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A method is presented for preparing core-shell silica-polypeptide composite particles with variable and controllable shell growth. The procedure is demonstrated using poly(carbobenzoxy-L-lysine) and poly(benzyl-L-glutamate); after deprotection, these can lead to the most common basic and acidic homopolypeptides, poly(L-lysine) and poly(L-glutamic acid). Control over shell thickness is made possible by sequential addition of N-carboxyanhydride peptide monomer to surfaces that have been functionalized with an amino initiator combined with a surface passivation agent. This results in a series of particles having different shell thicknesses. Variation of shell thickness was evident both in light scattering and in thermogravimetric assays. The shells were visible by transmission electron microscopy; these images along with light scattering measurements suggest the polymers in the shells are highly solvated.

Ludox density gradients were used to enrich for Escherichia coli mutants with conditional growth defects and alterations in membrane composition. A temperature-sensitive mutant named Lud135 was isolated with mutations in two related, nonessential genes: yghB and yqjA . yghB harbors a single missense mutation (G203D) and yqjA contains a nonsense mutation (W92TGA) in Lud135. Both mutations are required for the temperature-sensitive phenotype: targeted deletion of both genes in a wild-type background results in a strain with a similar phenotype and expression of either gene from a plasmid restores growth at elevated temperatures. The mutant has altered membrane phospholipid levels, with elevated levels of acidic phospholipids, when grown under permissive conditions. Growth of Lud135 under nonpermissive conditions is restored by the presence of millimolar concentrations of divalent cations Ca 2+ , Ba 2+ , Sr 2+ , or Mg 2+ or 300 to 500 mM NaCl but not 400 mM sucrose. Microscopic analysis of Lud135 demonstrates a dramatic defect at a late stage of cell division when cells are grown under permissive conditions. yghB and yqjA belong to the conserved and widely distributed dedA gene family, for which no function has been reported. The two open reading frames encode predicted polytopic inner membrane proteins with 61% amino acid identity. It is likely that YghB and YqjA play redundant but critical roles in membrane biology that are essential for completion of cell division in E. coli .

The marine bacterium Vibrio vulnificus is a human pathogen that can spontaneously switch between virulent opaque and avirulent translucent phenotypes. Here, we document an additional form, the rugose variant, which produces copious biofilms and which may contribute both to pathogenicity of V. vulnificus and to its survival under adverse environmental conditions.

Cephaleuros parasiticus Karsten, an endophyte of Magnolia grandiflora L. has been examined with light, and scanning and transmission electron microscopy. The discoid thalli are composed of filaments which ramify throughout the leaf tissues beneath the epidermis. Algal filaments do not penetrate host cells, but do produce black leaf spots which have been mistaken for those caused by the fungus Glomerella cingulate (Ston.) Spauld. and Schrenk. Two distinct thallus types occur, often simultaneously on a single leaf. One bears clusters of zoosporangiate branches which seasonally emerge through the ventral (and rarely, dorsal) surface of the leaf. In contrast, the other thallus type bears gametangia which break through the dorsal leaf surface. Zoosporangia and gametangia have never been found on the same thallus. The zoosporangia are smaller than, but almost identical in shape to, those of C. virescens Kunze. Simple plasmodesmata are present in crosswalls and acetolysis indicates that little or no sporopollenin is present in the cell walls. The ultrastructure of biflagellate gametes and quadriflagellate zoospores is virtually indistinguishable from that reported for C. virescens and similar to that reported for Phycopeltis and Trentepohlia. In both gametes and zoospores there are keeled flagella, overlapping and parallel basal bodies, two 3-layered multilayered structures with microtubular splines, and two medial compound microtubular roots. Pyrenoids, eyespots, flagellar and body scales, striated roots (or rhizoplasts), and distal bands are absent. Two presumptive mating structures are present in each biflagellate gamete, and flagellar collars occur in both types of motile cells. The extreme similarity in motile cell ultrastructure revealed in this interspecific comparison parallels that similarity revealed in intergeneric comparisons.

