Richard M. Franklin

Structure and synthesis of a lipid-containing bacteriophage

Abstract Detailed procedures are given for the purification of bacteriophage PM2, and some of the difficulties met in purifying this virus are discussed. The structure of the purified virus was examined by electron microscopy of intact virus and of virus dissociated under acid or alkaline conditions or by nonionic detergents. The virus is icosahedral in shape with spikes or fibers radiating from the vertices. The diameter of negatively stained particles was 61.4 mμ. In thin sections a bilayer could be seen. This may be either a membrane or two lipoprotein shells. This bilayer was 73 A thick but the diameter of the particle was now 56.1mμ, indicating some shrinkage during embedding. A number of stages in dissociation of the virion were observed under the conditions mentioned above. Structures which may be interpreted as cores were seen, and these were very labile once the outer envelope was removed.

Structure and synthesis of a lipid-containing bacteriophage: XII. The fatty acids and lipid content of bacteriophage PM2

Abstract The fatty acid composition of bacteriophage PM2 and its host Pseudomonas BAL-31 were investigated by gas-liquid chromatography. Nearly identical compositions were found in the virus and its host. Although no unusual fatty acids were present, an unusually high content (> 50%) of monounsaturated 16-carbon fatty acid(s) (C16:1) was found; the other major fatty acids were C16:0, C18:1 and a cyclopropane C17. The lipid-phosphorus content was evaluated as 4.88 μg/mg of virion; this value taken together with the fatty acid and phospholipid composition was used to estimate a phospholipid content of 118 μg/mg of virion. The total lipid content of PM2 was estimated by two different methods as 12.6 ± 0.4% and 14.0 ± 1.5%. These values indicate that the lipid content of PM2 is not sufficient to form a continuous lipid bilayer.

Structure and synthesis of a lipid-containing bacteriophage. V. Phospholipids of the host BAL-31 and of the bacteriophage PM2.

Abstract The major phospholipids of Pseudomonas BAL-31 grown in synthetic medium are phosphatidylethanolamine (75%) and phosphatidylglycerol (23%). The phospholipid composition remains relatively constant during different growth stages and in different growth media. Bacteriophage PM2 contains the same phospholipids as the host BAL-31, but with an altered percent composition (28% phosphatidylethanolamine and 65–68% phosphatidylglycerol). Phosphatidic acid is a trace component in uninfected cells, and appears to be present in virus (2–4%). The cellular phospholipid composition shifts during infection with bacteriophage PM2. Lysophosphatidylethanolamine and phosphatidic acid increase from trace amounts to as much as 18–22% of the infected cell phospholipid content, and at the same time phosphatidylglycerol increases from 23% to 36%. Phosphatidylethanolamine decreases from 75% to about 43% of the total phospholipid in infected cells. The dissimilarity between bacteriophage and host cell phospholipid compositions is discussed in terms of the probable biogenesis of the viral membrane.

Structure and synthesis of a lipid-containing bacteriophage: I. Growth of bacteriophage PM2 and alterations in nucleic acid metabolism in the infected cell

Abstract The one-step growth curve was measured for bacteriophage PM2 in Pseudomonas BAL-31, grown in synthetic medium at 25°. All virus was completed and found inside the cell prior to release. The eclipse period was 35–37.5 min, the latent period 40–45 minutes, and the burst size 684 PFU/cell. Cellular DNA synthesis stopped soon after infection but cellular DNA remained stable and there was considerable, but reduced, synthesis of cellular RNA throughout the course of infection. The rate of DNA synthesis was much higher in the infected cell than in the control cell and this proved to be viral DNA, according to hybridization tests. Viral DNA continued to be synthesized up to the time of cell lysis, even though synthesis of infectious virus stopped long before this time. Hybridization tests were also used to demonstrate the synthesis of viral mRNA, which continued up to the time of cell lysis.

Polyribosomes of Escherichia coli: I. Effects of monovalent cations on the distribution of polysomes, ribosomes and ribosomal subunits

Two different types of polyribosome profiles were obtained from Escherichia coli lysed and centrifuged in the presence of the chlorides of various monovalent cations. The type I profile observed in LiCl, NaCl, CsCl, N(CH3)4Cl and N(C2H5)4Cl consisted of 58 to 69% polysomes, small amounts of 70 s monomers sedimenting as a shoulder on the 50 s subunit peak, 12 to 26% 50 s subunits and 6 to 16% 30 s subunits. The type II profile observed in KCl, RbCl and NH4Cl consisted of 66 to 69% polysomes, 12 to 15% 70 s monomers, 12% 50 s subunits and 6 to 7% 30 s subunits. On the basis of these observations, an attempt was made to ascertain which of these types of profiles is most representative of the true in vivo distribution of polysomes, ribosomes and ribosomal subunits in exponentially growing E. coli.

