion sites and growth of cell surface of Salmonella typhimurium.

Salmonella typhimurium contains three "major proteins" or "porins" (34K, 35K, and 36K) in the outer membrane. A mutant strain producing only the 35K porin was first grown in media containing high concentrations of NaCl to "repress" the porin synthesis and then was shifted into a medium without NaCl. The newly made porin molecules were then labeled with the ferritin-coupled antibody at various times after the shift, and the samples were examined by whole-mount, freeze-etching, and thin-section electron microscopy. These experiments showed that newly inserted porins appeared as discrete patches uniformly distributed over the surface of the cell and, furthermore, that the sites of adhesion between the inner and outer membrane were most probably the pathway by which the newly made porin molecules appeared on cell surface. The 34K and 36K porins were also inserted in the same manner, since the appearance of new porins at discrete sites all over the cell surface was also observed when cells with wild-type porin phenotype were treated with unlabeled antibody to block existing antigenic sites, subsequently regrown, and labeled with the ferritin-coupled antibody. Since porins comprise a major portion of the densely packed, relatively immobile, "protein framework" of the outer membrane, these results lead us to conclude that the outer membrane grows predominantly by diffuse intercalation rather than by the zonal growth mechanism.     

Bacteriophage-resistant mutants of Salmonella typhimurium deficient in two major outer membrane proteins.

Mutants resistant to bacteriophages (P221 and PH105 or PH51) were isolated from a rfa strain of Salmonella typhimurium. They were found deficient in separate 33,000- to 36,000-dalton band proteins (major band proteins). Double mutants derived from both types of mutants were deficient in both of the bands. The growth behavior of all the mutants was normal. The outer membrane of the mutants appeared to be more wrinkled than normal and formed vesicles in many of the mutants. In freeze-fractured cells, changes were seen in the outer membrane (particleless patches in the concave fracture face, the particles themselves being smaller than normal). These changes were more marked in the double mutants.     

Use of a nonpenetrating substrate analogue to study the molecular mechanism of the outer membrane dicarboxylate transport system in Escherichia coli K12

We have recently demonstrated that a cell-surface dicarboxylate-binding protein (DBP) is involved in the outer membrane dicarboxylate transport system in Escherichia coli K12. The present report deals with our findings relating to the mode of action of this protein, and the identity and properties of the outer membrane integral protein which is involved in the translocation of dicarboxylic acids across the hydrophobic regions of the outer membrane. By the use of a nonpenetrating succinate analogue, aspartate-dextran, and through reconstitution studies with purified DBP, the cell-surface DBP is found to play an important role in succinate influx but not efflux. Transport studies with major outer membrane protein mutants indicate that the matrix protein (also referred to as protein I or porin) is the only outer membrane integral protein actively involved in the outer membrane dicarboxylate transport system. In the absence of a functional DBP, porin translocates succinate in a relatively less efficient and nonspecific manner. A tentative working model is proposed for this transport system. In this model, the cell-surface DBP is depicted as the substrate recognition component of the otherwise nonspecific porin channel. Together, this "DBP-porin channel complex" forms an efficient, specific transport channel for dicarboxylic acids.     

Porin channels in Escherichia coli: studies with beta-lactams in intact cells

Wild-type Escherichia coli K-12 produces two porins, OmpF (protein 1a) and OmpC (protein 1b). In mutants deficient in both of these "normal" porins, secondary mutants that produce a "new" porin, protein PhoE (protein E), are selected for. We determined the properties of the channels produced by each of these porins by measuring the rates of diffusion of various cephalosporins through the outer membrane in strains producing only one porin species. We found that all porin channels retarded the diffusion of more hydrophobic cephalosporins and that with monoanionic cephalosporins a 10-fold increase in the octanol-water partition coefficient of the solute produced a 5- to 6-fold decrease in the rate of penetration. Electrical charges of the solutes had different effects on different channels. Thus, with the normal porins (i.e., OmpF and OmpC proteins) additional negative charge drastically reduced the penetration rate through the channels, whereas additional positive charge significantly accelerated the penetration. In contrast, diffusion through the PhoE channel was unaffected by the presence of an additional negative charge. We hypothesize that the relative exclusion of hydrophobic and negatively charged solutes by normal porin channels is of ecological advantage to E. coli, which must exclude hydrophobic and anionic bile salts in its natural habitat. The properties of the PhoE porin are also consistent with the recent finding (M. Argast and W. Boos, J. Bacteriol. 143:142-150, 1980; J. Tommassen and B. Lugtenberg, J. Bacteriol. 143:151-157, 1980) that its biosynthesis is derepressed by phosphate starvation; the channel may thus act as an emergency pore primarily for the uptake of phosphate and phosphorylated compounds.     

