Rapid Method for Isolation of Large Quantities of Outer Membrane from Escherichia coli K-12 and Its Application to the Study of Envelope Mutants

A rapid method for the isolation of large quantities of bacterial outer membrane is described. This cell envelope component was removed from plasmolyzed cells of Escherichia coli K-12 by lysozyme-ethylenediaminetetra-acetic acid treatment, aggregated by lowering the pH to 5.0, and recovered by centrifugation. Aggregates of membrane fragments were clearly identified in an electron microscope. A criterion of homogeneity of the preparation was obtained by isopycnic sucrose gradient centrifugation. A single band appeared at a density of 1.24 g/cc. The cytoplasmic membrane marker, succinate dehydrogenase activity, was 40 times lower in the outer membrane preparation than in complete cell envelope preparations. A rich activity was, however, found for the outer membrane marker, phospholipase A. The compositions of outer membranes from a transductant pair were compared. One transductant was a chain-forming, antibiotic-supersensitive envA strain, whereas the other contained the envA(+) allele. The envA strain showed a slightly modified protein pattern and a lower relative content of phosphatidylglycerol.     

Isolation of Bacteriophages T2 and T4 Attached to the Outer Membrane of Escherichia coli

Phage T2 or T4 was adsorbed to Escherichia coli, and the outer (L) membrane was then isolated with the phage still attached in their usual postinjection appearance. T2 was readily inactivated by isolated cell walls but very poorly by purified L membrane. T4 was inactivated by neither.     

Involvement of Outer Membrane Protein TolC, a Possible Member of the mar-sox Regulon, in Maintenance and Improvement of Organic Solvent Tolerance of Escherichia coli K-12

Escherichia coli mutants with improved organic solvent tolerance levels showed high levels of outer membrane protein TolC and inner membrane protein AcrA. The TolC level was regulated positively by MarA, Rob, or SoxS. A possible mar-rob-sox box sequence was found upstream of the tolC gene. These findings suggest that tolC is a member of the mar-sox regulon responsive to stress conditions. When a defective tolC gene was transferred to n-hexane- or cyclohexane-tolerant strains by P1 transduction, the organic solvent tolerance level was lowered dramatically to the decane-tolerant and nonane-sensitive level. The tolerance level was restored by transformation of the transductants with a wild-type tolC gene. Therefore, it is evident that TolC is essential for E. coli to maintain organic solvent tolerance.     

Metalloadsorption by Escherichia coli Cells Displaying Yeast and Mammalian Metallothioneins Anchored to the Outer Membrane Protein LamB

Yeast (CUP1) and mammalian (HMT-1A) metallothioneins (MTs) have been efficiently expressed in Escherichia coli as fusions to the outer membrane protein LamB. A 65-amino-acid sequence from the CUP1 protein of Saccharomyces cerevisiae (yeast [Y] MT) was genetically inserted in permissive site 153 of the LamB sequence, which faces the outer medium. A second LamB fusion at position 153 was created with 66 amino acids recruited from the form of human (H) MT that is predominant in the adipose tissue, HMT-1A. Both LamB¹⁵³-YMT and LamB¹⁵³-HMT hybrids were produced in vivo as full-length proteins, without any indication of instability or proteolytic degradation. Each of the two fusion proteins was functional as the port of entry of lambda phage variants, suggesting maintenance of the overall topology of the wild-type LamB. Expression of the hybrid proteins in vivo multiplied the natural ability of E. coli cells to bind Cd²⁺ 15- to 20-fold, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions.     

