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Most copper bioleaching plants operate with a high concentration of sulfate salts, caused by the continuous addition of sulfuric acid and the recycling of the leaching solution. Since the bacteria involved in bioleaching have been generally isolated at low sulfate concentrations, the bacterial population present in the high-sulfate (150 g l −1 ) leaching solution, employed in a copper production plant, was investigated. The iron-oxidizing bacteria able to grow in the leaching solution were enriched by several batch cultivations and, after serial dilution, an abundant bacterial strain was isolated. This strain, called LA, exhibited a relatively constant rate of iron-oxidation in media containing sulfate ions at concentrations ranging from 10 to 150 g l −1 . Culture collection strains of Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans showed limited abilities to grow at sulfate ion concentrations higher than 70 g l −1 . In spite of its tolerance to high sulfate concentrations, strain LA was as sensitive to NaCl as A. ferrooxidans . Comparative sequence analysis of the 16S rRNA gene of strain LA indicated that it is phylogenetically related to strains described as Leptospirillum ferrooxidans . Bacterial community DNA restriction patterns of 16S rRNA genes suggested that strain LA was a minor component of the bacterial population present in leaching solution, but is abundant in ore leached with this solution.

Analysis of the 16S rDNAs obtained from cultures of single colonies of either type collection strains or environmental strains of the genus Vibrio revealed the presence of polymorphism in every one of the strains examined. Polymorphism was detected by visualization of heteroduplexes produced after 16S rDNA PCR amplification, a procedure that allows for the screening of a large number of isolates. Amplified 16S rDNAs obtained from both Vibrio parahaemolyticus and an environmental strain were cloned. Their nucleotide sequences revealed differences of up to 2% among 16S rDNAs from the same strain. Polymorphic sites were concentrated in a recognized variable stem–loop of bacterial 16S rRNA that contained in some cases up to 83% of the total mismatches observed. Most of the substitutions present in the stem–loop region showed compensating base covariation. The accumulation of so many compensating changes in the stem–loop region implies that the divergence of the different versions of this stem–loop is relatively ancient. This divergence could be the result of either a selection process or a lateral transfer of independently evolved genes.

An archaeon was detected in the leaching solution from a commercial copper production plant and in copper sulfide ores leached with the solution. The leaching solution in this plant contains a high concentration of sulfate salts. Analysis of the microbial population by polymerase chain reaction-amplification of archaeal 16S rDNAs indicated the presence of a single sequence type. Comparison of the nucleotide sequence of the polymerase chain reaction product with available reference sequences suggested that this archaeon corresponds to a new species of a novel genus and family within the order Thermoplasmales. This archaeon grows in synthetic media but it has not been possible to obtain isolates free of chemolithotrophic bacteria.

Analysis of the 16S rRNA genes retrieved directiy from different environments has proven to be a powerful tool that has greatly expanded our knowledge of microbial diversity and phylogeny. It is shown here that sequence similarity between 80 and 100% among 16S rDNAs can be estimated by the electrophoretic migration of their heteroduplexes. This was measured by hybridization and electrophoresis in polyacrylamide gels of the product obtained after PCR amplification of almost the entire 16S rRNA gene from different bacterial species. These heteroduplexes were also observed after amplification of samples containing DNA from two or more bacterial species and a procedure was applied to identify reliably heteroduplexes among the amplification products. The electrophoretic migration of the heteroduplexes observed after PCR was used to detect the presence of 16S rDNAs with different sequences in DNA extracted from both a mixture of two bacterial species and samples containing a natural bacterial community.

Most copper bioleaching plants operate with a high concentration of sulfate salts caused by the continuous addition of sulfuric acid and the recycling of the leaching solution. Since the bacteria involved in bioleaching have been generally isolated at low sulfate concentrations, the bacterial population in ores leached with the high-sulfate solution (1.25 M) employed in a copper production plant was investigated. The complexity of the original population was assessed by the length pattern of the spacer regions between the 16S and 23S rRNA genes, observed after PCR amplification of the DNA extracted from the leached ore. Six main spacers were distinguished by electrophoretic migration, but they could be further resolved into eight spacers by nucleotide sequence homology. The degree of homology was inferred from the electrophoretic migration of the heteroduplexes formed after hybridization. One of the spacers was indistinguishable from that found in Thiobacillus thiooxidans, four could be related to Thiobacillus ferrooxidans, and three could be related to Leptospirillum ferrooxidans. Only five of the spacers in the original sample could be recovered after culturing in media containing different inorganic energy source. Altogether, the results indicate that the bacteria in the leached ore formed a community composed of at least three species: a fairly homogeneous population of T. thiooxidans strains and two heterogeneous populations of T. ferrooxidans and L. ferrooxidans strains.