Identification of porin-like polypeptide(s) in the boundary membrane of oilseed glyoxysomes

A 36-kDa polypeptide of unknown function was identified by us in the boundary membrane fraction of cucumber seedling glyoxysomes. Evidence is presented in this study that this 36-kDa polypeptide is a glyoxysomal membrane porin. A sequence of 24 amino acid residues derived from a CNBr-cleaved fragment of the 36-kDa polypeptide revealed 72% to 95% identities with sequences in mitochondrial or non-green plastid porins of several different plant species. Immunological evidence indicated that the 36-kDa (and possibly a 34-kDa polypeptide) was a porin(s). Antiserum raised against a potato tuber mitochondrial porin recognized on immunoblots 34-kDa and 36-kDa polypeptides in detergent-solubilized membrane fractions of cucumber seedling glyoxysomes and mitochondria, and in similar glyoxysomal fractions of cotton, castor bean, and sunflower seedlings. The 36-kDa polypeptide seems to be a constitutive component because it was detected also in membrane protein fractions derived from cucumber leaf-type peroxisomes. Compelling evidence that one or both of these polypeptides were authentic glyoxysomal membrane porins was obtained from electron microscopic immunogold analyses. Antiporin IgGs recognized antigen(s) in outer membranes of glyoxysomes and mitochondria. Taken together, the data indicate that membranes of cucumber (and other oilseed) glyoxysomes, leaf-type peroxisomes, and mitochondria possess similar molecular mass porin polypeptide(s) (34 and 36 kDa) with overlapping immunological and amino acid sequence similarities.     

Ion channel activities in the Escherichia coli outer membrane.

The electrical properties of Escherichia coli cells were examined by the patch-clamp technique. Giant cells or giant spheroplasts were generated by five different methods. By electron micrographic and other criteria we determined that the patches are most likely from the outer membrane. We regularly observed currents through at least two types of channels in this membrane. The first current is mechanosensitive and voltage-dependent, and can be observed in single gene mutants of the known major porins (ompF, ompC, phoE, lamB); this channel may represent a minor porin or a new class of outer membrane protein. The possible identity of the second, voltage-sensitive channel with one of the known outer membrane proteins is being explored. The high-resistance seals consistently formed on these patches and the presence of gated ion channels suggest that most of the pores of the outer membrane are not statically open, as commonly held, but are closed at rest and may be openable by physiological stimuli.     

Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface.

We studied the reactivity of 66 anti-Escherichia coli B/r porin monoclonal antibodies (MAbs) with several E. coli and Salmonella typhimurium strains. Western immunoblots showed complete immunological cross-reactivity between E. coli B/r and K-12; among 34 MAbs which recognized porin in immunoblots of denatured outer membranes of E. coli B/r, all reacted with OmpF in denatured outer membranes of E. coli K-12. Extensive reactivity, although less than that for strain B/r (31 of 34 MAbs), occurred for porin from a wild-type isolate, E. coli O8:K27. Only one of the MAbs reacted with porin in denatured outer membranes of S. typhimurium. Even with immunochemical amplification of the Western immunoblot technique, only six MAbs recognized S. typhimurium porin (OmpD), demonstrating that there is significant immunological divergence between the porins of these species. Antibody binding to the bacterial surface, which was analyzed by cytofluorimetry, was strongly influenced by lipopolysaccharide (LPS) structure. An intact O antigen, as in E. coli O8:K27, blocked adsorption of all 20 MAbs in the test panel. rfa+ E. coli K-12, without an O antigen but with an intact LPS core, bound seven MAbs. When assayed against a series of rfa E. coli K-12 mutants, the number of MAbs that recognized porin surface epitopes increased sequentially as the LPS core became shorter. A total of 17 MAbs bound porin in a deep rough rfaD strain. Similar results were obtained with S. typhimurium. None of the anti-E. coli B/r porin MAbs adsorbed to a smooth strain, but three antibodies recognized porin on deep rough (rfaF, rfaE) mutants. These data define six distinct porin surface epitopes that are shielded by LPS from reaction with antibodies.     

Porin activity in the osmotic shock fluid of Escherichia coli.

