Nontoxigenic variants were isolated from Clostridium botulinum type A strain 190L after treatment with detergents such as deoxycholate, sodium dodecyl sulfate, Tween 80 and Brij-58. Deoxycholate was most effective for obtaining the variants. The variants exhibited a markedly increased frequency of sporulation compared with the oligosporogenic parent strain. The cell wall of the parent strain was composed of an outer layer and an inner layer, whereas that of the variants lost the outer layer. After treatment with mitomycin C the parent strain was subjected to lysis and produced bacteriophages with a hexagonal head and a contractible tail, while the nontoxigenic variants did not yield bacteriophages or phagelike structures. There appears to be a close relationship among the toxigenic and sporogenic properties, formation of the outer cell wall layer and lysogeny.

View full abstract

To establish the mode of gene expression specified by transposon, we investigated the correlation among the homology of the DNA sequence, the extent of transposon-specific transcription, the specific activity of penicillinase (PCase) per cell, and the transposition frequency by using two ampicillin resistance transposons (TnAs), Tn2601 and Tn2602. Although both the TnAs specify the so-called type I PCase, Tn2602 always conferred 10-to 20-fold higher PCase activity per cell than Tn2601 regardless of the kind of replicon carrying TnA. The transposition frequency of Tn2602 also was 8 to 50 times higher than that of Tn2601 in all combinations of donor and recipient plasmids examined. As a result, the transposability expressed by the TnA was thought to correlate with the productivity of PCase in the cell specified by the corresponding TnA. The level of TnA-specific transcription of Tn2602 was noticeably higher than that of Tn2601, whereas the two TnAs shared a high degree (more than 90%) of DNA sequence homology. These results suggest that the difference in rates of transcription of the two transposons plays a key role in determining the difference in the productivity of PCase and the transposition-protein (s) of Tn2601 and Tn2602.

View full abstract

The structure of the skeleton of spores of Bacillus megaterium was examined after ashing in a plasma asher and the elemental composition of the ashed whole spores was determined with an analytical electron microscope. All spores were ashed in situ although they shrank by about 15%. Even P and S, in addition to metals, were recovered well from ashed samples. Ash was rich in the core and the coat, and poor in the cortex. Ca, P, S, and Mg were detected in the core and coat of the spore of B. megaterium QM B 1551. Ca in the core was markedly decreased by germination or autoclaving. In the spore of B. megaterium ATCC 19213, almost all of the ash was detected in the core and its elemental composition was similar to that of the core of the strain QM B1551 spore.
These results suggest strongly that the core is the site of Ca associated with dipicolinic acid.

View full abstract

The ultrastructure of a hexagonal array in the exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A strain 190L was studied by electron microscopy of negatively stained exosporium fragments using optical diffraction and filtration. The exosporium was composed of three or more lamellae showing an equilateral, hexagonal periodicity. Images of the single exosporium layer from which the noise had been filtered optically revealed that the hexagonally arranged, morphological unit of the exosporium was composed of three globular subunits about 2.1 nm in diameter which were arranged at the vertices of an equilateral triangle with sides of about 2.4 nm. The morphological units were arranged with a spacing of about 4.5 nm. The adjacent globular subunits appeared to be interconnected by delicate linkers.

View full abstract

Coliphage HK243 can form plaques on Escherichia coli C and K-12, but not B. The plaques are 1-2 mm in diameter and are opaque areas which clear upon exposure to chloroform vapor. During one-step growth, the eclipse and the latent periods are 20 and 30 min, respectively. Phage-infected cells continue to produce cell-free plaque-forming units for as long as 80 min after the end of the latent period, although at high multiplicities of infection (MOI) most cells lyse. No lysogenic bacteria have been found among survivors, so HK243 is considered a virulent phage. Some of the cells surviving a high MOI challenge are maltose negative and resistant to both HK243 and coliphage lambda. This fact has made possible the isolation of lambda-resistant mutants of lambda-lysogens. However, no serological cross-reaction between the phages lambda and HK243 has been detected. Genetic data involving three essential loci and a locus controlling plaque morphology suggest a circular linkage map. The virions are tadpole-shaped with an icosahedral head 68 nm long which is attached to a flexible tail 131 nm long. The phage has a linear, duplex DNA genome of molecular weight approximately 44 × 10⁶ and a base composition of 33% adenine, 31% thymine, 16% guanine, and 20% cytosine.

