Protein Composition of the Cell Wall and Cytoplasmic Membrane of Escherichia coli

Envelope preparations obtained by passing Escherichia coli cells through a French pressure cell were separated by sucrose density gradient centrifugation into two distinct particulate fractions. The fraction with the higher density was enriched in fragments derived from the cell wall, as indicated by the high content of lipopolysaccharide, the low content of cytochromes, and the similar morphology of the fragments and intact cell walls. The less-dense fraction was enriched in vesicles derived from the cytoplasmic membrane, as indicated by the enrichment of cytochromes, the enzymes lactic and succinic dehydrogenase and nitrate reductase, and the morphological similarity of the vesicles to intact cytoplasmic membrane. Both fractions were rich in phospholipid. The protein composition was compared by mixing the cytoplasmic membrane-enriched fraction from a (3)H-labeled culture with the cell wall-enriched fraction from a (14)C-labeled culture and examining the resulting mixture by gel electrophoresis. Thirty-four bands of radioactive protein were resolved; of these, 27 were increased two- to fourfold in the cytoplasmic membrane-enriched fraction, whereas 6 were similarly increased in the cell wall-enriched fraction. One of the proteins which is clearly localized in the cell wall is the protein with a molecular weight of 44,000, which is the major component of the envelope. This protein accounted for 70% of the total protein of the cell wall, and its occurrence in the envelope from spheroplasts suggests that it is a structural protein of the outer membranous component of the cell wall.     

Distribution of Lipids in the Wall and Cytoplasmic Membrane Subfractions of the Cell Envelope of Escherichia coli

Cell wall and membrane subfractions of the cell envelope of Escherichia coli have been isolated by a procedure involving particle electrophoresis and sucrose gradient density centrifugation. The lipid content of each fraction has been investigated. The individual phospholipids of both fractions are quantitatively similar except that the proportion of lysophosphatidylethanolamine is greater in the wall than in the membrane. Fatty acid analysis of the phospholipids of each fraction revealed that the wall phospholipids contain a greater proportion of palmitic acid. Coenzyme Q is almost exclusively localized in the cell membrane.     

FtsL, an Essential Cytoplasmic Membrane Protein Involved in Cell Division in Escherichia coli

We have identified a gene involved in bacterial cell division, located immediately upstream of the ftsI gene in the min 2 region of the Escherichia coli chromosome. This gene, which we named ftsL, was detected through characterization of TnphoA insertions in a plasmid containing this chromosomal region. TnphoA topological analysis and fractionation of alkaline phosphatase fusion proteins indicated that the ftsL gene product is a 13.6-kDa cytoplasmic membrane protein with a cytoplasmic amino terminus, a single membrane-spanning segment, and a periplasmic carboxy terminus. The ftsL gene is essential for cell growth and division. A null mutation in ftsL resulted in inhibition of cell division, formation of long, nonseptate filaments, ultimate cessation of growth, and lysis. Under certain growth conditions, depletion of FtsL or expression of the largest ftsL-phoA fusion produced a variety of cell morphologies, including Y-shaped bacteria, indicating a possible general weakening of the cell wall. The FtsL protein is estimated to be present at about 20 to 40 copies per cell. The periplasmic domain of the protein displays a sequence with features characteristic of leucine zippers, which are involved in protein dimerization.     

Relationship between Cell Wall, Cytoplasmic Membrane, and Bacterial Motility

High-resolution electron microscopy of polarly flagellated bacteria revealed that their flagella originate at a circular, differentiated portion of the cytoplasmic membrane approximately 25 nm in diameter. The flagella also have discs attaching them to the cell wall. These attachment discs are extremely resistant to lytic damage and are firmly bound to the flagella. The cytoplasm beneath the flagellum contains a granulated basal body about 60 nm in diameter, and a specialized polar membrane. The existence of membrane-bound basal bodies is shown to be an artifact arising from adherence of cell wall and cytoplasmic membrane fragments to flagella in lysed preparations. Based on structures observed, a mechanism to explain bacterial flagellar movement is proposed. Flagella are considered to be anchored to the cell wall and activated by displacement of underlying cytoplasmic membrane to which they are also firmly attached. An explanation for the membrane displacement is given.     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by Triton X-100

