Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine.

Bupivacaine, a local anesthetic and cationic amphiphile, forms stable liposomal-like structures upon direct mixing with plasmid DNA in aqueous solutions. These structures are on the order of 50-70 nm as determined by scanning electron microscopy, and are homogeneous populations as analyzed by density gradient centrifugation. The DNA within these structures is protected from nuclease degradation and UV-induced damage in vitro. Bupivacaine:DNA complexes have a negative zeta potential (surface charge), homogeneous nature, and an ability to rapidly assemble in aqueous solutions. Bupivacaine:DNA complexes, as well as similar complexes of DNA with other local anesthetics, have the potential to be a novel class of DNA delivery agents for gene therapy and DNA vaccines.

Polymeric display of immunogenic epitopes from herpes simplex virus and transmissible gastroenteritis virus surface proteins on an enteroadherent fimbria.

The strong immunogenicity of bacterial fimbriae results from their polymeric and proteinaceous nature, and the protective role of these immunogens in experimental or commercial vaccines is associated with their capacity to induce antiadhesive antibodies. Fimbria-mediated intestinal colonization by enteropathogens typically leads to similar antibody responses. The possibility of taking advantage of these properties was investigated by determining whether enteroadhesive fimbriae, like the 987P fimbriae of enterotoxigenic Escherichia coli, can serve as carriers for foreign antigens without losing their adhesive characteristics. Random linker insertion mutagenesis of the fasA gene encoding the major 987P subunit identified five different mutants expressing wild-type levels of fimbriation. The linker insertion sites of these mutants were used to introduce three continuous segments of viral surface glycoproteins known to be accessible to antibodies. These segments encode residues 11 to 19 or 272 to 279 of herpes simplex virus type 1 (HSV-1) glycoprotein D [gD(11-19) and gD(272-279), respectively] or residues 379 to 388 of the transmissible gastroenteritis virus (TGEV) spike protein [S(379-388)]. Studies of bacteria expressing fimbriae incorporating mutated FasA subunits alone or together with wild-type FasA subunits (hybrid fimbriae) indicated that foreign epitopes were best exported and displayed on assembled fimbriae when they were inserted near the amino terminus of FasA. Fimbriated bacteria expressing FasA subunits carrying the HSV gD(11-19) or the TGEV S(379-388) epitope inserted between the second and third residues of mature FasA elicited high levels of foreign epitope antibodies in all rabbits immunized parenterally. Antibodies against the HSV epitope were also shown to recognize the epitope in the context of the whole gD protein. Because the 987P adhesive subunit FasG was shown to be present on mutated fimbriae and to mediate bacterial attachment to porcine intestinal receptors, polymeric display of foreign epitopes on 987P offers new opportunities to test the potential beneficial effect of enteroadhesion for mucosal immunization and protection against various enteric pathogens.

The mouse pale ear (ep) mutation is the homologue of human Hermansky-Pudlak syndrome.

The recessive mutation at the pale ear (ep) locus on mouse chromosome 19 was found to be the homologue of human Hermansky-Pudlak syndrome (HPS). A positional cloning strategy using yeast artificial chromosomes spanning the HPS locus was used to identify the HPS gene and its murine counterpart. These genes and their predicted proteins are highly conserved at the nucleotide and amino acid levels. Sequence analysis of the mutant ep gene revealed the insertion of an intracisternal A particle element in a protein-coding 3' exon. Here we demonstrate that mice with the ep mutation exhibit abnormalities similar to human HPS patients in melanosomes and platelet-dense granules. These results establish an animal model of HPS and will facilitate biochemical and molecular analyses of the functions of this protein in the membranes of specialized intracellular organelles.

Borrelia burgdorferi DNA in the urine of treated patients with chronic Lyme disease symptoms. A PCR study of 97 cases.

