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RNA-binding proteins (RBPs) form highly diverse and dynamic ribonucleoprotein complexes, whose functions determine the molecular fate of the bound RNA. In the model organism Sacchromyces cerevisiae, the number of proteins identified as RBPs has greatly increased over the last decade. However, the cellular function of most of these novel RBPs remains largely unexplored. We used mass spectrometry-based quantitative proteomics to systematically identify protein-protein interactions (PPIs) and RNA-dependent interactions (RDIs) to create a novel dataset for 40 RBPs that are associated with the mRNA life cycle. Domain, functional and pathway enrichment analyses revealed an over-representation of RNA functionalities among the enriched interactors. Using our extensive PPI and RDI networks, we revealed putative new members of RNA-associated pathways, and highlighted potential new roles for several RBPs. Our RBP interactome resource is available through an online interactive platform as a community tool to guide further in-depth functional studies and RBP network analysis (https://www.butterlab.org/RINE)., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background and Objectives: Hepatocellular carcinoma (HCC) is often associated with chronic hepatitis due to hepatitis-B or -C viruses. Active specific immunotherapy (ASI) with autologous dendritic cells (DC) presenting antigens from autologous tumor stem cell (TC) lines is associated with promising long-term survival in metastatic cancer, but hepatitis patients were excluded. ASI might benefit high-risk primary HCC patients following surgical resection, but first it is important to show that ASI does not exacerbate hepatitis., Methods: Previously untreated HCC patients with a solitary lesion > 5 cm, or three lesions with at least one > 3 cm, or more than three lesions, underwent surgical resection from which autologous TC lines were established. Irradiated TC were incubated with autologous DC to create DC-TC. After one course of trans-arterial chemoembolization therapy (TACE), three weekly subcutaneous injections of DC-TC suspended in granulocyte-macrophage colony stimulating factor were administered. Patients were monitored for eight weeks., Results: HCC cell lines were established within five weeks for 15/15 patients. Eight patients, all with chronic hepatitis B, were treated. There was no increase in hepatic transaminases, hepatitis B antigens, or viral DNA., Conclusion: Autologous DC-TC did not exacerbate HBV in these HCC patients. A phase II efficacy trial is being planned., (© 2015 Wiley Periodicals, Inc.)

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.

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 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.

Bdellovibrio bacteriovorus 109J attached to both capsulated and non-capsulated Escherichia coli K29 cells. Electron microscopy revealed penetration of the thick polysaccharide capsule without any major disintegration of the neighbouring capsular matrix. The capsule remained intact during bdelloplast formation and lysis was unaffected by capsulation of the prey cell. This study shows that, in contrast to its effect on bacteriophage penetration and its protective activities against immune defence mechanisms, the capsule of E. coli does not serve as a barrier against invasion by B. bacteriovorus.

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.

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.

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.

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.

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.

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 (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.

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.

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.

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.

The effect of ethylenediaminetetraacetate (EDTA) on the envelope of two strains of Escherichia coli (B and Cla) was studied with freeze-fracturing methods. Untreated cells showed the outer membrane's outer surface with a fine texture of randomly spaced depressions of about 4.5-nm diameter; small areas with symmetrical arrangements of structural surface elements were also observed. The outer membrane's fracture plane revealed a random distribution of particles on its "concave" plane, only occasionally interrupted by particle-free areas. The "convex" aspect of the outer membrane's fracture plane showed only a few scattered particles. The cleavage plane of the inner membrane was often interrupted by many localized elevated plateaus, at which the cleaving process had, for short distances, switched to the outer membrane. The effects of EDTA treatment were mainly seen in the structure of the freeze-etched outer membrane: (i) the pits as well as the symmetrical surface elements of the outer membrane's outer surface had disappeared; (ii) a number of plateaus (about 20 to 50/cell) were seen at which a cleavage plane within the inner membrane had switched to the hydrophobic portion of the outer membrane (outer membrane's fracture plane). These plateaus were also visible in untreated cells; however, EDTA treatment apparently caused an increased exposure of plateaus. Surface areas, exposed by freeze-etching, revealed the underlying plateaus as elevations in the surface contour of the cell, suggesting a slower etching rate in the zones of the plateaus relative to the rest of the outer membrane. Well-defined, particle-free patches in the outer membrane's fracture plane, concave, were more frequent and larger in size after EDTA treatment than in the controls. In the presence of glycerol, the cells often cleaved in the outer membrane's fracture plane, but isolated plateaus were rarely observed. After metabolic poisoning of cells for 15 to 25 min at 37 degrees C, the plateaus had widened. These data suggest that the material of the plateaus has a slow rate of lateral diffusion. Placement of EDTA-treated cells in fresh medium at 37 degrees C caused, after 3 to 5 min, the reoccurrence of the pitted surface structure. We propose that the plateaus represent localized zones, at which newly synthesized lipopolysaccharide has been inserted.

