Structure and synthesis of a lipid-containing bacteriophage. A polynucleotide-dependent polynucleotide-pyrophosphorylase activity in bacteriophage PM2.

A polymerase activity is associated with protein IV, a protein which is associated with the DNA in bacteriophage PM2. The native enzyme unit is probably a dimer. Manganese ions are required for the polymerisation reaction and there is a well-defined Mn2+ optimum at 2.5 mM. The pH optimum is at 8.1, the temperature optimum at 28 degrees C. The activity is a polynucleotide-pyrophosphorylating reaction in the presence of ribo- or deoxyribonucleoside triphosphates. The polymerisation reaction is stimulated in the presence of nuclei- acids or polynucleotides as effectors. The product is not covalently linked to the effector.     

Erythrocyte spectrin maintains its segmental motions on oxidation: a spin-label EPR study.

The segmental motions of cross-linked erythrocyte skeletal protein (spectrin-actin-protein 4.1) samples, labeled with nitroxide spin labels, were monitored by conventional first-harmonic and saturation transfer second-harmonic electron paramagnetic resonance methods. Skeletal proteins were extracted from human red blood cells and treated with three oxidative reagents (diamide, hydrogen peroxide, and phenylhydrazine) to cross-link sulfhydryl groups and with one fixative reagent (glutaraldehyde) to cross-link lysine residues. The treatments provided extensive cross-linking between spectrin-actin-protein 4.1 molecules, as determined by gel electrophoresis, and surface charge modification, as determined by pl measurements. However, segmental motions of the cross-linked skeletal proteins remained generally similar to those in normal skeletal proteins. Both the weakly immobilized and the strongly immobilized motions were similar in cross-linked and control samples. Small differences in some motional components were detected. In some cases, faster mobilities were observed, with approximately 5% of the strongly immobilized motions converted to the weakly immobilized motions in the cross-linked samples. It is often believed that the consequence of membrane protein oxidation is restricted protein dynamics, giving membrane rigidity. However, our studies provide needed experimental evidence to indicate that segmental motions are maintained with very little modification even in the presence of extensive cross-linking. Thus cross-linking does not restrict the internal molecular flexibility that gives rise to segmental motions.     

Effect of lipid alkyl chain perturbations on the assembly of bacteriophage PM2.

The lipid-containing bacteriophage PM2 can produce infectious virus in cultures infected at temperatures up to 31.5 degrees C, but not at 34 degrees C. Its host, Pseudomonas BAL-31, grows at 34 degrees C and cultures infected at that temperature undergo lysis. Sucrose-gradient analysis shows that 34 degrees C lysates contain no PM2-like particles. Temperature-shift experiments establish that the thermally sensitive process is late in infection when virus assembly is taking place. Adamantanone, a small hydrophobic molecule that perturbs membrane hydrocarbon zones, prevents the production of infective virus. Concentrations which prevent virus production have no effect on host-cell growth or stability of mature virions. Adamantanone exerts its effects late in the infectious cycle, and lysates amde in its presence contain no PM2-like particles. These experiments, carried out at 25 degrees C, indicate that adamantanone prevents the assembly of stable PM2 virus. Spin-label studies suggest that the lipid alkyl chains of the host-cell membrane are in an "ordered" state at temperatures below about 33 degrees C and undergo a transition to a "disordered" state above that temperature. Furthermore, the addition of adamantanone perturbs the hydrocarbon zones, producing a greater degree of disorder even below 25 degrees C. Our findings suggest that the cell membrane can function and grow with the lipid alkyl chains in either the "ordered" or "disordered" state, but that the "ordered" state must be maintanined for PM2 assembly to occur.     

The role of specific DNA base damages in the X-ray-induced inactivation of bacteriophage PM2