Osmotic shock fluid of Escherichia coli exhibited pore-forming activity. This activity could be followed by an in vitro assay based on the conductivity increase for ions due to the presence of pores in black lipid membranes. The histogram (the distribution of conductivity increments in a single pore experiment) obtained with osmotic shock fluid from E. coli was identical to the histogram obtained by detergent-solubilized porin isolated from the outer membrane. The osmotic shock fluid from porin-negative mutants also exhibited pore activity, although the histogram and ion specificity were different from those of porin. Antibodies raised against detergent-solubilized porin were able to form precipitin lines by the Ouchterlony immunodiffusion technique when shock fluids, but not detergent-solubilized porin, were used. These antibodies prevented the formation of pores when shock fluids contained porin but not when shock fluids obtained from porin-negative mutants were used. Macroscopic membrane conductivity of shock fluids due to porin exhibited a concentration dependence, in contrast to detergent-solubilized porin. These results indicate that the hydrodynamic properties of periplasmic or "soluble" porin are different from those of the detergent-solubilized porin of the outer membrane. Periplasmic porin comprises about 0.7% of total protein in the osmotic shock fluid.     

Mutants defective in the 33K outer membrane protein of Salmonella typhimurium.

Salmonella typhimurium LT2 lines, if phenotypically rough, are fully sensitive to bacteriocin 4-59, produced by Salmonella canastel strain SL1712. Bacteriocin-resistant mutants fell into three classes. Those resistant to phage ES18 and to albomycin proved to be mutants of class chr (equivalent to tonB of Escherichia coli); these mutants still adsorb the bacteriocin and so are classified as tolerant. Another class of (incompletely) tolerant mutants was resistant to phage PH51; their envelope fractions lacked the band corresponding to outer membrane protein 34K, known to serve for adsorption of phage PH51. A third class of mutants, which did not adsorb the bacteriocin, was unaltered in sensitivity to phages. Their envelopes lacked the 33K band, indicating absence of the outer membrane protein 33K, considered to correspond to outer membrane protein II* of E. coli, which in that species is determined at locus ompA (formerly tolG or con). Phage P22 HT105/1 cotransduced the 33K S. typhimurium gene (to be called ompA, to accord with E. coli usage) with pyrD+ at about 30% frequency when the donor allele was ompA+ or one ompA, but at only 3 to 11% when the donor allele was another ompA. When the donor carried either of two long deletions of the put (proline utilization) operon, phage P22 HT105/1 cotransduced put (and ompA+) with pyrD+ at low frequency. The cotransduction data indicate that ompA of S. typhimurium is located between pyrD and put, nearer the former. This corresponds to the map position of ompA in E. coli K-12.     

Altered antibiotic transport in OmpC mutants isolated from a series of clinical strains of multi-drug resistant E. coli

Antibiotic-resistant bacteria, particularly gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate). We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane.     

Copper sensitivity in an envelope mutant of Escherichia coli and its suppression by ColV, I-K94

A phage K3-resistant isolate from Escherichia coli P678-54 was devoid of both the OmpA and OmpC proteins but had high levels of the OmpF protein. Associated with these changes, the strain showed increased sensitivity to inhibition by detergents and greatly increased sensitivity to Cu²⁺. Introduction of the ColV, I-K94 plasmid into this mutant produced a derivative with markedly increased resistance to Cu²⁺ ions but unchanged detergent sensitivity. Analysis of membranes showed that the ColV, I-K94⁺ derivative had essentially no OmpF protein in its outer membrane. A ca 36 K outer membrane protein was present which resembled the OmpC protein in size and failure to dissociate in SDS at low temperature. It was distinct from the OmpC protein, however, in failing to allow either tetracycline uptake or the adsorption of T4-type phages. The possible significance of OmpF porin derepression (and its reversal by ColV, I-K94) for enterobacterial survival in aquatic situations is discussed.     

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.     

An essential role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer membrane

The preprotein translocase of the outer mitochondrial membrane (TOM complex) contains one essential subunit, the channel Tom40. The assembly pathway of the precursor of Tom40 involves the TOM complex and the sorting and assembly machinery (SAM complex) with the non-essential subunit Mas37. We have identified Sam50, the second essential protein of the mitochondrial outer membrane. Sam50 contains a beta-barrel domain conserved from bacteria to man and is a subunit of the SAM complex. Yeast mutants of Sam50 are defective in the assembly pathways of Tom40 and the abundant outer membrane protein porin, while the import of matrix proteins is not affected. Thus the protein sorting and assembly machinery of the mitochondrial outer membrane involves an essential, conserved protein.     

