Isolation and characterization of two hydrocarbon-degrading Bacillus subtilis strains from oil contaminated soil of Kuwait.

Two strains of hydrocarbon-utilizing bacteria were isolated from soil samples of the Kuwait Burqan oil field at a temperature of 37 degrees C. The bacteria were motile endospore-forming rods with slight differences in their metabolic patterns and 16S rRNA sequence. Vegetative cells of the strains designated as AHI and AHII had an ultrastructure typical of gram-positive bacteria and showed gram-positive staining. The bacteria did not show pigmentation. Best growth was observed at 37 degrees C at neutral pH and NaCl concentrations in the range of 5-10 g per l. Both strains were obligatory aerobic and developed on synthetic media with either Diesel fuel, n-decan or naphthalene as the sole carbon and energy source. No specific growth factors were required. On the basis of their morphological, physiological and biochemical features, as well as their 16S rRNA analysis and electron microscope study, both strains were assigned to the species of Bacillus subtilis.

Phylogenetic positions of novel aerobic, bacteriochlorophyll a-containing bacteria and description of Roseococcus thiosulfatophilus gen. nov., sp. nov., Erythromicrobium ramosum gen. nov., sp. nov., and Erythrobacter litoralis sp. nov.

We analyzed the 16S ribosomal DNAs of three obligately aerobic, bacteriochlorophyll a-containing bacteria, "Roseococcus thiosulfatophilus," "Erythromicrobium ramosum," and new isolate T4T (T = type strain), which was obtained from a marine cyanobacterial mat. "Roseococcus thiosulfatophilus" is a member of the alpha-1 subclass of the Proteobacteria and is moderately related to Rhodopila globiformis, Thiobacillus acidophilus, and Acidiphilium cryptum (level of sequence similarity, 90%). "Erythromicrobium ramosum" and isolate T4T are closely related to Erythrobacter longus and Porphyrobacter neustonensis (level of sequence similarity, 95%). These organisms are members of the alpha-4 subclass of the Proteobacteria. Strain T4T is a motile, red or orange bacterium. The major carotenoids are bacteriorubixanthinal and erythroxanthin sulfate. In vivo measurements revealed bacteriochlorophyll absorption maxima at 377, 590, 800, and 868 nm. Strain T4T grows in the presence of 5 to 96/1000 salinity and uses glucose, fructose, acetate, pyruvate, glutamate, succinate, and lactate as substrates. On the basis of its distinct phylogenetic position and phenotypic characteristics which are different from those of Erythrobacter longus, we propose that strain T4T should be placed in a new species of the genus Erythrobacter, Erythrobacter litoralis. The descriptions of "Roseococcus thiosulfatophilus" and "Erythromicrobium ramosum" are emended.

Bacteriochlorophyllc formation and chlorosome development inChloroflexus aurantiacus.

The dependence of chlorosome development on bacteriochlorophyll (BChl)c synthesis was studied with the phototrophic green bacteriumChloroflexus aurantiacus. By selecting defined culture conditions, three possibilities could be identified. Upon addition of 5-aminolevulinic acid, cells of resting cultures increased their specific BChlc contents as well as the volumes of already existing chlorosomes. The number of chlorosomes, however, remained constant. Serine-limited chemostat cultures grown under steady state conditions exhibited constant rates of synthesis of both BChlc as well as of chlorosomes. The volume of the latter remained constant, as well. Upon addition of ALA to chemostat cultures, chlorosomes were synthesized at the same rate as before but their volumes increased as a consequence of increased BChlc incorporation. In chlorosomes isolated from resting cultures supplied with ALA the amounts of all of the polypeptides increased only slightly, if at all. Moreover, the ratio of all of the chlorosomal polypeptides remained largely constant. These results show that chlorosomes may incorporate newly synthesized BChlc without concomitant formation of chlorosomal polypeptides. This means that there was no obvious coordination of polypeptide and BChlc synthesis. On this basis, it appears unlikely that one of the chlorosomal polypeptides functions as an apoprotein of a presumed BChlc holochrome complex.