Two types of X-ray-induced base damages, alkali-labile sites and thymine ring saturation products, were quantitated in PM2 DNA irradiated in the phage capsid under oxic and anoxic conditions. The extent of formation of these base damages was compared with the number of single- and double-strand breaks and lethal hits produced under the same conditions. The individual inactivation efficiencies of alkali-labile sites and thymine ring saturation products were determined by selectively inducing each of these damages in isolated PM2 DNA by chemical means in vitro and determining the rate of biological inactivation of the treated DNA by transfection. For each lethal X-ray hit induced in oxic conditions there were 1.06 alkali-labile sites, 0.40 thymine ring saturation products, 2.09 singe-strand breaks and 0.11 double-strand breaks in the PM2 genome. In anoxic conditions, the respective number of lesions was 1.00, 0.19, 1.73 and 0.09. The individual inactivation efficiencies of thymine ring saturation products and alkali-labile sites were found to be essentially equal, 7-8 lesions per lethal event in the PM2 genome. Alkali-labile sites and thymine ring saturation products together accounted for 15-20% of the biological inactivation of X-irradiated bacteriophage PM2. The presence or absence of oxygen during irradiation did not affect the contribution to inactivation made by alkali-labile sites, but the contribution by thymine ring saturation products to inactivation was about 2-fold higher in oxic compared with anoxic conditions. With the 4 lesions measured, we have accounted for some 28-34% of the lethal events in X-irradiated PM2 phage, most of the remaining events being caused by as yet unidentified base damages.     

Effect of lipid alkyl chain perturbations on the assembly of bacteriophage PM2

The lipid-containing bacteriophage PM2 can produce infectious virus in cultures infected at temperatures up to 31.5 °C, but not at 34 °C. Its host, Pseudomonas BAL-31, grows at 34 °C and cultures infected at that temperature undergo lysis. Sucrose-gradient analysis shows that 34 °C lysates contain no PM2-like particles. Temperature-shift experiments establish that the thermally sensitive process is late in infection when virus assembly is taking place.Adamantanone, a small hydrophobic molecule that perturbs membrane hydrocarbon zones prevents the production of infective virus. Concentrations which prevent virus production have no effect on host-cell growth or stability of mature virions. Adamantanone exerts its effects late in the infectious cycle, and lysates made in its presence contain no PM2-like particles. These experiments, carried out at 25 °C, indicate that adamantanone prevents the assembly of stable PM2 virus.Spin-label studies suggest that the lipid alkyl chains of the host-cell membrane are in an “ordered” state at temperatures below about 33 °C and undergo a transition to a “disordered” state above that temperature. Furthermore, the addition of adamantanone perturbs the hydrocarbon zones, producing a greater degree of disorder even below 25 °C. Our findings suggest that the cell membrane can function and grow with the lipid alkyl chains in either the “ordered” or “disordered” state, but that the “ordered” state must be maintained for PM2 assembly to occur.     

Left-handed Z-DNA regions are present in negatively supercoiled bacteriophage PM2 DNA

Bacteriophage PM2 DNA is a 10-kb covalently closed circular (ccc) molecule with a reported superhelical density of sigma = -0.12. Here we describe the binding of anti-Z-DNA antibodies to PM2 form I DNA under high and low salt conditions. The binding to PM2 DNA has been demonstrated by competitive radioimmunoassay (RIA), retardation of the DNA:antibody complexes in agarose gels and visualization by electron microscopy. The antibody binding is dependent on the degree of negative supercoiling. Thus, PM2 form II and form III did not bind the antibody. The low salt RIA results indicated the presence of 200-400 bp of left-handed DNA per PM2 molecule. This could reduce the effective superhelical density to sigma = -0.04 to -0.08, a range comparable with those found for other ccc DNAs in vivo. Electron microscopy revealed that a maximum of 22 antibody molecules bind to PM2. Single-site restriction with HpaII of the fixed DNA:antibody complex showed a cluster of four to five antibody molecules bound near one end of the linear DNA molecule. The evidence presented indicates that PM2 DNA contains regions of left-handed conformation under physiological conditions (low salt concentration) as well as at high salt concentrations. In addition, electrophoretic analyses of PM2 topoisomers indicate the presence of left-handed regions at superhelical densities less than that of isolated PM2 DNA.     

Calcium requirement for assembly of the lipid-containing bacteriophage PM2

The bacteriophage PM2 requires extracellular Ca²⁺ at concentrations greater than 3 · 10⁻⁴ M for the production of viable virus, whereas the host cell Pseudomonas BAL-31 grows normally in medium containing 3 · 10⁻⁵ M Ca²⁺ (low calcium). Virus attachment occurs normally in low calcium, the infected cultures partially lyse, but no infectious virus particles are released. Sucrose gradient analysis shows that lysates made in low calcium contain no PM2-like particles. The addition of calcium very late in the infectious cycle completely restores virus production to cultures infected in low calcium, whereas removal of calcium after infection prevents virus production. Our experiments indicate that Ca²⁺ is essential for some process late in the lytic cycle, such as the final assembly of stable, infectious PM2 particles.     