Interrelated Effects of Cold Shock and Osmotic Pressure on the Permeability of the Escherichia coli Membrane to Permease Accumulated Substrates

Permease studies are generally carried out by incubating cells in growth medium with labeled substrate, collecting the cells on microporous membrane filters, and washing them free from extracellular radioactivity with ice-cold medium. Studies of thiomethylgalactoside, valine, and galactose accumulation indicate that in several strains of Escherichia coli the bacterial membrane is exquisitely sensitive to isosmotic cold shock. Substrate pools formed at 25 C may suffer almost total loss if the cells are rapidly chilled to approximately 0 C during sampling. In glycerol-grown cells, this rapid efflux of substrate is prevented or minimized if the cells are subjected at the moment of cold shock to a simultaneous hyperosmotic transition. Because of this protective effect, the apparent size of a permease accumulated substrate pool is extremely sensitive to the osmotic composition of the incubation medium and may appear to be increased as much as 10-fold when the osmolarity is reduced from approximately 0.3 to 0.1 osmolar. These differences vanish when sampling and washing are carried out with medium at room temperature. It is suggested that isosmotic cold shock causes crystallization of the liquid-like lipids within the membrane. The hydrophilic channels created in this process would facilitate the rapid efflux of permease accumulated substrates. The imposition of a simultaneous hyperosmotic transition by dehydrating the cell periphery would cause increased lipid interaction, thus preserving the integrity of the cells membrane.     

Lambda Receptor in the Outer Membrane of Escherichia coli as a Binding Protein for Maltodextrins and Starch Polysaccharides

The starch polysaccharides amylose and amylopectin are not utilized by Escherichia coli, but are bound by the bacteria. The following evidence supports the view that the outer membrane lambda receptor protein, a component of the maltose/ maltodextrin transport system is responsible for the binding. (i) Amylose and amylopectin both inhibit the transport of maltose into E. coli. (ii) Both polysaccharides prevent binding of non-utilizable maltodextrins by the intact bacterium, a process previously shown to be dependent on components of the maltose transport system (T. Ferenci, Eur. J. Biochem., in press). (iii) A fluorescent amylopectin derivative, O-(fluoresceinyl thiocarbamoyl)-amylopectin, has been synthesized and shown to bind to E. coli in a reversible, saturable manner. Binding of O-(fluoresceinyl thiocarbamoyl)-amylopectin is absent in mutants lacking the lambda receptor, but mutations in any of the other components of the maltose transport system do not affect binding as long as lambda receptor is present. (iv) Using the inhibition of lambda receptor-dependent O-(fluoresceinyl thiocarbamoyl)-amylopectin binding as an assay, the affinities of the lambda receptor for maltodextrins and other sugars have been estimated. The affinity for dextrins increases with increasing degree of polymerization (K(d) for maltose, 14 mM; for maltotetraose, 0.3 mM; for maltodecaose, 0.075 mM). Maltose and some other di- and trisaccharides are inhibitory to amylopectin binding, but only at concentrations above 1 mM.     

Diglyceride Kinase Mutants of Escherichia coli: Inner Membrane Association of 1,2-Diglyceride and Its Relation to Synthesis of Membrane-Derived Oligosaccharides

Mutants of Escherichia coli defective in diglyceride kinase contain 10 to 20 times more sn-1,2-diglyceride than normal cells. This material constitutes about 8% of the total lipid in such strains. We now report that this excess diglyceride is recovered in the particulate fraction, primarily in association with the inner, cytoplasmic membrane. The diglyceride kinase of wild-type cells was recovered in the same inner membrane fractions. The conditions employed for the preparation of the membranes did not appear to cause significant redistribution of lipids and proteins. The biochemical reactions leading to the formation of diglyceride in E. coli are not known. To determine whether diglyceride formation requires concurrent synthesis of the membrane-derived oligosaccharides (H. Schulman and E. P. Kennedy, J. Biol. Chem. 252:4250-4255, 1977), we have constructed a double mutant defective in both the kinase (dgk) and phosphoglucose isomerase (pgi). When oligosaccharide synthesis was inhibited in this organism by growing the cells on amino acids as the sole carbon source, the diglyceride was no longer present in large amounts. When glucose was also added to the medium, the pgi mutation was bypassed, oligosaccharide synthesis resumed, and diglyceride again accumulated. These findings suggest that diglyceride may arise during the transfer of the sn-glycero-1-P moiety from phosphatidylglycerol (and possibly cardiolipin) to the oligosaccharides. In wild-type cells the kinase permits the cyclical reutilization of diglyceride molecules for phospholipid biosynthesis.     