The presence of Borrelia burgdorferi DNA was established by PCR from urine samples of 97 patients clinically diagnosed as presenting with symptoms of chronic Lyme disease. All patients had shown erythema chronica migrans following a deer tick bite. Most of the patients had been antibiotic-treated for extended periods of time. We used three sets of primer pairs with DNA sequences for the gene coding of outer surface protein A (OspA) and of a genomic sequence of B. burgdorferi to study samples of physician-referred patients from the mideastern USA. Controls from 62 healthy volunteers of the same geographic areas were routinely carried through the procedures in parallel with patients' samples. Of the 97 patients, 72 (74.2%) were found with positive PCR and the rest with negative PCR. The 62 healthy volunteers were PCR negative. It is proposed that a sizeable group of patients diagnosed on clinical grounds as having chronic Lyme disease may still excrete Borrelia DNA, and may do so in spite of intensive antibiotic treatment.

Ordered translocation of 987P fimbrial subunits through the outer membrane of Escherichia coli.

The 987P fimbria of enterotoxigenic Escherichia coli is a heteropolymeric structure which consists essentially of a major FasA subunit and a minor subunit, the FasG adhesin. The latter harbors the binding moiety for receptor molecules on piglet intestinal epithelial cells. In this study, anti-FasF antibody probes were developed and used to demonstrate that the FasF protein represents a new minor fimbrial component. FasF was identified in highly purified fimbriae, and its sequence demonstrated significant levels of similarity with that of FasA. Immune electron microscopy localized both the FasG and FasF proteins at the fimbrial tip as well as at broken ends and at various intervals along the fimbrial length. The presence of these minor proteins in purified 987P fimbriae was corroborated by enzyme-linked immunosorbent assay inhibitions. Finally, the use of nonfimbriated fasG, fasF, and fasA mutants indicated that subunit translocation through the outer membrane follows a specific order, FasG being the first, FasF being the second, and FasA being the third type of exported subunit. Since fimbriae are thought to grow from the base, FasG is proposed to be a tip adhesin and FasF is proposed to be a linker molecule between the adhesin and the fimbrial shaft. Moreover, export of FasG (or FasF) in the absence of FasF (or FasA) indicates that during the process of fimbrial biogenesis in the outer membrane, translocating events precede the initiation of subunit heteropolymerization.

Physical and functional characterization of the Bacillus subtilis spoIIM gene.

The spoIIM locus of Bacillus subtilis is the most recently discovered of six genetic loci in which mutations can prevent the synthesis of a normal asymmetric septum or prevent migration of the septal structure to engulf the forespore compartment of the sporangium. Ultrastructure studies of a spoIIM mutant confirmed a block prior to the completion of engulfment. Introduction of a spoIIM mutation into a panel of strains containing lacZ fusions belonging to different regulatory classes allowed us to determine that the spoIIM gene product is required for the efficient expression of genes transcribed by sigma G-associated RNA polymerase but is not required for the expression of sigma F-controlled genes, including spoIIIG, which encodes sigma G. The results of complementation studies, gene disruption analysis, and DNA sequencing revealed that the spoIIM locus contains a single sporulation-essential gene encoding a polypeptide with a predicted molecular mass of 24,850 Da. The predicted spoIIM gene product is highly hydrophobic and very basic, and it does not exhibit significant homology to sequence files in several major data bases.

Zones of membrane adhesion in the cryofixed envelope of Escherichia coli.

