Membrane biogenesis in Escherichia coli: effects of a secA mutation

In Escherichia coli K-12, temperature-sensitive mutations in the secA gene have been shown to interfere with protein export. Here we show that the effect of a secA mutation is strongly pleiotropic on membrane biogenesis. Freeze-fracture experiments as well as cryosections of the cells revealed the appearance of intracytoplasmic membranes upon induction of the SecA phenotype. The permeability barrier of the outer membrane to detergents was lost. Two alterations in the outer membrane may be responsible for this effect, namely the reduced amounts of outer membrane proteins, or the reduction of the length of the core oligosaccharide of the lipopolysaccharide, which was observed in phage-sensitivity experiments and by SDS-polyacrylamide gel electrophoresis. Phospholipid analysis of the secA mutant, grown under restrictive conditions, revealed a lower content of the negatively charged phospholipid cardiolipin and of 18:1 fatty acid compared to those of the parental strain grown under identical conditions. These results are in line with the hypothesis that protein export and lipid metabolism are coupled.     

Monoclonal antibodies against the capsular K antigen of Escherichia coli (O9:K30(A):H12): characterisation and use in analysis of K antigen organisation on the cell surface

Monoclonal antibodies were produced against the capsular antigen of Escherichia coli serotype K(A)30, using a mouse hybridoma system. The antibodies also recognised the chemically identical capsular polysaccharide produced by Klebsiella K20. Chemical modification of the K30 polysaccharide indicated that the glucuronic acid residues found in the E. coli K30 capsular antigen were important in the epitope recognised by these antibodies. Use of the antibodies as molecular probes revealed the presence of two discrete forms of the K30 antigen. One form was comprised of high molecular weight polysaccharide, present as a surface capsular layer. The second form of the antigen was of low molecular weight and was associated with lipopolysaccharide fractions from cell surface polysaccharide extracts. Separation of lipopolysaccharide fractions using gel chromatography in the presence of detergent showed that the low molecular weight K-antigenic fraction comigrated with a lipopolysaccharide lipid A core fraction present in encapsulated E. coli K30 bacteria but absent in acapsular mutants.     

Isolation and Characterization of Outer and Cytoplasmic Membranes from Spheroplasts of Acetobacter aceti

The spheroplast membrane of Acetobacter aceti IFO 3284 was separated into outer and cytoplasmic membranes by alkaline sucrose density gradient centrifugation after treatment of the cells with lysozyme in sucrose-EDTA, pH 8.0. The cytoplasmic membrane, which was transparent and red colored, showed a specific gravity of 1.15 g/ml, and a number of protein components. High contents of heme b and heme c, and high enzyme activities of various membrane-bound primary dehydrogenases, which are characteristics of acetic acid bacteria, were found in the cytoplasmic membrane fraction. On the other hand, the outer membrane, which was white and turbid when homogenized, exhibited a high content of 2-keto-3-deoxyoctonate, and only four major polypeptides were observed on SDS-polyacrylamide gel electrophoresis. The outer membrane showed a specific gravity of 1.25 g/ml due to its high lipopolysaccharide content. A predominant species of the outer membrane proteins, tentatively designated as AI, was found to be heat-modifiable in SDS solution. The Al peptide on SDS-polyacrylamide gel showed varied migration, from a position corresponding to 31,000 daltons to one of 37,000 daltons, when heated at over 60°C and then subjected to gel electrophoresis.     

Temporal sequence of the recovery of traits during phenotypic curing of a Cytophaga johnsonae motility mutant.

The lack of cell translocation and the resulting formation of nonspreading colonies of mutants of the gram-negative gliding bacterium Cytophaga johnsonae have been correlated with the loss of cell surface features of the organism. These cell surface traits include the ability to move polystyrene-latex beads over the cell surface and the ability to be infected by bacteriophages that infect the parent strain. In order to assess whether these traits reflect structures or functions that actually play a role in gliding, we studied a mutant (21A2I) selected for its inability to form spreading colonies; it is deficient in sulfonolipid, lacks bead movement ability, and is resistant to at least one bacteriophage. The provision of cysteate (a specific sulfonolipid precursor) restores lipid content and gliding to the mutant; hence, the lipids are necessary for motility. Growth with cysteate also restores bead movement and phage sensitivity. In order to determine the temporal relationship of these traits, we undertook a kinetic study of the appearance of them after addition of cysteate to the mutant. One predicts that appearance of a trait essential for cell translocation will either precede or accompany the appearance of this ability, while a nonessential trait need not do so. Sulfonolipid synthesis was the only trait that appeared before gliding; this is consistent with its established importance for motility. Bead movement and phage sensitivity first appeared only after gliding started, suggesting that the machinery involved in those processes is not necessary, at least for the initiation of gliding.     