Mutant OmpF porins of Yersinia pseudotuberculosis with deletions of external loops: Structure–functional and immunochemical properties

Recombinant mutant OmpF porins from Yersinia pseudotuberculosis outer membrane were obtained using site-directed mutagenesis. Here we used four OmpF mutants where single extracellular loops L1, L4, L6, and L8 were deleted one at a time. The proteins were expressed in Escherichia coli at levels comparable to full-sized recombinant OmpF porin and isolated from the inclusion bodies. Purified trimers of the mutant porins were obtained after dialysis and consequent ion-exchange chromatography. Changes in molecular and spatial structure of the mutants obtained were studied using SDS–PAGE and optical spectroscopy (circular dichroism and intrinsic protein fluorescence). Secondary and tertiary structure of the mutant proteins was found to have some features in comparison with that of the full-sized recombinant OmpF. As shown by bilayer lipid membrane technique, the pore-forming activity of purified mutant porins was identical to OmpF porin isolated from the bacterial outer membrane. Lacking of the external loops mentioned above influenced significantly upon the antigenic structure of the porin as demonstrated using ELISA.     

Comparison of chloroplast DNAs by specific fragmentation with EcoRI endonuclease

Summary Mutants ofEscherichia coli K12, deficient in up to three major outer membrane proteinsb,c andd have been constructed. Mutants that lack the lipopolysaccharide sugar heptose are deficient in proteinb. All heptose-deficient strains are supersensitive to lysozyme, various antibiotics and detergents. They excrete the periplasmic enzyme ribonuclease I. Mutants deficient in proteinsc and/ord have the same sensitivity towards these compounds as the parent strain. Cells of single, double and triple mutants are all rod-shaped. Electrophoretic analysis of cell evelope proteins indicates that in some mutants the protein deficiency is partially compensated for by increased amounts of one or two of the other major outer membrane proteins. Heptose-deficient strains have an increased amount of 2-keto-3-deoxyoctonate.     

High-affinity binding sites involved in the import of porin into mitochondria.

The specific recognition by mitochondria of the precursor of porin and the insertion into the outer membrane were studied with a radiolabeled water-soluble form of porin derived from the mature protein. High-affinity binding sites had a number of 5-10 pmol/mg mitochondrial protein and a ka of 1-5 X 10(8) M-1. Binding was abolished after trypsin pretreatment of mitochondria indicating that binding sites were of protein-aceous nature. Specifically bound porin could be extracted at alkaline pH but not by high salt and was protected against low concentrations of proteinase K. It could be chased to a highly protease resistant form corresponding to mature porin. High-affinity binding sites could be extracted from mitochondria with detergent and reconstituted in asolectin-ergosterol liposomes. Water-soluble porin competed for the specific binding and import of the precursor of the ADP/ATP carrier, an inner membrane protein. We suggest that (i) binding of precursors to proteinaceous receptors serves as an initial step for recognition, (ii) the receptor for porin may also be involved in the import of precursors of inner membrane proteins, and (iii) interaction with the receptor triggers partial insertion of the precursor into the outer membrane.     

Effect of Temperature on the Distribution of Membrane Particles in Streptococcus faecalis as Seen by the Freeze-Fracture Technique

When cells of Streptococcus faecalis ATCC 9790 were incubated at temperatures above 10 C before being frozen for freeze-fracture, a random distribution of particles was observed on the outer fracture face of the freeze-cleaved cell membrane. However, when cells were incubated below 10 C before freezing, particleless patches were seen on this membrane surface. The size of the patches produced on chilling could be increased by centrifugation or by storing the chilled cells overnight at about 3 C. Patch formation appeared readily reversible, since the medium and large patches that formed on chilling could not be observed in cells warmed for 10 s at 25 C. However, during the transition from the patch to patchless state, smaller patches not seen in the chilled cells were observed. This suggested that the smaller patches might have been intermediate forms produced by the fragmentation of larger patches on warming.     

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.     

Structure-function correlation of outer membrane protein porin from Paracoccus denitrificans

Porins from outer membrane of Gram-negative bacteria have a highly stable structure. Our previous studies on porin from Paracoccus denitrificans showed that the outer membrane protein porin is extremely stable toward heat, pH, and chemical denaturants. The major question we have addressed in this paper is whether the high stability of porin is a consequence of the beta-barrel structure and whether it is required for its function. To explain this we have analyzed two cases: first, we used porin wild-type and mutants and compared their structure and function; second, we compared the activity of porin preheated to different temperatures. Structural changes were monitored by infrared spectroscopy. We observed that the structural stability of porin is not equivalent to functional activity as minor alteration in the structure can result in drastic differences in the activity of porins.     

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.