Previous methods for cheese sample processing for electron microscopy involves several days. Our objective was to identify a suitable yet rapid method. The rapid method involved an initial fixation of cheese in a solution of 2% glutaraldehyde and 1% OsO4 in 0.05M cacodylate buffer for 10 min. This was followed by a second fixation in 2 % glutaraldehyde and 2 % OsO4 in 0.05M buffer for an additional 20 min. Sample fixation by the rapid method was attained in a total of 30 min compared to 17 h in the control, i.e., the previous method, which required 15 h (overnight) fixation in 1% glutaraldehyde and 2 h fixation in 1 % OsO4. After fixation, en block staining with aqueous uranyl acetate was conducted for 30 min. This was followed by ethanol dehydration and infiltration in resin. Additionally, the rapid method was conducted with LR White resin compared to Spurr's epoxy resin in the control. The culture bacteria, protein matrix and dispersed fat globules were clearly visible and were unaltered by the rapid method.

Summary We used DNA macroarray analysis to identify genes that respond to the status of the intracellular acetyl phosphate (acP) pool. Genes whose expression correlated negatively with the ability to synthesize acP (i.e. negatively regulated genes) function primarily in flagella biosynthesis, a result consistent with observations that we published previously (Prus and Wolfe, 1994, Mol Microbiol 12: 973‐984). In contrast, genes whose expression correlated positively with the ability to synthesize acP (i.e. positively regulated genes) include those for type 1 pilus assembly, colanic acid (capsule) biosynthesis and certain stress effectors. To our knowledge, this constitutes the first report that these genes may respond to the status of the intracellular acP pool. Previously, other researchers have implicated flagella, type 1 pili, capsule and diverse stress effectors in the formation of biofilms. We therefore tested whether cells altered in their ability to metabolize acP could construct normal biofilms, and found that they could not. Cells defective for the production of acP and cells defective for the degradation of acP could both form biofilms, but these biofilms exhibited characteristics substantially different from each other and from biofilms formed by their wild-type parent. We confirmed the role of individual cell surface structures, the expression of which appears to correlate with acP levels, in fim or fli mutants that cannot assemble type 1 pili or flagella respectively. Thus, the information gained by expression profiling of cells with altered acP metabolism indicates that acP may help to co-ordinate the expression of surface structures and cellular processes involved in the initial stages of wild-type biofilm development.

Described here is a simple and versatile technique for capturing small samples of the thin biofilm that is located at the meniscus of almost every air-water interface (AWI), an electrostatically distinct aquatic domain/habitat. The method uses a microscope slide (and other supporting surfaces) coated in the lab with a collodion membrane, which has a strong affinity for the upper surface of the AWI biofilm. The structural integrity of the biofilm is maintained during the capture process, and components of the biofilm are effectively separated from the subtending liquid. The captured thin biofilm can be analyzed in many ways including almost any form of light, electron, and atomic force optics; and spatially significant molecular analyses may be performed on the captured biofilm or its components.

Described here is a simple and versatile technique for capturing small samples of the thin biofilm that is located at the meniscus of almost every air-water interface (AWI), an electrostatically distinct aquatic domain/habitat. The method uses a microscope slide (and other supporting surfaces) coated in the lab with a collodion membrane, which has a strong affinity for the upper surface of the AWI biofilm. The structural integrity of the biofilm is maintained during the capture process, and components of the biofilm are effectively separated from the subtending liquid. The captured thin biofilm can be analyzed in many ways including almost any form of light, electron, and atomic force optics; and spatially significant molecular analyses may be performed on the captured biofilm or its components.