Extraction and characterization of lipopolysaccharide from Pseudomonas pseudomallei

The best yield of lipopolysaccharide (LPS) of Pseudomonas pseudomallei GIFU 12046 was obtained by extraction of defatted cells by phenol/chloroform/petroleum ether. The LPS showed a smooth character on SDS-polyacrylamide gel electrophoresis and contained D-glucose, L-glycero-D-manno-heptose, and D-glucosamine as the main sugar components, and 3-hydroxypalmitic acid as an amide-linked fatty acid. The growth conditions did not affect the electrophoresis profile and chemical composition of LPS. 2-Keto-3-deoxyoctonic acid was not detectable, and mild acid hydrolysis could not liberate free lipid A, suggesting that the linkage between inner core and lipid A was stable against acid hydrolysis, and the structure of this region is similar to that of P. cepacia, which has close taxonomic relationship with P. pseudomallei.     

Structure of the O-chain of the lipopolysaccharide of Haemophilus pleuropneumoniae serotype 1

By phenol-water extraction an aqueous-phase soluble cellular lipopolysaccharide was isolated from Haemophilus pleuro-pneumoniae serotype 1. It was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, hydrolysis, methylation, and both one- and two-dimensional 1H and 13C nuclear magnetic resonance studies to be an S-type lipopolysaccharide, which could be cleaved to yield a lipid A and an O-chain polysaccharide identified as a high molecular weight branched polymer of a tetrasaccharide repeating unit having the structure: (Formula: see text).     

Absence of a characteristic cell wall lipopolysaccharide in the phototrophic bacterium Chloroflexus aurantiacus.

Two strains of the gliding phototrophic bacterium Chloroflexus aurantiacus were investigated for the presence of lipopolysaccharide (LPS). With both strains, all fractions of hot phenol-water extracts and the extracted cell residues from whole cells or cell homogenates were found to be free from characteristic LPS constituents, such as 3-hydroxy fatty acids, 2-keto-3-deoxyoctonate, heptoses, or O-chain sugars. Phenolchloroform-petroleum ether extracts were also free from precipitable LPS. A lipid A fraction could not be obtained, and there was no hint for glucosamine as a possible lipid A backbone amino sugar. Absence of LPS was confirmed by sodium deoxycholate gel electrophoresis.     

Lipopolysaccharide-specific bacteriophage for Klebsiella pneumoniae C3.

Bacteriophage FC3-1 is one of several specific bacteriophages of Klebsiella pneumoniae C3 isolated in our laboratory. Unlike receptors for other Klebsiella phages, the bacteriophage FC3-1 receptor was shown to be lipopolysaccharide, specifically the polysaccharide fraction (O-antigen and core region). We concluded that capsular polysaccharide, outer membrane proteins, and lipid A were not involved in phage binding. Mutants resistant to this phage were isolated and were found to be devoid of lipopolysaccharide O-antigen by several criteria but to contain capsular material serologically identical to that of the wild type. The polysaccharide fraction was concluded to be the primary phage receptor, indicating that it is available to the phage.     

