Isolation and Characterization of Supercoiled Circular Deoxyribonucleic Acid from Beta-Hemolytic Strains of Escherichia coli

Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated by cesium chloride centrifugation in the presence of ethidium bromide from a naturally occurring beta-hemolytic Escherichia coli strain (SC52). The open circular forms have contour lengths of 2.25 ± 0.1 μm, 24.0 ± 0.3 μm, and 29.5 ± 0.5 μm. The beta-hemolytic character of E. coli SC52 can be transferred by conjugation to a nonhemolytic recipient strain. Analysis of the supercoiled DNA of the hemolytic recipient demonstrated that the two large supercoiled DNA molecules of E. coli SC52 are transferred during this event, too. A beta-hemolytic laboratory E. coli strain and several of its derivatives have been shown to contain at least one circular DNA molecule, slightly larger in size than those isolated from E. coli SC52 and its conjugant. The possible significance of these DNA molecules for hemolysin production and transfer is discussed.     

Isolation of Circular Deoxyribonucleic Acid from Salmonella typhosa Hybrids Obtained from Matings with Escherichia coli Hfr Donors

Heterozygous, partial diploid Salmonella typhosa hybrids obtained from matings with Escherichia coli K-12 Hfr strains were observed to contain supercoiled, circular deoxyribonucleic acid (DNA) when examined by the dye-buoyant density method. Examination of one such S. typhosa hybrid after its loss, by segregation, of the inherited E. coli genetic markers revealed a concurrent loss of its supercoiled circular DNA. Subsequent remating of this segregant with various E. coli Hfr strains resulted in the reappearance of the circular DNA. Molecular weight determinations of circular DNA molecules isolated from a number of S. typhosa partial diploid hybrids were made by sucrose density gradient ultracentrifugation and electron microscopy. These studies revealed a range of molecular sizes among the various hybrids examined, but each hybrid exhibited only a single characteristic size for its contained circular DNA. The range of size is consistent with the presence in each hybrid of a different length of E. coli chromosome. It was concluded that the E. coli Hfr genetic segments transferred to these S. typhosa hybrids were conserved, in the diploid state, in the form of supercoiled, circular DNA molecules.     

Studies on Circular Deoxyribonucleic Acid I. Isolation of Bovine Papilloma Virus and Characterization of Its Deoxyribonucleic Acid

A simple method is described for the isolation of bovine papilloma virus and its deoxyribonucleic acid (DNA). As found with other representatives of this virus group, this DNA preparation contains two components, I and II, as shown by sedimentation and electron microscopic studies. Component I is a fast-sedimenting, twisted, circular DNA molecule and represents usually 70 to 90% of the DNA in the mixture. The direction of the twist in the superstructure is right-handed. Component II originates from I by one or more single-strand breaks and is the "relaxed" circular from of the viral DNA.     

Isolation and Characterization of Plasmid Deoxyribonucleic Acid from Streptococcus mutans

A small plasmid with a molecular weight of approximately 3.0 x 10(6) and present to the extent of about 16 copies per chromosomal genome equivalent was isolated from Streptococcus mutans strain LM-7.     

Isolation of Covalently Closed Circular Deoxyribonucleic Acid from Bacteria Which Produce Exocellular Nuclease

Reproducible yields of covalently closed circular (plasmid) deoxyribonucleic acid were obtained from mutants defective for extracellular nuclease but not from the corresponding wild-type strain of Serratia marcescens     

Lactose-Fermenting Salmonella from Dried Milk and Milk-Drying Plants

A study of 552 salmonella cultures revealed that 86 (15.6%) of the cultures fermented lactose. These had been isolated from dried milk products and milk-drying plants. Acid and gas were produced in lactose broth. Solid media containing lactose as the key ingredient for the differential reaction were not satisfactory for recognizing salmonella colonies. No problem was encountered in selecting salmonella colonies when bismuth sulfite agar was used.     

