Characterization of a Sinorhizobium isolate and its extracellular polymer implicated in pollutant transport in soil.

A bacterium isolated from soil (designated 9702-M4) synthesizes an extracellular polymer that facilitates the transport of such hydrophobic pollutants as polynuclear aromatic hydrocarbons, as well as the toxic metals lead and cadmium in soil. Biolog analysis, growth rate determinations, and percent G+C content identify 9702-M4 as a strain of Sinorhizobium meliloti. Sequence analysis of a 16S rDNA fragment gives 9702-M4 a phylogenetic designation most closely related to Sinorhizobium fredii. The extracellular polymer of isolate 9702-M4 is composed of both an extracellular polysaccharide (EPS) and a rough lipopolysaccharide. The EPS component is composed mainly of 4-glucose linkages with monomers of galactose, mannose, and glucuronic acid and has pyruval and acetyl constituents. The lipid fraction and the negative charge associated with carbonyl groups of the exopolymer are thought to account for the binding of polynuclear aromatic hydrocarbons and cationic metals.     

The role of nitric oxide in cancer

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including va-sodilatation, neurotransmission and macrophage-mediated immunity. The family of nitric oxide synthases (NOS) comprises inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Interestingly, various studies have shown that all three isoforms can be involved in promoting or inhibiting the etiology of cancer. NOS activity has been detected in tumour cells of various histogenetic origins and has been associated with tumour grade, proliferation rate and expression of important signaling components associated with cancer development such as the oestrogen receptor. It appears that high levels of NOS expression (for example, generated by activated macrophages) may be cytostatic or cytotoxic for tumor cells, whereas low level activity can have the opposite effect and promote tumour growth. Paradoxically therefore, NO (and related reactive nitrogen species) may have both genotoxic and angiogenic pro     

Gap junction structures: Analysis of the x-ray diffraction data

Models for the spatial distribution of protein, lipid and water in gap junction structures have been constructed from the results of the analysis of X-ray diffraction data described here and the electron microscope and chemical data presented in the preceding paper (Caspar, D. L. D., D. A. Goodenough, L. Makowski, and W.C. Phillips. 1977. 74:605-628). The continuous intensity distribution on the meridian of the X-ray diffraction pattern was measured, and corrected for the effects of the partially ordered stacking and partial orientation of the junctions in the X-ray specimens. The electron density distribution in the direction perpendicular to the plane of the junction was calculated from the meridional intensity data. Determination of the interference function for the stacking of the junctions improved the accuracy of the electron density profile. The pair-correlation function, which provides information about the packing of junctions in the specimen, was calculated from the interference function. The intensities of the hexagonal lattice reflections on the equator of the X-ray pattern were used in coordination with the electron microscope data to calculate to the two-dimensional electron density projection onto the plane of the membrane. Differences in the structure of the connexons as seen in the meridional profile and equatorial projections were shown to be correlated to changes in lattice constant. The parts of the junction structure which are variable have been distinguished from the invariant parts by comparison of the X-ray data from different specimens. The combination of these results with electron microscope and chemical data provides low resolution three- dimensional representations of the structures of gap junctions.     

An ultrastructural study of iron and manganese deposition associated with extracellular polymers of pedomicrobium-like budding bacteria

Morphological characteristics of two Pedomicrobium-like budding bacteria are described. A structured surface layer was regularly observed on strain 868. Ruthenium red- and Alcian blue-staining polymers were found on both strains.When either strain was grown in the presence of iron or manganese, the corresponding oxides accumulated on their surfaces. In thin sections iron oxides appeared as fine threads, arrays of particles or dense coatings, depending on the source of iron. Manganese oxides appeared as branching filaments or convoluted ribbons. Both metal oxides stained with ruthenium red. Extraction of the oxides followed by ruthenium red staining revealed that polyanionic polymers previously deposited on the cells were associated with the metals.Treatment of cultures with glutaraldehyde, HgCl₂, or heat, inhibited manganese but not iron deposition, suggesting that iron oxides accumulated by passive, non-biological processes. Manganese oxides apparently accumulated under control of a biological manganese-oxidizing factor. Incomplete inhibition of manganese deposition observed in cell suspensions suggested that, if the oxidizing factor was an enzyme, it was unusually stable.Based on these results, possible mechanisms of iron and manganese deposition in association with extracellular polymers are suggested.     

Micromorphology of Cryptococcus neoformans

Fine details of the internal and external morphology of Cryptococcus neoformans as seen in ultrathin sections are described and illustrated with electron micrographs. The capsule characteristic of this species contained microfibrils (30 to 40 A in diameter) that appeared to radiate from the cell wall and to coil and intertwine in various directions. These thin, uniformly structured, electron-dense filaments are believed to represent complex polysaccharide molecules. The internal morphology of C. neoformans was in many ways similar to that of yeasts studied by other authors. The cell was uninucleate with a single nucleolus. The nuclear envelope, a pair of unit membranes interrupted by pores, was typical of that found in eucaryotic organisms. Smooth endoplasmic reticulum, mitochondria, vacuoles, storage granules, and ribosomes were consistent features of the cytoplasm. In addition, C. neoformans presented membranous organelles derived from the plasma membrane and comparable to bacterial mesosomes and mitochondria of an annulate type.     

