Effect of iron and other nutrient limitations on the pattern of outer membrane proteins in the cyanobacterium Synechococcus PCC7942

When cells of Synechococcus PCC7942 were subjected to either iron or magnesium limitation, there was an appearance of specific proteins in the outer membrane (isolated as the cell wall fraction). Under iron limitation outer membrane polypeptides of M _r 92000, 48000–50000 and 35000 appeared. Specific iron-limited outer membrane proteins (IRMPs) of M _r 52000 and 36000 were also induced in iron-limited cultures of Synechocystis PCC6308. Under magnesium limitation polypeptides of M _r 80000, 67000, 62000, 50000, 28000 and 25000 appeared in the outer membrane. phosphate limitation caused minor changes in the outer membrane protein pattern, with polypeptides of M _r 32000 and one of over 100000 being induced, whereas calcium limitation had no apparent affect.Abbreviations EDDA ethylenediaminedihydroxyphenyl acetic acid - IRMP iron-regulated outer membrane protein - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane sulphonic acid - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - PMSF phenylmethylsulphonyl fluoride     

Isolation, characterization and protein and polar lipid compositions of Paracoccus denitrificans outer membrane

Sucrose density gradient centrifugation of Paracoccus denitrificans strains ATCC 13543 and ATCC 17741 cell envelopes plus poly-β-hydroxybutyrate, isolated from organisms broken using a French pressure cell, revealed three bands of densities: I, 1.16 g/ml; II, 1.19 g/ml; III, 1.24 g/ml. On the basis of chemical and enzymatic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis the bands were identified as: I, cytoplasmic membrane; II, poly-β-hydroxybutyrate; III, outer membrane plus poly-β-hydroxybutyrate. Poly-β-hydroxybutyrate was removed by increased low-speed centrifugation before deposition of cell envelopes. Density gradient centrifugation of cell envelopes gave a simple pattern of two bands, cytoplasmic and outer membranes. In both strains outer membranes showed a broad protein band at M_r 70 000–83 000 upon SDS-polyacrylamide gel electrophoresis of samples solubilized at 25°C, which was not present in samples solubilized at 100°C, where a single major band was present of M_r 32 000 in strain ATCC 13543 and 35 000 in strain ATCC 17741. The major outer membrane protein stained positively for lipid in both strains, as did an M_r 70 000 protein, which was the second major protein in strain ATCC 17741. The second major outer membrane protein of stain ATCC 13543 had an M_r of 20 000 in unheated samples but 23 000 in heated samples. This protein was not present in strain ATCC 17741. Quantitative data on the polar lipid compositions of cell envelope fractions are presented.     

Physical Determinants of β-Barrel Membrane Protein Folding in Lipid Vesicles

The spontaneous folding of two Neisseria outer membrane proteins, opacity-associated (Opa)₆₀ and Opa₅₀ into lipid vesicles was investigated by systematically varying bulk and membrane properties. Centrifugal fractionation coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility assays enabled the discrimination of aggregate, unfolded membrane-associated, and folded membrane-inserted protein states as well as the influence of pH, ionic strength, membrane surface potential, lipid saturation, and urea on each. Protein aggregation was reduced with increasing lipid chain length, basic pH, low salt, the incorporation of negatively charged guest lipids, or by the addition of urea to the folding reaction. Insertion from the membrane-associated form was improved in shorter chain lipids, with more basic pH and low ionic strength; it is hindered by unsaturated or ether-linked lipids. The isolation of the physical determinants of insertion suggests that the membrane surface and dipole potentials are driving forces for outer membrane protein insertion and folding into lipid bilayers.     