Nature of Lactose-fermenting Salmonella Strains Obtained from Clinical Sources

Six of seven lactose-fermenting (lac(+)) Salmonella strains obtained from clinical sources were found to be capable of transferring the lac(+) property by conjugation to Salmonella typhosa WR4204. All of the six S. typhosa strains which received the lac(+) property transferred it in turn to S. typhimurium WR5000 at the high frequencies typical of extrachromosomal F-merogenotes. These six lac elements were also transmissible from S. typhosa WR4204 to Proteus mirabilis and to some strains of Escherichia coli K-12; moreover, they were capable of promoting low frequency transfer of chromosomal genes from S. typhimurium WR5000 to S. typhosa WR4204. One of these lac elements was shown also to be capable of promoting low frequency chromosome transfer in E. coli K-12. E. coli K-12 strains harboring these lac elements exhibited sensitivity to the male specific phage R-17. Sensitivity to R-17 was not detected in Salmonella strains containing the elements. Examination of the lac elements in P. mirabilis by cesium chloride density gradient centrifugation showed that each element had a guanine plus cytosine content of 50%. The sizes of the elements varied from 0.8 to 3% of the total Proteus deoxyribonucleic acid. The amount of beta-galactosidase produced by induced and uninduced cultures of S. typhimurium WR5000 and S. typhosa WR4204 containing the lac elements was lower than that produced by these strains with the F-lac episome. The heat sensitivity of beta-galactosidase produced by the lac elements in their original Salmonella hosts indicated that the enzyme made by these strains differs from E. coli beta-galactosidase.     

Characterization of the Polydisperse Closed Circular Deoxyribonucleic Acid Molecules of Bacillus megaterium

The polydisperse circular deoxyribonucleic acid (DNA) molecules which comprise up to 30% of the total extractable DNA of Bacillus megaterium strain 216 have been purified and partially characterized. Banding in cesium chlorideethidium bromide by "gradient relaxation" in a fixed-angle rotor provided good resolution of circular and chromosomal DNAs for preparative separations. Renaturation studies on purified circular DNA failed to reveal a rapidly renaturing fraction, and DNA-DNA hybridization studies indicated that the majority of the chromosomal nucleotide sequences are represented in the heterogeneous-size population of circular molecules. It is concluded that the circular DNA of B. megaterium does not represent typical bacterial plasmid DNA. The possibility that the circular DNA molecules are the result of the expression of a defective bacteriophage is discussed.     

Method for Isolation of Deoxyribonucleic Acid from Mycobacteria

A relatively gentle and efficient procedure for obtaining deoxyribonucleic acid from mycobacteria by the use of glycine and lysozyme is described.     

Deoxyribonucleic Acid Characterization of Bdellovibrios

The guanine plus cytosine (GC) content of the deoxyribonucleic acid (DNA) of 11 isolates of host-dependent (H-D) bdellovibrios and 18 host-independent (H-I) derivatives was determined from thermal denaturation curves and buoyant densities in CsCl. The H-D and respective H-I cultures have GC contents which are identical within the limits of experimental error. Most cultures of Bdellovibrio bacteriovorus, including the holotype culture, have 50.4 +/- 0.9 moles% GC in their DNA; two bdellovibrio isolates of presently uncertain nomenclatural status contain DNA of about 43% GC. Optical melting profiles of all the DNA from all of these organisms are particularly steep, indicating little compositional heterogeneity. Chromatography of acid hydrolysates of Bdellovibrio nucleic acids reveal no unusual components. The DNA content per cell of one H-I derivative is about one-third the amount per Escherichia coli cell growing at a comparable rate.     

Transformation of Salmonella typhimurium by Plasmid Deoxyribonucleic Acid

A modified transformation procedure that is effective for the introduction of plasmid deoxyribonucleic acid at high frequency into Salmonella typhimurium, as well as into Escherichia coli, is described. Transformed bacteria acquire a circular deoxyribonucleic acid species having the genetic and molecular characteristics of the transforming plasmid.     