The aim of this study was to evaluate the role of the intestinal microbiota in soybean meal enteritis. Three groups of Atlantic salmon (Salmo salar) were fed for 35 days with different diets: a control diet (CD) containing 510 g kg �1 fishmeal, diet 1 (D1) containing 378 g kg �1 of soybean meal and diet 2 (D2) containing D1 supplemented with two viable lactic acid bacteria (LAB). As expected, the fish fed with D1 showed clear signs of distal intestinal inflammation during the study compared with the fish fed CD. For the fish fed with D2, the addition of LAB diminished the inflammation at day 28, but did not abolish it. Microbiological analysis demonstrated that specific bacterial groups were not correlated with the development of enteritis, but were correlated with the three diets. Microbacterium, Pseudomonas, Lactococcus lactis sp. cremoris and Aeromonas VIa were correlated with the CD, Aeromonas VIb and Sporosarcina aquimarina were correlated with D1, and Alcaligenes, Acinetobacter, L. lactis sp. lactis and Carnobacterium maltaromaticum were correlated with D2. Shewanella was not affected by the diet and was present in all fish intestines. Our study suggests that LAB may modulate intestinal inflammation; however, the role of the microbiota in the aetiology of enteritis in fish still requires further study.

Soybean meal (SBM) is an important protein source in animal feed. However, the levels of SBM inclusion are restricted in some animal species by the presence of antinutritional factors (ANFs), including non-starch polysaccharides (NSPs) and α-galactosides (GOSs). The aim of this study was to reduce the soybean meal NSPs and GOSs by solid-state fermentation (SSF) using a combination of cellulolytic bacteria isolated from different environments (termites, earthworms, corn silage and bovine ruminal content). To analyse the key enzymatic activities, the isolates were grown in minimal media containing NSPs extracted from SBM. The selected bacterial strains belonged to the genera Streptomyces, Cohnella and Cellulosimicrobium. SSF resulted in a reduction of nearly 24% in the total NSPs, 83% of stachyose and 69% of raffinose and an increase in the protein content. These results suggest that cellulolytic bacteria-based SSF processing facilitates SBM nutritional improvement. In addition, the use of fermented SBM in animal diets can be recommended.

Analysis by radioimmunoprecipitation of serum samples from 27 different human immunodeficiency virus type 1 (HIV-1)-infected individuals residing in Chile showed that the sera of 26% of these individuals also react with glycoprotein gp125 of HIV type 2 (HIV-2). This cross-reaction seems to reflect a qualitative difference among infected individuals, because the titer of antibodies against gp120 of HIV-1 in the cross-reacting samples did not differ significantly from that in the non-cross-reacting samples. Most of the HIV-1-seropositive sera, including many that did not react with gp125 of HIV-2, reacted with gp140, the precursor of HIV-2 glycoproteins. The observed cross-reactions allowed us to distinguish three groups of HIV-1-infected individuals: (i) those whose sera react with both gp140 and gp125, (ii) those whose sera react with gp140, and (iii) those whose sera react with neither of these glycoproteins. The possible cause and significance of these differences is under study.

PCR amplification of the spacer region between the 16S and 23S rRNA genes is commonly employed for the analysis of bacterial communities. In this analysis, the intergenic spacers are amplified by PCR using primers complementary to conserved regions in the 3' 16S rDNA and 5' 23S rDNA. By this method, the observation of every bacterial population may be limited by several causes. To explore the extent of bacterial populations overlooked by this method, we have used an empirical approach. In a sample containing about 50 colonies, we tested the capability to amplify by PCR the spacers from each colony. We also examined the ability to observe the spacers from each colony in the product obtained after amplification of the DNA extracted from the whole sample, as it is usually performed by this method. Contrarily to our expectations that a significant fraction of colonies would not yield amplification products, spacers were successfully amplified from every colony of two different samples examined. Overall, our results suggest that in spite of well-based theoretical limitations, the analysis of bacterial communities by amplification of the spacer regions can render a comprehensive representation of the more abundant bacterial clades in the sample.

