Chemical basis of rough and smooth variation in mycobacteria.

Rough and smooth colony variants of Mycobacterium kansasii were compared with respect to surface glycolipid composition. Thin-layer chromatography of the native glycolipid antigens, gas chromatography of the constituent sugars, and in situ probing with an appropriate monoclonal antibody by colony dot blot enzyme-linked immunosorbent assay and immunogold labeling demonstrated that all M. kansasii strains of smooth colony morphology contain on their surfaces the recently described trehalose-containing lipooligosaccharides, whereas all rough variants were devoid of such surface antigens. Yet all strains, rough and smooth, contained another glycolipid, the M. kansasii-specific phenolic glycolipid. Previous studies by others had shown that the rough forms of M. kansasii persist longer than smooth variants in experimentally infected mice. Therefore, this study may provide some insight into the question of the chemical basis of pathogenesis in certain mycobacteria.     

Occurrence of cytochromes in R and S yeast mutants of the genusSaccharomyces

Visible region of an absorption spectrum was followed in cells of original strains and of rough mutants ofSaccharomyces cerevisiae andS. cerevisiae var.ellipsoideus. It was found that there are no substantial differences in relative content of cytochromesb andc in aerobically grown rough and smooth yeast forms, in spite of the fact that both forms differ substantially in the metabolic oxygen quotient. If the cytochromes present were not reduced in washed cells by dithionite or by substrate addition, the rough forms exhibited a lower cytochrome b:c ratio than the smooth forms. Under anaerobic conditions of cultivation, the rough forms retained a typical aerobic spectrum, lacking, however, the cytochromea and a₃ band; the ratio of cytochromesb andc was changed in favour of cytochromeb (from the original 1.7: 1 up to 3.4: 1). The inability of the rough mutants to produce anaerobic cytochrome spectrum represented by cytochrome b₁ was connected with their inability to reproduce under anaerobic conditions.     

Sensitivity of Rough Gram-negative Bacteria to the Bactericidal Action of Serum

Three Salmonella minnesota rough mutants were found to be more sensitive to the bactericidal action of antibody and complement than was the parent smooth strain. The antibodies involved were shown to be against components which are all present in the smooth parent strain and are not identical with the lipopolysaccharides isolated by the phenol-water extraction procedure. The lipopolysaccharide of the smooth strain was shown to confer resistance by blocking access of antibody to the sensitive antigens.     

Differences Between Brucella Antigens Involved in Indirect Hemagglutination Tests with Normal and Tanned Red Blood Cells

Brucella antigens capable of sensitizing normal and tanned sheep red blood cells for indirect hemagglutination were compared with antigens involved in agglutination, gel diffusion, and immunoelectrophoresis. Hyperimmune rabbit sera, before and after absorption with various antigenic preparations from smooth and rough B. abortus, were used in the tests. Normal erythrocytes could be sensitized with an NaOH-treated ether-water extract (EW-T) of smooth Brucella. Tanned erythrocytes could be sensitized with a water-soluble extract from ultrasonically disrupted smooth or rough Brucella. The EW-T produced a single precipitation band and the water-soluble antigens produce 6 to 23 bands in immunoelectrophoresis with unabsorbed sera. After absorption of antisera with water-soluble extracts from smooth or rough Brucella cells or from smooth or rough cell walls, the hemagglutinins for sensitized tanned erythrocytes and the precipitins for water-soluble antigens were removed. Absorption with living smooth or rough Brucella cells or with EW-T did not remove these antibodies. The precipitins and hemagglutinins for the antigen EW-T, and agglutinins for smooth cells, were absorbed by smooth antigens but not by rough antigens. It appears that the antigens which sensitize tanned erythrocytes and diffuse through agar gels are present in both smooth and rough forms and may be situated in the cytoplasm or in the internal part of the cell wall, whereas the agglutinogen and the antigen which attaches to normal erythrocytes are surface antigens found only on the smooth Brucella cell.     