The envelopes of Escherichia coli B and E. coli K29 were examined using cryofixation and freeze substitution. Emphasis was directed toward the question whether membrane adhesion zones (which connect inner membrane (IM) and outer membrane (OM) after plasmolysis in 10-20% sucrose) can be visualized with the use of cryotechniques. Plasmolysis in 10-20% sucrose was observed to have no effect on cell viability. We found that simple plunge-freezing methods preserve adhesion sites, whereas these sites were not observed after impact-freezing. Also, plasmolysis "bays," visible in light microscopic preparations of living cells, were seen to be maintained intact after plunge-freezing. Employment of photocrosslinking with UV-flashes before or after plasmolysis showed a significant increase in the number of adhesion areas compared to noncrosslinked specimens. To control the contact speed of the specimen during immersion into the cryogen, a hollow rotor was constructed in which the cryogenic liquid is moving at desired high speeds. Adhesion sites presented themselves in the plasmolyzed cell as sites of close contact of the outer and inner membrane, an arrangement that would leave very limited space for peptidoglycan layers at the contact site of the two membranes. Adhesion sites may occur either as single, isolated sites or within stretches of IM/OM apposition where they appear to function as "spot welds" between the two membranes. Exposure of cells to sucrose concentrations of 35% caused rupture of adhesions with cytoplasmic fragments remaining attached to the envelope. The cryofixation procedures described here do not presently yield the number of membrane adhesions obtainable with conventional aldehyde fixation. However, since the combination of millisecond photocrosslinking and cryofixation of plasmolyzed cells resulted in a higher membrane stabilization and in an increase of the number of adhesion sites, this combination appears to be a useful tool for the analysis of sensitive membrane structures.

Tubular spinae are long-distance connectors between bacteria.

The marine pseudomonad D71 (NCMB 2018) ['Spinomonas maritima'] can be induced to produce long tubular surface appendages (spinae) in a growth medium of low osmolarity. In general, spina-carrying cells show these appendages with open distal ends. We examined cultured cells by scanning and transmission electron microscopy, using both critical-point drying and thin sectioning after embedding with agarose protection. By scanning electron microscopy, spinae were observed that connected cells over distances of several micrometers. Ultrathin sections often revealed an additional layer outside the outer membrane, resembling an S-layer. The inner and outer cell membranes were often joined at spina-insertion areas. Furthermore, evidence was found in ultrathin sections for uninterrupted tubes connecting two cells over a distance of up to 7 microns. We propose, therefore, that spinae form the framework for wide open cell clusters; we hypothesize that these spinae might also permit an exchange of cell-to-cell signals.

Lanthanide accumulation in the periplasmic space of Escherichia coli B.

Treatment of growing Escherichia coli B with lanthanide ions [lanthanum(III), terbium(III), and europium(III)] and subsequent aldehyde-OsO4 fixation caused areas of high contrast to appear within the periplasm (the space between inner and outer membrane of the cell envelope). X-ray microanalysis of ultrathin sections of Epon-embedded or acrylic resin-embedded cells revealed the presence of the lanthanide and of phosphorus in the areas, whose contrast greatly exceeded that of other stained structures. Comparatively small amounts of the lanthanide were also present in the outer membrane and in the cytoplasm. The distribution of the periplasmic areas of high contrast was found to be random and not clustered at areas of current or future septum formation. Irregular cell shapes were observed after lanthanide treatment before onset of fixation. In contrast to glutaraldehyde-OsO4 fixation, glutaraldehyde used as the sole fixer caused a scattered distribution of the lanthanide. Cryofixation (slam-freezing) and freeze substitution revealed a lanthanum stain at both the periplasm and the outer part of the outer membrane. Deenergization of the cell membrane by either phage T4 or carbonyl cyanide m-chlorophenylhydrazone abolished the metal accumulation. Furthermore, addition of excess calcium, administered together with the lanthanide solution, diminished the quantity and size of areas of high contrast. Cells grown in media of high NaCl concentration revealed strongly stained areas of periplasmic precipitates, whereas cells grown under low-salt conditions showed very few high-contrast patches in the periplasm. Terbium treatment (during fixation) enhanced the visibility of the sites of inner-outer membrane contact (the membrane adhesion sites) in plasmolized cells, possibly as the result of an accumulation of the metal at the adhesion domains. The data suggest a rapid interaction of the lanthanides with components of the cell envelope, the periplasm, and the energized inner membrane.

Automated electrokinetic analysis; description and application in virology and cell biology.