The heteroduplex mobility assay (HMA) reveals sequence dissimilarity between DNA by measuring the retarded migration of the hybrid or heteroduplex using polyacrylamide gel electrophoresis. Heterogeneity in some cultures of toxic dinoflagellates of the genus Alexandrium (Halim) Balech was observed during comparison of the amplified D1–D2 region of the large subunit rRNA gene (rDNA) using this method. HMA also allowed grouping of clones obtained from toxic bloom events in the Chilean, southernmost Pacific within the Asian Southern Pacific lineage of A. catenella (Whedon et Kofoid) Balech. The applied methodology provides a rapid and simple tool for use in assessing heterogeneity as well as for molecular grouping of strains among the genus Alexandrium.

Extensive bacterial growth was observed when copper sulfide ores were leached with 0.6 N sulfuric acid. The bacterial population developed in this condition was examined by characterization of the spacer regions between the 16S and 23S rRNA genetic loci obtained after PCR amplification of the DNA extracted from the leached ore. The spacers observed had the sizes found in strains of "Leptospirillum ferrooxidans" and Thiobacillus thiooxidans, except for a larger one, approximately 560 bp long, that was not observed in any of the strains examined, including those of Thiobacillus ferrooxidans. The bacteria with this last spacer were selected after culturing in mineral and elemental sulfur media containing 0.7 N sulfuric acid. The spacer and the 16S ribosomal DNA of this isolate were sequenced and compared with those in species commonly found in bioleaching processes. Though the nucleotide sequence of the spacer showed an extensive heterologous region with T. thiooxidans, the sequence of its 16S rDNA gene indicated a close relationship (99.85%) with this species. These results indicate that a population comprised of bacterial strains closely related to T. thiooxidans and of another strain, possibly related to "L. ferrooxidans," can develop during leaching at high sulfuric acid concentration. Iron oxidation in this condition is attributable to "L. ferrooxidans" and not T. ferrooxidans, based on the presence of spacers with the "L. ferrooxidans" size range and the absence of spacers characteristic of T. ferrooxidans.

When MA104 cells were infected with SA11 rotavirus, 12 protein classes, absent in mock-infected cells, could be distinguished by polyacrylamide gel electrophoresis. At least two of these proteins were glycosylated, and their synthesis could be blocked with tunicamycin. The oligosaccharides of both glycoproteins were cleaved by endo-beta-N-acetylglucosaminidase H, suggesting that they were residues of the "high-mannose" type. Of the 12 viral polypeptides observed in infected cells, 1 was probably the apoprotein of one of these glycoproteins; 5, including 1 glycoprotein, were structural components of the virions, whereas the other 6, including a second and possibly third glycoprotein, were nonstructural viral proteins. When the 11 double-stranded RNA genome segments of SA11 were translated, after denaturation, in an RNA-dependent cell-free translation system, at least 11 different polypeptides were synthesized. Ten of these polypeptides had electrophoretic migration patterns equal to those of viral proteins observed in tunicamycin-treated infected cells. Nine of the 11 double-stranded RNA genome segments were resolved by polyacrylamide gel electrophoresis and were translated individually. Two were not resolved from each other and therefore were translated together. Correlation of each synthesized polypeptide with an individual RNA segment allowed us to make a probable gene-coding assignment for the different SA11 genome segments.

Substantial differences in the RNA electrophoretic patterns of rotaviruses were observed when the conditions for gel electrophoresis were varied. Electropherotypes which seem alike under one set of conditions, may appear significantly different under other conditions such as distinct proportions of acrylamide and bisacrylamide or different running buffer. Different patterns were observed for the same rotavirus RNA sample even when different voltages were applied to otherwise identical runs; these differences were probably caused by the increasing amount of heat generated at higher voltage. Although the conditions of electrophoresis did not have as pronounced an effect on ‘double-stranded’ as on ‘single-stranded’ RNA. precautions should be taken to obtain comparable and reproducible results. A standard methodology for ‘electropherotyping’ of rotaviruses is therefore suggested.

A DNA form with restricted binding of intercalating dyes (propidium iodide or ethidium bromide) has been found in bacteriophage φX-infected cells during the period of single-stranded DNA synthesis. In the electron microscope, this DNA form is seen to be a double-stranded DNA ring with two single-stranded DNA tails protruding from the same portion of the ring; it is composed of a linear φX DNA strand, longer than one φX genome, and a single-stranded ring complementary to φX DNA. Base-pairing of these two tails in partially complementary regions restricts unwinding of the double-stranded DNA ring and consequently intercalation and binding of the dyes. It is postulated that these molecules originate from a previously reported precursor of φX DNA, namely a double-stranded ring with a single-stranded tail, by branch migration.

