Characterization of novel, phenol-soluble polypeptides which confer rigidity to the sheath of Methanospirillum hungatei GP1.

Treatment of the Methanospirillum hungatei GP1 sheath with 90% (wt/vol) phenol resulted in the solubilization of a novel phenol-soluble group of polypeptides. These polypeptides were purified by the removal of insoluble material by ultracentrifugation and represented approximately 19% of the mass of the sheath. The phenol-insoluble material resembled untreated sheath but had lost its rigidity and cylindrical form. Recombination of phenol-soluble and phenol-insoluble fractions by dialysis to remove phenol resulted in cylindrical reassembly products. Although bona fide sheath (complete with the 2.8-nm lattice) was not produced, a role for the phenol-soluble polypeptides in the maintenance of sheath rigidity is implied. The phenol-soluble polypeptides have limited surface exposure as detected by antibodies on intact sheath; therefore, they are not responsible for the 2.8-nm repeat occurring on the outer face of the sheath. However, longitudinal and transverse linear labeling by protein A-colloidal gold on the outer and inner faces, respectively, occurred with monoclonal antibodies specific to the phenol-soluble polypeptides. Restricted surface exposure of phenol-soluble polypeptides on the sheath highlighted molecular defects in sheath architecture. These lattice faults may indicate sites of sheath growth to accommodate cell growth or division (longitudinal immunogold label) and filament division (transverse immunogold label). The identification of a second group of polypeptides within the infrastructure of the sheath suggests that the sheath is a trilaminar structure in which phenol-soluble polypeptides are sandwiched between sodium dodecyl sulfate-beta-mercaptoethanol-EDTA-soluble polypeptides (G. Southam and T. J. Beveridge, J. Bacteriol. 173:6213-6222, 1991) (phenol-insoluble material).     

Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution.

The majority of Pseudomonas aeruginosa strains synthesize two antigenically distinct types of lipopolysaccharide (LPS), namely, a serotype-specific B-band LPS and a common antigen A-band LPS. A-band LPS consists of uncharged poly-D-rhamnan, which does not bind uranyl ions and is difficult to stain for electron microscopy; the highly charged B-band LPS is more easily visualized. We selected two wild-type strains, PAO1 (serotype O5) and IATS O6 (serotype O6), generated isogenic mutants from them, and examined the distribution of LPS on the surface of these organisms by freeze-substitution and electron microscopy. On PAO1 cells, which express both A-band and B-band LPSs, a 31- to 36-nm-wide fringe extending perpendicularly from the outer membrane was observed. A fine fibrous material was also observed on the surface of serotype O6 (A+ B+) cells, although this material did not form a uniform layer. When the LPS-deficient mutants, strains AK1401 (A+ B-), AK 1012 (A- B-), rd7513 (A- B-), and R5 (an IATS O6-derived rough mutant; A- B-), were examined, no extraneous material was apparent above the bilayer. However, an asymmetrical staining pattern was observed on the outer leaflet of the outer membrane of each of these mutants, presumably conforming to the anionic charge distribution of the core region of the rough LPS. In all cases, expression of the LPS types was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. When optical densitometry on electron microscopy negatives was used to analyze the outer membrane staining profiles, subtle differences in the degrees of core deficiency among rough mutants were detectable. This is the first time an electron microscopy technique has preserved the infrastructure produced in the outer membrane by its constituent macromolecules. We conclude that freeze-substitution electron microscopy is effective in the visualization of LPS morphotypes.     

Evaluation of freeze-substitution and conventional embedding protocols for routine electron microscopic processing of eubacteria.