Outer Penetration Barrier of Escherichia coli K-12: Kinetics of the Uptake of Gentian Violet by Wild Type and Envelope Mutants

Wild-type strains of Escherichia coli K-12 adsorb gentian violet to the cell surface, but the dye is not transported into the cytoplasm. However, when some mutants that have an altered outer membrane are exposed to gentian violet, the dye is also found in the ribosomal fraction. The transport into the cytoplasm is inhibited at 0 C and requires that the concentration of gentian violet exceeds a threshold value. The initial rate of uptake as well as the amount of gentian violet found in the cytoplasm increases with the concentration of the dye in the medium. The rate of transport of the dye into the cytoplasm is much lower for stationary mutant cells than for exponentially growing cells. The rate of uptake into the cytoplasm increases with increasing deficiency of carbohydrate in the lipopolysaccharide (carbohydrate content lpsB > lpsA > galU). However, other components are also responsible for the barrier since an envA mutant which is not altered in the lipopolysaccharide carbohydrates show an extremely rapid uptake of the dye. The rate of uptake for the envA mutant was the highest found and the same as that of spheroplasts. Growth in the presence of agents affecting the murein sacculus, e.g., lysozyme and sublethal concentrations of penicillin, increased the rate of uptake of gentian violet. Brief treatments with tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid drastically impaired the barrier function. Inhibition of protein synthesis by chloramphenicol also opened the barrier to gentian violet. In conclusion, the outer part of the bacterial envelope is a penetration barrier for gentian violet and probably also for other substances. The lipopolysaccharide, the murein and also other components are important for the function of this barrier. Resistance to gentian violet was found to be inversely correlated to the rate of penetration of the dye into the cytoplasm.     

Using Codon Usage to Predict Genes Origin: Is the Escherichia coli Outer Membrane a Patchwork of Products from Different Genomes?

Analysis of the codon usage of genes coding for the structuralcomponents of the outer membrane in Escherichia coli, is consistentwith the requirement for high expression of these genes. Becauseporins (which constitute the major protein component of theouter membrane), and LPS (which constitute the major outermostconstituent of the outer membrane), are synthesized from genesdisplaying widely different codon usage, it is possible to investigatethe origin of the outer membrane. The analysis predicts thatthe outer membrane might originate from a genome other thanthe genome coding for the major part of the cell. Such a specialorigin would explain in structural terms, the likely lethalityof porins if they were inadvertently inserted within the innermembrane, giving rise to the Gram-negative bacterial type, havingan envelope comprising two membranes, instead of a single cytoplasmicmembrane and a murein sacculus.     

Transport of Vitamin B12 in Escherichia coli: Common Receptor Sites for Vitamin B12 and the E Colicins on the Outer Membrane of the Cell Envelope

The first step in the transport of cyanocobalamin (CN-B(12)) by cells of Escherichia coli was shown previously to consist of binding of the B(12) to specific receptor sites located on the outer membrane of the cell envelope. In this paper, evidence is presented that these B(12) receptor sites also function as the receptors for the E colicins, and that there is competition between B(12) and the E colicins for occupancy of these sites. The cell strains used were E. coli KBT001, a methionine/B(12) auxotroph, and B(12) transport mutants derived from strain KBT001. Colicins E1 and E3 inhibited binding of B(12) to the outer membrane B(12) receptor sites, and CN-B(12) protected cells against these colicins. Half-maximal protection was given by CN-B(12) concentrations in the range of 1 to 6 nM, depending upon the colicin concentration used. Colicin E1 competitively inhibited the binding of (57)Co-labeled CN-B(12) to isolated outer membrane particles. Functional colicin E receptor sites were found in cell envelopes from cells of only those strains that possessed intact B(12) receptors. Colicin K did not inhibit the binding of B(12) to the outer membrane receptor sites, and no evidence was found for any identity between the B(12) and colicin K receptors. However, both colicin K and colicin E1 inhibited the secondary phase of B(12) transport, which is believed to consist of the energy-coupled movement of B(12) across the inner membrane.     