PagP activation in the outer membrane triggers R3 core oligosaccharide truncation in the cytoplasm of Escherichia coli O157:H7

The Escherichia coli outer membrane phospholipid:lipid A palmitoyltransferase PagP is normally a latent enzyme, but it can be directly activated in outer membranes by lipid redistribution associated with a breach in the permeability barrier. We now demonstrate that a lipid A myristate deficiency in an E. coli O157:H7 msbB mutant constitutively activates PagP in outer membranes. The lipid A myristate deficiency is associated with hydrophobic antibiotic sensitivity and, unexpectedly, with serum sensitivity, which resulted from O-antigen polysaccharide absence due to a cytoplasmically determined truncation at the first outer core glucose unit of the R3 core oligosaccharide. Mutational inactivation of pagP in the myristate-deficient lipid A background aggravated the hydrophobic antibiotic sensitivity as a result of losing a partially compensatory increase in lipid A palmitoylation while simultaneously restoring serum resistance and O-antigen attachment to intact lipopolysaccharide. Complementation with either wild-type pagP or catalytically inactive pagPSer77Ala alleles restored the R3 core truncation. However, the intact lipopolysaccharide was preserved after complementation with an internal deletion pagPDelta5-14 allele, which mostly eliminates a periplasmic amphipathic alpha-helical domain but fully supports cell surface lipid A palmitoylation. Our findings indicate that activation of PagP not only triggers lipid A palmitoylation in the outer membrane but also separately truncates the R3 core oligosaccharide in the cytoplasm. We discuss the implication that PagP might function as an apical sensory transducer, which can be activated by a breach in the outer membrane permeability barrier.     

Hydrogen production during fermentation of acetoin and acetylene by Pelobacter acetylenicus

SDS-polyacrylamide gel electrophoresis of outer membrane (OM) proteins of different mucoid strains of P. aeruginosa revealed a protein of about 54 kDa that was absent in nonmucoid strains. This 54 kDa protein was expressed under iron-restricted and iron sufficient growth conditions. Electrophoretic mobility of the 54 kDa protein was modified by the solubilization temperature as well as by the addition of lipopolysaccharide and alginate prior to electrophoresis. Treatment of OMs with octylglucoside/KCl or SDS completely extracted the 54 kDa protein at low temperatures. The possible role of this protein in biosynthesis and/or excretion of bacterial alginate is discussed.     

The occurrence of glycine in bacterial lipopolysaccharides

Abstract The aminoacyl analysis of endotoxic lipopolysaccharides (LPS) isolated from several bacteria revealed essential amounts of glycine, among the inherent LPS components. Significant amounts of the glycine was detected in lipopolysaccharides isolated from over 30 strains of Escherichia, Salmonella, Hafnia, Citrobacter and Shigella species. Glycine as a single amino acid was found only in a core part of LPS. Molar ratio of glycine in core oligosaccharide fraction ranged from 0.2 to 0.6 per 3 heptoses. The oligosaccharide enriched in glycine was isolated using the HPLC. The amino acid appeared to be terminally located in a core oligosaccharide. The labelling of the lipopolysaccharide cores was achieved when the bacteria were cultivated in the presence of radioactive [¹⁴C]glycine. The labelled core oligosaccharide released the radioactivity during treatment with mild alkali or acid (0.1 M NaOH or HCl, 100°C, 4 h). The radioactivity in SDS-polyacrylamide gel electrophoresis migrated exclusively with LPS. The results indicate that amino acid is an integral constituent of core oligosaccharide in lipopolysaccharide.     

Physicochemical surface properties ofKlebsiella pneumoniae

The following cell surface physicochemical characteristics were investigated inKlebsiella pneumoniae: surface charge, surface hydrophobicity by different methods, and accessibility of the lipid fraction of the outer membrane. The capsular polysaccharide, as well as the O-antigen repeating units of the lipopolysaccharide (LPS), conferred a hydrophilic, negatively charged surface to the bacterium, and a barrier to the dye congo red, which binds sites within the lipid fraction of the outer membrane (OM).     

Structural studies of the O-chain of the phenol-phase soluble lipopolysaccharide from Haemophilus pleuropneumoniae serotype 2

The phenol-phase soluble cellular lipopolysaccharide that was isolated by the phenol-water extraction from Haemophilus pleuropneumoniae serotype 2 was shown to be of the S type by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, hydrolysis, methylation, specific degradations, and both one- and two-dimensional 1H and 13C nuclear magnetic resonance studies. It could be cleaved to yield a lipid A and an O-chain polysaccharide. This O-polysaccharide was identified as a high molecular weight unbranched linear polymer of a pentasaccharide repeating unit having the structure: (Formula: see text).     