Freeze-substitution and more conventional embedding protocols were evaluated for their accurate preservation of eubacterial ultrastructure. Radioisotopes were specifically incorporated into the RNA, DNA, peptidoglycan, and lipopolysaccharide of two isogenic derivatives of Escherichia coli K-12 as representative gram-negative eubacteria and into the RNA and peptidoglycan of Bacillus subtilis strains 168 and W23 as representative gram-positive eubacteria. Radiolabeled bacteria were processed for electron microscopy by conventional methods with glutaraldehyde fixation, osmium tetroxide postfixation, dehydration in either a graded acetone or ethanol series, and infiltration in either Spurr or Epon 812 resin. A second set of cells were simultaneously freeze-substituted by plunge-freezing in liquid propane, substituting in anhydrous acetone containing 2% (wt/vol) osmium tetroxide, and 2% (wt/vol) uranyl acetate, and infiltrating in Epon 812. Extraction of radiolabeled cell components was monitored by liquid scintillation counting at all stages of processing to indicate retention of cell labels. Electron microscopy was also used to visually confirm ultrastructural integrity. Radiolabeled nucleic acid and wall components were extracted by both methods. In conventionally embedded specimens, dehydration was particularly damaging, with ethanol-dehydrated cells losing significantly more radiolabeled material during dehydration and subsequent infiltration than acetone-treated cells. For freeze-substituted specimens, postsubstitution washes in acetone were the most deleterious step for gram-negative cells, while infiltration was more damaging for gram-positive cells. Autoradiographs of specimens collected during freeze-substitution were scanned with an optical densitometer to provide an indication of freezing damage; the majority of label lost from freeze-substituted cells was a result of poor freezing to approximately one-half of the cell population, thus accounting for the relatively high levels of radiolabel detected in the processing fluids. These experiments revealed that gram-positive and gram-negative cells respond differently to freezing; these differences are discussed with reference to wall structure. It was apparent that the cells frozen first (ie., the first to contact the cryogen) retained the highest percentage of all radioisotopes, and the highest level of cellular infrastructure, indicative of better preservation. The preservation of these select cells was far superior to that obtained by more conventional techniques.     

Binding of metals to cell envelopes of Escherichia coli K-12.

As representative of gram-negative bacteria, the isolated and purified envelopes of an Escherichia coli K-12 strain were used to determine metal-binding capacity. The envelopes were suspended in 5 mM metal solutions for 10 min and 23 degrees C, separated and washed by centrifugation, and analyzed for metal by either atomic absorption or X-ray fluorescence spectroscopy. Of 32 metals tested, large amounts (> 0.9 mumol/mg [dry weight]) of Hf and Os, intermediate amounts (0.1 to 0.4 mumol/mg [dry weight]) of Pb, Zn, Zr, Fe III, Mn, Mo, Mg, Co, and Ce IV, and small amounts (< 0.1 mumol/mg [dry weight]) of Na, K, Rb, Ca, Sr, Cu, Sc, La, Pr, Sm, U, Fe II, Ru, Ni, Hg, Pt, Pd, Au, and In were detected Li and V were not bound to the envelopes. Electron microscopy of unstained, thin-sectioned material provided an electron-scattering profile for localizing the bound metal within the envelope. Energy-dispersive X-ray analysis of thin sections detected all metals in single envelope vesicles. These data suggest that most metal deposition occurred at the polar head group regions of the constituent membranes or along the peptidoglycan layer. No leaching of envelope components was detected by monitoring radioactive probes within the lipopolysaccharide and peptidoglycan layers during metal uptake experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from metal-loaded envelopes, or protein and carbohydrate determinations on the wash fluids. These results suggest that membrane integrity was not disturbed under these ionic conditions.     

Nutritional requirements for conidial germination and hyphal growth of Beauveria bassiana

Nutritional requirements for germination and growth of the entomopathogenic fungus Beauveria bassiana are not complex. For germination to occur, a utilizable source of carbon must be present; however, a nitrogen source is needed for continued hyphal growth, otherwise lysis ensues. Compounds that can serve as utilizable carbon-energy sources for germination include glucose, N-acetylglucosamine, glucosamine, chitin, starch, lanolin, hydrocarbons in crude oil, and some longer-chain fatty acids. Both organic and inorganic sources of nitrogen are readily utilized for growth. Conidia undergo active metabolism soon after being placed in a suitable growth medium, indicating that conidia are released from their state of dormancy several hours before emergence of the germ tube can be observed. Because of the nutritional versatility of B. bassiana, this fungus should be able to survive and be infective in several types of natural environments.     