Antigenic relationship between Pseudomonas Bal-31 membranes and bacteriophage PM2

Summary Antisera against bacteriophage PM2 and against membranes of its host cell, Pseudomonas BAL-31, were prepared. Cross-reactivity between these two antigens and both antisera was found by immunodiffusion, complement fixation and viral neutralization experiments. Anti-membrane sera up to a dilution of 1/100 were able to neutralize 60% of the infective capacity of PM2. This neutralizing capacity was partially abolished by the presence of Pseudomonas BAL-31 membranes. It is concluded that similar antigenic determinants are present in the PM2 phage and in the host membrane.     

Insights into virus evolution and membrane biogenesis from the structure of the marine lipid-containing bacteriophage PM2

Recent, primarily structural observations indicate that related viruses, harboring no sequence similarity, infect hosts of different domains of life. One such clade of viruses, defined by common capsid architecture and coat protein fold, is the so-called PRD1-adenovirus lineage. Here we report the structure of the marine lipid-containing bacteriophage PM2 determined by crystallographic analyses of the entire approximately 45 MDa virion and of the outer coat proteins P1 and P2, revealing PM2 to be a primeval member of the PRD1-adenovirus lineage with an icosahedral shell and canonical double beta barrel major coat protein. The view of the lipid bilayer, richly decorated with membrane proteins, constitutes a rare visualization of an in vivo membrane. The viral membrane proteins P3 and P6 are organized into a lattice, suggesting a possible assembly pathway to produce the mature virus.     

X-ray diffraction and flash photolysis studies of M intermediate of lattice-contracted purple membrane

The effects of cross-linking and lattice contraction of purple membrane (PM) on the photodynamics of bacteriorhodopsin (bR) and on the tertiary structure were studied by flash photolysis and X-ray diffraction. To get a contracted lattice form of PM, native PM, and/or PM cross-linked by glutaraldehyde were treated with deoxycholate or Triton X-100. Part of the Triton-treated cross-linked PM was further incubated with Bio-Beads SM-2 to remove Triton X-100. In the modified PM, several long-lived components of the M intermediate appeared, the features of which were related to the environment of bR. Also, X-ray diffraction studies using synchrotron radiation were performed on the modified PM under intense light irradiation (lambda greater than 500 nm) in which 40-80% of bR was photoconverted to the M state. In the Triton-treated cross-linked PM dispersed in 0.25% Triton X-100, the unit cell of membrane crystalline lattice was enlarged from 58.8 to 59.8 A and the crystalline order decreased with irradiation. The analysis of X-ray diffraction patterns suggests that light-induced conformational changes of bR correlated with the Triton content of the environment and an increase of substitution disorder was caused by these changes, but the average location of bR was unchanged. However, the other modified PM showed no significant changes of diffraction, upon light irradiation.     

Interaction between bacteriophage PM2 protein IV and DNA.

The interaction between DNA and the structural protein IV of bacteriophage PM2 was studied by co-sedimentation, filter binding and electron microscopy. The co-sedimentation data and the sigmoid-shaped filter binding curve were interpreted in terms of co-operative binding. At a given DNA/protein input ratio, some DNA molecules were associated with a large amount of protein IV while others had no detectable protein bound to them. Electron microscopic examination of DNA-protein IV mixtures showed highly condensed DNA molecules alongside uncomplexed native DNA. Dissociation experiments revealed the presence of two types of complexes. Type I dissociated rapidly while type II had a long half-life. Dissociation of complexes obtained with increasing protein/DNA ratios suggested that the type I complex was a precursor of type II complex. Protein IV binds equally well to superhelical, relaxed or linear DNA as well as to single-stranded DNA. These observations lead to a model for the interaction and for the consequent alterations in the DNA structure.     

State of aggregation of the (Ca2+ + Mg2+)-ATPase studied using chemical cross-linking

We have studied cross-linking of the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum and in reconstituted systems, using glutaraldehyde, cupric-1,10-phenanthroline and 3,3'-dithiobis (sulphosuccinimidylpropionate). All reagents produce extensive cross-linking, forming aggregates too large to enter polyacrylamide gels. Only traces of cross-linked dimeric ATPase species are formed. Saturation transfer electron spin resonance spectra of spin-labelled sarcoplasmic reticulum cross-linked with glutaraldehyde are also consistent with the formation of extensively cross-linked aggregates in the membrane. The results are interpreted in terms of dynamic clusters of ATPase molecules in the membrane, probably in the form of rows of ATPase molecules.     