View full abstract

Migration of peritoneal exudate cells (PEC), which were prepared from mice immunized against mouse adenovirus (M-Ad), was inhibited upon exposure to the antigenic extract of M-Ad-infected cells. This inhibition was shown to be blocked when infected cells or their extracts were pretreated with antiserum against M-Ad-induced cell surface (S) antigen (s) or with antisera against alloantigens of infected cells. Immune spleen cell-mediated cytolysis of M-Ad-infected cells was also blocked in the presence of anti-S, anti-alloantigen or anti-β₂m serum. Immunofluorescent antibody staining of S antigen (s) was blocked when infected cells were pretreated with anti-alloantigen or anti-β₂m serum, whereas it was not blocked when they were pretreated with anti-mouse immunoglobulin or anti-Thy-1.2 serum. Conversely, immunofluoresent antibody staining of alloantigens was blocked when infected cells were pretreated with anti-S serum. These findings indicate that S and alloantigens are associated with each other or at least located very close to each other.

View full abstract

The topological relationship on the mouse adenovirus (M-Ad) -infected cell surface between virus-induced specific cell surface (S) antigens and serologically defined major histocompatibility antigens (H-2) was analyzed by the cap formation technique. Rhodamine-isothiocyanate (RITC) -labeled anti-S serum failed to stain the surface of virus-infected lymphoid cells which were pretreated with anti-H-2 serum and fluorescein isothiocyanate (FITC) -labeled anti-mouse immunoglobulin serum (anti-M-Ig) to cap the appropriate H-2 antigens. Conversely, the capping of the S antigens by pretreatment with anti-S followed by FITC anti-M-Ig serum induced cocapping of H-2 antigens. The β₂microglobulins (β₂m) were also shown to be cocapped with S antigens by anti-β₂m or by anti-S serum. The S antigens, however, did not cocap with mouse-immunoglobulins or Thyl. 2 antigens on virusinfected B or T lymphocytes, respectively.
To further elucidate the molecular relationship between S and H-2 antigens, radio-iodinated virus-infected cells were solubilized with Nonidet P40 (NP40) and S antigens were precipitated with anti-S serum. When the precipitates were analysed with sodium dodecyl sulfate polyacrylamide gel electrophoresis, two major peaks were seen at positions of molecules of about 45, 000 and 12, 000 daltons both of which corresponded with molecules which were observed when NP40 extracts of virus-infected or uninfected cells were precipitated with anti-H-2 serum. Sequential immunoprecipitation analysis of infected cell extracts showed that S antigens were coprecipitated with either H-2K or H-2D antigens. These results suggest that the S antigens are somehow associated with H-2K or H-2D antigens separately.

View full abstract

A synthetic bacterial cell wall constituent, muramyl dipeptide (MDP), was found to induce the enhancement of macrophage spreading and attachment on glass or plastic surfaces. Macrophages exposed to bacterial lipopolysaccharide or lymphokine-containing cell supernatants showed similar enhancement. This finding supports the view that MDP activates macrophages. MDP was also found to enhance the viability of macrophages but to inhibit ³H-thymidine incorporation by macrophages.

View full abstract

Depletion of macrophages from immune spleen cells by treatment with carbonyl iron and magnet or by in vivo treatment with carrageenan enhanced the in vivo secondary cell-mediated cytotoxic response against a syngeneic Friend virus-induced leukemia, FBL-3 cells of C57BL/6 mice. However, further depletion of macrophages by passing the carbonyl iron-treated immune spleen cells through a nylon wool column abrogated the cytotoxic response. The addition of splenic macrophage-enriched preparations from either FBL-3-immune or normal mice suppressed the cytotoxic response of immune spleen cells treated with carbonyl iron and magnet. This suppressive effect of splenic macrophages presented a marked contrast with the enhancing effect of normal peritoneal macrophages on the same cell-mediated cytotoxic response, indicating regulation of the generation of killer T cells against a syngeneic tumor by functionally distinct macrophages. The suppressed cell-mediated cytotoxic response against FBL-3 cells by immune spleen cells was augmented by the addition of indomethacin to the culture medium, and this augmentation with indomethacin was greatly decreased by depletion of phagocytic cells from the immune spleen by treatment with carbonyl iron and magnet. The mechanisms of regulation of the cell-mediated cytotoxic response with soluble factors released from macrophages are discussed.

View full abstract