The electrophoretic mobility (EPM) of selected macromolecules in solution was shown to be accurately determined using an automated electrokinetic analyzer, the PenKem S3000. In addition, the S3000 was used to monitor the effects of T4 phage infection on the EPM of Escherichia coli B. EPM, expressed as the ratio of velocity in microns/sec to field strength in V/cm, was measured for calf thymus DNA, for pneumococcal capsular polysaccharide serotype 3 (PCP-3), and for bovine serum albumin (BSA) unbound in solution; values of -3.05, -2.736 and -1.176, respectively, were obtained. The EPM of these macromolecules remained the same when they were bound to latex beads. The S3000 may therefore be suitable for measurement of the EPM of unbound macromolecules. The EPM of T4 phage in solution was measured to be -1.203. However, both the zwitterionic latex-bound T4 phage as well as T4 phage disrupted by ultrasonication exhibited an EPM of approximately -2.50, suggesting to us that binding to zwitterionic latex may cause release of phage DNA. The notion that phage DNA is responsible for the increased negative charge was supported by the observation that the EPM of E. coli B increased to the level of free DNA within 5 min when E. coli B (the host cell for phage T4) had been exposed to 10 phage particles per cell. Electronmicrographs of phage infected E. coli B cells showed numerous strands of free DNA at the bacterial surface. It is concluded that the S3000 not only measures the EPM of macromolecules in solution but that the instrument can be used also to monitor the behavior of the host cell surface in response to attachment of viral particles.

The electrophoretic mobility of gram-negative and gram-positive bacteria: an electrokinetic analysis.

The electrophoretic mobility (EPM) of a variety of Gram-negative and Gram-positive bacteria was measured with a Penkem S3000 analyser. Under standard growth conditions and neutral pH all cells displayed a negative EPM. The polysaccharide capsules of Escherichia coli strains K1, K5, K29 and K30 generated the highest EPM; to a lesser and varying degree O-antigens with charged groups and core lipopolysaccharides also contribute to the net EPM. Very little negative EPM was measured in suspension cultures of the gliding bacterium Cytophaga U67. No difference in the EPM was observed between rapidly growing and stationary-phase E. coli B. De-energization of the cell membranes by carbonyl cyanide m-chlorophenylhydrazone (CCCP) did not affect the EPM of wild-type and deep rough mutants of E. coli; and the EPM of Cytophaga U67 and Acholeplasma laidlawii remained unaltered by CCCP when measured in their respective growth media. Extrusion of filamentous bacteriophage f1 from cells of its host, E. coli A95, caused a shift to a higher negative EPM. We also measured a variety of Gram-positive strains, all of which displayed different EPMs. When membrane fractions of E. coli were adsorbed to latex spheres, characteristic differences between the EPM of beads coated with either inner or outer membrane were observed. The results suggest that the rapid EPM analysis is a useful tool to study the net electric charge of microorganisms and to examine changes of surface properties during interaction of cells with viruses, proteins (antibody) and charged antibiotics.

Localization of penicillin-binding protein 1b in Escherichia coli: immunoelectron microscopy and immunotransfer studies.

We report the localization of penicillin-binding protein 1b (PBP 1b) in Escherichia coli KN126 and in an overproducing construct containing plasmid pHK231. We used PBP 1b-specific antiserum for the immunoelectron microscopy of ultrathin sections of whole cells and for immunoelectrophoresis of cytoplasm and isolated membrane fractions. We studied ultrathin sections of both glutaraldehyde-fixed cells that had been embedded after progressively lowering the temperature and cryofixed cells that had been freeze-substituted in Lowicryl K4M and HM20. Most of the PBP 1b-specific label was observed in the inner membrane (IM) and the adjacent cytoplasm, much less was observed in the outer membrane (OM); appreciable amounts were also seen in the bulk cytoplasm. Distribution and intensity of label were both temperature dependent: temperature shift-up to 37 degrees C, causing PBP 1b overproduction in the construct, showed a statistically highly significant increase in label of the IM, including a cytoplasmic zone (of at least 30 nm in depth) adjacent to the IM, a zone we termed the membrane-associated area. Concomitant with the temperature shift-up, a decrease in label density was observed in the bulk cytoplasm. Increased label was also found in IM-OM contact areas (zones of membrane adhesion). The periplasm did not show significant label. Western blotting (immunoblotting) revealed PBP 1b in most of the isolated membrane fractions; however, the highest label density was found in membrane fractions of intermediate density, supporting the suggestion of an increased concentration of PBP 1b in the membrane adhesion zones. In summarizing, we propose that PBP 1b is present in the membrane-associated area of the cytoplasm, from where proteins (such as PBP 1b or thioredoxin) gain access to their specific insertion sites in the envelope. The use of several methods of immunoelectron microscopy provided the first unequivocal evidence for localization of PBP 1b at membrane adhesion sites. Since such sites are specifically labeled with anti-PBP 1b serum, we hypothesize that they contain parts of the machinery for assembly and growth of the murein layer.