A quantitative analysis of DNA extraction and purification from compost

We quantified both DNA and humic acid concentrations during the extraction and purification of DNA from compost. The DNA extraction method consisted of bead-beating with SDS for cell lysis, poly(ethylene glycol)-8000 precipitation for preliminary DNA purification, and chromatography on a 10-ml Sephadex G-200 column for final DNA purification. Direct microscopic observation of pre- and post-lysis samples revealed that 95.3+/-2.3% of native cells was lysed. Sixty-three percent of the original DNA was lost during purification, resulting in a final DNA yield of 18.2+/-3.8 microg DNA/g of wet compost. The humic acid content was reduced by 97% during the purification steps resulting in a final humic acid concentration of 27+/-4.7 ng humic acid/microl. The purified DNA fragments were up to 14 kbp in size and were sufficiently free of contaminants to allow both restriction enzyme digestion by four different enzymes and PCR amplification of 16S rDNA.     

Structure and carbohydrate analysis of the exopolysaccharide capsule of Pseudomonas putida G7

The aromatic hydrocarbon-degrading bacterium, Pseudomonas putida G7, produces exopolymers of potential interest in biotechnological applications. These exopolymers have been shown to have significant metal-binding ability . To initiate the study of the metal–polymer interactions, we explored the physical and chemical nature of the P. putida G7 exopolysaccharide, a major component of the exopolymer. A capsular structure was observed by light microscopy surrounding both planktonic and attached cells in biofilms after immunofluorescence staining with polyclonal antiserum raised against planktonic cells. Further work with planktonic cells showed that the immunostained capsule remained associated with young (log phase) cells, whereas older (stationary phase) cells lost their capsular material to the external milieu. Visualization of frozen, hydrated stationary phase cells by cryo-field emission scanning electron microscopy (cryoFESEM) revealed highly preserved extracellular material. In contrast, conventional scanning electron microscopy (SEM) of stationary phase cells showed rope-like material that most probably results from dehydrated and collapsed exopolymer. Both capsular and released exopolymers were separated from cells, and the released extracellular polysaccharide (EPS) was purified. Deoxycholate–polyacrylamide gel electrophoresis (PAGE) and silver/alcian blue staining of the partially purified material showed that it contained both EPS and lipopolysaccharide (LPS). Further purification of the EPS using a differential solubilization technique to remove LPS yielded highly purified EPS. Gas chromatography–mass spectrometry revealed that the purified EPS contained the monosaccharides, glucose, rhamnose, ribose, N-acetylgalactosamine and glucuronic acid. The structural and chemical properties of the P. putida EPS described here increase our understanding of the mechanisms of toxic metal binding by this well-known Proteobacterium.     

Formation of Metallogenium-like structures by a manganese-oxiding fungus

Structures resembling Metallogenium spp. were observed in agar and in liquid cultures of a Mn-oxidizing basidiomycetous fungus only when Mn²⁺ was oxidized. Fungal viability was necessary for formation of the structures; Mn²⁺ concentration and the presence or absence of agar in the medium were important factors determining their morphology. Slide cultures revealed no identifiable cells in any stage of development. Fluorescent dyes that stained nucleic acids and polysaccharides in the fungal hyphae did not stain the Metallogenium-like structures. Likewise, Rhodamine 123, a fluorescent probe for membrane potential, stained fungal mitochondria, but did not stain the structures. Thin sections through the structures showed no biological membranes or other cellular features. Only the characteristic ultrastructure of biological Mn oxides were observed in serial thin sections. In agar, unfixed structures disappeared permanently during reduction of Mn oxides with hydroxylamine. Glutaraldehyde fixation stabilized these structures. Fixed structures lost most of their original phase density during reduction with hydroxylamine, but continuous microscopic observations showed that their phase density could be restored by staining with Coomassie blue. Structures that formed in liquid medium did not require stabilization with glutaraldehyde during reduction of Mn oxides. They, too, lost their original phase density during reduction with hydroxylamine; phase density could be restored by staining with cationic colloidal iron or Coomassie blue. The results suggest that the Metallogenium-like structures were formed as a result of Mn oxidation associated with exopolymers produced by the fungus.Non-standard abbreviations HEPES (N-hydroxyethylpiperazine-N-2-ethane sulfonic acid) - DAPI (4,6-diamidino-2-phenylindole) - PIPES (piperazine-N,N-bis[2-ethane sulfonic acid])     

Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta aurantia.

The outer membrane of Spirochaeta aurantia was isolated after cells were extracted with sodium lauryl sarcosinate and was subsequently purified by differential centrifugation and KBr isopycnic gradient centrifugation. The purified outer membrane was obtained in the form of carotenoid-containing vesicles. Four protein species with apparent molecular weights of 26,000 (26K), 36.5K, 41K, and 48.5K were readily observed as components of the vesicles. The 36.5K protein was the major polypeptide and constituted approximately 90% of the outer membrane protein observed on sodium dodecyl sulfate-polyacrylamide gels. Under mild denaturing conditions the 36.5K major protein exhibited an apparent molecular weight of approximately 90,000. This, together with the results of protein cross-linking studies, indicates that the 36.5K polypeptide has an oligomeric conformation in the native state. Reconstitution of solubilized S. aurantia outer membrane into lipid bilayer membranes revealed the presence of a porin, presumably the 36.5K protein, with an estimated channel diameter of 2.3 nm based on the measured single channel conductance of 7.7 nS in 1 M KCl.