Summary Analysis of cells infected with simian rotavirus SA11 at late times of infection indicated that the particles were associated with membranes and the cytoskeleton. Although a large amount of cellular and non-structural viral proteins were released at these times, probably by cellular lysis, only virus with an outer layer was found outside the cells, while virus without an outer layer remained associated with the cells, probably with membranes and the cytoskeleton. Inhibition of glycosylation by tunicamycin did not abolish cell lysis but inhibited the liberation of particles and the non-glycosylated precursors of the structural and non-structural viral glycoproteins. These results indicate that immature virus was tightly associated with the structural matrix of the cell.

The primary structure of the trypsin cleavage site in the outer layer protein VP3 of rotavirus SA11 was determined. This cleavage enhances the infectivity of rotavirus SA11. Both VP8, one of the polypeptides generated by the cleavage, and VP3 had their alpha-NH2 blocked. Only VP5, the other polypeptide produced by the cleavage, was susceptible to sequential Edman degradation, indicating that it contained the new alpha-NH2 terminus generated by trypsin hydrolysis. The results indicated that purified VP5 is composed of two polypeptides with the following amino acid sequence at their N terminus: (a) ??VYTRAQPNQDAVVSKTS...; (b) AQPNQDAVVSKTS.... Sequencing of the DNA complementary to ds RNA segment 4 revealed a nucleotide sequence encoding the amino acid sequences indicated above, with only one different amino acid. From these results, the amino acid sequence of the site cleaved by trypsin was extended to cover the C termini (present in VP8). The following sequence, which contains two sites (indicated with asterisks) and can be cleaved by trypsin was deduced: ... VPVSIVSR*NIVYTR*AQPNQDIVVSKTS....

In 1983, we isolated a porcine rotavirus (strain YM) that was prevalent in several regions of Mexico, as judged by the frequency of its characteristic electropherotype. By a focus reduction neutralization test, rotavirus YM was clearly distinguished from prototype rotavirus strains belonging to serotypes 1 (Wa), 2 (S2), 3 (SA11), 4 (ST3), 5 (OSU), and 6 (NCDV). Minor, one-way cross-neutralization (1 to 5%) was observed when antisera to the various rotavirus strains were incubated with rotavirus YM. In addition, the YM virus was not neutralized by neutralizing monoclonal antibodies with specificity to serotypes 1, 2, 3, and 5. The subgroup of the virus was determined to be I by enzyme-linked immunosorbent assay. To characterize the serotype-specific glycoprotein of the virus at the molecular level, we cloned and sequenced the gene coding for VP7. Comparison of the deduced amino acid sequence with reported homologous sequences from human and animal rotavirus strains belonging to six different serotypes further supported the distinct immunological identity of the YM VP7 protein.

The oxidation of ferrous iron and elemental sulfur by Thiobacillus ferrooxidans that was absorbed and unabsorbed onto the surface of sulfur prills was studied. Unadsorbed sulfur-grown cells oxidized ferrous iron at a rate that was 3 to 7 times slower than that of ferrous iron-grown cells, but sulfur-grown cells were able to reach the oxidation rate of the ferrous iron-adapted cells after only 1.5 generations in a medium containing ferrous iron. Bacteria that were adsorbed to sulfur prills oxidized ferrous iron at a rate similar to that of unadsorbed sulfur-grown bacteria. They also showed the enhancement of ferrous iron oxidation activity in the presence of ferrous iron, even though sulfur continued to be available to the bacteria in this case. An increase in the level of rusticyanin together with the enhancement of the ferrous iron oxidation rate were observed in both sulfur-adsorbed and unadsorbed cells. On the other hand, sulfur oxidation by the adsorbed bacteria was not affected by the presence of ferrous iron in the medium. When bacteria that were adsorbed to sulfur prills were grown at a higher pH (ca. 2.5) in the presence of ferrous iron, they rapidly lost both ferrous iron and sulfur oxidation capacities and became inactive, apparently because of the deposition of a jarosite-like precipitate onto the surface to which they were attached.

The growth of Thiobacillus ferroxidans in a copper-containing ore suspension incubated in shake flasks was studied by determining the number of colony-forming units both in solution and attached to ore particles. The amounts of iron and copper released from the ore under experimental conditions were also determined. The total ferrous iron either released from the minerals or generated by reduction of the ferric iron in the minerals or generated by reduction of the ferric iron in the minerals could account for the observed growth of bacteria in solution. Only a small fraction of the total colony-forming units - about 500 per mg ore - was found to be associated with the ore particles throughout the experiments. However, the rapid development of these colonies when ore particles were plated suggested that they were produced by a number of bacteria associated with each ore particle. Accordingly, when the amount of bacteria attached to ore particles was determined by monitoring the formation of ferric iron in the plates, the percentage of the total activity associated with attached bacteria was found to be between 1 and 10%.