Mats consisting of the large sulfide-oxidizing bacterium, Beggiatoa, were collected from the sediment/water interface at several locations in the Gulf of Mexico. The collection sites were associated with the presence of petroleum hydrocarbons or the microbial breakdown products of the hydrocarbons. The morphologies of the mats varied with the nature of the underlying sediments, and some mats were pigmented either yellow or orange instead of the usual white. At one site, beggiatoas were found that had a diameter of nearly 200 μm, making them the largest prokaryotic organism known. In filaments with a diameter of over approximately 10 μm the cytoplasm was restricted to a thin layer immediately underlying the cell membrane, and the majority of the cell consisted of a vacuole with unknown contents. Beggiatoa filaments often rotated as they moved by gliding. Parallel rows of 15 nm diameter pores were found on the surface of the beggiatoas. The pores may have been wound in a spiral fashion around the cell. These pores may be involved in the gliding motility of the bacteria by the motion imparted by the excretion of slime through the pores. Several structures with the typical morphology of prokaryotic cells but lacking a cell wall were found within the vacuolar and cytoplasmic portions of the hollow beggiatoas. Some of these internal “symbionts” ultrastructurally resembled methanotrophic bacteria like those that have been seen in animals taken from vent areas. Other symbionts ultrastructurally resembled autotrophic bacteria with carboxysome-like structures. These internal symbionts may enable the Beggiatoa to grow in different environments on different carbon sources. They also provide important evidence for the endosymbiotic theory of the evolution of internal organelles of eukaryotic organisms. © 1996 Wiley-Liss, Inc.

In the green marine alga Dunaliella tertiolecta, a CO2-concentrating mechanism is induced when the cells are grown under low-CO2 conditions (0.03% CO2). To identify proteins induced under low-CO2 conditions the cells were labelled with 35SO4 2−, and seven polypeptides with molecular weights of 45, 47, 49, 55, 60, 68 and 100 kDa were detected. The induction of these polypeptides was observed when cells grown in high CO2 (5% CO2 in air) were switched to low CO2, but only while the cultures were growing in light. Immunoblot analysis of total cell protein against pea chloroplastic carbonic anhydrase polyclonal antibodies showed immunoreactive 30-kDa bands in both high- and low-CO2-grown cells and an aditional 49-kDa band exclusively in low-CO2-grown cells. The 30-kDa protein was shown to be located in the chloroplast. Western blot analysis of the plasmamembrane fraction against corn plasma-membrane AT-Pase polyclonal antibodies showed 60-kDa bands in both high- and low-CO2 cell types as well as an immunoreactive 100-kDa band occurring only in low-CO2-grown cells. These results suggest that there are two distinct forms of both carbonic anhydrase and plasma-membrane ATPase, and that one form of each of them can be regulated by the CO2 concentration.

Microbial mats were collected from a variety of sites near hydrocarbon vents along the slope in the northern Gulf of Mexico and, for comparison, from Warm Mineral Springs, Florida, USA. A predominant microorganism in each of the mats was the giant bacterium,Beggiatoa. Diameters of the bacterial filaments ranged from about 6 µm to approximately 200 µm. The latter organisms are the largest prokaryotic organisms yet found. All filaments over about 10 µm in diameter contained a large central vacuole, producing a cell with the cytoplasm as a cylindrical tube underlying the cytoplasmic membrane. Sulfur globules were confined to this peripheral layer. Push cores often contained pyrite tubules whose appearance is suggestive of aBeggiatoa origin. Determinations ofδ13C inBeggiatoa mats from vents along the Louisiana slope yielded values in the range of −26.6 to −27.9‰ (PDB), suggesting an unusually high degree of isotope fractionation (−24.9‰) relative to the carbon source in the ambient seawater, which is typical of sulfur-oxidizing chemoautotrophs. The presence of SO (elemental sulfur) within cells ofBeggiatoa resulting from oxidation of H2S supports the importance of bacterial sulfate reduction processes in the underlying vents for the sustenance of theBeggiatoa mats.

High-level expression offtsA, an essential cell division gene inEscherichia coli, inhibits cell septation and causes the formation of filaments that develop spherical bulges up to 4 μm in diameter. These bulges may emanate from septation sites, since they were evenly spaced in relation to one another and to the cell poles. Electron microscopic examination of thin sections through the bulged regions reveals large electron-lucent, plate-like regions that appear to originate from the membrane. In addition, these bulging filamentous cells contain more hexosamine per mass than control cells. A model is proposed that the electron-lucent regions observed by electron microscopy are caused by peptidoglycan accumulation stimulated by the overexpression offtsA.