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.

The viability of resting suspensions of Escherichia coli K12 Ymel exposed to air plus 300 psi (1 psi = 6.895 kPa) oxygen (hyperbaric oxygen) decreased as an apparent first-order process after an initial period of constant viability. Control suspensions exposed to air plus 300 psi nitrogen (hyperbaric nitrogen) did not lose viability over the 96 h of the experiment. It was observed that a decrease in the refractive index of the cells preceded the loss of viability in hyperbaric oxygen. This finding together with electron micrographs, which showed extensive loss of ribosomal particles in bacteria incubated in hyperbaric oxygen, led us to suspect that ribosome injury or disassociation might be important in hyperbaric oxygen toxicity. In support of this we found that cellular RNA, labeled with [5-3H]uridine, was much more rapidly and more completely degraded in hyperbaric oxygen than in hyperbaric nitrogen. Furthermore, a far greater proportion of RNA was degraded than was DNA or protein. A direct assay for ribosome particles by sucrose gradient centrifugation showed that only 34% of the 70S ribosome particles was lost during the first 24 h in hyperbaric nitrogen whereas in hyperbaric oxygen 99.6% of the 70S particles was degraded during the same period. In hyperbaric oxygen the rate of viability loss between 24 and 72 h was equal to the rate of 70S ribosome degradation during the first 24 h. If 70S ribosome disassociation in hyperbaric oxygen continues at the same rate after first 24 h, then cumulative 70S ribosome disassociation or injury may lead to and provide an explanation for irreversible bacterial cell injury and the loss of viability.

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.

At areas of adhesion between outer membrane (OM) and inner membrane (IM) in gram-negative bacteria, newly synthesized membrane constituents are inserted, and bacteriophage infection occurs. We describe here the isolation of these sites from cell membrane fractions of Salmonella anatum. Sucrose density gradients yielded membrane vesicles of the OM and IM; their mutual cross-contamination was low, as measured by 2-keto-3-deoxyoctonate and beta-NADH-oxidase activities. To mark the areas of lipopolysaccharide synthesis in the envelope (the adhesion sites), we infected S. anatum with phage epsilon 15, which causes a rapid change (conversion) in the cell's O-antigenic composition from serogroup E1 to E2; lipopolysaccharide of type E2 also serves as receptor for phage epsilon 34. We found that the fractions of intermediate density (Int. M) from briefly converted cells bound both phage epsilon 34 and E2-specific antibody. In the electron microscope, epsilon 34 was seen to have absorbed with a high degree of significance to the Int. M fraction of briefly converted cells, but not to the Int. M fraction of unconverted cells. Furthermore, the Int. M fractions of briefly converted cells coagglutinated anti-E2-coated Staphylococcus aureus, whereas the OM and IM fractions showed comparatively little agglutination. In addition, Int. M material exhibited elevated phospholipase A1 and A2 activities comparable to those of the OM fraction; the IM was essentially phospholipase free. Our data indicate that this membrane fractionation allows one to isolate from Int. M regions a variety of activities associated with adhesion sites.

The mode of transport of the complex toxin molecule of Vibrio cholerae (which has a mol. wt of 84000 and consists of several subunits) across the inner and outer membranes of V. cholerae is not known. In this study we found two peptides in the outer and inner membranes of V. cholerae which may be the form in which the toxin subunits are transported across the membrane. We examined two growth conditions: aerobic growth at 37 degrees C, when most of the synthesized toxin is membrane-bound; and anaerobic growth at 37 degrees C, when little toxin remains membrane-bound, the toxin being released into the growth medium. When V. cholerae was grown aerobically at 37 degrees C, the outer and the inner membranes contained two peptides with mol. wts of approximately 22000 and 6000 which were not found in the outer or the inner membrane of anaerobically grown cells. Sodium deoxycholate, which releases membrane-bound toxin, released several peptides including the 22000 and the 6000 mol. wt peptides. Trypsin also released the 22000 and 6000 mol. wt peptides. Purified cholera toxin had three kinds of peptides, of mol. wt 21000 (A1 peptide), 11000 (B subunit) and 5000 (A2 peptide). We postulate that the membrane peptides may be precursors of the A subunit of the toxin molecule.