Sheath and outer membrane components from the cyanobacterium Fischerella sp. PCC 7414

A purified sheath fraction and an outer membrane fraction were obtained from the cyanobacterium Fischerella sp. PCC 7414. The sheath had a fine structure with osmiophilic fibers running in parallel to the cell surface in two distinct layers. The sheath fraction contained mainly neutral sugars (Glc, Man, Gal, Xyl, Fuc, 2-O-methylhexose), GlcN, uronic acids, and minor components such as amino acids, sulfate, phosphate, and fatty acids. The protein moiety was removable from the sheath fraction by treatment with boiling sodium dodecyl sulfate. The presence of three different 3-hydroxy fatty acids (3-OH-14:0, 3-OH-16:0, 3-OH-18:0) in addition to GlcN indicated the presence of lipopolysaccharide in the outer membrane. One major (M_r 50,000) and two minor (M_r 54,000 and 65,000) proteins were detected as constituents of the outer membrane.Abbreviations A₂pm diaminopimelic acid - GLC gas-liquid chromatography - GlcN glucosamine - Ino inositol - MurN muramic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Outer membrane proteins induced upon iron deprivation of Paracoccus denitrificans

Deprivation of two strains of Paracoccus denitrificans of iron elicited substantial siderophore production, and the associated cell envelope protein changes were examined. In strain DSM 413 four new outer membrane proteins in the M_r range 84000 to 76000 were induced, and an M_r 23000 protein disappeared, upon iron deprivation. In strain DSM 65 three new outer membrane proteins in the M_r range 81000 to 76000 were induced.     

Neisseria proteomics for antigen discovery and vaccine development

Neisseria meningitidis (meningococcus) is a major causative organism of meningitis and sepsis and Neisseria gonorrhoeae (gonococcus) is the causative organism of the sexually transmitted disease gonorrhea. Infections caused by meningococci are vaccine-preventable, whereas gonococcal vaccine research and development has languished for decades and the correlates of protection are still largely unknown. In the past two decades, complementary ‘omic’ platforms have been developed to interrogate Neisseria genomes and gene products. Proteomic techniques applied to whole Neisseria bacteria, outer membranes and outer membrane vesicle vaccines have generated protein maps and also allowed the examination of environmental stresses on protein expression. In particular, immuno-proteomics has identified proteins whose expression is correlated with the development of human natural immunity to meningococcal infection and colonization and following vaccination. Neisseria proteomic techniques have produced a catalog of potential vaccine antigens and investigating the functional and biological properties of these proteins could finally provide ‘universal’ Neisseria vaccines.     

Variations in the cytoplasmic region account for the heterogeneity of the chicken MHC class I (B-F) molecules

Molecular variation among major histocompatibility complex (MHC) class I (B-F) proteins from B-homozygous chickens is apparently caused by C-terminal variation. Analysis of the total B-F protein pool revealed substantial heterogeneity with two or three molecular mass constituents, each being comprised by several isoelectric focusing variants. This heterogeneity could not be reduced by enzymatic deglycosylation. By contrast, proteolytic removal of a small (M _r 1000–4000) fragment from the chain resulted in the generation of a M _r 36 000 fragment, common to all the molecular mass variants. Unlike the parent proteins, the M _r 36 000 fragment derived from isolated variants yielded identical, simple patterns in two-dimensional gel electrophoresis and identical finger prints in peptide mapping. This, together with N-terminal amino acid sequencing, as well as comparison of hydrophobicity properties of fragments obtained by gradual proteolytic digestion, indicated that the small peptide responsible for the major B-F heterogeneity was situated in the intracellular, C-terminal part.     

Amino terminal sequence of heavy and light chains from ratfish immunoglobulin

The ratfish,Callorhinchus callorhinchus, a representative of the Holocephali, has a natural serum hemagglutinin (M _r 960 000), composed of heavy (M _r 71000), light (M _r 22 500), and J (M _r 16 000) chains. To approach the mechanisms that generate diversity at this level of evolution, the amino terminal sequence of the heavy and light chains was determined by automated microsequencing. The chains are unblocked and have modest internal sequence heterogeneity. The heavy chains show sequence similarity with the terminal region of the heavy chain from the horned shark,Heterodontus francisci, and other species. In contrast to the heavy chain, the ratfish light chains display low sequence similarity with their shark kappa counterparts. However, their similarity with the variable region of the chicken lambda light chains is about 75%.     