Role of outer membrane lipopolysaccharides in the protection of Salmonella enterica serovar Typhimurium from desiccation damage

The ability to survive desiccation between hosts is often essential to the success of pathogenic bacteria. The bacterial outer membrane is both the cellular interface with hostile environments and the focus of much of the drying-induced damage. This study examined the contribution of outer membrane-associated polysaccharides to the survival of Salmonella enterica serovar Typhimurium in air-dried blood droplets following growth in high and low osmolarity medium and under conditions known to induce expression of these polysaccharides. Strains lacking the O polysaccharide (OPS) element of the outer membrane lipopolysaccharide were more sensitive to desiccation. Lipopolysaccharide core mutation further to OPS loss did not result in increased susceptibility to drying. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed lipopolysaccharide profiles that supported the hypothesis that OPS expression is required for optimal drying resistance in S. Typhimurium. The role of O antigen in Salmonella spp. in maintaining a hydrated layer around the dried cell or in slowing the rate of dehydration and rehydration is discussed.     

Problem-solving test: submitochondrial localization of proteins

Terms to be familiar with before you start to solve the test: mitochondria, outer membrane, inner membrane, intermembrane space, mitochondrial matrix, mitochondrial fraction, cell fractionation by differential centrifugation, pellet, supernatant, detergents, phenol, cytosolic fraction, integral and peripheral membrane proteins, hypotonic solution, SDS-polyacrylamide gel electrophoresis, Western blotting.     

Isolation and characterization of the lipopolysaccharides from Bradyrhizobium japonicum.

The lipopolysaccharide (LPS) of Bradyrhizobium japonicum 61A123 was isolated and partially characterized. Phenol-water extraction of strain 61A123 yielded LPS exclusively in the phenol phase. The water phase contained low-molecular-weight glucans and extracellular or capsular polysaccharides. The LPSs from B. japonicum 61A76, 61A135, and 61A101C were also extracted exclusively into the phenol phase. The LPSs from strain USDA 110 and its Nod- mutant HS123 were found in both the phenol and water phases. The LPS from strain 61A123 was further characterized by polyacrylamide gel electrophoresis, composition analysis, and 1H and 13C nuclear magnetic resonance spectroscopy. Analysis of the LPS by polyacrylamide gel electrophoresis showed that it was present in both high- and low-molecular-weight forms (LPS I and LPS II, respectively). Composition analysis was also performed on the isolated lipid A and polysaccharide portions of the LPS, which were purified by mild acid hydrolysis and gel filtration chromatography. The major components of the polysaccharide portion were fucose, fucosamine, glucose, and mannose. The intact LPS had small amounts of 2-keto-3-deoxyoctulosonic acid. Other minor components were quinovosamine, glucosamine, 4-O-methylmannose, heptose, and 2,3-diamino-2,3-dideoxyhexose. The lipid A portion of the LPS contained 2,3-diamino-2,3-dideoxyhexose as the only sugar component. The major fatty acids were beta-hydroxymyristic, lauric, and oleic acids. A long-chain fatty acid, 27-hydroxyoctacosanoic acid, was also present in this lipid A. Separation and analysis of LPS I and LPS II indicated that glucose, mannose, 4-O-methylmannose, and small amounts of 2,2-diamino-2,3-dideozyhexose and heptose were components of the core region of the LPS, whereas fucose, fucosmine, mannose, and small amounts of quinovosamine and glucosamine were components of the LPS O-chain region.     

Symbiont of the stink bug Plautia stali synthesizes rough-type lipopolysaccharide

The structures and biosynthesis of lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, have been studied extensively in cultured bacteria such as Escherichia coli. In contrast, little is known about the structures and biosynthesis of the LPS of unculturable bacteria, including insect symbionts, many of which are Gram-negative bacteria. A brown-winged green bug, Plautia stali, is known to harbor a single species of gamma-proteobacterium in the posterior mid-gut caeca. To characterize the features of its LPS, we analyzed the genome sequence of the symbiont, and identified the putative genes involved in LPS synthesis. Genes involved in the synthesis of lipid A and the core oligosaccharide were found in the genome, but waaL, which encodes the O-antigen ligase, was not. Furthermore, we characterized the LPS of this symbiont using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Toll-like receptor 4 (TLR4) stimulation assays. Consistent with the genomic analysis, the SDS-PAGE analysis suggested that the symbiont had rough-type LPS, which lacked the O-antigen. The TLR4 stimulation assay demonstrated that LPS purified from the symbionts activated NF-κB-dependent reporter expression, indicating the existence of a bioactive lipid A portion in the LPS. These results suggest that the P. stali symbiont produces rough-type LPS.     