PM2 is a lipid-containing bacteriophage that grows on BAL3 1, a marine pseudomonas host [ 11. This bacterial strain is able to utilize lactose, among several other sugars, as the only carbon source [2]. The existance of both a P-galactosidase activity and a /3galactoside transporting activity, tentatively identitied as a lactose permease activity, has been described in BAG3 1 cells [3]. The present work is a study of the /3-galactoside transporting activity at different times after infection of the host cells with PM2. The results obtained show that prior to cell lysis there is a significant increase in transport activity, reflected in an increase in the maximum transport rates. The /3-galactosidase activity remains unaffected prior to cell lysis to decrease slightly thereof. These results are discussed in terms of possible modifications of lipid membrane composition produced by PM2 infection.

Phosphatidylethanolamine is the only phosphatide present in purified bacteriophage PM2 when obtained from bacteria grown and infected in minimal medium. The growth cycle of bacteriophage PM2 shows the basic general features described for virulent bacteriophages. PM2 infection of Pseudomonas BAL-31 causes a pronounced increase in deoxyribonucleic acid synthesis, but no detectable effect on the incorporation of phosphorus into lipid. When 32 P-prelabeled bacteria are infected with PM2, the specific activity of the phosphatide phosphorus in the virus is almost the same as that in the host bacteria labeled before infection. It is concluded that the viral phosphatide is not synthesized de novo after infection, but it probably is derived from preexisting cellular phosphatidylethanolamine.

Shortly after infection with bacteriophage PM2, host DNA synthesis is inhibited while synthesis of the supercoiled viral DNA begins at a low rate. This rate increases at about ten minutes after infection to reach a value that is thereafter maintained until lysis of the cells. Pulse-chase experiments suggest the existence of several precursors of supercoiled PM2 DNA. Two of them, one which sediments as nicked circular PM2 DNA (21 s) and another, appearing earlier in time, sedimenting 1.15 times faster (24 s), have been partially characterized. The 24 s component seems to contain linear single strands both longer and shorter than the strands of PM2 DNA. It has been purified, examined in the electron microscope and shown to consist of double-stranded rings of the circumference of PM2 DNA, with one double-stranded DNA branch of distinct length, but always shorter than PM2 DNA. From these characteristics the 24 s precursor seems to be a PM2 DNA molecule with a growing chain in accordance with the hypothesis that chain growth involves addition of nucleotides to the end of one parental strand while a second strand is initiated de novo. The pulse-labeled precursor which sediments like nicked circular PM2 DNA does not contain label in single-strand rings but only in open strands. Experiments based on transfer of the infected cell from heavy to light medium suggest a mechanism of replication which involves random selection of the viral DNA molecule to be duplicated from among these present in the cell.

1. 1. After γ-irradiation (0.9–18.0·10 4 R) of ribomononucleotides in aqueous solution a decrease in absorbancy at 260 mμ of at least two different components is observed. This decrease is an exponential function of the dose. 2. 2. Alkaline and enzymic hydrolysis with RNAase or phosphodiesterase of the irradiated samples produces an hyperchromic effect. 3. 3. Paper chromatography shows that the irradiated product has a lower R F than the mononucleotides. After alkaline or phosphodiesterase hydrolysis the R F approaches again that of the controls. 4. 4. The irradiated samples were chromatographed on a DEAE-cellulose column. Stronger anions (polymers) with a nucleic acid-like absorption spectrum were separated from the original mononucleotides by elution with increasing concentrations of NH 4 HCO 3 . 5. 5. The polymers formed by radiation decrease or disappear if the samples are incubated with alkali or enzymes before adsorption on the column. 6. 6. There is a correlation between the size of the polymers formed and the dose of γ-rays used. 7. 7. Irradiated samples excited at 310 mμ have a fluorescence at 400 mμ, not shown by the mononucleotides, directly related to the dose and decreasing after alkaline hydrolysis. 8. 8. Irradiated deoxyribomononucleotides (14.4·10 4 R) show a decrease in absorbancy at 260 mμ, which is partially reversed after incubation with deoxyribonuclease. 9. 9. The polymerization through phosphodiester bonds after γ-radiation of ribomononucleotides is discussed.