Expanding the paradigm for the outer membrane: Acinetobacter baumannii in the absence of endotoxin

Asymmetry in the outer membrane has long defined the cell envelope of Gram‐negative bacteria. This asymmetry, with lipopolysaccharide (LPS) or lipooligosaccharide (LOS) exclusively in the outer leaflet of the membrane, establishes an impermeable barrier that protects the cell from a number of stressors in the environment. Work done over the past 5 years has shown that Acinetobacter baumannii has the remarkable capability to survive with inactivated production of lipid A biosynthesis and the absence of LOS in its outer membrane. The implications of LOS‐deficient A. baumannii are far‐reaching – from impacts on cell envelope biogenesis and maintenance, bacterial physiology, antibiotic resistance and virulence. This review examines recent work that has contributed to our understanding of LOS‐deficiency and compares it to studies done on Neisseria meningitidis and Moraxella catarrhalis; the two other organisms with this capability.     

Isolation and characterization of the outer membrane of Acinetobacter calcoaceticus

A method for the separation of the outer membrane (OM) from the cytoplasmic membrane (CM) of Acinetobacter calcoaceticus 69/V grown on different carbon sources is described. The contamination of the OM with CM was less than 10%. Independent of the carbon source, five protein bands with apparent molecular weights of 47 000, 33000, 21 000, 19 000 and 12 000 were found by solubilization at 37°C and six bands at 100°C (apparent M_r 53 000, 47 000, 38 000, 26 000, 21000, 12000). Three proteins were modifiable by heat. With the periodic acid-Schiff procedure the bands with apparent M_r of 33 000 and 12 000 were made visible. After growth on d,l-carnitine an additional two non-heat-modifiable protein bands with apparent M_r between 40 000 and 45 000 were detected. By cultivation on acetate and peptone as carbon source one additional band (M_r 15 000) from OM of cells could be found.     

Expression of high affinity iron uptake systems by clinical isolates of Klebsiella

Seventeen isolates of Klebsiella aerogenes, K. pneumoniae, K. oxytocum and K. edwardsii were examined for their ability to express iron-regulated outer membrane proteins (IROMPs) and high affinity iron-chelating agents (siderophores). In response to iron deprivation, all strains induced at least 4 IROMPs in the approximate M_r range 70 000–85 000 and the phenolate siderophore enterobactin. Six strains also produced the hydroxamate siderophore aerobactin. The Klebsiella enterobactin receptor was identified as an 81 000 M_r iron-repressible outer membrane (OM) protein which appears to be highly conserved and shows considerable antigenic homology with that of Escherichia coli.     

Leishmania tropica: Surface antigens of intracellular and flagellate forms

Promastigotes and amastigotes of Leishmania tropica were surface-radioiodinated using the lactoperoxidase technique. Detergent lysates of the labeled organisms were analyzed by two-dimensional gel electrophoresis. Analysis of radioiodinated promastigote membrane proteins revealed six major and some minor acidic polypeptides. Analysis of the amastigote membrane proteins revealed six major proteins, mostly acidic, and some poorly resolved basic proteins. Four of the major membrane proteins appeared to be common to the two parasitic forms (M_r 67,000, M_r 50,000, M_r 68,000, and M_r 80,000). These polypeptides were recognized by antipromastigote antibodies as well as antibodies from CBA/H mice that had recovered from infection. Peptide mapping confirmed their homology in the two parasite forms. One polypeptide appeared to be specific for the promastigote (M_r 50,000) and two polypeptides appeared to be specific for the amastigote form of the parasite (M_r 94,000 and M_r 43,000).     