Visualization of the bacterial polysaccharide capsule.

The highly hydrated capsule of E. coli strains is composed of a large number of polysaccharide fibers of which the thinnest measure about 2 nm in width. The fibers may span the entire distance from the outer membrane to the outer rim of the capsule and show a propensity to associate with each other to form thicker filaments. Presence of thick filaments may also indicate a partial collapse of the capsular organization due to removal of water. The in vivo capsule represents a relatively open structure with the negatively charged polysaccharide fibers permitting the binding of large quantities of water and ions, and providing intracellular space for diffusing molecules to access the envelope membranes even in conditions of high cell density. Negative charge and steric hindrance of the polysaccharide strands protect the cells against attack by a large variety of harmful macromolecules and against infection by most bacteriophages. Two types of procedure have been most successful in maintaining the size and overall structure of the capsule: (a) the interaction of cationic molecules with the in vivo capsule, and (b) the use of antibody to stabilize capsules for subsequent dehydration and plastic embedding. A further type of potentially useful procedure, cryofixation and cryosubstitution, has shown interesting results in a number of cases. These techniques are expected to play a significant role in structural studies in the near future. The sites of export of capsular antigen have been described in earlier conventional electron microscopic studies. Data obtained from the recent technique of "on-section" labeling support the model that both the capsular antigen and the O antigen are assembled at junctions of the inner and outer membrane. It is anticipated that one will be able to discern in greater ultrastructural detail the membranes at which the antigen is translocated. Novel membrane fixation and isolation techniques will have to be established and employed in a combination of sensitive microscopic techniques and immuno- and enzyme localization methods. These developments will make it possible to explore questions pertaining to the maintenance and structural organization of microbial capsules and the functional interaction of polysaccharides with natural surfaces, man-made substances and drugs.

Association of thioredoxin with the inner membrane and adhesion sites in Escherichia coli.

The intracellular localization of thioredoxin in Escherichia coli was determined by immunoelectron microscopy and correlated to previous biochemical data which had suggested that thioredoxin resides at inner-outer membrane adhesion sites. Since a considerable amount of thioredoxin was lost during preparation of cells for electron microscopy, we immobilized the protein with the heterobifunctional photoactivatable cross-linker p-azidophenacylbromide before the cells were fixed with aldehyde and embedded in Lowicryl K4M. Thin sections were labeled with affinity-purified antithioredoxin antiserum and protein A-gold complexes. Densities of immunolabel in a designated membrane-associated area and in the rest of the cytoplasm were compared and the data were statistically evaluated. Wild-type strain W3110 and strain SK3981, an overproducer of thioredoxin, exhibited increased labeling at the inner membrane and its adjacent cytoplasmic area. In contrast, the more centrally located cytoplasm of both strains showed much lower label density. This label distribution did not change with cell growth or in the stationary phase. Immunolabel was often found at bridges between the inner and outer membranes; this result is consistent with a model which places at least a portion of the thioredoxin at membrane adhesion sites, corresponding to an osmotically sensitive cytoplasmic compartment bounded by a hybrid inner-outer membrane (C.A. Lunn and V. Pigiet, J. Biol. Chem. 257:11424-11430, 1982; C.A. Lunn and V. Pigiet, J. Biol. Chem. 261:832-838, 1986). Specific label was absent in the periplasmic space.

Effects of bacteriophage fd infection on Escherichia coli HB11 envelope: a morphological and biochemical study.

