Further characterization of herpes virus persistence.

Infection of cell cultures with herpes simples virus type 1 (HSV-1) under standard culture conditions yielded persistently infected cells capable of continued growth in the presence of virus and of forming colonies in agarose. The ability of an infected culture to yield cells able to survive HSV-1 infection was directly related to the presence of S phase cells (cells actively engaged in DNA synthesis) at the time of infection. Only when very high multiplicities of infecting virus (greater than 10) were used did cultures fail to yield persistently infected cells capable of colonial growth in agarose. Cell clones derived from colonies grown in agarose established cell cultures which possessed all the characteristics of HSV-1 persistently infected cultures. When cultures were cloned a second time in agarose, as a rare event, cell cultures which did not immediately liberate infectious virus could be isolated. These cell cultures, however, possessed an increased resistance to superinfection by HSV-1. On continued cultivation they reverted to persistence as exhibited by the sudden onset of virus cytopathic effects and release of infectious virus.

Immunological detection of O6-methylguanine in alkylated DNA.

Antibodies to O6-methyldeoxyguanosine were produced in rabbits and utilized in a radioimmunoassay to detect this nucleoside at picomole levels. The specificity of the antibodies was demonstrated by the use of nucleoside analogues as inhibitors in the radioimmunoassay. The antibodies cross-reacted with O6-methylguanosine, O6-methylguanine, and O6-ethylguanosine. There was 10(4) to 10(6) times less sensitivity to inhibition by deoxyadenosine, deoxyguanosine, and guanosine than by O6-methyldeoxyguanosine. The radioimmunoassay also detected O6-methylguanine in DNA alkylated by agents known to produce O6-methylguanine, such as N'-methyl-N-nitrosourea. DNA alkylated with dimethyl sulfate, which does not produce O6-methylguanine in DNA, cross-reacted with the antibodies to a very limited extent. Such an assay system for modified nucleic acid components would be very useful in following the production, persistence, and repair of these lesions in a variety of cells and tissues treated with a broad spectrum of carcinogens and suspected carcinogens.

Flow microfluorometric analysis of herpesvirus infected BHK-21 and BALB/3T3 cell cultures.

The interaction of herpes simplex type 1 with two eukaryotic cell lines (BHK-21 and BALB/3T3) was investigated by flow microfluorometric analysis after cell staining with mithramycin. Uninfected, lytically infected and persistently infected cell populations were examined. Viral replication within an infected cell population could be detected via FMF analysis. The low levels of viral replication occurring within persistently infected cell populations were also detectable. A marked degree of correlation was noted between morphological observations of infected cultures and the FMF profiles obtained. The results deomonstrate the great potential of this technique for the early detection and analysis of viral infection.

In vitro-labeled DNA for detecting viral genomes in multiple sclerosis: I. Papovaviruses.

Papovaviruses appear to be neurotropic and one, JC virus, is implicated as the cause of one type of demyelinating disease, progressive multifocal leukoencephalopathy. To investigate whether human papovaviruses play a role in multiple sclerosis, radioactively labeled DNA from BK virus, human papilloma virus, and simian virus 40 was used as a probe in order to detect related unlabeled DNA sequences in DNA isolated from multiple sclerosis brain and/or spinal cord. Labeled viral probes were denatured and DNA allowed to reassociate in the presence of excess unlabeled DNA from multiple sclerosis tissue or from controls. The reassociation rate of the probe is proportional to the concentration of viral DNA present, and an increase in the reassociation rate of the probe over that of control reactions would indicate the presence of unlabeled viral DNA in multiple sclerosis cellular DNA. However, addition of DNA derived from multiple sclerosis patients did not increase rates of reassociation of viral probes. Known human papovaviruses probably have no role in the pathogenesis of multiple sclerosis.

Selective inhibition of Bacillus subtilis sporulation by acridine orange and promethazine.