Treatment of a partially purified preparation of cell walls of Escherichia coli with Triton X-100 at 23 C resulted in a solubilization of 15 to 25% of the protein. Examination of the Triton-insoluble material by electron microscopy indicated that the characteristic morphology of the cell wall was not affected by the Triton extraction. Contaminating fragments of the cytoplasmic membrane were removed by Triton X-100, including the fragments of the cytoplasmic membrane which were normally observed attached to the cell wall. Treatment of a partially purified cytoplasmic membrane fraction with Triton X-100 resulted in the solubilization of 60 to 80% of the protein of this fraction. Comparison of the Triton-soluble and Triton-insoluble proteins from the cell wall and cytoplasmic membrane fractions by polyacrylamide gel electrophoresis after removal of the Triton by gel filtration in acidified dimethyl formamide indicated that the detergent specifically solubilized proteins of the cytoplasmic membrane. The proteins solubilized from the cell wall fraction were qualitatively identical to those solubilized from the cytoplasmic membrane fraction, but were present in different proportions, suggesting that the fragments of cytoplasmic membrane which are attached to the cell wall are different in composition from the remainder of the cytoplasmic membrane of the cell. Treatment of unfractionated envelope preparations with Triton X-100 resulted in the solubilization of 40% of the protein, and only proteins of the cytoplasmic membrane were solubilized. Extraction with Triton thus provides a rapid and specific means of separating the proteins of the cell wall and cytoplasmic membrane of E. coli.     

Adsorption of Bacteriophages to Adhesions Between Wall and Membrane of Escherichia coli

In plasmolyzed Escherichia coli, wall and membrane adhered to one another at 200 to 400 localized areas. This number of specialized wall areas per cell was of the same order of magnitude as the total number of bacteriophage receptors. When bacteriophages T1 to T7 were adsorbed to the bacteria, they were seen to attach almost exclusively to these areas. Comparisons of the number of adsorbed phage particles observed in ultrathin sections and the expected number of phages per cell were in agreement. These results suggest a sharing of receptive areas by the various phages. Adsorption to the wall-membrane associations would permit the virus to release its nucleic acid at an area closest to the cell's protoplasmic contents.     

Alterations in the Cytoplasmic Membrane Proteins of Various Chlorate-Resistant Mutants of Escherichia coli

Chlorate-resistant mutants corresponding to each known genetic locus (chlA, chlB, chlC, chlD, chlE) were isolated from Escherichia coli K-12. All these mutants showed decreased amounts of membrane-bound nitrate reductase, cytochrome b, and formic dehydrogenase, but all had normal succinic dehydrogenase activity. Proteins from the cytoplasmic membranes of these mutants were compared to those of the wild type-on polyacrylamide gels. The addition of nitrate to wild-type anaerobic cultures caused increased formation of three membrane proteins. These same proteins, along with one other, were missing in varying patterns in mutants altered at the different genetic loci. One of the missing proteins was found to be the enzyme nitrate reductase, although this protein was present in some mutants lacking nitrate reductase activity. None of the others has been identified.     

Cell Division of Escherichia coli: Control by Membrane Organization

Cells of certain strains of Escherichia coli, after transfer from 37 to 45 C and incubation for 16 min, were observed to swell and subsequently divide synchronously. This swelling and the resulting stretching of the membrane are proposed to be the basis for the synchronous division. Four lines of evidence support this hypothesis. First, osmotic protection by the addition of either sodium chloride or sucrose at the time of heat shock prevents both swelling and synchrony. Second, a mutant neither swelled nor divided synchronously after heat shock. Third, cells grown for several generations with 10% sucrose in the medium swelled and divided synchronously upon transfer to medium without sucrose. Fourth, the mutant not synchronized by heat shock also swelled and underwent synchronous division after the osmotic shift. A tentative model is suggested for the normal control of division, based on membrane configuration at the septation site.     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by the Ionic Detergent Sodium-Lauryl Sarcosinate

The sensitivity of the outer and cytoplasmic membranes of Escherichia coli to detergent was examined by isopycnic sucrose density gradient centrifugation. Sodium lauryl sarcosinate (Sarkosyl) was found to disrupt the cytoplasmic membrane selectively under conditions in which Triton X-100 and dodecyl sodium sulfate solubilized all membrane protein. These results were verified by gel electrophoresis; membrane proteins solubilized by Sarkosyl were identical to those of the cytoplasmic membrane. The presence of Mg(2+) during treatment with Sarkosyl was found to afford partial protection of the cytoplasmic membrane from dissolution.     

Isolation and Characterization of an Escherichia coli Bacteriophage Requiring Cell Wall Galactose

A new coliphage, designated U3, has been selected for the ability to discriminate the presence of galactose in the cell wall of Escherichia coli. U3 attacks E. coli K-12 cells that are able to incorporate galactose into their cell walls, but mutants blocked in the synthesis of uridine diphosphogalactose, the precursor of cell wall galactose, are completely resistant to the phage. U3 is a small, tail-less, approximately spherical phage resembling X174 in its physical properties. Its diameter by electron microscopy is 21 to 22 nm, and its particle weight is approximately 4 × 10⁶ daltons. Like X174, U3 appears to have a single-stranded deoxyribonucleic acid genome and has at least four cistrons.     