Capsular polysaccharide (CPS) is a major virulence factor in Vibrio vulnificus , and encapsulated strains have an opaque, smooth (OpS) colony morphology, while nonencapsulated strains have a translucent, smooth (TrS) colony morphology. Previously, we showed that OpS and TrS parental strains can yield a third colony type, rugose (R), and that the resulting strains, with the OpR and TrR phenotypes, respectively, form copious biofilms. Here we show that while OpR and TrR strains both produce three-dimensional biofilm structures that are indicative of rugose extracellular polysaccharide (rEPS) production, OpR strains also retain expression of CPS and are virulent in an iron-supplemented mouse model, while TrR strains lack CPS and are avirulent. Chlorine resistance assays further distinguished OpR and TrR isolates as exposure to 3 μg/ml NaOCl eradicated both OpS and OpR strains, while both TrS and TrR strains survived, but at rates which were significantly different from one another. Taken together, these results further emphasize the importance of CPS for virulence of V. vulnificus and establish a correlation between CPS expression and chlorine sensitivity in this organism. Using reverse transcriptase PCR, we also identified a nine-gene cluster associated with both CPS and rEPS expression in V. vulnificus , designated the wcr ( c apsular and r ugose polysaccharide) locus, with expression occurring primarily in R variants. The latter results set the stage for characterization of functional determinants which individually or collectively contribute to expression of multiple EPS forms in this pathogen.

Larvae of the mayfly ( Drunella grandis [Eaton]) from Diamond Fork Creek, Utah, were covered with a heavy growth of the sulfide-oxidizing bacterium Thiothrix. The bacterium did not seem to harm the mayfly, but the Thiothrix trichomes were parasitized by three morphologically distinct bacteria, two of which were cytoplasmic and one of which was probably periplasmic. At least two of the parasites destroyed the cytoplasmic contents of the Thiothrix sp., thus killing the host cell. Attempts to obtain the parasites in pure culture were unsuccessful.

Spermatozoa from diploid and tetraploid Pacific oysters (Crassostrea gigas) were examined after anisotonic fixation. Morphological anomalies, such as membrane rupture, detached tails, and the formation of tail vesicles (typically associated with damage attributable to procedures such as cryopreservation) were observed; the Mantel-Haenszel Chi-square test indicated a strong association between the anomalies and fixative osmolality (P

The ionizing-radiation-resistant fractions of two soil bacterial communities were investigated by exposing an arid soil from the Sonoran Desert and a nonarid soil from a Louisiana forest to various doses of ionizing radiation using a 60 Co source. The numbers of surviving bacteria decreased as the dose of gamma radiation to which the soils were exposed increased. Bacterial isolates surviving doses of 30 kGy were recovered from the Sonoran Desert soil, while no isolates were recovered from the nonarid forest soil after exposure to doses greater than 13 kGy. The phylogenetic diversities of the surviving culturable bacteria were compared for the two soils using 16S rRNA gene sequence analysis. In addition to a bacterial population that was more resistant to higher doses of ionizing radiation, the diversity of the isolates was greater in the arid soil. The taxonomic diversity of the isolates recovered was found to decrease as the level of ionizing-radiation exposure increased. Bacterial isolates of the genera Deinococcus , Geodermatophilus , and Hymenobacter were still recovered from the arid soil after exposure to doses of 17 to 30 kGy. The recovery of large numbers of extremely ionizing-radiation-resistant bacteria from an arid soil and not from a nonarid soil provides further ecological support for the hypothesis that the ionizing-radiation resistance phenotype is a consequence of the evolution of other DNA repair systems that protect cells against commonly encountered environmental stressors, such as desiccation. The diverse group of bacterial strains isolated from the arid soil sample included 60 Deinococcus strains, the characterization of which revealed nine novel species of this genus.