Attachment of Blastocrithidia triatomae (trypanosomatidae) by flagellum and cell body in the midgut of the reduviid bug Triatoma infestans.

The pathology and attachment of Blastocrithidia triatomae in two regions of the midgut of the reduviid bug Triatoma infestans (the stomach and the small intestine) were investigated by electron microscopy. In both regions the extracellular-membrane layers and the apical microvilli were often reduced, and some cells of the intestinal wall were vacuolated. In the stomach two types of attachment occurred: Flagella with intraflagellar swellings lay over and between the apices of the microvilli of the stomach cells, and in microvilli-free regions the cell body of B. triatomae seemed to be anchored to the host cell by corrugations of both cells in the attachment zone, a hitherto unknown mode of attachment of trypanosomatids in the intestinal tract. In the small intestine flagellar expansions held several microvilli, and rarely an unaltered flagellum seemed to be anchored between the microvilli. In both midgut regions, the flagellum of some epimastigotes was inserted into the apex of an intestinal cell.

Ultrastructural and electron spectroscopic analyses of cyanobacteria and bacteria.

The extracellular sheath material and some intracellular cell components of cyanobacteria and phosphate-accumulating sewage bacteria were analysed by electron spectroscopic imaging (ESI) and electron energy-loss spectroscopy (EELS). The specimens were embedded in water-soluble Nanoplast resin without any previous fixation and ultrathin sections were examined in a Zeiss CEM 902 microscope. A high sulphur content was detected in the inner sheath of the cyanobacterium Gloeothece. The elemental composition of some cell components and inclusion bodies, such as carboxysomes and cyanophycin, was determined by ESI and EELS. In addition, the phosphate content in specific granules of phosphate-accumulating sewage bacteria was estimated by EELS and nuclear magnetic resonance spectroscopy.

Fine-structural and chemical analyses on inner and outer sheath of the cyanobacterium Gloeothece sp. PCC 6909.

Cells of the unicellular cyanobacterium Gloeothece sp. PCC 6909 are surrounded by an inner (enclosing 1-2 cells) and an outer (enclosing cell groups) sheath. Using conventional Epon-embedding in combination with ruthenium-red staining, the inner and outer sheaths appeared similar and displayed multiple bands of electron-dense subunits. However, embedding in Nanoplast resin to avoid shrinkage led to the detection of two distinct zones (inner and outer zone) each with several distinct layers. The zone delimited by the electron-dense thick inner sheath layer, and the zone enclosed by the thin electron-dense outer sheath layer, are composed of a homogeneous material of little electron-contrast. Whereas the outer zone appears to be of even contrast, the inner zone is characterized by a distinct electron-transparent layer. Element distribution analysis revealed that the electron-transparent layer contained relatively large amounts of sulfur, carbon, and oxygen but only little nitrogen. Inner and outer sheath fractions were isolated by differential mechanical cell breakage and centrifugation. The outer sheath fraction was less hydrated than the inner one. The two fractions differed little in their contents of uronic acids, carbohydrate and protein, although the outer sheath fraction contained less sulfate. A soluble polysaccharide with a chemical composition similar to that of inner and outer sheath fractions was also obtained from the culture supernatant.

Further electron microscopic studies on the expression of Escherichia coli group II capsules.

The de novo expression of Escherichia coli K1, K5, and K12 capsules was analyzed with immunoelectron microscopy in temperature upshift experiments, with upshift from 18 degrees C (capsule restrictive) to 37 degrees C (capsule permissive). Newly produced capsular polysaccharides appeared at the cell surface atop membrane adhesion sites (Bayer's junctions). After plasmolysis of the bacteria at an early expression stage, the capsular polysaccharides were labeled at discrete sites in the periplasm by the immunogold technique. After temperature upshift in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or chloramphenicol, the polysaccharides were labeled in the cytoplasm.

Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli.

The capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three gene regions responsible for polymerization and surface expression have been defined (I. S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1310, 1988). In this report, we describe the immunoelectron microscopic analysis of recombinant bacteria expressing the K5 antigen and of mutants defective in either region 1 or region 3 gene functions, as well as the biochemical analysis of the K5 capsular polysaccharide. Whereas the K5 clone expressed the K5 polysaccharide as a well-developed capsule in about 25% of its population, no capsule was observed in whole mount preparations and ultrathin sections of the expression mutants. Immunogold labeling of sections from the region 3 mutant revealed the capsular K5 polysaccharide in the cytoplasm. With the region 1 mutant, the capsular polysaccharide appeared associated with the cell membrane, and, unlike the region 3 mutant polysaccharide, the capsular polysaccharide could be detected in the periplasm after plasmolysis of the bacteria. Polysaccharides were isolated from the homogenized mutants with cetyltrimethylammonium bromide. The polysaccharide from the region 1 mutant had the same size as that isolated from the capsule of the original K5 clone, and both polysaccharides were substituted with phosphatidic acid. The polysaccharide from the region 3 mutant was smaller and was not substituted with phosphatidic acid. These results prompt us to postulate that gene region 3 products are involved in the translocation of the capsular polysaccharide across the cytoplasmic membrane and that region 1 directs the transport of the lipid-substituted capsular polysaccharide through the periplasm and across the outer membrane.

Evidence for phagosome-lysosome fusion in Mycobacterium leprae-infected murine Schwann cells.

Murine Schwann cells were infected with viable armadillo-derived Mycobacterium leprae in vitro, and the lysosomal marker enzyme, acid phosphatase, was stained by the Gomori reaction. Electron microscopic analysis revealed that Schwann cells infected with M. leprae possess acid phosphatase and that lysosomes fuse with infected phagosomes.

Nonfimbrial, mannose-resistant adhesins from uropathogenic Escherichia coli O83:K1:H4 and O14:K?:H11.

Nonfimbrial, mannose-resistant hemagglutinins (nonfimbrial adhesions [NFA] NFA-1 and NFA-2) were extracted from two agar-grown urinary isolates of Escherichia coli strains 827 (O83:K1:H4) and 54 (O14:K?:H11). The proteins were purified to homogeneity by ammonium sulfate precipitation and column chromatography. Nonfimbrial adhesins are soluble proteins, which tend to form aggregates of molecular weight above 10(6). NFA-1 and NFA-2 consist of subunits of 21,000 and 19,000 molecular weight, respectively. Both hemagglutinins caused hemagglutination of human erythrocytes and bound to human kidney cell monolayers. The binding of bacteria and hemagglutinins was assessed by using suitable antisera as detectors in an enzyme-linked immunosorbent assay. NFA-1 and NFA-2 inhibited the adherence of their respective strains to human kidney cells in a linear dose response. NFA-2 also inhibited heterologous strain adherence, but NFA-1 did not. Hemabsorption of bacterial suspension with erythrocytes at 4 degrees C, followed by differential centrifugation, enabled us to obtain a bacterial suspension lacking nonfimbrial adhesins in the supernatant and an adhesin-enriched bacterial suspension that was eluted from erythrocytes at 40 degrees C. Bacteria eluted from erythrocytes exhibited a higher adherence capacity than unfractionated cells. Bacteria of the fraction lacking adhesins did not adhere to human kidney cells. Electron microscope examinations showed the presence of an extracellular capsule-like layer in adhering E. coli 827, but not in nonadhering bacteria. E. coli 54 did not express the adhesin as a capsule. We conclude that E. coli 827 and 54 produce extracellular adhesins consisting of soluble proteins which are differently expressed and antigenically distinct. The adhesins seem to share a common receptor and mediate the adherence of two uropathogenic E. coli strains to epithelial cells.