Characteristics of intracellular proteolytic activities ofBacillus megaterium

Intracellular proteolytic activities ofB. megaterium KM occur soluble in the cytoplasm and periplasm and insoluble in the membrane. Two proteolytic enzymes were found in the cytoplasmic fraction by gel filtration on Sephadex G 150 and by polyacrylamide gel electrophoresis. The first enzyme called C_I was stable, had a relative molecular mass ofM _r=105000 (M=105 kg/mol) and was inhibited by EDTA and PMSF, whereas the second, designated C_II, was labile and had a relative molecular mass ofM _r=46000 (M=46 kg/mol). Because of its lability it could not be characterized in detail. In the “periplasm” only a single proteolytic enzyme P (M _r=28000;M=28 kg/mol) inhibited by EDTA could be demonstrated. The extracellular enzyme exhibited similar properties. The membrane proteolytic activity was sensitive to PMSF and EDTA. The membrane enzymes have not yet been solubilized. In cells of the mutant KM 12 that does not produce the extracellular proteinase, only one type of proteinase, in all its properties identical with the cytoplasmic proteinase C_I, could be demonstrated.     

Characterization of membrane permeability alterations induced in vero cells by Clostridium perfringens enterotoxin

Alterations in plasma membrane permeability induced by Clostridium perfringens enterotoxin were studied using Vero (African green monkey kidney) cells which were radioactively labeled with four markers of different molecular size. The markers were α-amino[¹⁴C]isobutyric acid (M_r 103), ³H-labeled nucleotide (M_r approx. 300), ⁵¹Cr label (M_r approx. 3000) and [³H]RNA (M_r > 25 000). Over a 2 h period, enterotoxin caused significant release of aminoisobutyric acid, nucleotides and ⁵¹Cr label but not RNA. The effects of enterotoxin on label release were dose- and time-dependent. The rate of release of markers was dependent upon their size. Permeability alterations could be detected within 15 min with a high dose of enterotoxin. Gel chromatography of released material was used to determine that markers of M_r 3000 but not 25 000 leaked from permeabilized cells. It was concluded that enterotoxin is producing functional ‘holes’ of limited size in the membrane. Permeability changes due to enterotoxin treatment differed between confluent and non-confluent (growing) cells. We propose that the primary action of the enterotoxin is to interact with the plasma membrane and produce functional ‘holes’ of defined size. The resultant alterations in membrane permeability cause the loss of essential cellular substances which inhibits processes such as macromolecular synthesis and eventually leads to cell deterioration and death.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (M_r 600 000) containing the three short chains (M_r 200 000) but lacking the long chain M_r 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

Biochemical complexity of serum HLA class I molecules

Human serum was found to contain a variety of class I-like molecules by Western blotting with anti-class I heavy chain reagents: major bands usually are observed around M _r 44 000, 40 000, and 35 000–37 000. HLA-A24-positive individuals are distinguished by higher serum levels of M _r 44 000 and 40 000 class I-like molecules than those found in HLA-A24-negative individuals. The M _r 44 000 serum molecules are probably intact class I molecules that have been shed from the cell membrane, because they contain both a transmembrane segment (TM), as deduced from detergent-binding experiments, and a cytoplasmic tail (CT), as inferred from reactivity with an antipeptide serum specific for the cytoplasmic domain of class I antigens (RaCT). The M _r 35 000 and 37 000 molecules contain neither a TM nor a CT region and therefore are probably proteolytic breakdown products of cellular and/or serum M _r 44 000 molecules, although the existence of Q10-like molecules in man cannot be ruled out. The M _r 40 000 molecules do not contain a TM region. M _r 40 000 molecules reactive with the RaCT serum were found in the minority (2/13) of sera tested. We conclude that alternative splicing resulting in a precise excision of the TM exon plays a minor role in the generation of serum HLA class I antigens.Abbreviations used in this paper B2m beta-2 microglobulin - BSA bovine serum albumin - EBV-BLCL Epstein-Barr virus-transformed B lymphoblastoid cell line - mAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Helix pomatia agglutinin binds specifically to the Golgi apparatus in cultured human fibroblasts and reveals two Golgi apparatus-specific glycoproteins