A method has been developed for collecting air-water interface (AWI) microbes and biofilms that enables analysis of the same sample with various combinations of bright-field and fluorescence light microscopy optics, scanning and transmission electron microscopy (TEM), and atomic force microscopy. The identical sample is then subjected to molecular analysis. The sampling tool consists of a microscope slide supporting appropriate substrates, TEM grids, for example, that are removable for the desired protocols. The slide with its substrates is then coated with a collodion polymer membrane to which in situ AWI organisms adhere upon contact. This sampling device effectively separates the captured AWI bacterial community from the bulk water community immediately subtending. Preliminary data indicate that the AWI community differs significantly from the water column community from the same sample site when both are evaluated with microscopy and with 16S ribosomal DNA sequence-based culture-independent comparisons. This microbe collection method can be used at many levels in research and teaching.

This paper reports transmission electron microscopy (TEM) and immunofluorescence evidence for phragmoplast-mediated cytokinesis in the green alga, Trentepohlia (Ulvophyceae, Chlorophyta). This type of cytokinesis is normally found only in land plants and some charophycean green algae. Earlier TEM work documented a phragmoplast in another trentepohlialean alga, Cephaleuros. Numerous molecular studies have shown that both Trentepohlia and Cephaleuros are clearly not in the charophycean (streptophyte) lineage of green algae. Also, details of vegetative cytokinesis in Trentepohlia and Cephaleuros indicate differences from the processes and structures observed in the streptophytes. Parallel evolution could be the explanation for a phragmoplast-mediated cytokinesis in both the chlorophycean Trentepohliales and the streptophyte lineage of charophycean green algae and land plants.

The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The most prominent protein in the pyrenoid is the enzyme ribulose-1,5-bisphosphate carboxylaseoxygenase (Rubisco). We have investigated whether the pyrenoid is present in strains of Chlamydomonas reinhardtii Dangeard containing mutations in the chloroplast rbcL gene. The mutants examined include a nonsense mutant lacking Rubisco, 18-7G, a missense mutant with an inactive Rubisco, 10-6C, and a temperature-sensitive mutant, 68-4PP, which contains Rubisco at room temperature but lacks the protein at 35°C. Normally, each C. reinhardtii cell has one chloroplast containing one large pyrenoid. In the nonsense mutant and 68-4PP at the non-permissive temperature no pyrenoid was observed. In the other strains, even those with an inactive Rubisco, the pyrenoid appeared normal. These results indicate that the presence of the Rubisco protein is necessary for the formation of a normal pyrenoid in C. reinhardtii. It is also clear that the Rubisco does not have to be active for normal pyrenoid formation, as strains 10-C and F-60 had morphologically normal pyrenoids.

The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The pyrenoid contains the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase and is sometimes surrounded by a carbohydrate sheath. We have observed in the unicellular green alga Chlamydomonas reinhardtii Dangeard that the pyrenoid starch sheath is formed rapidly in response to a decrease in the CO2 concentration in the environment. This formation of the starch sheath occurs coincidentally with the induction of the CO2-concentrating mechanism. Pyrenoid starch-sheath formation is partly inhibited by the presence of acetate in the growth medium under light and low-CO2 conditions. These growth conditions also partly inhibit the induction of the CO2-concentrating mechanism. When cells are grown with acetate in the dark, the CO2-concentrating mechanism is not induced and the pyrenoid starch sheath is not formed even though there is a large accumulation of starch in the chloroplast stroma. These observations indicate that pyrenoid starch-sheath formation correlates with induction of the CO2-concentrating mechanism under low-CO2 conditions. We suggest that this ultrastructural reorganization under lowCO2 conditions plays a role in the CO2-concentrating mechanism C. reinhardtii as well as in other eukaryotic algae.