The T cell-specific serine proteinase TSP-1 is associated with cytoplasmic granules of cytolytic T lymphocytes.

This study describes the localization of the previously purified T cell-specific serine proteinase, termed TSP-1 (M. M. Simon et al., EMBO J. 1986. 5: 3267), within cytoplasmic granules of cytolytic T cell lines (CTLL). Subcellular fractionation of disintegrated CTLL (ruptured by nitrogen cavitation) was accomplished by Percoll density gradient centrifugation of cell lysates (postnuclear supernatant). Individual fractions were tested for proteinase activity on chromogenic peptide substrates and for the presence of TSP-1 by Western blot analysis. In addition, each fraction was assayed for cytolytic activity against sheep red blood cells (SRBC), for protein and for additional marker enzymes to assess the enrichment for cellular organells. All serine enzyme-type molecules including TSP-1 expressed by CTLL were identified by labeling cell lysates or gradient fractions with the serine proteinase-specific affinity ligand tritiated diisopropyl fluorophosphate [( 3H]DFP) in the presence or in the absence of class-specific or enzyme-specific proteinase inhibitors and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data demonstrate that the Percoll gradient fraction, which was shown by morphological examination in the electron microscope to be highly enriched for cytoplasmic granules, also contained greater than 80% of proteinase activity in addition to the granule-associated structures cytolysin and arylsulfatase. The identity of the granule-associated proteinase in two independent cell lines, CTLL HY3-Ag3 and CTLL 1.D.9, with the serine proteinase TSP-1 is indicated by its specificity for the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide, its sensitivity to class-specific as well as TSP-1-specific enzyme inhibitors and by its reactivity with a polyvalent TSP-1-specific rabbit antiserum. Both CTLL contain a [3H]DFP-labeled protein that migrates with a molecular mass of 60 kDa under nonreducing conditions and with 30 kDa under reducing conditions and which can be inactivated by the TSP-1-specific inhibitor H-D-Pro-Phe-Arg-chloromethylketone. CTLL HY3-Ag3 (a long-term culture CTLL with natural killer-like activity) but not CTLL 1.D.9 (an antigen-specific short-term cultured CTLL) express in addition a further [3H]DFP-binding protein which migrates with 27 kDa under nonreducing or reducing conditions. No substrate specificity was found for this molecule. The possible function of the granule-associated serine proteinase TSP-1 is discussed.

Race cultivar-specific differences in callose deposition in soybean roots following infection with Phytophthora megasperma f.sp. glycinea.

Primary roots of soybean (Glycine max (L.), Merrill, cv. Harosoy 63) seedlings were inoculated with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea and total callose was determined at various times after inoculation. From 4 h onward, total callose was significantly higher in roots showing the resistant rather than the susceptible response. Local callose deposition in relation to location of fungal hyphae was determined in microtome sections by its specific fluorescence with sirofluor and was quantified on paper prints with an image-analysis system. Callose deposition, which occurs adjacent to hyphae, was found soon after inoculation (2, 3 and 4 h post inoculation) only in roots displaying the resistant response, and was also higher at 5 and 6 h after inoculation in these resistant roots than in susceptible roots. Early callose deposition in the incompatible root-fungus reaction could be a factor in resistance of soybean against P. megasperma.

Skin reactivity of unsensitized monkeys upon challenge with staphylococcal enterotoxin B: a new approach for investigating the site of toxin action.

The correlation between skin tests and emetic responses in unsensitized monkeys was used to elucidate the cellular site of action of staphylococcal enterotoxin B (SEB). Evidence is presented that SEB administered intradermally provoked immediate-type skin reactions associated with mild degranulation of cutaneous mast cells. The cytoplasma showed signs of synthetic and metabolic activity, with formation of vesicles and increased prominence of mitochondria. Carboxymethylation of histidine residues of SEB altered the molecule (cSEB) from more alkaline components to more acidic species with increased microheterogeneity. This modification caused a loss in toxicity and completely abrogated the skin-sensitizing activity without changing the immunological specificity. cSEB, however, could compete with SEB for binding sites on the target cell surface. Previously, compound 48/80-treated skin sites behaved refractively to challenge with SEB, indicating that mediators from cutaneous mast cells are required for SEB-induced skin reactions. Skin reactions as well as emetic responses challenged with SEB were completely inhibited by H2 receptor antagonists and calcium channel blockers but not by H1 antihistamine or competitive antagonists of serotonin. This new approach provides a model for investigating the mechanisms of SEB action.