Immunogenic endotoxin associated protein from a rough strain of Salmonella

Abstract A multimolecular complex of polypeptides found associated with the lipopolysaccharide endotoxin in Salmonella , reffered to as endotoxin-associated protein (EP), has been extracted from a rough strain of Salmonella typhimurium which does not synthesize 0 antigens. Since standard methods of extraction applicable to smooth strains of Salmonella were not successful for this rough strain, two modified procedures were developed. The resulting products were similar to smooth EP in terms of their biochemical, physical and mitogenic properties. When the immunogenicity of the rough EP was characterized by a protection assay in mice challenged with virulent Salmonella , it was found that the rough EP preparations were protective; however, they were not as active as the EP from a smooth strain of S. typhimurium .     

Immunogenic endotoxin associated protein from a rough strain of Salmonella

A multimolecular complex of polypeptides found associated with the lipopolysaccharide endotoxin in Salmonella, referred to as endotoxin-associated protein (EP), has been extracted from a rough strain of Salmonella typhimurium which does not synthesize 0 antigens. Since standard methods of extraction applicable to smooth strains of Salmonella were not successful for this rough strain, two modified procedures were developed. The resulting products were similar to smooth EP in terms of their biochemical, physical and mitogenic properties. When the immunogenicity of the rough EP was characterized by a protection assay in mice challenged with virulent Salmonella, it was found that the rough EP preparations were protective; however, they were not as active as the EP from a smooth strain of S. typhimurium.     

Lipopolysaccharide-Defective Mutants of the Wilt Pathogen Pseudomonas solanacearum

Lipopolysaccharide (LPS)-defective mutants of Pseudomonas solanacearum were used to test the hypothesis that differences in LPS structure are associated with the ability or inability of different strains to induce a hypersensitive response (HR) in tobacco. To obtain these mutants, LPS-specific bacteriophage of P. solanacearum were isolated and used to select phage-resistant mutants of the virulent, non-HR-inducing strain K60. The LPS of 24 of these mutants was purified and compared with that of K60 and its HR-inducing variant, B1. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, LPS from K60 and other smooth strains separated into many evenly spaced bands that migrated slowly, whereas LPS from B1 and most phage-resistant strains separated into one to three bands that migrated rapidly. Carbohydrate analysis showed that the LPS of the phage-resistant strains lacked O-antigen sugars (rhamnose, xylose, and N-acetylglucosamine) and could be grouped into (i) those that had all core sugars (rhamnose, glucose, heptose, and 2-keto-3-deoxyoctonate), (ii) those that had no core rhamnose, and (iii) those that lacked all core sugars except for 2-keto-3-deoxyoctonate. The LPS composition of 10 of the rough, phage-resistant mutants was similar to that of the HR-inducing strain, B1, yet none of them induced the HR. Only 2 of 13 mutant strains tested caused wilting of tobacco, and these had rough LPS but produced large amounts of extracellular polysaccharide, unlike most LPS-defective mutants. The evidence did not support the hypothesis that the initial interaction between rough LPS and tobacco cell walls is the determining factor in HR initiation.     

Effect of Heat on Spores of Rough and Smooth Variants of Bacillus stearothermophilus

Spores of variants of Bacillus stearothermophilus were subjected to activating and lethal temperatures. Spore suspensions which were incubated longer contained a higher percentage of spores of the rough variant. The effect of sublethal heat on spore suspensions containing mixed variants (rough and smooth) was difficult to measure at sublethal temperatures (110 C), since the rough variant was not as heat-resistant. While the rough variant was activated in a shorter time, the smooth variant was not activated; when the smooth variant was activated, the rough was killed. A higher percentage of the smooth variant was forced into dormancy after being held at 50 C for 30 hr than the rough variant. When mixed populations were subjected to a lethal temperature (120 C), the curves only reflected the smooth variant. Since the curves which represented the smooth variant or mixtures containing the smooth variant were not linear, this was thought to be due to activation overbalancing the lethal effect. This research emphasized the importance of variants in explaining differences in spore resistance among spore suspensions of the same strain.     

Antigenic determinants of murein lipoprotein and its exposure at the surface of Enterobacteriaceae.