Phage fd-infected host bacteria revealed three characteristic changes in their envelope. (i) The preferred cleavage plane during freeze-fracturing shifted from the inner to the outer membrane (OM). (ii) The total lipids of the OM of the infected cells increased by 25% without major alterations in the relative concentration of phospholipids. We propose that such an increase would to some extent contribute to the change in the freeze-fracture behavior of the OM; however, additional factors will have to play a role in the apparent fracture resistance of the inner membrane. (iii) Ultrathin sectioning and immunolabeling methods revealed that extrusion of fd phages takes place at membrane adhesion sites of the infected cells.

Capsule of Escherichia coli K29: ultrastructural preservation and immunoelectron microscopy.

The polysaccharide capsule of Escherichia coli K29 fully surrounds the microorganism and thus occupies an extracellular space ca. 20 times larger in volume than that of the decapsulated cell. Since more than 95% of the capsule consists of water, dehydration for electron microscopy causes the material to collapse. We describe here a method for embedding the capsule in an uncollapsed form. Dehydration of gelatin-enrobed, glutaraldehyde-fixed cells was performed in dimethyl formamide. The cells were embedded in Lowicryl K4M with the "progressive lowering of temperature" method and UV polymerization. In ultrathin sections, the capsule can be identified by its low electron contrast. It occupies a layer 3/4 micron thick thick and shows fibrous strands embedded in a fine granular matrix. The thin strands extend radially from the cell wall and transverse the capsule. The entire capsule domain, as well as the outer membrane, binds specific anticapsular antibody, whereas the periplasmic space and most of the inner membrane lack capsule-specific immunostain.

Phosphoglycerides and phospholipase C in membrane fractions of Escherichia coli B.

The phospholipid composition and the phospholipase C activity of envelope fractions of Escherichia coli B were determined with special consideration of fractions containing sites at which an attachment of inner and outer membranes had been observed in the electron microscope (Int.M). Phosphoglycerides labeled with [14C]palmitic acid and [3H]serine were extracted from membrane fractions and identified by two-dimensional thin-layer chromatography. The amount of phosphatidylethanolamine was highest in the outer membrane, whereas the amounts of phosphatidylglycerol and cardiolipin were highest in the inner membrane. The Int.M fractions were observed to have concentrations of phospholipids intermediate to those of the inner and outer membranes. This result supports the assumption that a concentration gradient of inner membrane-outer membrane lipids might exist at the membrane contact sites. The highest phospholipase C activity was detected in the inner membrane and Int.M fractions. The presence of phospholipase C and other lipolytic enzymes in the Int.M fractions suggests a possible involvement of adhesion sites in lipid metabolism, adding a further set of activities to the function of these domains.

Measurement of the rotational behaviour of virus particles during adsorption to the host cell surface.

The rotational diffusion of bacteriophage epsilon 15 was measured before and after virus adsorption to outer membrane vesicles of the host Salmonella anatum. The virus capsid was labeled with eosin isothiocyanate, and the decay of transient dichroism following dye excitation by pulses of plane-polarized light was measured. From the data, the rotational diffusion constant of the unadsorbed virion and its hydrodynamic diameter were estimated and found to be consistent with electron microscopic measurements of the capsid dimensions. Addition of outer membrane vesicles of S. anatum to the virus suspension revealed the immobilization of the virus particles on the membrane surface.

Crystallographic studies of Escherichia coli citrate synthase.

The citrate synthase from Escherichia coli B has been crystallized in a cubic space group with a unit cell spacing of 220 A. X-ray diffraction, electron microscopy, symmetry considerations, and low resolution projection Patterson syntheses are consistent with a model proposed in which 24 tetrameric molecules of Mr = 188,000 +/- 12,000 occupy the unit cell. The space group is apparently P23, although at low resolution the observed systematic absences in reflections are consistent with the space group P43n, a space group not allowed for asymmetric molecules. Estimates of VM suggest that in the true space group, P23, two tetrameric molecules occupy the asymmetric unit.