Immunoelectron microscopic studies on the localization of peptidoglycan peptide subunit pentapeptides in bacterial cell walls.

The peptide subunit pentapeptide H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH2 of peptidoglycan was localized in the cell walls of several Gram-positive bacteria employing the indirect immunoferritin technique. Specific antibodies to the D-alanyl-D-alanine moiety of non-crosslinked peptide subunit pentapeptide were raised in rabbits by immunization with synthetic immunogen albumin-(CH2CO-Gly-L-Ala-L-Ala-D-Ala-D-Ala-OH)39. Specificity of these antibodies for the peptide subunit pentapeptide and not for the peptide subunit tetrapeptide was corroborated in a Farr-type radio-active hapten binding assay. Specificity of labelling with ferritin was established by immunoelectron microscopic controls, and by the excellent correlation between specific labelling of cells with ferritin and the particular peptidoglycan primary structure of bacterial strains investigated. Cells of Lactobacillus gasseri, Streptococcus pyogenes and Staphylococcus aureus revealing non-crosslinked peptide subunit pentapeptides in their peptidoglycans could specifically be labelled. Lactobacillus acidophilus and Bacillus subtilis, on the contrary, missing such pentapeptides, failed in labelling. The implication of this method to possibly localize the points of attack of penicillin or cycloserine is discussed.

Fusion of liposomes and chromatophores of Rhodopseudomonas capsulata: effect on photosynthetic energy transfer between B875 and reaction center complexes.

The photosynthetic chromatophore membranes of Rhodopseudomonas capsulata were fused with liposomes to investigate the effects of lipid dilution on energy transfer between the bacteriochlorophyll-protein complexes of this membrane. Phosphatidylcholine-containing liposomes were mixed with chromatophores at pH 6.0 to 6.2, and the mixture was fractionated on discontinuous sucrose gradients into four membrane fractions with lipid-to-protein ratios that varied 11-fold. Freeze-fracture electron microscopy revealed that the fractions contained closed vesicles formed by the fusion of liposomes to chromatophores. Particles with 9-nm diameters on the P fracture faces did not appear to change in size with increasing lipid content, but the number of particles per membrane area decreased proportionally with increases in the lipid-to-protein ratio. The bacteriochlorophyll-to-protein ratios, electrophoretic polypeptide profiles on sodium dodecyl sulfate-polyacrylamide gels, and light-induced absorbance changes at 595 nm caused by photosynthetic reaction centers were not altered by fusion. The relative fluorescence emission intensities due to the B875 light-harvesting complex increased significantly with increasing lipid content, but no increases in fluorescence due to the B800-B850 light-harvesting complex were observed. Electron transport rates, measured as succinate-cytochrome c reductase activities, decreased with increased lipid content. The results indicate an uncoupling of energy transfer between the B875 light-harvesting and reaction center complexes with lipid dilution of the chromatophore membrane.

Electron microscopic study showing antibody-independent binding of C1q, a subcomponent of the first component of complement, to serum-sensitive salmonellae.

Effective serum-mediated killing of sensitive gram-negative bacteria requires all the complement components. In the preimmune phase the antibody-independent interaction of the first component of complement, C1, with the bacteria might be especially important. Electron microscopic studies showed that the C1 subcomponent C1q binds only to the serum-sensitive R form of Salmonella minnesota and not to the serum-resistant S form.

Isolation of surface lectins of GH3 cells from whole cells and isolated plasma membranes.

