Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01.

The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [14C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35,000 is a porin, responsible for the size-dependent permeability of the outer membrane.     

Glycine and Triton X-100 enhanced secretion of recombinant α-CGTase mediated by OmpA signal peptide in Escherichia coli

OmpA signal peptide mediated cgt gene from Paenibacillus macerans JFB05-01 was cloned and expressed in E. coli BL21 (DE3). The effects of glycine and Triton X-100 on extracellular production of α-cyclodextrin glycosyltransferase (α-CGTase) were investigated. When supplemented with Gly or Triton X-100 to the culture media individually, the secreted extracellular enzyme reached 32 or 33 U/mL at 48 h of cultivation, respectively. When supplemented with Gly and Triton X-100 together, the extracellular α-CGTase activity reached 48 U/mL after 48 h cultivation, which was 20-fold of the control group without any additives. Analysis of membrane permeability demonstrated that addition of glycine and Triton X-100 enhanced the permeability of both outer and inner membrane. The potential mechanism of the enhanced protein secretion was discussed.     

Parallel effects of freezing and osmotic stress on the ATPase activity and protein composition of the plasma membrane of winter rye seedlings

The objective of this study was to determine the influence of freezing versus hypertonic stress on the ATPase activity and polypeptide profile of the plasma membrane of nonacclimated winter rye leaves (Secale cereale L. cv Puma). Exposure of leaves to hypertonic sorbitol solutions resulted in a similar extent of injury as did freezing to subzero temperatures that resulted in equivalent osmotic stresses. When isolated with a two-phase partition system of aqueous polymers, the plasma membrane fractions of control, frozen, or hypertonically stressed leaves were of similar purity as judged by the distribution of marker enzyme activities. When assayed in the presence of Triton X-100 (0.05% w/w), ATPase activity was decreased only slightly in plasma membrane fractions isolated from either frozen or hypertonically stressed leaves. In contrast, the specific ATPase activity of the plasma membrane fractions assayed in the absence of Triton X-100 increased following freezing or hypertonic stress. As a result, the Triton X-100 stimulation of the ATPase activity decreased significantly from sixfold in control leaves to threefold in lethally stressed leaves and reflects an increase in the permeability of the plasma membrane vesicles. The increased permeability was also manifested as a decrease in H⁺-transport following exposure to freezing or hypertonic stress. Both freezing and hypertonic exposure at subzero temperatures altered the polypeptide profile of the plasma membrane, but with the exception of one polypeptide, there was no difference between the two treatments.     

DNA synthetic activity of nuclei isolated from differentiating cardiac muscle and association of DNA polymerase alpha with the outer nuclear membrane

[³H]dTMP incorporation into DNA of nuclei isolated from differentiating cardiac muscle of the rat has been characterized. Nuclei prepared at different times during the terminal phase of differentiation by a procedure not involving a detergent (Triton X-100) wash show a progressively diminished capacity to support in vitro [³H]dTMP incorporation; this diminution parallels the loss of DNA polymerase α from cardiac muscle. The rate of incorporation of [³H]dTMP into DNA of nuclei washed twice with 0.5% Triton X-100 does not correlate with the in vivo DNA synthetic activity. As determined by electron microscopy the Triton X-100 wash removes the outer nuclear membrane; the pellet obtained by centrifuging the Triton X-100 extract of these nuclei consists of circular membrane vesicles. The predominant DNA polymerase activity in these preparations was characterized using pH optimum, N-ethylmaleimide sensitivity, and correlation to in vivo DNA synthetic activity as criteria. DNA polymerase α activity predominated in the non-Triton X-100-extracted nuclei and in the outer nuclear membrane fraction; DNA polymerase β activity was the predominant activity observed in Triton X-100-extracted nuclei. These data emphasize that the procedure which is used to isolate nuclei from proliferating cells can greatly influence the nature of the DNA synthetic activity that is observed in vitro, suggest that DNA polymerase α is associated with the outer nuclear membrane, and add support to the idea that this enzyme is involved in eukaryotic DNA replication.     