Deletion of lolB, Encoding an Outer Membrane Lipoprotein, Is Lethal for Escherichia coli and Causes Accumulation of Lipoprotein Localization Intermediates in the Periplasm

Outer membrane lipoproteins of Escherichia coli are released from the inner membrane upon the formation of a complex with a periplasmic chaperone, LolA, followed by localization to the outer membrane. In vitro biochemical analyses revealed that the localization of lipoproteins to the outer membrane generally requires an outer membrane lipoprotein, LolB, and occurs via transient formation of a LolB-lipoprotein complex. On the other hand, a mutant carrying the chromosomal lolB gene under the control of the lac promoter-operator grew normally in the absence of LolB induction if the mutant did not possess the major outer membrane lipoprotein Lpp, suggesting that LolB is only important for the localization of Lpp in vivo. To examine the in vivo function of LolB, we constructed a chromosomal lolB null mutant harboring a temperature-sensitive helper plasmid carrying the lolB gene. At a nonpermissive temperature, depletion of the LolB protein due to loss of the lolB gene caused cessation of growth and a decrease in the number of viable cells irrespective of the presence or absence of Lpp. LolB-depleted cells accumulated the LolA-lipoprotein complex in the periplasm and the mature form of lipoproteins in the inner membrane. Taken together, these results indicate that LolB is the first example of an essential lipoprotein for E. coli and that its depletion inhibits the upstream reactions of lipoprotein trafficking.     

Mechanisms of Resistance in Escherichia coli to the Sideromycin Antibiotic No. 216: Isolation and Characterization of the Resistant Mutants

Escherichia coli easily developed resistance to a new antimicrobial agent of the sideromycin group, No. 216, by spontaneous mutation. Most of the No. 216-resistant mutants tested proved not to be cross-resistant to E. coli phages T1, T5, and φ80. On the other hand, these phage-resistant mutants were cross resistant to No. 216. The initial site for binding of No. 216 to the sensitive cells was located, at the ton A gene product (Ton A-protein) of the outer membrane. However, unlike the phage-resistant mutants, ton A protein (78K-protein) in most No. 216-resistant mutants was intact and these mutants were possessed a particular 87K protein in the outer membrane. It is suggested that No. 216 is taken up by ton A protein and then penetrates into the cell by way of a particular transport system and that a highly mutable portion may exist in this reaction system.     

Organic Solvents as Probes for the Structure and Function of the Bacterial Membrane: Effects of Ethanol on the Wild Type and an Ethanol-Resistant Mutant of Escherichia coli K-12

The effects of ethanol on the growth of a wild-type Escherichia coli K-12 are described. These effects include a reduction of the steady-state growth rate and an interference with the division process. They appear as an immediate response to the addition of ethanol and are rapidly reversed by removal of ethanol. Mutants were selected that could grow at a concentration of ethanol that stopped wild-type growth. The growth of one of the mutants we studied (strain S9L100) is stimulated by the presence of ethanol, methanol, or dimethyl sulfoxide. This strain exhibits pleiotropic growth defects including abnormal cell division and morphology. It also appears to have an altered lac permease function which is not due to a mutation in the Y gene itself. We conclude that this mutant has an altered membrane and that the membrane defect may be the cause of the abnormal growth properties. The use of compounds which serve as general membrane perturbants and mutants resistant to these perturbants form a system accessible to both genetic and physical-biochemical techniques.