The diffusion constant of bacteriophage phiX174 was determined by laser light self-beat spectroscopy. The method allows one to establish the diffusion constant in comparatively short time and with high accuracy. From the diffusion constant D(37) = 1.96 +/- 0.08 x 10(-7) cm(2)/s, the size of the virus particles within their aqueous milieu can be calculated. For phiX174, a diffusional diameter of 31.4 +/- 1.0 nm was found, in good agreement with measurements of diameters of freezeetched (32.3 +/- 1.8 nm) and negatively stained particles (33.8 +/- 2.1 nm), provided that the entire spikes of the virion are included. Other isometric viruses may show a complex interaction of the virion with the surrounding water and its ions.

A lysogenic strain of Pseudomonas aeruginosa was cultured from the dialysis fluid of a patient on chronic hemodialysis treatment whose blood contained hepatitis B surface antigen (HB8Ag). When this bacterium was incubated for 4 to 7 days with serum containing HB8Ag or with purified HB8Ag, a loss of the HB8Ag-specific immunological reactivity was observed. Bacteriophages can be induced from the isolated P. aeruginosa with mitomycin C; the phages, after purification on CsCl gradients, also lyse P. aeruginosa strain 25102 (ATCC). Subsequent to gradient centrifugation of the lysate, a fraction was found with a density around 1.40 g/ml that inactivated HB8Ag after a 4-h incubation at 37 C as determined by counterelectrophoresis and hemagglutination inhibition. The activity was not found in appreciable amounts in other gradient fractions. The electron microscope shows that the active fraction contains envelope vesicles of 45 to 60 nm in diameter. In spite of their loss in HB8Ag activity, the HB8Ag particles (22nm) appeared morphologically intact. These findings suggest that an enzyme(s) is present in the vesicle fraction which inactivates antigenic determinants on HB8Ag particles. Thus, the presence of these bacteria in environments such as feces, dialysis tanks, and contaminated drinking water may prevent the detection of HB8Ag.

After collision with their host cells, virus particles may remain mobile on cell surfaces until they become attached at firm binding sites. We propose that a virion will arrive within a typical median time at such a site, generating a membrane signal such as an increased membrane fluorescence in cells labeled with the voltage-sensitive dyes 8-anilino-1-naphthalene-sulfonate (Mg-salt) (ANS), N-phenylnaphthylamine (NPA), or 3,3'-dipentyl-2,2'-oxacarbocyanine (di-O-C5[3]). We found that the time span between virus adsorption and fluorescence response varies widely among phages and also depends on bacterial strain, metabolic state, and type of dye. di-O-C5[3]-labeled cells react within 1 sec to uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells labeled with ANS and NPA react to CCCP in 4-6 sec. Bacteriophages T4, T5, chi, and BF23, added to ANS-labeled cells, change the fluorescence in 9-15 sec. T-even ghosts cause a response at identical times. Baseplate-defective phage mutant T412- and isolated adsorption organelles of smaller viruses fail to cause an effect. di-O-C5[3]-labeled cells respond to T4 at a multiplicity of infection greater than or equal to 40 within 1 sec. A longer time (8 sec) is required at lower multiplicities. The receptor-degrading phages epsilon 15, epsilon 34, c 341, and K29 need the longest time (1 min for ANS) to cause a fluorescence increase. We suggest that the delayed fluorescence response is concomitant with the surface "walk" of the virion, which is terminated at an injection site. T4 tail sheath contraction coincides with the onset of the membrane fluorescence response.