Antimicrobial activity of some alkyl esters of gallic acid (3,4,5,-trihydroxybenzoic acid) against Escherichia coli NCTC 5933 with particular reference to n-propyl gallate

The growth inhibitory and bactericidal activities of eight alkyl esters of gallic acid towards Escherichia coli NCTC 5933 have been determined. A previously suggested role for gallic acid and its esters as shikimate antimetabolites could not be substantiated. No induction of gross changes in cell morphology was observed. Bactericidal activity was accompanied only be very slight leakage of general ionic materials from the bacteria. Propyl gallate did not appear to uncouple oxidative phosphorylation from respiration as indicated by its failure to stimulate proton translocation across the cytoplasmic membrane.     

Analysis of neonatally induced tolerance of H-2 alloantigens

There is a considerable amount of evidence, confirmed and extended by our studies, in favor of clonal deletion of alloantigen-reactive cells in neonatally induced transplantation tolerance. We have demonstrated in adult mice bearing long-standing skin allografts that lymphocytes specifically reactive with the tolerated H-2 alloantigens are undetectable by mixed lymphocyte and graftversus-host reactions, and in cell-mediated lympholysis. In addition, lymphoid cells capable of suppressing the reactivity of syngeneic normal lymphocytes in these assays similarly escape detection. Moreover, putative precursors of T cells specific for the tolerated antigens cannot be activated polyclonally with concanavalin A (Con A), nor can they be identified among thymocytes ofH-2-tolerant mice. Since the tolerant state can be adoptively transferred with lymphohematopoietic cells to adult, syngeneic mice, we infer that transplantation tolerance is maintained by an active process that achieves specific clonal deletion at an early stage in the ontogeny of alloreactive T lymphocytes.     

Sites of metal deposition in the cell wall of Bacillus subtilis

Amine and carboxyl groups of the cell wall of Bacillus subtilis were chemically modified individually to neutralize their electrochemical charge for determination of their contribution to the metal uptake process. Mild alkali treatment removed ca. 94% of the constituent teichoic acid (expressed as inorganic phosphorus) and allowed estimation of metal interaction with phosphodiester bonds. Chemical modifications of amine functions did not reduce the metal uptake values as compared to native walls, whereas extraction of teichoic acid caused a stoichiometric reduction in levels. In contrast, alteration of carboxyl groups severely limited metal deposition of most of the metals tested. X-ray diffraction and electron microscopy suggested, in this case, that the form and structure of the metal deposit could be different from that found in native walls. The observations suggest that carboxyl groups provide the major site of metal deposition in the B. subtilis wall.     

Surface arrays on the cell wall of Spirillum metamorphum.

A complex and easily disrupted arrangement of macromolecules was present on the outer (lipopolysaccharide) membrane of the cell wall of Spirillum metamorphum. Separation of the arrays from the cell and spontaneous reassembly into regularly structured complexes usually occurred during preparation for electron microscopy. Freeze etchings, thin sections, and optical diffraction analysis of negatively stained fragments indicated that they consisted of two sets of a thin layer which was studied with 3-nm particles arranged in a loose (OL). The OSL consisted of a hexagonal arrangement of 8-nm disks and the OL of a thin layer which was studied with 3-nm particles arranged in a loose rectangular manner. The OSL of reassembled fragments displayed numerous broken delta-linkers between units and a center-to-center spacing of half the expected distance, which suggests that an interdigitation of two OSL arrays had occurred. The observations combined with freeze etchings and thin sections of whole cells suggested a possible reassembly mechanism. The normal surface arrangement of these layers on cells was thought to consist of the OL overlying one set of OSL which was loosely adherent to a thin amorphous backing layer.