At 15 degrees C, phi X174-infected cells make single-stranded viral DNA fragments, varying in size from 0.2 to 0.9 times that of phi X DNA. In non-deproteinized lysates, this single-stranded DNA is found associated with proteins in particles sedimenting heterogeneously with an S20, w average of 80 to 90S. These particles do not differ appreciably from mature virus in polypeptide composition. Chase experiments, at 37 degrees C, of the label incorporated into this DNA at 15 degrees C suggest that both the single-stranded DNA fragments and the 80 to 90S particles are not precursors of virions but are defective end products.

Electron microscopy after negative staining of SA11-infected cell homogenates revealed that most of the viral particles are associated with membrane-like material. Many of the particles seemed to be fully enveloped in a membrane. This association could also be detected by the observed cosedimentation of viral proteins and cell membranes. Pulse-chase experiments showed that viral glycoproteins rapidly associate with membranes, whereas most of the structural proteins appearing in the soluble fraction immediately after the pulse were slowly chased into the membrane fraction. The membranes could be further fractionated into at least four fractions differing in density and containing a different distribution of viral proteins. Also, the distribution of label into each of these membrane fractions changed after long chase periods. The inhibition of glycosylation with tunicamycin yielded viral particles without an outer layer, but did not affect the described association with membranes. The possible relationship of this finding to the maturation of the virion is discussed.

The nucleotide sequences for several complementary DNA clones of the rotavirus genome were determined. When the sequences obtained from different clones for the same regions (16,000 bases) were compared, differences in eight base positions were observed. These discrepancies, approximately 1 in 2,000 bases, may be due to differences in individual RNA genomes resulting from multiple passages; infidelity of DNA synthesis in the cloning procedure; or both factors. Whatever the cause, this frequency of base substitution found in sequences of complementary DNA obtained from the same isolate should be considered when comparing DNA sequences obtained from independent isolates. On the other hand, the frequency of base changes observed suggests that the rotavirus genome is very conserved since the virus used for cDNA synthesis has been continuously passaged for 6 years without plaque purification.

Growth kinetics of Thiobacillus ferrooxidans in batch cultures, containing prills of elementary sulfur as the sole energy source, were studied by measuring the incorporation of radioactive phosphorus in free and adsorbed bacteria. The data obtained indicate an initial exponential growth of the attached bacteria until saturation of the susceptible surface was reached, followed by a linear release of free bacteria due to successive replication of a constant number of adsorbed bacteria. These adsorbed bacteria could continue replication provided the colonized prills were transferred to fresh medium each time the stationary phase was reached. The bacteria released from the prills were unable to multiply, and in the medium employed they lost viability with a half-life of 3.5 days. The spreading of the progeny on the surface was followed by staining the bacteria on the prills with crystal violet; this spreading was not uniform but seemed to proceed through distortions present in the surface. The specific growth rate of T. ferrooxidans ATCC 19859 was about 0.5 day −1 , both before and after saturation of the sulfur surface. The growth of adsorbed and free bacteria in medium containing both ferrous iron and elementary sulfur indicated that T. ferrooxidans can simultaneously utilize both energy sources.

Procedures are described for the isolation in pure form of the bacterial virus PM2. This virus isolated from seawater is shown to contain lipids by its sensitivity to ethyl ether and by chemical analysis of the purified virus. The virus has a buoyant density in CsCl of 1.28 g cm −3 and a sedimentation constant in 1 M NaCl of 230 S ; it contains 14% by weight of DNA and a minimum of 10% by weight of lipids. Phosphatidylethanolamine is the main lipid present in weight percent. The virus is disrupted in 0.05% Sarkosyl or sodium dodecyl sulfate. Analysis in acrylamide gel electrophoresis indicates the presence in the virus of at least two proteins, probably lipoproteins since they can be stained with Black Sudan. The virus appears to be a polyhedral object 60 mμ in diameter; the coat seems to have an unusual structure, in that an apparent double-layered wall can be observed in the electron microscope.

The DNA molecules isolated from mature bacteriophage PM2 are closed double-stranded rings of a molecular weight of six million. Direct evidence for the circularity and supercoiling of PM2 DNA was obtained by electron microscopy. Other properties, such as sedimentation behavior at neutral and alkaline pH, unusually high buoyant density in alkaline CsCl, and stability upon heating, are consistent with this structure. Only the four common nucleotides are found after hydrolysis and chromatographic analysis of PM2 DNA.

Vegetative supercoiled DNA of bacteriophage PM2 has a lower superhelix density than does the viral supercoiled PM2 DNA for which it is a precursor.