This report describes the structure, size, and shape of the uncollapsed polysaccharide capsule of Escherichia coli strain Bi 161/42 [O9:K29(A):H-], its ultrastructural preservation as well as the filamentous components of the isolated capsular material. In a temperature-sensitive mutant, sites were localized at which capsular polysaccharide is "exported" to the cell surface. The highly hydrated capsule of the wild-type cells was visible in the uncollapsed state after freeze-etching, whereas dehydration in greater than or equal to 50% acetone or alcohol caused the capsule to collapse into thick bundles. This was prevented by pretreatment of the cell with capsule-specific immunoglobulin G; the capsule appeared as a homogeneous layer of 250- to 300-nm thickness. The structural preservation depended on the concentration of the anti-capsular immunoglobulin G. Temperature-sensitive mutants, unable to produce capsular antigen at elevated temperatures, showed, 10 to 15 min after shift down to permissive temperature, polysaccharide strands with K29 specificity appearing at the cell surface at roughly 20 sites per cell; concomitantly, capsule-directed antibody started to agglutinate the bacteria. The sites at which the new antigen emerged were found in random distribution over the entire surface of the organism. Spreading of purified polysaccharide was achieved on air-water interfaces; after subsequent shadow casting with heavy metal, filamentous elements were observed with a smallest class of filaments measuring 250 nm in length and 3 to 6 nm in width. At one end these fibers revealed a knoblike structure of about 10-nm diameter. The slimelike polysaccharides from mutants produced filamentous bundles of greater than 100-microns length, with antigenic and phage-receptor properties indistinguishable from those of the wild-type K29 capsule antigen.

We studied the fine structure of the envelope of Escherichia coli auxotroph K1060 after the cells were grown in the presence of one of the following fatty acids; oleic, palmitelaidic, or elidic acid. The cells were freeze-fractured after exposure to temperatures above and below the lipid phase transition range. As judged by freeze-etching methods, we observed that below the transition range the fracture plane of the inner membrane showed the typical aggregation of intramembranous particles (IMP) and concomitant development of areas devoid of IMP. In these areas we found a regular arrangement of equally spaced ridges, often intersected at 90 degrees by arrays of similar ridges. The ridges were composed of spherical particles measuring 4 to 5 nm in diameter. Formation and melting of these arrays took place within 15 to 30s after temperature shift-down or shift-up, respectively. Fixation in glutaraldehyde prevented these changes. The outer-membrane fracture plane revealed ordered areas to a lesser degree; these were discernible only by the regular arrangement of the IMP of the concave fracture plane. We interpret the data by suggesting that the pattern of ridges in E. coli K1060 is analogous to the band patterns described for artificial liposomes, and that the particles, possibly proteins, are lined up or extruded along the ridges during membrane lipid crystallization.

Binding of either ferritin (F) or cationized ferritin (CF) was employed to indicate the surface charge of the envelope of mainly two Salmonella typhimurium strains (395 MR10, a Rd-mutant, and LT2-M1, a UDP-galactose-4-epimerase-less mutant). Lowering the pH from 7 to 4 decreased binding of CF, but increased binding of F. At low concentrations, the distribution of CF on S. typhimurium 395 MR10 was in general random, with individual ferritin molecules often forming clusters of two or three particles. At ionic strengths of 0.25M NaCl, ferritin produced distinctive, larger clusters at relatively few sites (10-50/cell). Addition of galactose to cultures of growing S. typhimurium, LT2-M1 reduced the binding of CF in 1-10 min, and numerous ferritin-free areas became visible. Possibly this is caused by a pluri-focal reduction in the negative cell surface charge that was generated at the multiple sites of export of new, smooth-type lipopolysaccharide, which either exhibits lesser charge or masks a preexisting surface charge. Dividing cells may show unequal charges on the prospective daughter cells, and the difference in the capacity for ferritin adsorption of both daughter cells is sharply separated at the division site.

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.

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.

The surface of freeze-etched E. coli strain GR467, a heptose-deficient ("deep rough") mutant derived from CR34, was studied by electron microscopy. The outer membrane of GR467 has an increased ratio of phospholipid to protein, mainly due to a decreased protein content. Freeze-etched CR34 showed structural features indistinguishable for wild-type E. coli, i.e., the primary cleavage occurring in the inner membrane with only minor appearance of cleavage within the outer membrane. In contrast to this, in mutant GR467 most of the freeze-cleavages had taken place along a new plane, presumably in a hydrophobic region of the outer membrane. In this cleavage plane numerous particles were seen. Often the cleavage extended over the entire exposed cell surface; occasionally only a few large plateaus were visible, around which the next deeper cleavage plane, that of the protoplasmic or inner membrane, was discernible. Two spontaneous revertants (R11 and R16) with protein and lipid A levels similar to wild-type cells showed mostly freeze fractures with wild-type characteristics, and only a few cells had retained fracturing properties of GR467. A partial revertant revealed intermediate characteristics. Thus, there appears to be a morphological correlation with the chemical data relating the amount of outer membrane protein with the heptose content of the lipopolysaccharide.