Lectins localized in the plasma membranes seem to be of special importance for the intercellular interaction mechanisms. We describe the isolation of mannose-binding proteins by Triton X-100 extraction and affinity chromatography on agarose-bound mannose. The isolation procedure was performed with whole GH3 cells as well as with isolated plasma membranes. For the isolation of plasma membranes of GH3 cells a mechanical pump was used for the disruption. After differential centrifugation an enriched plasma membrane fraction was achieved by discontinuous sucrose gradient centrifugation. The whole fractionation procedure was controlled by total balance sheets for the marker enzymes of the different cell organelles. The plasma membrane fraction was further characterized by gel electrophoresis and electron microscopy. The SDS gel electrophoresis patterns of the proteins, resulting from the Triton X-100 extraction and the affinity chromatography, are nearly identical for whole cells as well as for the enriched plasma membrane fraction. Therefore we presume these mannose-specific proteins to be plasma membrane bound, showing the molecular properties of integral proteins and having a molecular weight of Mr 67 000, 57 000, 47 000.

The differentiation of the photosynthetic apparatus and the intracytoplasmic membrane in cells of Rhodopseudomonas capsulata upon variation of light intensity.

In cells of Rhodopseudomonas capsulata, grown at growth limiting light intensity (7 W x m-2), the cellular bacteriochlorophyll (Bchl) content increased 13-fold, the Bchl concentration of membranes 3.4 fold, the concentration of reaction centers in membranes 1.6-fold, the size of the photosynthetic unit twofold, the concentration of carotenoids in membranes 2.4 fold and the number of intracytoplasmic membrane vesicles, bearing the photosynthetic apparatus, 6..3 fold in comparison with cells grown at 2000 W x m-2 light intensity. Thus, the variation of incident caused mainly a variation of the amount of intracytoplasmic membrane vesicles per cell, but also a variation of the size and concentration of photosynthetic units in the membrane system.

Differences in the architecture of cytoplasmic and intracytoplasmic membranes of three chemotrophically and phototrophically grown species of the Rhodospirillaceae.

Freeze-fracture faces of membranes of either chemotrophically or phototrophically grown Rhodospirillum rubrum, Rhodopseudomonas sphaeroides, and Rhodospirillum tenue were analyzed. All three species differed from each other with respect to size as well as numerical density (number per square micrometer) of intramembrane particles. In R. rubrum the number of particles on exoplasmic fracture faces of the cytoplasmic membrane stayed nearly constant (about 900 particles per microns2), but on the plasmic fracture face there were 4,700 and 6,264 particles per microns2, respectively, under chemotrophic and phototrophic conditions. The increase in number was largely a result of an enhanced occurrence of particles 10 nm in diameter. This diameter corresponds to the mean diameter of the predominant class of particles visible on the plasmic fracture faces of intracytoplasmic membrane formed under phototrophic conditions. In R. sphaeroides the number of particles on both of the fracture faces of cytoplasmic membranes stayed nearly constant. The mean diameter of articles appeared to be slightly increased under phototrophic conditions. Particles of cytoplasmic and intracytoplasmic membranes of phototrophically grown cells were of similar diameter. The number of particles, however, on plasmic fracture faces of intracytoplasmic membranes (6,674/microns2) was significantly higher than that on cytoplasmic membranes (5,708/microns2). R. tenue, on the other hand, which does not produce intracytoplasmic membranes, showed on exoplasmic fracture faces 543 and 3,765 particles per micron2 under chemotrophic and phototrophic conditions, respectively, whereas the corresponding numerical densities of plasmic fracture faces were 4,043 and 3,711 particles per microns2. The increased number of articles on exoplasmic fracture faces was mainly the result of an increased occurrence of particles with diameters greater than or equal to 10 nm. The results are interpreted to allow for the different modes of intractyoplasmic membrane development in Rhodospirillum rubrum and Rhodopseudomonas sphaeroides, respectively.