Many cases of Pseudomonas aeruginosa infection are considered to be secondary superinfections, resulting from bacterial colonization. Such cases of superinfection with P. aeruginosa developing after administration of cephalosporin or penicillin are offering serious clinical problems. To make a fundamental analysis of the development of such superinfections, attempts were made to compare the growth patterns of Escherichia coli and P. aeruginosa in pure and mixed cultures and to determine the effects of cephalothin, cefazolin, cephalexin, and ampicillin on the growth patterns.
In mixed cultures, the growth of P. aeruginosa was markedly inhibited by E. coli. The higher the concentration of each of the cephalosporins and ampicillin added to the mixed culture, the smaller the population of E. coli sensitive to these agents. When the population of E. coli became smaller than that of P. aeruginosa, which is resistant to these agents, the latter was restored to the same population level as that in pure cultures.
Experimental bacterial colonization, by which the predominant population of E. coli was replaced by that of P. aeruginosa in mixed culture, was brought about more efficiently with the cephalosporins than with ampicillin. This might be accounted for by the difference in minimal inhibitory concentration for P. aeruginosa between ampicillin and the other three agents.

View full abstract

Rms 201-12 and Rms 201-46 are R mutants with increased copy number, and are derived from a conjugative plasmid Rms 201 that encodes resistance to five drugs, ampicillin (Apc), tetracycline (Tc), chloramphenicol (Cm), streptomycin (Sm), and sulfonamides (Sa). The mutants expressed increased levels of resistance to Apc, Sm, and Cm, and a decreased level of resistance to Tc than those of the parent Rms 201 plasmid.
When the Rms 201-12⁺ or Rms 201-46⁺ cells were inoculated onto plates containing a high concentration of Tc, colonies developed on the plate at a frequency of 10^-3 to 10^-4 after overnight incubation. The cells grown on the Tc plate carried a tet (Tc gene) -deleted R mutant besides tet-possessing Rms 201-12 or Rms 201-46, and we isolated the tet-deleted R mutant by purifying the R⁺ cells on drug-free plates. On the other hand, various deletion mutants possessing tet were isolated by prolonged culture of the cells. We have presented a circular gene order of Rms 201 by comparing the genetic markers of all deletion mutants derived from Rms 201, Rms 201-46, and Rms 201-12. The gene(s) regulating the copy number was closely linked to the rep gene. The gene(s) specifying entry exclusion was jointly lost with the tra region.

View full abstract

Cell packets of Micrococcus lysodeikticus (luteus) mutant strain MT grown in medium supplemented with trypsin consisted of a tetrad as the unit structure. An interstice was observed between the unit-tetrads, and a three dimensional structure of cell packets was maintained by the cell wall-bridge along the rim of the cell packets which linked each unit-tetrad. This unique structure of strain MT cell packets seemed to occur when the cell separation was suppressed locally, i.e., when the cross wall inside the initial site of cell separation was cut off, while the wall outside the initial site of separation was not cut off but remained as a joint of the daughter cells.
The mechanism of cell wall-bridge formation is discussed in connection with cell separation.

View full abstract

Growth and sporulation were examined for 30 auxotrophs of Bacillus subtilis in a chemically defined medium with suboptimal amounts of nutrients. All strains except for some adenine-requiring mutants could not overtake sporulation stage II when amino acids, vitamins, or bases were limited, whereas they sporulated fairly well without limitation.
Abnormal structures, a cell with thickened cell wall and a cell with several refractile bodies, were found in some strains after the vegetative growth stopped.

View full abstract

TMV binding substance (R) was isolated from a tobacco leaf membrane fraction and was purified by extraction with organic solvents and by column chromatography. Experimental results suggest that the binding of R with TMV results in inactivation of TMV. When tobacco leaves were inoculated with the R-TMV complex, it was found that the formation of polysome containing infecting viral RNA was inhibited. Model experiments showed that the mode of R-TMV adsorption to the membrane is different from that of TMV adsorption and that stripping of coat protein from TMV by SDS was inhibited by R. A possible explanation for the mechanism of this inhibition by R is that the R-TMV complex follows a pathway which does not lead to establishment of infection. Although less efficient, R was still active when it was applied after virus inoculation. Due to its affinity to coat protein, R might also interfere with a later process of viral multiplication.