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.

We have confirmed and extended the observation of Terawaki et al. that the R factor, Rts1, alters the growth of its host at 42 C. In all media tested there was a period during which total cell numbers increased linearly, while viable counts remained constant. During this period the rate of precursor incorporation per cell particle into deoxyribonucleic acid, ribonucleic acid, and protein declined steadily. These patterns were a consequence of the accumulation of increasing numbers of cells which had lost colony-forming ability. A temperature shiftdown experiment showed that the colony formers could, after a lag, go on to divide normally, whereas most of the noncolony formers could not undergo even a limited number of divisions after shiftdown. The number of normal divisions which occurred after shiftup of Rts1 cells to 42 C was medium dependent. In rich medium there were, on the average, two or three doublings; in glucose medium, one; and in glycerol medium, only a fraction of a doubling. Even in glucose medium, however, no increase in viable counts was observed during growth at 42 C if the cells were first starved for glucose for 1 h at 42 C. A temperature shiftdown from 42 C to 27 C during glucose starvation reversed the effect of starvation at 42 C alone. These results are consistent with the hypothesis that the thermosensitive Rts1 component(s) responsible for the host effects is present at permissive temperature, but can undergo a reversible temperature-induced alteration which then interferes with some essential host function. The detrimental effects of this R factor on its host were also reflected in a heightened sensitivity to kanamycin and actinomycin D at 42 C. Electron microscope observations revealed changes in the appearance of the cell membrane. Membranous invaginations were noted at discrete sites in the cell.

Australia antigen [Au(1)], a particle associated with viral hepatitis, was isolated from the plasma of a patient with chronic anicteric hepatitis and leukemia who had received radioactive phosphorus. We have found that the immunoreactivity and appearance of Au(1) in the electron microscope were not altered by treatment with enzymes including trypsin, pronase, lipase, phospholipase C, ribonuclease, deoxyribonuclease, amylase, and neuraminidase. In contrast, other serum constituents were degraded by these enzymes. Therefore, treatment of the patient's plasma with many enzymes was exploited as an initial step for the isolation of Au(1). Subsequently, Au(1) was purified from the enzyme-treated (32)P-labeled plasma by gel filtration through Sephadex G-200 and centrifugation through sucrose and in cesium chloride gradients. There were no detectable human serum components in the purest fractions, as tested by immunoelectrophoresis and immunodiffusion. The density of the purified Au(1) was 1.21 in CsCl. The particle measured about 200 A in diameter, was predominantly spherical in shape and appeared to be composed of subunits. Nucleic acids were not detected by spectrophotometric, radiochemical, and chemical analyses. Immunoreactivity of purified Au(1) was destroyed by heating for 1 hr at 85 degrees C but was stable at 56 degrees C. Treatment with Carnoy's solution (3 parts ethanol:1 part glacial acetic acid) followed by pronase disrupted the particles as seen with the electron microscope. These findings, combined with other published information on Australia antigen and viral hepatitis, suggest that the bulk of Australia antigen in the blood of this patient is an incomplete virus or virus capsid.

The rate of survival after osmotic shocks was found to be dependent on the state of growth. When growing logarithmically, Escherichia coli was about 20 to 100 times more sensitive to an abrupt decrease of the environmental osmotic pressure than when it was in the stationary phase. Subjecting rapidly growing cells to such a treatment caused fingerlike extrusions to emerge from the bacterial wall. Our results suggest that underneath these extrusions the rigid layer of the wall contains weak areas which appear as discontinuities or gaps when viewed in an electron microscope. After exposure to osmotic shock, the gaps became wider. We concluded that the gaps represent sites of mucopolymer synthesis where the rigid structure has temporarily been opened by hydrolytic enzymes to allow for the insertion of new wall material into the older portions of the wall.