A plasmid, pDLL4, was isolated from a Tn5tac1 mutagenesis experiment with plasmid pZAQ. When pDLL4 was transformed into wild-type rod-shaped cells, it caused cells in the population to become curved (C-shaped or convoluted). The Tn5tac1 transposon was integrated within the carboxyl end of the ftsA gene in pDLL4. This mutation was designated ftsAc. Subcloning ftsAc DNA into another plasmid vector verified that the curved-cell phenotype was caused by the expression of this altered gene. DNA sequence analysis of the ftsAc mutation revealed that the transposition event changed the DNA so that the last 28 amino acids of the FtsA protein were lost and 5 new amino acids were added. A radioactive peptide band corresponding to this truncated FtsAc protein was identified by a T7 promoter-T7 polymerase protein labeling system. Observations of thin sections of these curved cells with an electron microscope revealed aggregates of striated cylindrical structures traversing the cytoplasm. The ends of these aggregates appear to be at or near the cell membrane. The linear periodicity of the cylinders was approximately 11 nm, and the diameter of a cylinder was about 15 nm. Aggregates of as many as five cylinders were arrayed diagonally to the long axis of the curved cells, a finding that suggests that some type of internal organization may be causing the curved cell shape.

Cytokinesis in apical cells of actively growing cultures of Cephaleuros parasiticus Karsten sporangiate thalli was examined with transmission electron microscopy. A massive, interzonal cytokinetic microtubule spindle is anchored at its poles to the medial surfaces of the daughter nuclei at telophase. Later, the daughter nuclei are widely separated and no longer associated with the interzonal spindle; however, the spindle retains its shape and becomes a distinct phragmoplast with an array of vesicles, presumably derived from dictyosomes, aligned in the division plane. Fusion of the vesicles gives rise to a thin cell plate. Some bundles of microtubules in the spindle appear to mark the sites of plasmodesmata formation, but no endoptasmic reticulum is directly involved in plasmodesmata formation. No infurrowing or phycoplast array of microtubules is involved in the cytokinesis. The relationship, if any. between the metaphase-anaphase mitotic microtubule system and the interzonal cytokinetic spindle has not been determined. Cephaleuros parasiticus isone of only four green algae now known to contain a higher plant-like phragmoplast and cytokinetic process. The observations reported can be interpreted as very strong evidence for a phylogenetic affinity between the Trentepohliaceae and the Charophyceae, but consideration of ulvophycean features of the Trentepohliaceae such as motile cell ultrastructure and life histories precludes unequivocal assignment of the family to either the Charophyceae or Ulvophyceae.

The structure and orientation of polymer and clay platelets in multilayered, micrometer-thick nanocomposite films was investigated by means of scanning electron microscopy (SEM), atomic force microscopy (AFM), optical microscopy and small-angle neutron scattering (SANS). Microscopic and scattering methods complementarily measured the morphology and shear-induced orientation of polymer and platelets in films when spread layer by layer from a network-like polymer clay solution. During the drying process, the polymer clay network collapsed and clay platelets oriented inside the network with the clay surface normal perpendicular to the spread direction (x–z plane). On nanometer length scales, SANS and AFM yielded structure and orientation of platelets and polymer in and perpendicular to the spread direction of the film. SEM investigations led to the observation of unexpected morphology on the micron length scale. SEM did not detect boundaries between single spread, micron-thick film layers but, surprisingly, showed a highly ordered and layered structure of the film. Polarized light microscopy showed differences in birefringence in each plane.

Transmission electron microscope studies of the quadriflagellate zoospores of the parasitic, subaerial green alga Cephaleuros virescens Kunze have provided a basis for comparison between these motile cells and biflagellate gametes previously examined.1 As seen in cross-section (Fig. 1), the four basal bodies form a trapezoid in which the two upper basal bodies are closer together than the two lower basal bodies. The basal bodies are parallel, overlapping, and interdigitated. Two flagella are inserted into either side of the apical papilla and terminate in diagonally opposed basal bodies. The bilaterally keeled flagella (Fig. 2) are often closely appressed thereby creating a biflagellate appearance. Each of the four basal bodies is associated with a microtubular spline which extends posteriorly beneath the plasmalemma. A densely stained flagellar cap is present at the end of each basal body and together with the terminal region of its spline, forms a multilayered structure (MLS). Although the MLSs associated with the two lower basal bodies (Fig. 3) are virtually identical to those found in the gametes, those associated with the upper basal bodies are morphologically different or positioned in such a manner that “typical” sectional views cannot be obtained. The observations reveal that although minor differences exist, the zoospores of Cephaleuros are similar to those of Phycopeltis,2 a related genus.