Structure and assembly of lipid-containing viruses, with special reference to bacteriophage PM2 as one type of model system.

The simpler lipid-containing viruses (influenza, Semliki Forest, PM2) may have a phospholipid bilayer sandwiched between an outer shell of protein and an internal nucleocapsid possessing helical or icosahedral symmetry. Extensive physical and chemical studies have enabled us to form a more detailed picture of the structure of bacteriophage PM2 and controlled stepwise degradation of the virion has helped us to localize the four viral proteins. The surface protein (II) of PM2 is basic and interacts with the acidic phosphatidylglycerol of the bilayer to stabilize the membrane. The nucleocapsid protein (III) has proteolipid characteristics and may interact with the phospholipids in a hydrophobic fashion. The spikes are formed from protein I and the fourth protein (IV) is closely associated with the DNA. It is possible to reassemble the virus by reversing the degradation steps. Assembly has been especially useful in revealing the processes whereby the proteins and lipids interact to form the bilayer. Furthermore, results of in vivo studies of phospholipid synthesis and both in vivo and in vitro studies of viral protein synthesis have enabled us to form a reasonably complete picture of the biosynthesis of PM2.     

Control of membrane morphogenesis in bacteriophage PM2

The regulation of membrane formation in bacteriophage PM2 serves as a simple model for changes in membrane structure in eukaryotic cells. Prior to Pseudomonas host lysis, wild-type virions mature to an icosahedral morphology at the inner face of the cytoplasmic membrane. The proliminary charcterization of two temperature-sensitive mutants of PM2 is described. In cells infected at the restrictive temperature with ts 1, an abundance of “empty” virus-size membrane vesicles are seen. Synthesis of DNA is also reduced in ts 1 infected cells. The preponderance of vesicles is not sen in cells infected with wil-type virus or with ts 1 at the permissive temperature. The “empty” appearance of the viral membranes suggests that viral DNA is not encapsulated. The major viral capsid protein (MW 26,000) is located just out side the viral membrane and normallyl sediments with host and virus membranes; insted, large amounts of capsid protein can be precipitated from the supernatant with TCA. Compared to cells infected with wild type virus, cells infected with is 5 at th restrictive temperature produce inside the cell an aboundance of virus-soze membrane vesicles. Taken Together, These results with viral mutants suggest that formation of a viral membrane of the proper size does not require a DNA core around which to form, or an outer scaffolding of coat protein against which to form a spherical bilayer.     

The conformation of cross-linked actin.S-1 in the presence and absence of ATP

Electron microscopy studies have shown that the structure of the complex of myosin subfragment 1 (S-1) cross-linked to actin with 1-ethyl-3-[3-(dimethyl-amino) propyl] carbodiimide is very different in the presence and absence of ATP (Craig, R., Greene, L. E., and Eisenberg, E. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3247-3251). More recent studies have found that the structure of the cross-linked complex between S-1 modified extensively with N-ethylmaleimide (NEM.S-1) and actin resembles that of the rigor complex both in the presence and absence of ATP, whereas the structure of the cross-linked complex between S-1 modified with N',N'-p-phenylenedimaleimide (pPDM.S-1) and actin resembles that of the cross-linked actin.S-1 complex in the presence of ATP. In the present study, we have obtained biochemical evidence supporting these results. The conformation of the different cross-linked actin.S-1 complexes was determined by studying their effect on the troponin-tropomyosin-actin complex (regulated actin). The basis of this probe for conformation is that S-1.ATP, which is in the weak-binding conformation, interacts very differently with regulated actin than S-1 or S-1.ADP, which are in the strong-binding conformation. We find that both in the presence and absence of ATP, cross-linked NEM.S-1 appears to be in the strong-binding conformation, whereas cross-linked pPDM.S-1 appears to be shifted toward the weak-binding conformation. In contrast, cross-linked unmodified S-1 appears to be in the strong-binding conformation in the presence of ADP and the weak-binding conformation in the presence of ATP. Therefore, in agreement with electron microscopy studies, the cross-linked actin.S-1 complex appears to be able to alternate between the weak-binding and strong-binding conformation during the cross-bridge cycle.     