Acquisition of Sequences Homologous to Host Deoxyribonucleic Acid by Closed Circular Simian Virus 40 Deoxyribonucleic Acid

The synthesis of closed circular simian virus 40 (SV40) deoxyribonucleic acid (DNA) containing sequences homologous to host cell DNA depends upon the conditions under which the cells are infected. When BS-C-1 monkey cells were infected with non-plaque-purified virus at low multiplicity of infection [MOI, 0.032 plaque-forming units (PFU)/cell], little, if any, of the SV40 DNA extracted from the infected cells hybridized to host DNA; but when increasingly higher multiplicities were used (in the range 0.16 to 3,000 PFU/cell), an increasingly greater amount of the extracted SV40 DNA hybridized to host DNA. The same effect was observed when the closed circular SV40 DNA was extracted from purified virions (grown at low and high MOI) rather than from the infected cell complex. When the cells were infected at high MOI with plaque-purified virus (11 viral clones were tested), none of the SV40 DNA extracted from the cells hybridized detectably with host cell DNA. However, plaque-purified virus that was serially passaged, undiluted, induced the synthesis of virus DNA which again showed extensive homology to host DNA. It is suggested that, under certain circumstances, recombination occurs between viral and host DNA during lytic infection which results in the incorporation of host DNA sequences into closed circular SV40 DNA.     

Deoxyribonucleic Acid Adenine and Cytosine Methylation in Salmonella typhimurium and Salmonella typhi

The methylations of adenine in the sequence -GATC- and of the second cytosine in the sequence - [Formula: see text] - were studied in Salmonella typhimurium and in Salmonella typhi. The study was carried out by using endonucleases which restrict the plasmid pBR322 by cleavage at the sequences -GATC- (DpnI and MboI) and - [Formula: see text] - (EcoRII). The restriction patterns obtained for this plasmid isolated from transformed S. typhimurium and S. typhi were compared with those of pBR322 isolated from Escherichia coli K-12. In E. coli K-12, adenines at the sequence -GATC- and the second cytosines at - [Formula: see text] - are met hylated by enzymes coded for by the genes dam and dem, respectively. From comparison of the restriction patterns obtained, it is concluded that S. typhimurium and S. typhi contain genes responsible for deoxyribonucleic acid methylation equivalent to E. coli K-12 genes dam and dcm.     

Composite Circular Forms of R Factor Deoxyribonucleic Acid Molecules

Two R factors, one (R15) conferring resistance to streptomycin and sulfonamide (SM(r)SU(r)) and the other (222/R3) to streptomycin, sulfonamide, and chloramphenicol (SM(r)SU(r)CM(r)), were transferred to a Proteus mirabilis strain, and deoxyribonucleic acid (DNA) extracted from these strains was subjected to density-gradient centrifugation. R15-DNA formed a single satellite band at a density of 1.709 g cm(-3). Electron microscopy of samples from this band showed circular molecules of one type, with a contour length of 18 mum (35 x 10(6) daltons). 222/R3-DNA formed a satellite band with three peaks at densities 1.708, 1.711 and 1.717 g cm(-3). Electron micrographs revealed circular structures from each band with contour lengths, respectively, of 29 (54 x 10(6) daltons), 36 (68 x 10(6) daltons), and 6 mum (12 x 10(6) daltons). "Supertwisted" forms of several molecular species were found. It is suggested that 222/R3 DNA comprises either a single 36-mum molecule or two individual molecules, 29 and 6 mum in length, and that this may reflect the evolutionary development of R factors.     