Murein lipoprotein from the outer membrane of Escherichia coli could be fixed to erythrocytes without pretreatment of the erythrocytes. Passive hemagglutination or immune hemolysis could thus be used as sensitive assays to determine antibodies against lipoprotein. In rabbit antisera prepared against whole cells of E. coli, Salmonella, Arizona, and Shigella antibodies against lipoprotein were present. The respective titers were lowest in encapsulated smooth strains and highest in rough mutants. Antisera against deep rough mutants showed even higher anti-lipoprotein titers than anti-R-lipopolysaccharide titers. Correspondingly,absorption of lipoprotein antibodies with enterobacterial strains was most pronounced with deep rough mutants and lowest with smooth strains. Lipoprotein becomes increasingly an immunogen as well as an antigen the more sugar residues are missing in the lipolysaccharide on the cell surface. In wild-type cells lipoprotein is buried in the outer membrane; its exposure in mutant cells is related to defects at the cell surface.     

Exopolysaccharide mutants of Rhizobium loti are fully effective on a determinate nodulating host but are ineffective on an indeterminate nodulating host.

By Tn5 mutagenesis of Rhizobium loti PN184 (NZP2037 str-1) and selection for nonfluorescence of colonies on Calcofluor agar, eight independently generated expolysaccharide (EPS) mutants (three smooth and five rough) were isolated. The parent strain, PN184, was found to produce an acidic EPS. This EPS was produced. with reduced O acetylation, by the smooth EPS mutants but not by the rough EPS mutants. Lipopolysaccharide was isolated from all mutants and was identical to that of PN184 as defined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All mutants were resistant to lysis by R. loti bacteriophage phi 2037/1. Cosmids that complemented the mutations in the rough EPS mutants were isolated from a pLAFR1 gene library of NZP2037 by complementation of the nonfluorescent phenotype. The genes identified were shown to be unlinked and located on the chromosome. All mutants were fully effective when inoculated onto Lotus pedunculatus, a determinate nodulating host, but were ineffective, inducing the formation of very small nodules or tumorlike growths, when inoculated onto Leucaena leucocephala, an indeterminate nodulating host. These results, obtained in an isogenic Rhizobium background, support suggestions that acidic EPS is required for effective nodulation of indeterminate nodulating legumes but is not required for effective nodulation of determinate nodulating legumes.     

Immunochemistry of the surface carbohydrate antigens of Bacteroides fragilis and definition of a common antigen

The components extracted by aqueous phenol from whole cells of Bacteroides fragilis were analysed by SDS-PAGE and immunoblotting and shown to consist of a series of strain-specific, cross-reactive and common antigens. Regularly-spaced ladder patterns on silver-stained gels indicated that in most strains the LPS was present as a predominantly smooth type, but with chain lengths of varying molecular mass, ranging within each particular strain from essentially rough forms to long chain-length smooth forms. The rough form of the LPS at the gel front possessed an antigen common to most of the strains investigated. Another antigen, which migrated behind the rough LPS on SDS gels, was common to all strains of the species. The smooth LPS forms and the other high molecular mass components were strain-specific antigens. Previously published methods are not capable of producing pure LPS or capsular polysaccharide for this organism.     

Transformation of Salmonella typhimurium with plasmid DNA: differences between rough and smooth strains.

Lipopolysaccharide-defective mutants of Salmonella typhimurium were transformed by plasmid DNA with a Ca2+ treatment method. Only those mutants with an Rc or Rd2 chemotype, due to galE or rfaF mutations, respectively, gave efficiencies greater than 10(5) transformants per microgram of DNA, frequencies 8- to 630-fold higher than with smooth strains or other rough mutants.     

Spontaneous excision of the O-polysaccharide wbkA glycosyltranferase gene is a cause of dissociation of smooth to rough Brucella colonies

The brucellae are Gram-negative pathogens that cause brucellosis, a zoonosis of worldwide importance. The genus Brucella includes smooth and rough species that differ in that they carry smooth and rough lipopolysaccharides, respectively. Brucella abortus, B. melitensis, and B. suis are typical smooth species. However, these smooth brucellae dissociate into rough mutants devoid of the lipopolysaccharide O-polysaccharide, a major antigen and a virulence determinant encoded in regions wbo (included in genomic island-2) and wbk. We demonstrate here the occurrence of spontaneous recombination events in those three Brucella species leading to the deletion of a 5.5-kb fragment carrying the wbkA glycosyltranferase gene and to the appearance of rough mutants. Analysis of the recombination intermediates suggested homologous recombination between the ISBm1 insertion sequences flanking wbkA as the mechanism generating the deletion. Excision of wbkA was reduced but not abrogated in a recA-deficient mutant, showing the existence of both RecA-dependent and -independent processes. Although the involvement of the ISBm1 copies flanking wbkA suggested a transpositional event, the predicted transpositional joint could not be detected. This absence of detectable transposition was consistent with the presence of polymorphism in the inverted repeats of one of the ISBm1 copies. The spontaneous excision of wbkA represents a novel dissociation mechanism of smooth brucellae that adds to the previously described excision of genomic island-2. This ISBm1-mediated wbkA excision and the different %GC levels of the excised fragment and of other wbk genes suggest that the Brucella wbk locus is the result of at least two horizontal acquisition events.     