Summary Fluorochrome-coupled Helix pomatia agglutinin (HPA), but not other lectin-conjugates with the same nominal specificity, bound specifically to the Golgi apparatus in cultured human fibroblasts, revealing a cytoplasmic juxtanuclear reticular structure. Unlike other Golgi-binding lectins the HPA-conjugates did not bind to the cell surface membrane or pericellular matrix. Experiments with ³⁵S-methionine-labeled cells showed that HPA recognized two glycoproteins of M_r 170000 and 400000 among the secreted products of fibroblasts and two major cellular glycoproteins of M_r 40000 and M_r 180000 in Triton X-100 extracts of the cells. The two cellular HPA-binding polypeptides were also found in cells depleted of secretory products and in cells pulselabeled shortly with ³⁵S-methionine and then chased with methionine containing medium up to 12 h. These findings suggest that the two cellular glycoproteins recognized by HPA are retained in the Golgi apparatus and are therefore not precursors of secretory proteins. The results suggest that there are two endogenous, Golgi apparatus-specific glycoproteins in cultured human fibroblasts with terminal non-reducing O-glycosidic N-acetyl galactosaminyl residues.     

Structure and expression of the mouse homologue of the XK gene

 The human Kx blood group antigen is carried by a 37 000 M _r apparent molecular mass membrane polypeptide which is deficient in rare individuals with the McLeod syndrome. The X-linked human XK gene is transcribed in many tissues including adult skeletal muscle and brain, sieges of disorders observed in McLeod syndrome. We report here the cloning of the orthologous mouse XK mRNA. Comparison of XK from human and mouse revealed 80% sequence similarity at the amino acid level. The mouse XK gene is organized in two exons and is expressed in many tissues, but its expression pattern is slightly different from that of the human gene. The presence in mouse erythrocyte membrane of a 43 000 M _r Kx-related protein was demonstrated by immunoblotting with a rabbit antiserum directed against the human protein. With non-reduced samples, a 140 000 M _r species was detected instead of the 43 000 M _r protein, suggesting that, as demonstrated in the Kx polypeptide might be complexed with another protein in mouse red cells, presumably the homologue of the human Kell protein of 93 000 M _r. Received: 22 February 1999 / Revised: 8 June 1999     

The high-molecular-weight proteins of bovine brain plain synaptic vesicles

High molecular mass polypeptides (M _r >100,000) of plain synaptic vesicles from bovine cerebral cortex were separated using porous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Four major bands, ofM _r 262,000, 249,000, 216,000, and 173,000, were resolved. Investigations into the membrane association of theM _r 216,000 and 173,000 proteins by means of solubilization experiments and Sepharose 4B chromatography indicate that the former is a peripheral protein and the latter is more firmly attached, possibly an integral protein. Finally, theM _r 216,000 protein was shown to be highly enriched in synaptic vesicles compared to other brain subfractions. It thus appears to be specifically associated with synaptic vesicles and therefore may have an important role specific to synaptic vesicle function or structure.     

Enzyme antigens associated with pigeon breeder's disease. I. Isolation and characterization of basic hydrolases

A survey of the hydrolytic enzymes present in pigeon dropping extracts (PDE) has shown that this material contains a variety of proteolytic and nonproteolytic activities. These enzymes were separated into their basic and acidic components by chromatography on DEAE-cellulose. Staining of immunoprecipitates with specific chromogenic substrates demonstrated the presence of antibodies in symptomatic breeders to several of the basic enzymes in PDE. Five distinct hydrolytic activities were isolated from the basic group of enzymes. Trypsin, elastase, and two forms of collagenase were the specific proteolytic activities isolated. A phospholipase was also purified from these preparations. The purified elastase consisted of a single polypeptide chain (M _r =22,000). The purified trypsin had a molecular weight (M _r =25,000) and charge similar to those reported for elastase and, like elastase, the trypsin from PDE appeared to be composed of a single polypeptide chain. Two molecular weight forms of collagenase were found; both hydrolyzed bovine collagen. The high-molecular-weight collagenase (M _r =51,000) was shown to be a glycoprotein consisting of two polypeptides (M _r =24,000). It was readily separated from the low-molecular-weight collagenase (M _r =15,000) by gel filtration. The phospholipase (M _r =99,000) appeared to be a dimer. The relevance of these enzymes to the development of pigeon breeder's disease is discussed.