The nature and location of Acholeplasma laidlawii membrane proteins investigated by two-dimensional gel electrophoresis

The high resolution, two-dimensional electrophoresis system for the separation of proteins described by O'Farrell, (O'Farrell, P.H. (1975) J. Biol. Chem. 250, 4007–4021) has been modified for the separation of Acholeplasma laidlawii proteins.Reproducible protein patterns have been obtained from A. laidlawii cell, membrane and soluble protein preparations. The isoelectric focusing of membrane proteins was greatly improved by removing the bulk of the membrane lipid before solubilizing the protein.A. laidlawii peripheral membrane proteins were removed from the membrane by low ionic strength washing and by treatment with EDTA. The effect of an exhaustive EDTA treatment and a rapid, warm EDTA treatment were compared. By comparing the protein patterns obtained in these ways it was possible to distinguish two separate groups of peripheral membrane proteins and one integral membrane protein group. The peripheral membrane proteins which were removed from the membrane at low ionic strength (group I) were also insoluble in Triton X-100, whereas additional peripheral membrane proteins extractable by subsequent EDTA treatment (group II) were soluble in Triton X-100.Exterior-facing membrane proteins were distinguished from the interiorfacing ones by lactoperoxidase-catalyzed iodination of intact cells and membranes. Group I peripheral membrane proteins faced the cell interior whereas group II proteins faced the cell exterior. We counted approximately 320 individual whole cell proteins. Of these, about 140 were membrane associated and a maximum of 40 proteins were iodinated after iodinationg intact cells.A. laidlawii was also grown in the presence of NaH₂³²PO₄ and whole cell proteins were separated by two-dimensional gel electrophoresis. One membrane protein and two soluble proteins were labelled.     

Outer membrane proteins of Escherichia coli. II. Heterogeneity of major outer membrane polypeptides

When E. coli outer membrane protein is dissolved in sodium dodecyl sulfate (SDS) solution and boiled briefly, a single major peak (peak B) with a molecular weight of 42,000 daltons is observed on SDS-containing polyacrylamide gels. If the protein is dissolved in SDS solution at 37 °C and applied to gels without further treatment, peak B disappears and two other major peaks appear: Peak A, which is composed of aggregates and migrates more slowly than peak B, and peak C which is composed of monomeric protein not fully reacted with SDS and which migrates faster than peak B. When cyanogen bromide peptides of protein from peak A and peak C were compared, it was evident that peak A and peak C contained entirely different polypeptides. This was further confirmed by differential labeling studies with methionine and leucine. The cyanogen bromide peptide profiles of protein from peak A suggested that this peak was composed of two polypeptides, and this was confirmed by electrophoresis in an alkaline gel system which resolves peak B into three subcomponents. Two of these were derived from peak A and the third was derived from peak C. These results indicate that the outer membrane of E. coli contains at least three nonidentical major polypeptides, each of which has a nearly identical molecular weight of about 42,000 daltons. These polypeptides are present in identical proportions in the soluble and insoluble fractions obtained when the outer membrane is treated with Triton X-100 plus EDTA.     

Absence of ferric enterobactin receptor modification activity in mutants of Escherichia coli K-12 lacking protein a.

The modification activity for the ferric enterobactin receptor in the Triton X-100 solubilized outer membrane of $\text{Escherichia}\text{coli}$ K-12 was adsorbed to a column of $\text{p}$-aminobenzamidine-//-sepharose and eluted with free benzamidine. Recombination of the dialyzed eluate with the filtrate from the column reinstituted conversion of the receptor from 81K to 81K¹, the latter exhibiting an apparent molecular weight of 74,000 daltons in sodium dodecyl sulfate polyacrylamide gel analysis. The eluate from the $\text{p}$-aminobenzamidine column was shown to contain a component, coincident on gels with both protein and modification activity, which by mutational and other analyses appears to be identical with protein $\text{a}$ of the outer membrane.     

Comparison of methods used to separate the inner and outer membranes of cell envelopes of Campylobacter spp

The outer membrane of Campylobacter coli, C. jejuni and C. fetus cell envelopes appeared as three fractions after sucrose gradient centrifugation. Each outer membrane fraction was contaminated with succinate dehydrogenase activity from the cytoplasmic membrane fraction. Similarly the inner membrane fraction was contaminated with 2-ketodeoxyoctonate and outer membrane proteins including the porin(s). The separation of these two membranes was not facilitated by variations in lysozyme treatment, cell age, presence or absence of flagella, or longer lipopolysaccharide chain length. Sodium lauroyl sarcosinate extraction resulted in an outer membrane fraction which contained some inner membrane contamination and produced multiple bands upon sucrose gradient centrifugation. Triton X-100 extraction removed the inner membrane from the outer membrane and Triton X-100/EDTA treatment extracted lipopolysaccharide-rich regions of the outer membrane which contained almost exclusively the Campylobacter porin(s). These data indicated that the inner and outer membranes of the Campylobacter cell envelope were very difficult to separate, possibly because of extensive fusions between these two membranes.     