Effect of polymyxin on the ultrastructure of the outer membrane of wild-type and polymyxin-resistant strain of Salmonella.

The effect of polymyxin on two sets of Salmonella mutants was studied by thin-section and scanning electron microscopy. Polymyxin (in increasing concentrations, starting just below bactericidal effect) caused the appearance of the previously described rodlike projections on the cell surface of wild-type (smooth, polymyxin-sensitive) bacteria. These projections seemed to involve the outer membrane of the cell wall. In rough mutants, which are deficient in lipopolysaccharide, the projections were much smaller and flat. Higher concentrations of polymyxin were required to produce morphological effects in polyxmin-resistant mutants of both smooth and rough forms. Furthermore, in these mutants polymyxin caused vesicle-like bulging of the total outer membrane quite different in appearance from the rodlike projections of the wild type.     

COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG : I. Isolation of Membrane Fractions

The subcellular components involved in the synthesis, transport, and discharge of secretory proteins in the guinea pig pancreatic exocrine cell have been isolated from gland homogenates by differential and gradient centrifugation. They include rough and smooth microsomes derived respectively from the rough endoplasmic reticulum and Golgi periphery, a zymogen granule fraction consisting mainly of mature zymogen granules and a smaller population of condensing vacuoles, and a plasmalemmal fraction. Membrane subfractions were obtained from the particulate components by treatment with mild (pH 7.8) alkaline buffers which extract the majority (>95%) of the content of secretory proteins, allowing the membranes to be recovered from the extracting fluid by centrifugation. The purity of the fractions was assessed by electron microscopy and by assaying marker enzymes for cross-contaminants. The rough and smooth microsomes were essentially free of mitochondrial contamination; the smooth microsomes contained <15% rough contaminants. The zymogen granule fraction and its derived membranes were free of rough microsomes and contained <3% contaminant mitochondria. The plasmalemmal fraction was heterogeneous as to origin (deriving from basal, lateral, and apical poles of the cell) and contained varying amounts of adherent fibrillar material arising from the basement membrane and terminal web. The lipid and enzymatic composition of the membrane fractions are described in the following reports.     

Surface Antigens of Smooth Brucellae

Surface antigens of smooth brucellae were extracted by ether-water, phenol-water, trichloroacetic acid, and saline and examined by immunoelectrophoresis and gel diffusion with antisera from infected and immunized rabbits. Ether-water extracts of Brucella melitensis contained a lipopolysaccharide protein component, which was specific for the surface of smooth brucellae and was correlated with the M agglutinogen of Wilson and Miles, a polysaccharide protein component devoid of lipid which was not restricted to the surface of smooth brucellae and was not correlated with the smooth agglutinogen (component 1), and several protein components which were associated with internal antigens of rough and smooth brucellae. Immunoelectrophoretic analysis of ether-water extracts of B. abortus revealed only two components, a lipopolysaccharide protein component, which was correlated with the A agglutinogen, and component 1. Component 1 from B. melitensis and B. abortus showed identity in gel diffusion tests, whereas component M from B. melitensis and component A from B. abortus showed partial identity with unabsorbed antisera and no cross-reactions with monospecific sera. Attempts to prepare monospecific sera directly by immunization of rabbits with cell walls or ether-water extracts were unsuccessful. Absorption of antisera with heavy fraction of ether-water extracts did not always result in monospecific sera. It was concluded (as has been described before) that the A and M antigens are present on a single antigenic complex, in different proportions depending upon the species and biotype, and that this component is a lipopolysaccharide protein complex of high molecular weight that diffuses poorly through agar gel. Components 1, A, and M were also demonstrated in trichloroacetic acid and phenol-water extracts. With all extracts, B. melitensis antigen showed greater diffusibility in agar than B. abortus antigens. After mild acid hydrolysis, B. abortus ether-water extract was able to diffuse more readily.     