Coordinated Rearrangements between Cytoplasmic and Periplasmic Domains of the Membrane Protein Complex ExbB-ExbD of Escherichia coli

1. Download : Download high-res image (239KB)
2. Download : Download full-size image

     

Cell Cycle-Specific Incorporation of Lipoprotein into the Outer Membrane of Escherichia coli

A cell cycle-specific incorporation of free lipoprotein into the outer membrane of Escherichia coli was observed, with a maximal rate of incorporation occuring at the time of septation.     

Bacteriophage T4D Receptor and the Escherichia coli Cell Wall Structure: Binding of Endotoxin-Like Particles to the Cell Wall

A variety of degradative treatments have been used to investigate the nature of the structure and components of the cell walls of Escherichia coli B. The binding and localization of the endotoxin-like particles found on the cell walls were of special interest because some of them are associated with the site where the inner tail tube of bacteriophage T4D penetrates the cell wall. Modified cell walls were obtained by heating a suspension of bacterial cells originally in 0.1 M phosphate, pH 7.0, after the addition of 12.5 M NaOH to a final concentration of 0.25 M. With regard to the endotoxin-like particles, it was found that: (i) at least part of them still remained bound to the modified cell wall after the alkali treatment; (ii) the subsequent incubation of alkali-treated cell walls with lysozyme destroyed the bacterial form and released a complex of endotoxin-like particles together with a fibrous material; (iii) on the other hand, treatment with 45% phenol at 70°C removed the endotoxin-like particles from the surface of the alkali-treated cell walls, but most of the fibrous material was left on the cell wall; and (iv) incubation of alkali-treated cell walls with 5 mM ethylenediaminetetraacetic acid at 20°C also removed the endotoxin-like particles, but did not disrupt the rodlike bacterial form. However, if the ethylenediaminetetraacetic acid treatment was performed at 55°C, the bacterium-like form was destroyed. These differential sensitivities to ethylenediaminetetraacetic acid suggested that loosely bound divalent metal ions normally hold these endotoxin-like particles on the cell wall surface, but that probably more tightly bound metal ions are involved in the determination of cell shape. Analysis of the protein components of the alkalitreated cell walls showed that only one protein was present in significant amounts, and this protein had an electrophoretic mobility similar to that of the Braun lipoprotein. This protein was released from the alkali-treated cell walls upon heating with 2% sodium dodecyl sulfate at 100°C. Phospholipids were also absent from this structure. The distribution of the remaining cell wall components on the alkali-treated cell walls is discussed.     

Recovery of Impaired Cell Division of UV-Irradiated Escherichia coli by Several Chemical Agents Affecting Cell Membrane

We examined the effect of various agents on the cell division of E. coli B(Sm^r) irradiated with ultraviolet (UV) light. It was found that the impaired cell division was reversed by one of various agents such as higher fatty acids, lower alcohols, terpineol, phenethyl alcohol, streptomycin, ribonuclease and EDTA. These agents, except RNase, showed the maximum activity of recovery just below a concentration causing a complete suppression of cell growth.     

Distribution of AC Electric Field-Induced Transmembrane Voltage in Escherichia coli Cell Wall Layers

On the basis of Gram-negative bacterium Escherichia coli models previously published in the literature, the transmembrane voltage induced by the application of an alternating current (AC) electric field on a bacterial suspension is calculated using COMSOL Multiphysics software, in the range 1–20 MHz, for longitudinal and transverse field orientations. The voltages developed on each of the three layers of the cell wall are then calculated using an electrical equivalent circuit. This study shows that the overall voltage on the cell wall, whose order of magnitude is a few tens of µV, is mainly distributed on inner and outer layers, while a near-zero voltage is found on the periplasm, due to its much higher electrical conductivity compared with the other layers. Although the outer membrane electrical conductivity taken in the model is a thousand times higher than that of the inner membrane, the voltage there is about half of that on the inner membrane, due to capacitive effects. It follows that the expression of protein complexes anchored in the inner membrane could potentially be disrupted, inducing in particular a possible perturbation of biological processes related to cellular respiration and proton cycle, and leading to growth inhibition as a consequence. Protein complexes anchored in the outer membrane or constituting a bridge between the three layers of the cell wall, such as some porins, may also undergo the same action, which would add another growth inhibition factor, as a result of deficiency in porin filtration function when the external environment contains biocides. Bioelectromagnetics. 2020;41:279–288 © 2020 Bioelectromagnetics Society.     