Identification of surface components of mammalian respiratory tract cilia

Cilia isolation methods were modified to retain respiratory tract ciliary membranes and to identify accessible surface components. Prior to isolation of cilia, halves of cow tracheae were treated with the extended spacer arm analog of N-hydroxysuccinimido-biotin (NHS-LC-biotin) to label accessible membrane constituents. Mechanical disruption of the epithelium and substitution of CHAPS for Triton X-100 provided a good yield of cilia with membranes and with minimal contamination. Subsequent extraction of these cilia with Triton X-100 solubilized the membranes and released soluble matrix proteins. Proteins of membrane + matrix and axoneme fractions were analyzed after electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The major biotin-labeled components in the membrane + matrix fraction were 105, 98, and 92 kd, were glycosylated, and remained with reconstituted, pelleted membrane vesicles along with the major non-biotinylated protein at 51 kd. Other membrane + matrix proteins at 126 and 76 kd bound streptavidin even from nonlabeled trachea, but remained soluble. Several biotin-labeled proteins distinct from those in the membrane fraction remained with Triton X-100-extracted axonemes. Streptavidin-colloidal-gold (SAG) particles appeared to bind randomly along the length of cilia. The peripheral join between A and B microtubules was a predominant nonspecific location of SAG on axonemes. Axonemes with biotin label also bound significant numbers of SAG to outer dynein arms, confirming the streptavidin reaction with separated proteins on transfers. These results suggest close association of the membrane with the axoneme in respiratory tract cilia and a membrane composition somewhat different from protozoan cilia.     

Effect of Ethylenediaminetetraacetic Acid, Triton X-100, and Lysozyme on the Morphology and Chemical Composition of Isolated Cell Walls of Escherichia coli

Extraction of a partially purified preparation of cell walls from Escherichia coli with the nonionic detergent Triton X-100 removed all cytoplasmic membrane contamination but did not affect the normal morphology of the cell wall. This Triton-treated preparation, termed the “Triton-insoluble cell wall,” contained all of the protein of the cell wall but only about half of the lipopolysaccharide and one-third of the phospholipid of the cell wall. This Triton-insoluble cell wall preparation was used as a starting material in an investigation of several further treatments. Reextraction of the Triton-insoluble cell wall with either Triton X-100 or ethylenediaminetetraacetic acid (EDTA) caused no further solubilization of protein. However, when the Triton-insoluble cell wall was extracted with a combination of Triton X-100 and EDTA, about half of the protein and all of the remaining lipopolysaccharide and phospholipid were solubilized. The material which remained insoluble after this combined Triton and EDTA extraction still retained some of the morphological features of the intact cell wall. Treatment of the Triton-insoluble cell wall with lysozyme resulted in a destruction of the peptidoglycan layer as seen in the electron microscope and in a release of diaminopimelic acid from the cell wall but did not solubilize any cell wall protein. Extraction of this lysozyme-treated preparation with a combination of Triton X-100 and EDTA again solubilized about half of the cell wall protein but resulted in a drastic change in the morphology of the Triton-EDTA-insoluble material. After this treatment, the insoluble material formed lamellar structures. These results are interpreted in terms of the types of noncovalent bonds involved in maintaining the organized structure of the cell wall and suggest that the main forces involved are hydrophobic protein-protein interactions between the cell wall proteins and to a lesser degree a stabilization of protein-protein and protein-lipopolysaccharide interactions by divalent cations. A model for the structure of the E. coli cell wall is presented.     

GABA receptor binding in bovine retina: effects of Triton X-100 and perchloric acid

In two synaptosomal fractions from bovine retina, Triton X-100 and sodium perchlorate specifically enhanced the high affinity binding of ³H-GABA to sites with pharmacological specificity similar to the GABA receptor. Maximal effects were noted at 0.05% Triton X-100 and 100 mM sodium perchlorate. In the fraction enriched in photoreceptor cell synaptosomes from the outer plexiform layer, Triton and perchlorate had similar effects in that two binding sites were observed: a higher affinity site (～20 nMK_D) and a lower affinity site (～200 nMK_D). However, in the fraction enriched in conventional sized synaptosomes primarily from the inner plexiform layer, the 20 nM site was virtually absent after Triton treatment, but was readily detectable in the presence of perchlorate. These results may suggest that ³H-GABA binding $\text{in vitro}$ is inhibited by an endogenous substance which is removed by Triton or perchlorate treatment. The difference in the sensitivity of the two fractions to Triton and perchlorate suggests that in retina this substance (whether it is a membrane peptide or GABA itself) is not uniformly distributed and/or uniformly sensitive to Triton and perchlorate.     