Two structurally similar compounds were found to inhibit sporulation in Bacillus subtilis 168. A dye, acridine orange, and an antischizophrenic drug, promethazine, blocked spore formation at concentrations subinhibitory to vegetative growth, while allowing synthesis of serine protease, antibiotic, and certain catabolite-repressed enzymes. The sporulation process was sensitive to promethazine through T2, whereas acridine orange was inhibitory until T4. The drug-treated cells were able to support the replication of phages phie and phi29, although the lytic cycles were altered slightly. The selective inhibition of sporulation by these compounds may be related to the affinity of some sporulation-specific genes to intercalating compounds.

Isolation, characterization, and activation of the magnesium dependent endodeoxyribonuclease from Bacillus subtilis.

A major endodeoxyribonulcease was isolated from a mutant of the transformable Bacillus subtilis 168. The magnesium-dependent endonuclease was purified approximately 750-fold to electrophoretic homogeneity. The enzyme had a molecular weight of about 31 000, as determined by gel filtration and polyacrylamide gel electrophoresis. The protein appears to be composed of two subunits. The nuclease was dependent on magnesium or maganese ions for hydrolytic activity. The purified nuclease degraded DNA from several species of Bacillus, as well as Escherichia coli DNA, alkylated, depurinated, and thymine-dimer containing B. subtilis DNA, and hydroxymethyluracil-containing phage DNA. The enzyme also hydrolyzed single-stranded DNA, although native DNA was the preferred substrate. However, the nuclease was unable to degrade ribosomal RNA. The cleavage products of the DNA hydrolysis have 5'-phosphate and 3'-hydroxyl ends. The enzyme could be activated in crude extracts by heat treatment or treatment with guanidine hydrochloride. The nuclease activity was inhibited by phosphate and by high concentrations of NaCl. A possible function for this endonuclease in bacterial transformation is discussed.

Effects of solar ultraviolet radiations on Bacillus subtilis spores and T7 bacteriophage.

Spores of Bacillus subtilis HA 101 and the DNA polymerase I-defective mutant HA 101 (59) F were exposed to selected wavelengths of solar ultraviolet light and space vacuum during the return of Apollo 16. In addition, coliphage T7 suspensions were exposed to solar ultraviolet radiation as part of the Microbial Response to Space Environment Experiment. Optical filters were employed to provide different energy levels at wavelengths 254 nm and 280 nm. Dose-response curves for lethal and mutagenic effects were compared with ground-based data. A close parallel was observed between the results of solar radiation and ground tests with spores of the two strains. However, significantly greater inactivation of T7 bacteriophage was observed after exposure to solar ultraviolet radiation.

Isolation of two acetyl esterases from extracts of Bacillus subtilis.

Acrylamide gel electrophoresis of crude cellular extracts of Bacillus subtilis revealed the presence of two acetyl esterases. Esterase A, the slower migrating enzyme, was found to be present in both vegetative and sporulating cells, whereas esterase B activity was more abundant after exponential growth ceased. Both esterases were present in the supernatant fraction of lysed spheroplasts and in a disrupted spore preparation. Of four pleiotropic asporogenous mutants tested, three exhibited decreased esterase B activity. Esterases A and B were partially purified by differential precipitation and co-chromatographed on diethylaminoethyl (DEAE)-cellulose (pH 7.5) and DEAE-Sephadex (pH 8.5). By employing gel filtration chromatography, the two esterases were separated, and molecular weights of 160,000 and 51,000 were estimated for esterases A and B, respectively. Esterase A was further purified to electrophoretic homogeneity by differential heating and preparative starch block electrophoresis. Sodium dodecyl sulfate-acrylamide gel electrophoresis of purified esterase A yielded a single protein band with a molecular weight of 31,000. The pI values of esterases A and B were determined to be 6.4 and 5.4, respectively.

Hyperprotease-producing mutants of Bacillus subtilis.

A number of mutants of Bacillus subtilis producing high levels of extracellular protease have been isolated. Analysis of culture supernatants of these mutants has shown that the total amount of proteolytic activity is elevated from 16- to 37-fold over the wild strain. The elevated activity was due to a simultaneous increase in both the neutral and alkaline protease. All of the mutants genetically analyzed were found linked to the argC4 marker by PBS-1 transduction analysis.