Novel sulfonolipid in the extremely halophilic bacterium Salinibacter ruber

Salinibacter ruber is an extremely halophilic bacterium, phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria. Electrospray mass analyses (negative ion) of the total lipid extract of a pure culture of S. ruber shows a characteristic peak at m/z 660 as the most prominent peak in the high-mass range of the spectrum. A novel sulfonolipid, giving rise to the molecular ion [M-H]- of m/z 660, has been identified. The sulfonolipid isolated and purified by thin-layer chromatography was shown by chemical degradation, mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance analysis to have the structure 2-carboxy-2-amino-3-O-(13'-methyltetradecanoyl)-4-hydroxy-18-methylnonadec-5-ene-1-sulfonic acid. This lipid represents about 10% of total cellular lipids, and it appears to be a structural variant of the sulfonolipids found as main components of the cell envelope of gliding bacteria of the genus Cytophaga and closely related genera (W. Godchaux and E. R. Leadbetter, J. Bacteriol. 153:1238-1246, 1983) and of diatoms (R. Anderson, M. Kates, and B. E. Volcani, Biochim. Biophys. Acta 528:89-106, 1978). Since this sulfonolipid has never been observed in any other extreme halophilic microorganism, we consider the peak at m/z 660 the lipid signature of Salinibacter. This study suggests that this novel sulfonolipid may be used as a chemotaxonomic marker for the detection of Salinibacter within the halophilic microbial community in saltern crystallizer ponds and other hypersaline environments.     

Effect of variations in lipopolysaccharide on the fluidity of the outer membrane of Escherichia coli.

The lipid hydrocarbon chains in the outer membrane of gram-negative bacteria appear from previous experiments to be less mobile than in the cytoplasmic membrane. To determine whether lipopolysaccharide, a unique outer membrane component, is a cause of this restricted mobility, outer membranes differing in the amount of lipopolysaccharide, and the length of the polysaccharide side chain, were prepared from Escherichia coli J5. Cytoplasmic membranes were prepared for comparison. The probes, 5- and 12-doxylstearate, were introduced into these membranes, electron spin resonance spectra were analyzed, and the order parameter (S) and empirical motion parameter (tau0) were calculated. Outer membrane preparations containing long chain lipopolysaccharide were much less fluid by these criteria than were preparations containing short chain lipopolysaccharide. Removing about 40% of the lipopolysaccharide from the former preparations greatly increased their fluidity. The lipid in the cytoplasmic membrane preparations was more fluid than in the outer membrane and cytoplasmic membranes were similar to each other regardless of the composition of the outer membrane. These results indicate that lipopolysaccharide, and especially the polysaccharide portion, directly or indirectly causes the restricted mobility of the lipid hydrocarbon chains observed in the outer membrane.     

Molecular characteristics of rat brain glucose transporter: a novel species with Mr 45,000

The glucose transporter of rat brain was examined by the use of cytochalasin B, a potent inhibitor. The dissociation constants (Kd) of D-glucose-inhibitable cytochalasin B binding in various membrane fractions were about 100 nM. Solubilization and partial purification of glucose transporter were carried out by procedures of DE 52 column chromatography, Bio Gel HT column chromatography and Sepharose CL-6B column chromatography from postnuclear membrane fraction. Purified transporter, reconstituted in lipid vesicles, showed D-glucose-specific transport activity with a Michaelis constant (Km) of 7 mM. The molecular weight was estimated to be about 200K by gel filtration in the presence of 0.1% Triton X-100. The subunit molecular weight was estimated to be 45K by SDS-polyacrylamide gel electrophoresis after photoaffinity labeling using [3H]cytochalasin B as a covalent probe, indicating that rat brain glucose transporter is a tetramer.