Separation and reconstitution of sodium-dependent glucose transport activity from renal brush-border membranes using gel-filtration chromatography

Pig kidney brush-border membrane vesicles were solubilized using a final concentration of 1% Triton X-100, found optimal for quantitative reconstitution of d-glucose transport into liposomes. Using reconstituted proteoliposomes, selective permeability towards d-glucose compared to other sugars tested was shown as well as the main features of d-glucose transport in native membranes, namely sodium dependence and phlorizin inhibition of d-glucose accumulation. After removal of Triton X-100 from the detergent extract, some membrane proteins (about 40%), which are insoluble in the absence of detergent, were isolated. Among these proteins resolubilized by 1% Triton X-100, the component catalyzing the d-glucose transport was located by gel-filtration chromatography separation, using reconstitution of transport as the assay. The active fraction displayed a molecular size of 50 Å; when analyzed on SDS polyacrylamide gel electrophoresis, it contained one major protein subunit with an apparent molecular weight close to 65 000. We conclude that this protein fraction is involved in d-glucose transport by renal brush borders.     

Dissociation and Reassembly of Escherichia coli Outer Membrane and of Lipopolysaccharide, and Their Reassembly onto Flagellar Basal Bodies

Purified lipopolysaccharide vesicles dissociate when treated with ethylenediaminetetraacetic acid (EDTA) and then reassemble when dialyzed against Mg(2+). Purified outer, lipopolysaccharide membrane (L membrane) is partially dissociated by treatment with EDTA and fully dissociated upon further treatment with Triton X-100. Both the partially and fully dissociated L membrane can be reassembled by dialysis against Mg(2+). Reassembly of lipopolysaccharide or L membrane in the presence of intact flagella results in specific attachment of flagellar basal bodies to vesicles via the L and sometimes the M ring. Lipopolysaccharide and L membrane appear to be composed of substructures bound together by both Mg(2+) (divalent cation)-mediated and hydrophobic bonds.     

Solubilization,purification and reconstitution of Ca-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: Preservation of native structure and function of the enzyme by DHPC

The properties of Ca²⁺-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca²⁺-ATPase with a greater specific activity than solubilization with C₁₂E₈ or Triton X-100. DHPC was determined to be superior to C₁₂E₈; while that the C₁₂E₈ was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca²⁺-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C₁₂E₈ and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca²⁺ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C₁₂E₈ and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca²⁺-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C₁₂E₈ and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca²⁺ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca²⁺-ATPase retained more organized and native secondary conformation compared to C₁₂E₈ and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C₁₂E₈ and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca²⁺-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C₁₂E₈ and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.     

Functioning of the colicin A lysis protein is affected by Triton X-100, divalent cations and EDTA

The colicin A lysis protein, Cal, is synthesized at the same time as colicin A by Escherichia coli harbouring plasmid pColA after induction by mitomycin C. Its function in the induced bacteria involves the release of colicin A, quasi-lysis, the death of the producing cells and the activation of the outer membrane phospholipase A. We have found that these various functions are affected differently by treatment of the induced cells with Triton X-100, divalent cations or EDTA. Triton X-100 and EDTA caused increased quasi-lysis and a higher level of mortality of the producing cells, but while Triton X-100 enhanced the release of colicin A, EDTA reduced it. Divalent cations protected the cells against both killing and quasi-lysis without greatly affecting colicin release. The effects of these agents were similar for both wild-type and phospholipase A mutants and depended only on the presence of a functional cal gene.     