View full abstract

Treatment of herpes simplex virus type 1 (HSV-1) -infected human embryo lung (HEL) cells with phosphonoacetic acid (PAA) resulted in complete inhibition of HSV DNA replication. DNA was extracted from PAA-treated HEL cells infected with HSV-1 and centrifuged in a neutral CsCl density gradient. The HSV DNA sequences in the nuclei of PAA treated cells at 24 hr post infection banded at the same density as free HSV DNA (1.725 g/cm³), but a significant amount of viral DNA sequences were detected in the regions of cell DNA (1.700 g/cm³) as well as in the intermediate fractions as determined by hybridization with ³H HSV complementary RNA. The viral DNA sequences of lower density did not change in density by recentrifugation in a CsCl density gradient, but did change to the density of free viral DNA after treatment with EcoR 1 restriction endonuclease. When the DNA from the nuclei of PAA treated cells was analyzed in an alkaline glycerol gradient, more than 95% of the viral DNA sequences were found in the free viral DNA fractions. Since the viral and cellular hybrid DNA represented approximately 33% of the total viral DNA sequences, it is concluded that some of the HSV DNA sequences in PAA treated, infected cells are associated with cell DNA by alkali-labile bonds

View full abstract

Electron microscopy revealed multi-layered membranes within the cytoplasmic inclusion (accumulation of nucleocapsids) produced by rabies virus. When infected BHK cells were maintained at 31 C, an enhancement in production of these membranes occurred in approximately 60% of inclusion-containing cells. Multi-layered membranes were composed of an alternate array of two different layers; an electron-dense, thin membrane and a less dense layer which was thicker. SDS-polyacrylamide gel electrophoresis and immune electron microscopy of isolated multi-layered membrane preparations demonstrated that the structures contained viral G and M₂ polypeptides. Our observations suggest that these membranous structures are not a degenerative product of rabies virus infection but rather are related to the replication of viral envelope constituents, although they represent themselves to be an abortive form of viral assembly.

View full abstract

The size of genomic RNA in PR-RSV A passaged in chick embryo fibroblasts (CEF) or quail embryo fibroblasts (QEF) was determined by gel electrophoresis. The results showed that 3 undiluted passages resulted in heterogeneity of RNA. The heterogeneity of the smaller incomplete RNAs in the virus stock was decreased by diluted passage or cloning, but RNA of the b subunit size and a subunit RNA of complete genome size were relatively stable.
These heterogeneous RNAs were characterized by hybridization analysis. The RNAs from 4 peaks hybridized with both cDNA_total and cDNA_src to appreciable extents, indicating that they were derived from viral RNA and that at least some of them contained the src sequence.
This finding and the failure to isolate a td mutant from the undiluted-passaged virus stock or from some subclones that had a and b subunits of RNA indicate that the td virus was only a minor constituent of the incomplete virus population caused by undiluted passages.
Some viruses with incomplete RNA in the virus stock could produce foci with the aid of td B77 or RAV-60. The emergence of rd viruses by undiluted passages was indicated.

View full abstract

The antigenicity of human erythrocytes of four different ABO blood groups and sheep erythrocytes of unknown blood type from different individual sheep were analyzed in terms of their cross-reactivity with antibody-producing cells (plaque-forming cells, PFC) and serum antibody in immunized C57BL/6 and C3H/He mice. The antigenicity of human erythrocytes of different ABO blood groups in the C57BL/6 mice, as determined by the number of specific PFC, was, in decreasing order, AB= A> B=O (p<0.005). The efficiency of immunogenicity of the human erythrocytes in terms of their cross-reactivity with PFC was, in order, AB= A= B>O, and the degree of reactinogenicity was, in order, AB> A ≥ B > O. The order of antigenicity of sheep erythrocytes from different animals, SRBC #1-#6, was #1 (= #2) > #3 = #4= #5> #6 in C57BL/6 mice and #1= #2 = #3= #4= #6> # 5 in C3H/He mice, determined by the number of specific PFC (p<0.01). The cross-reactivity of SRBC #1 -#6 with PFC demonstrates that the order of immunogenicity of SRBC was #1= #2= #3= #4= #5> #6 in C57BL/ 6 mice and #1= #2= #3 = #4 = #6 > #5 in C3H/He mice, and that of their reactinogenicity was #1> #2= #3= #4= #5 > #6 in C57BL/6 mice and #1>#4=#6>#2=#3>#5 in C3H/He mice. The cross -reactivity at the antibody level was indicative of the immunologic characteristics of blood cells of low antigenicity (human group O erythrocytes and SRBC #5 and # 6). SRBC #5 and #6 were somewhat opposed to each other regarding antigenicity in C57BL/6 and C3H/He mice. This signifies the presence of different immunogenic components on SRBC #5 and #6. The production of anti-SRBC #1 antibody reached its peak on the third day after secondary immunization. That of anti-SRBC #1, cross-reactive with SRBC #6, occurred after a longer latent period, reaching its peak on day 6. This indicates that SRBC #1 possesses more than one kind of immunogenic component or immunogenic determinant group on its surface.

View full abstract