Porosity of the Yeast Cell Wall and Membrane

The limiting sizes of molecules that can permeate the intact cell wall and protoplast membrane of Saccharomyces cerevisiae were determined from the inflection points in a triphasic pattern of passive equilibrium uptake values obtained with a series of inert probing molecules varying in molecular size. In the phase identified with the yeast protoplast, the uptake-exclusion threshold corresponded to a monodisperse ethylene glycol of molecular weight = 110 and Einstein-Stokes hydrodynamic radius (r(ES)) = 0.42 nm. In the cell wall phase, the threshold corresponded to a polydisperse polyethylene glycol of number-average molecular weight ( M(n)) = 620 and average radius (r(ES)) = 0.81 nm. The third phase corresponded to complete exclusion of larger molecules. The assessment of cell wall porosity was confirmed by use of a second method involving analytical gel chromatographic analyses of the molecular weight distribution for a single polydisperse polyglycol before and after uptake by the cells, which indicated a quasi-monodisperse threshold for the cell wall of M(n) = 760 and r(ES) = 0.89 nm. The results were reconciled with two situations in which much larger protein molecules previously have been reported able to penetrate the yeast cell wall.     

Cytoplasmic protein mobility in osmotically stressed Escherichia coli

Facile diffusion of globular proteins within a cytoplasm that is dense with biopolymers is essential to normal cellular biochemical activity and growth. Remarkably, Escherichia coli grows in minimal medium over a wide range of external osmolalities (0.03 to 1.8 osmol). The mean cytoplasmic biopolymer volume fraction ((phi)) for such adapted cells ranges from 0.16 at 0.10 osmol to 0.36 at 1.45 osmol. For cells grown at 0.28 osmol, a similar phi range is obtained by plasmolysis (sudden osmotic upshift) using NaCl or sucrose as the external osmolyte, after which the only available cellular response is passive loss of cytoplasmic water. Here we measure the effective axial diffusion coefficient of green fluorescent protein (D(GFP)) in the cytoplasm of E. coli cells as a function of (phi) for both plasmolyzed and adapted cells. For plasmolyzed cells, the median D(GFP) (D(GFP)(m)) decreases by a factor of 70 as (phi) increases from 0.16 to 0.33. In sharp contrast, for adapted cells, D(GFP)(m) decreases only by a factor of 2.1 as (phi) increases from 0.16 to 0.36. Clearly, GFP diffusion is not determined by (phi) alone. By comparison with quantitative models, we show that the data cannot be explained by crowding theory. We suggest possible underlying causes of this surprising effect and further experiments that will help choose among competing hypotheses. Recovery of the ability of proteins to diffuse in the cytoplasm after plasmolysis may well be a key determinant of the time scale of the recovery of growth.     

Murein Synthesis and Identification of Cell Wall Precursors of Temperature-Sensitive Lysis Mutants of Escherichia coli

A group of temperature-sensitive lysis mutants of Escherichia coli K-12 was studied. Mutants impaired in the synthesis of uridine diphosphate-N-acetylmuramyl (UDP-MurNAc)-pentapeptide or in the synthesis of murein amino acids were found. Their rate of murein synthesis at the restrictive temperature was decreased. A large number of mutants did not differ from the parent strain with respect to the rate of murein synthesis and the precursor pattern. The behavior of these mutants is discussed. It was impossible to accumulate UDP-MurNAc-pentapeptide in E. coli by the antibiotics penicillin and vancomycin. The hypothesis is put forward that the amount of this murein precursor is regulated by feedback inhibition.     

Reconstitution of Nitrate Reductase Activity and Formation of Membrane Particles from Cytoplasmic Extracts of Chlorate-Resistant Mutants of Escherichia coli

The reconstitution of nitrate reductase activity in mixtures of cytoplasmic fractions from the chlorate-resistant mutants chlA, B, C, and E which are lacking this activity was investigated, and the membrane-like particulate material which formed during this reconstitution was analyzed by polyacrylamide gel electrophoresis. When chlA and chlB extracts are incubated together, the cytoplasmic membrane proteins present in the particles which are formed are contributed by both mutants, and the proteins are essentially the same as the proteins in the cytoplasmic membrane fractions of the two mutants. Identical amounts of protein become particulate when cytoplasmic extracts of any of the mutant strains or wild-type strains are incubated at 32 C either singly or in mixtures, and the formation of particulate material does not appear to be a consequence of nitrate reductase reconstitution. Experiments with wild-type strains indicate that the membrane proteins in the cytoplasmic extract are derived from the cytoplasmic membrane during cell breakage. Reconstitution experiments involving various combinations of preincubated and unincubated extracts of the mutants have allowed a preliminary identification of three types of components which are necessary for the formation of active nitrate reductase: (i) a soluble factor present only in extracts from induced chlB; (ii) a different soluble factor which is missing in chlB but is present in extracts from wild-type, chlA, chlC, and chlE; and (iii) a complex including the nitrate reductase protein which is inactivated by preincubation of the mutant extracts.