MtrB Is Required for Proper Incorporation of the Cytochromes OmcA and OmcB into the Outer Membrane of Shewanella putrefaciens MR-1

When grown under anaerobic conditions, Shewanella putrefaciens MR-1 synthesizes multiple outer membrane (OM) cytochromes, some of which have a role in the use of insoluble electron acceptors (e.g., MnO₂) for anaerobic respiration. The cytochromes OmcA and OmcB are localized to the OM and the OM-like intermediate-density membrane (IM) in MR-1. The components necessary for proper localization of these cytochromes to the OM have not been identified. A gene replacement mutant (strain MTRB1) lacking the putative OM protein MtrB was isolated and characterized. The specific cytochrome content of the OM of MTRB1 was only 36% that of MR-1. This was not the result of a general decline in cytochrome content, however, because the cytoplasmic membrane (CM) and soluble fractions were not cytochrome deficient. While OmcA and OmcB were detected in the OM and IM fractions of MTRB1, significant amounts were mislocalized to the CM. OmcA was also detected in the soluble fraction of MTRB1. While OmcA and OmcB in MR-1 fractions were resistant to solubilization with Triton X-100 in the presence of Mg²⁺, Triton X-100 readily solubilized these proteins from all subcellular fractions of MTRB1. Together, these data suggest that MtrB is required for the proper localization and insertion of OmcA and OmcB into the OM of MR-1. The inability of MTRB1 to properly insert these, and possibly other, proteins into its OM likely contributes to its marked deficiency in manganese(IV) and iron(III) reduction. While the localization of another putative OM cytochrome (MtrF) could not be directly determined, an mtrF gene replacement mutant exhibited wild-types rates of Mn(IV) and Fe(III) reduction. Therefore, even if MtrF were mislocalized in MTRB1, it would not contribute to the loss of metal reduction activity in this strain.     

Isolation and characterization of transposon Tn5-induced mutants of Pseudomonas perfectomarina defective in nitrous oxide respiration.

Outer membranes of Haemophilus influenzae type b were fractionated to yield Triton X-100-insoluble material and lipopolysaccharide and phospholipids. Liposomes reconstituted from lipopolysaccharide and phospholipids were impermeable to sucrose (Mr, 342) and to a high-molecular-weight dextran (average Mr, 6,600). When the Triton X-100-insoluble material was introduced into the reconstituted liposomes, the vesicles became permeable to sucrose, raffinose (Mr, 504), and stachyose (Mr, 666) and fully retained dextrans of Mr greater than 1,500. Inulin (average Mr, 1,400) was tested for its efflux from the reconstituted outer membrane vesicles; 62% of the added inulin was trapped. The molecular weight exclusion limit for the outer membrane of H. influenzae type b was therefore estimated at approximately 1,400. A protein responsible for the transmembrane diffusion of solutes was purified from H. influenzae type b by extraction of whole cells with cetyl trimethyl ammonium bromide. When this extract was passed over DEAE-Sepharose, three protein-containing peaks (I, II, and III) were eluted. Peaks I and II contained mixtures of proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; when tested for their pore-forming properties, these proteins were unable to render liposomes of lipopolysaccharide and phospholipid permeable to sucrose. Peak III contained only one molecular species of protein of molecular weight 40,000; this protein acted as a porin in reconstituted vesicles. The molecular weight exclusion limit for 40,000-molecular-weight protein matched the estimate of approximately 1,400 which was determined for outer membranes. A series of homologous saccharides of increasing degree of polymerization was prepared from agarose by hydrolysis with beta-agarase and fractionation on gel filtration chromatography. These oligosaccharides of Mr, 936, 1,242, 1,548, and 1,854 were assayed for retention by the complete vesicles containing 40-kilodalton protein and lipopolysaccharide and phospholipids. All of these oligosaccharides were lost by efflux through the porin. Since the molecular conformation of the largest oligosaccharide is an elongated semirigid helix, it is suggested that the pore formed by the 40-kilodalton protein does not act as a barrier to the diffusion of this compound.     

Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl₂. Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.     

Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier.

A method for separating the outer and inner membranes of Pseudomonas aeruginosa PAO1 in the absence of added ethylenediaminetetraacetic acid was devised. The method yields two outer membrane fractions which show the same protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but differ substantially in their relative contents of phospholipids. One of these outer membrane fractions and the inner membrane fraction are less than 4% cross-contaminated, as judged by the content of typical inner and outer membrane markers. The outer membrane contains four major protein bands with apparent molecular weights of 37,000, 35,000, 21,000 and 17,000. Vesicles reconstituted from lipopolysaccharide and phospholipids were impermeable to all saccharides included in the vesicles during vesicle formation. When the vesicles contained outer membrane proteins, they fully retained only those saccharides of greater than 9,000 molecular weight, suggesting that the exclusion limit of the outer membrane of P. aeruginosa for saccharides is substantially larger than the figure (500 to 600 daltons) obtained for certain enteric bacteria. The advantages and potential disadvantages of having an outer membrane with a higher exclusion limit for hydrophilic substances are discussed.