Polyribosomes of Escherichia coli: II. Experiments to determine the in vivo distribution of polysomes, ribosomes and ribosomal subunits☆☆☆

Abstract A study was made to determine the most probable in vivo distribution of polysomes, ribosomes and ribosomal subunits in early log-phase Escherichia coli . Experimental conditions for obtaining a type I-polysome profile in which there is little or no 70 s monomers were compared to those for obtaining a type II profile in which 12 to 15% of the ribosomes are present as 70 s monomers. Polysome profiles of lysates of E. coli strains with decreasing endogenous ribonuclease activity (strains 3000, Q13, 1113B) were almost identical, which suggested that the 70 s monomers of a type II profile were not derived from polysomes by endogenous ribonuclease activity. Furthermore, purified ribonuclease II degraded neither the larger polysomes in a lysate nor isolated polysome dimers. Even in 60 m m -KCl, where one finds a large peak of ribosomes, addition of yeast RNA oligonucleotides to the lysate and to the sucrose gradients results in suppression of the monomer peak. The same effect can be obtained with higher concentrations of KCl in the lysate and the sucrose gradient. On the other hand, isolated 70 s monomers are stable when centrifuged through gradients containing RNA oligonucleotides or high KCl concentrations. These are all conditions which will inhibit the activity of E. coli RNase II but are also conditions which block initiation of protein synthesis. Polysome profiles were also examined in E. coli lysates to which various inhibitors of protein synthesis were added: chloramphenicol, dihydro-streptomycin, mikamycin, sparsomycin and tetracycline. The results indicated that the 70 s monomers observed in type II profiles were formed in vitro after cell lysis by association of native 50 s and 30 s ribosomal subunits to form 70 s complexes. Studies with trimethoprim and puromycin, which were used as in vivo inhibitors of protein synthesis, supported this hypothesis. Further support of this hypothesis came from analyzing the pulse-labeled RNA associated with the 70 s monomer in TMK § lysates. This RNA, which is mostly messenger RNA, should be degraded if the 70 s peak arises from breakdown of polysomes and should be heterogeneous and large if the 70 s peak represents artifactual 70 s monosemes formed with intact mRNA. The latter was found to be true. Also, it was possible to obtain a small amount of association of isolated 50 and 30 s subunits to form a 70 s complex when they were mixed with spheroplasts prior to lysis in TMK but not in TMNa. In conclusion, all of our experiments suggest that the type I profile is probably most representative of the in vivo distribution of polysomes, ribosomes and ribosomal subunits in early log-phase E. coli .

Structure and synthesis of a lipid-containing bacteriophage: VII. Structural proteins of bacteriophage PM2

Abstract The structural proteins of bacteriophage PM2 were investigated by SDS-polyacrylamide gel electrophoresis and by gel filtration on 6% agarose. By electrophoresis, four distinct polypeptides were identified and their molecular weights were estimated to be I = 42,700, II = 32,000, III = 12,250, and IV = 7660. By gel filtration in the presence of 6 M guanidine hydrochloride on 6% agarose, the molecular weights were estimated to be 43,000, 35,000, 12,500, and 4650. The percentage of the total protein corresponding to each polypeptide was estimated by gel electrophoresis of dissociated PM2 which had been uniformly labeled with acetate-14C. The moles of each polypeptide per mole of virion were: I = 90, II = 710, III = 560, and IV = 470.

Structure and synthesis of a lipid-containing bacteriophage: IV. Electron microscopic studies of PM2-infected Pseudomonas BAL-31

Abstract The ultrastructure of Pseudomonas BAL-31 infected with a lipid-containing bacteriophage, PM2, was studied. It was shown that although PM2 matures at the cell periphery, in close association with the cytoplasmic membrane, the virus did not seem to form by an inward budding process. The earliest visible particles, seen at 40–50 min after infection, were lined up in a single row along the cytoplasmic membrane and most of them lacked a dense inner core. After 55 min, additional layers of virus particles were apparent, and the majority contained a dense core. Serial sections indicated that the virus particles tended to occur in well delineated clusters, and that empty particles occurred predominantly at the periphery of these clusters. Just prior to lysis, many cell profiles contained 20–60 virus particles, in up to 5 layers. From kinetic studies, the particles with dense cores were formed at approximately the same rate that infective intracellular virus was formed. At no time were structures resembling buds seen in either infected cells or in cellular debris following lysis.

Structure and synthesis of a lipid-containing bacteriophage: VI. The spectrum of cytoplasmic and membrane-associated proteins in Pseudomonas BAL 31 during replication of bacteriophage PM2

Abstract After infection of Pseudomonas BAL-31 with the lipid-containing bacteriophage PM2, the rate of incorporation of amino acids into membrane-associated polypeptides increases and the incorporation into cytoplasm decreases, when compared to uninfected cells. When examined by SDS-acrylamide gel electrophoresis, at least 13 distinct newly synthesized membrane-associated viral-specific polypeptides can be demonstrated, while only one new polypeptide was observed in the cytoplasmic fraction after infection. Prelabeling experiments indicate that the membrane-associated peptides present in uninfected cells are conserved during infection, and thus the newly synthesized proteins probably do not displace membrane-associated proteins present prior to infection. In the presence of nalidixic acid, which inhibits both cellular and viral DNA synthesis, at least two viral specific membrane-associated polypeptides are not synthesized, and these probably correspond to two of the viral structural proteins.

Structure and synthesis of a lipid-containing bacteriophage: XI. Studies on the structural glycoprotein of the virus particle

Abstract The glycoprotein nature of one protein and the proteolipid characteristics of some proteins of the lipid-containing bacteriophage PM2 were studied. [ 3 H]Glucosamine-derived label can be incorporated into bacteriophage structural protein IV (MW 4650). The glycoprotein nature of this protein was confirmed (a) by its ability to be stained by the periodic acid-Schiff reagent and (b) by the presence of labeled amino sugars in thin-layer chromatograms of [ 3 H]glucosamine-labeled viral hydrolyzates. The predominant amino sugar is either glucosamine or mannosamine; attempts to distinguish between these two were unsuccessful. No neuraminic acid, fucose, or hexoses were detected in this glycoprotein. Both proteins III and IV have the solubility properties of proteolipids.