Distribution of Six Synaptic Membrane Antigens in Subcellular Fractions of Rat Brain Cortex

Abstract: The subcellular distribution in rat brain cortex of six synaptic membrane antigens (56K, 58K, 62K, 63K, 64K, 66K) was studied by rocket immunoelectrophoresis, using antiserum to a highly purified synaptic plasma membrane fraction. Initial analysis of the insoluble portion of subcellular fractions showed that these antigens were also present in smooth microsomes, rough microsomes, and synaptic vesicles; that only traces were present in synaptic junctions; and that none was present in nuclei, mitochondria, and myelin. A trace amount of activity was also present in synaptic vesicle cytosol, but none in whole brain cytosol. Quantitative measurements of synaptic plasma membranes, smooth microsomes, and synaptic vesicles showed that all six antigens were present in synaptic plasma membranes and smooth microsomes, but that the 66K antigen was absent from synaptic vesicles. The 56K, 58K, 62K, 63K, and 64K antigens were present in highest concentration in synaptic plasma membranes, whereas the 66K antigen content was highest in smooth microsomes. Only the 58K, 62K, and 63K antigens were detectable in the membrane fraction of whole brain. Their enrichments in synaptic plasma membranes were 10.9, 5.4, and 5.9, respectively. We conclude that the 56K, 58K, 62K, 63K and 64K antigens are primary components of synaptic plasma membranes. The presence of synaptic plasma membrane antigens in smooth microsomes and synaptic vesicles probably represents material being actively transported, consistent with the hypothesis that proteins of synaptic plasma membranes and synaptic vesicles are transported via smooth endoplasmic reticulum.     

Gloverins of the silkworm Bombyx mori: Structural and binding properties and activities

Gloverins are basic, glycine-rich and heat-stable antibacterial proteins (～14- kDa) in lepidopteran insects with activity against Escherichia coli, Gram-positive bacteria, fungi and a virus. Hyalophora gloveri gloverin adopts a random coil structure in aqueous solution but has α-helical structure in membrane-like environment, and it may interact with the lipid A moiety of lipopolysaccharide (LPS). Manduca sexta gloverin binds to the O-specific antigen and outer core carbohydrate of LPS. In the silkworm Bombyx mori, there are four gloverins with slightly acidic to neutral isoelectric points. In this study, we investigate structural and binding properties and activities of B. mori gloverins (BmGlvs), as well as correlations between structure, binding property and activity. Recombinant BmGlv1-4 were expressed in bacteria and purified. Circular dichroism (CD) spectra showed that all four BmGlvs mainly adopted random coli structure (>50%) in aqueous solution in regardless of pH, but contained α-helical structure in the presence of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), smooth and rough mutants (Ra, Rc and Re) of LPS and lipid A. Plate ELISA assay showed that BmGlvs at pH 5.0 bound to rough mutants of LPS and lipid A but not to smooth LPS. Antibacterial activity assay showed that positively charged BmGlvs (at pH 5.0) were active against E. coli mutant strains containing rough LPS but inactive against E. coli with smooth LPS. Our results suggest that binding to rough LPS is the prerequisite for the activity of BmGlvs against E. coli.     

Studies on Variants of Bacillus stearothermophilus Strain NCA 1518

The heat resistance, fermentation reactions, nutritional requirements, and phage sensitivity of 18 selected morphological variants of Bacillus stearothermophilus NCA 1518 were studied. It was found that when smooth variants mutated to rough colonial morphology, there was no concurrent change in fermentation reactions, nutritional requirements, or heat resistance. The smooth variant, and the rough mutants derived directly from it, presented a uniform pattern of biochemical capabilities which differed from the pattern presented by the rough variants isolated from the same stock culture. This led to the conclusion that the smooth and rough types previously observed in stocks of B. stearothermophilus NCA 1518 either were carried in the stock since the original isolation or represent a very profound and uncommon mutation, or that one of the variants has been introduced into the stock culture from an extraneous source sometime in the past.