Protease activities during the course of sporulation on Bacillus subtilis.

Three proteolytic enzymes have been isolated from sporulating cultures of Bacillus subtilis. These activities were, respectively, a protease inhibited by ethylenediaminetetraacetic acid (EDTA) but not phenylmethylsulfonyl fluoride (PMSF), a protease active on both protein and ester substrates, and an ester-active enzyme with low activity on proteins. The latter two enzymes were inhibited by PMSF but not by EDTA. The specific activity of each was determined both intra- and extra-cellularly during growth and sporulation in a single-defined medium. All three enzymes were shown to exhibit a rapid increase in specific activity at a time coinciding with the appearance of refractile bodies in cells.

Abortive infection of sporulating Bacillus subtilis 168 by phi 2 bacteriophage.

Bacteriophage phi2 is unable to replicate in Bacillus subtilis 168. Although some phage deoxyribonucleic acid (DNA) synthesis can occur, the DNA made is not biologically active and sedimentation analysis reveals that it is smaller in size than that of mature DNA or DNA isolated from phi2-infected permissive hosts. Messenger ribonucleic acid hybridizable with phi2 DNA is also synthesized in phi2-infected cells of 168. Mutants of 168 which are permissive hosts for phi2 have been isolated. These mutants are defective in sporulation and possess the phenotype of "early sporulation mutants." The majority map in two locations, one near the lys locus opposite the trp locus (spoA locus) and the other tightly linked to a phe locus.

Microbiological problems of manned space flight.

The hazard of infectious disease in long-duration manned space flight is discussed in relation to the unique conditions of spacecraft environment. Confinement in a small, enclosed volume, special problems of waste disposal and personal hygiene, zero gravity, and high oxygen content present special features that favor transmission of micro-organisms. Alterations in microbial flora will occur and these will influence host defenses against disease. Genetic variants of micro-organisms could be induced by and selected under the unique conditions of space flight. A reduction in the variety of microbial flora could result in "microbial shock" on re-entry. The influence of environmental conditions of the spacecraft on host defenses against infection is unpredictable in the absence of investigative work. However, pre-flight assessment of immune status is recommended. A graduated pre-flight isolation period is highly recommended to permit exchange of flora, to protect astronauts from new agents, and to allow most infections to emerge.

Mechanism of transfection with deoxyribonucleic acid from the temperate Bacillus bacteriophage phi-105.

Bacteriophage phi105 is a temperate phage for the transformable Bacillus subtilis 168. The infectivity of deoxyribonucleic acid (DNA) extracted from mature phi105 phage particles, from bacteria lysogenic for phi105 (prophage DNA), and from induced lysogenic bacteria (vegetative DNA) was examined in the B. subtilis transformation system. About one infectious center was formed per 10(8) mature DNA molecules added to competent cells, but single markers could be rescued from mature DNA by a superinfecting phage at a 10(3)- to 10(4)-fold higher frequency. Single markers in mature DNA were inactivated at an exponential rate after uptake by a competent cell. Prophage and vegetative DNA gave about one infectious center per 10(3) molecules added to competent cells. Infectious prophage DNA entered competent cells as a single molecule; it gave a majority of lytic responses. Single markers in sheared prophage DNA were inactivated at the same rate as markers in mature DNA. Prophage DNA was dependent on the bacterial rec-1 function for its infectivity, whereas vegetative DNA was not. The mechanism of transfection of B. subtilis with viral DNA is discussed, and a model for transfection with phi105 DNA is proposed.

Conditions for competence in the Bacillus licheniformis transformation system.

Some variables in the Bacillus licheniformis transformation system, such as inoculum size, initial cell density in transformation medium, and time of optimal competence, have been explored, and a methodology has been developed to obtain greater reproducibility and maximal competence for transformation. The transformation system in B. licheniformis has been shown to be similar to that of B. subtilis in a number of respects, including "dormancy" of transformed cells and the concentration of transforming deoxyribonucleic acid required for saturation.