Survival, Deoxyribonucleic Acid Breakdown, and Synthesis in Salmonella typhimurium as Compared with Escherichia coli B Strains

Salmonella typhimurium LT-2 was compared with radioresistant (B/r) and radiosensitive (B(s-2)) strains of Escherichia coli in respect to the survival, deoxyribonucleic acid (DNA) breakdown, and DNA synthesis after X irradiation. It is shown that S. typhimurium LT-2 is about four times more sensitive than E. coli B/r but less sensitive than B(s-2). The DNA breakdown is in S. typhimurium LT-2 lower than the postirradiation breakdown of DNA in both E. coli strains and DNA synthesis proceeds in this bacterium in spite of a much lower survival, as in the radioresistant E. coli B/r.     

Characterization of Excess Deoxyribonucleic Acid Synthesized by Pneumococci in the Presence of Polyadenylic Acid and Deoxyribonucleic Acid Precursors

Excess deoxyribonucleic acid (DNA) synthesized by cell suspensions of encapsulated pneumococci in the presence of polyadenylic acid plus all eight of the naturally occurring deoxyribonucleosides and deoxyribonucleotides has been characterized in several ways. The DNA represents complete molecules, is synthesized by a relatively large population at a steady rate, and is replicated in a semiconservative manner.     

Deoxyribonucleic Acid Synthesis in Bacteriophage SPO1-Infected Bacillus subtilis I. Bacteriophage Deoxyribonucleic Acid Synthesis and Fate of Host Deoxyribonucleic Acid in Normal and Polymerase-Deficient Strains

The effect of bacteriophage SPO1 infection of Bacillus subtilis and a deoxyribonucleic acid (DNA) polymerase-deficient (pol⁻) mutant of this microorganism on the synthesis of DNA has been examined. Soon after infection, the incorporation of deoxyribonucleoside triphosphates into acid-insoluble material by cell lysates was greatly reduced. This inhibition of host DNA synthesis was not a result of host chromosome degradation nor did it appear to be due to the induction of thymidine triphosphate nucleotidohydrolase. Examination of the host chromosome for genetic linkage throughout the lytic cycle indicated that no extensive degradation occurred. After the inhibition of host DNA synthesis, a new polymerase activity arose which directed the synthesis of phage DNA. This new activity required deoxyribonucleoside triphosphates as substrates, Mg²⁺ ions, and a sulfhydryl reducing agent, and it was stimulated in the presence of adenosine triphosphate. The phage DNA polymerase, like that of its host, was associated with a fast-sedimenting cell membrane complex. The pol⁻ mutation had no effect on the synthesis of phage DNA or production of mature phage particles.     

Host Specificity of Salmonella typhimurium Deoxyribonucleic Acid Restriction and Modification

The restriction and modification genes of Salmonella typhimurium which lie near the thr locus were transferred to a restrictionless mutant of Escherichia coli. These genes were found to be allelic to the E. coli K, B, and A restriction and modification genes. E. coli recombinants with the restriction and modification host specificity of S. typhimurium restricted phage λ that had been modified by each of the seven known host specificities of E. coli at efficiency of plating levels of about 10⁻². Phage λ modified with the S. typhimurium host specificity was restricted by six of the seven E. coli host specificities but not by the RII (fi⁻ R-factor controlled) host specificity. It is proposed that the restriction and modification enzymes of this S. typhimurium host specificity have two substrates, one of which is a substrate for the RII host specificity enzymes.     

Phenotypic Characterization and Deoxyribonucleic Acid Homologies of Pseudomonas solanacearum

Twenty-six strains and colony variants of Pseudomonas solanacearum belonging to four described biotypes were characterized, by using 169 phenotypic characters previously found useful in distinguishing among strains of other Pseudomonas species. Deoxyribonucleic acid (DNA) hybridization (intra- and interspecific DNA-DNA hybridizations) was performed by using the in vitro "DNA competition" technique. P. solanacearum appears to be a moderately homogeneous species, which is, at most, only remotely related to all other species of the genus studied to date. The four biotypes are not clearly distinct from one another with respect to nutritional characters or DNA homologies. Discrepancies between acid production and growth with some carbohydrates were noted. Difficulties were encountered in certain DNA competition experiments and some problems of the methodology are discussed.