Characterization of temperature-sensitive mutants of bacteriophage PM2: Membrane mutants

Summary In an effort to understand the genetic regulation of membrane morphogenesis, twenty-nine temperature-sensitive mutants of the membrane-containing bacteriophage PM2 were isolated. Characterization at restrictive temperature revealed groups showing no lysis (Groups I–IV), partial lysis (Groups V–VIII), and full lysis (Groups IX–XII) of the host Pseudomonas BAL-31. When the cell lysis data are considered in conjunction with data on stimulation of viral DNA synthesis, at least six mutant groups are defined. Analysis by gel electrophoresis of the pattern of viral proteins synthesized under restrictive conditions further divides the mutants into twelve groups. Temperature shift experiments delineate early, intermediate and late mutants. Complementation data support some of these groupings. The observed low levels of complementation and recombination are discussed in terms of gene product/genome restriction, bound to the membrane at the site of infection.It is of particular interest to membrane morphogenesis that under restrictive conditions late mutants in Groups II, III and IV make empty-appearing vesicles inside the cell that are the size of virus membranes as seen in thin sections of cells in the electron microscope. Mutants ts 1 (Group II) and ts 12 (Group III) show defects in their ability to incorporate into membranes viral structural proteins sp 13 and sp 6.6. The possibility is discussed that either of these proteins control the size and shape of the viral membrane.     

Isolation of the prohead core of bacteriophage T4 after cross-linking and determination of protein composition

The naked core of bacteriophage T4 was isolated ex vivo after cross-linking with either glutaraldehyde or dithiobis(succinimidyl propionate). The isolated particles appeared to be morphologically identical to the cores found in thin sections, to those demonstrated in in situ lysis preparations, and to core structures assembled in vitro. Treatment with glutaraldehyde provided core particles which were morphologically well preserved, whereas dithiobis(succinimidyl propionate)-induced cross-linking was reversible and allowed analysis of the protein composition of the isolated particles. The identity of the reversibly cross-linked particles with those obtained after irreversible cross-linking was suggested by their morphology and their similar sedimentation behavior. Immunolabeling confirmed the structural presence of the main core protein in both structures. Gel electrophoresis of reversibly cross-linked cores revealed the essential head proteins gp22, gp67, and gp21, the three internal proteins IPI, IPII, and IPIII, and a 17K protein.     

Oxidation of hydroxylamine by cytochrome P-460 of the obligate methylotroph Methylococcus capsulatus Bath.

An enzyme capable of the oxidation of hydroxylamine to nitrite was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The absorption spectra in cell extracts, electron paramagnetic resonance spectra, molecular weight, covalent attachment of heme group to polypeptide, and enzymatic activities suggest that the enzyme is similar to cytochrome P-460, a novel iron-containing protein previously observed only in Nitrosomonas europaea. The native and subunit molecular masses of the M. capsulatus Bath protein were 38,900 and 16,390 Da, respectively; the isoelectric point was 6.98. The enzyme has approximately one iron and one copper atom per subunit. The electron paramagnetic resonance spectrum of the protein showed evidence for a high-spin ferric heme. In contrast to the enzyme from N. europaea, a 13-nm blue shift in the soret band of the ferrocytochrome (463 nm in cell extracts to 450 nm in the final sample) occurred during purification. The amino acid composition and N-terminal amino acid sequence of the enzyme from M. capsulatus Bath was similar but not identical to those of cytochrome P-460 of N. europaea. In cell extracts, the identity of the biological electron acceptor is as yet unestablished. Cytochrome c-555 is able to accept electrons from cytochrome P-460, although the purified enzyme required phenazine methosulfate for maximum hydroxylamine oxidation activity (specific activity, 366 mol of O2 per s per mol of enzyme). Hydroxylamine oxidation rates were stimulated approximately 2-fold by 1 mM cyanide and 1.5-fold by 0.1 mM 8-hydroxyquinoline.     

The relation of single-stranded regions in bacteriophage PM2 supercoiled DNA to the early melting sequences.

Bacteriophage PM2 supercoiled DNA contains one to three small single-stranded regions that can be detected in the electron microscope after various treatments. The relative positions of these regions were mapped against the unique cleavage site for the restriction endonuclease R · HapII on PM2 DNA. Any of eight sharply defined regions of the genome may be single-stranded in supercoiled molecules. They are found in all possible combinations of three or less and at approximately the same frequency. A comparison of this map of supercoiled DNA with the alkaline denaturation pattern of nicked circular or linear PM2 DNA showed that these same regions were also the earliest melting regions in non-supercoiled DNA.