Map of DNA homology between the genomes of Salmonella bacteriophages P22 and L.

The genomes of temperate Salmonella typhimurium phages P22 and L share approximately 69% homology, as measured by DNA heteroduplex analysis. Alignment of the P22/L heteroduplex molecules with a P22 physical map places most of this homology between the capsid genes and genes in the vicinity of the prophage attachment sites. The degree of genetic relatedness between these phages and the lambdoid phages is also discussed.     

The activity of ant product of the Salmonella phage P22 against the closely related but heteroimmune phage L.

Summary P22 mutants defective in the early gene 24 are complemented by phage L in mixed infection. P22 12 ^- and P22 23 ^- mutants are not complemented by phage L. Gene function 24 of an L prophage is turned on by a superinfecting P22 24 ^- mutant and complements the missing function of the defective P22 phage. Since this transactivation of prophage gene 24 depends on a functional gene ant in the superinfecting P22 mutant, it indicates derepression for leftward directed gene expression in prophage L. On the contrary neither the rightward directed expression of gene 12 nor of gene 23 in prophage L can be turned on by superinfecting P22 24 ^- 12 ^- or P22 24 ^- 23 ^- mutants (and also not by P22 12 ^- and P22 23 ^-) to a degree sufficient for complementation of simultaneously superinfecting L virB 12 ^- or L virB 23 ^- mutants. The failure to detect release of repression for rightward directed gene expression of prophage L corresponds to the earlier observation (Prell, 1975) that P22 superinfecting L lysogens cannot release replication inhibition for simultaneously infecting phage L. The results are discussed with respect to the mechanism underlying the different action of P22 antirepressor in L and in P22 lysogens.     

Genetic studies of hybrids between coliphage lambda and salmonella phage P22: genetic analysis of the P22-lambda hybrid class.

Summary P22- hybrids which retain the protein coat of P22 have been isolated and characterized into two types. Type 1 hybrids which have the c through O-P genes of are unable to grow lytically on Salmonella typhimurium. On the other hand, type 2 hybrids which contain only the c region of , plated on S. typhimurium. Both hybrid types retained the generalized transducing and antigenic conversion capabilities of P22.     

lambdaimm P22dis: a hybrid of coliphage lambda with both immunity regions of Salmonella phage P22.

Summary Genetically marked and P22 phages were recombined in Escherichia coli-Salmonella typhimurium hybrid WR4028, a host sensitive to infection by both of these phages. Hybrid phages that acquired the immC region of P22, but retained the genes for the protein coat were selected on WR4027 (), a -immune, P22-resistant derivative of WR4028. In these immP22 hybrids, at least the c through P genes of were replaced with functionally related P22 genes. Phage recombinants with more extensive regions of the P22 genome were selected on the double lysogen WR4027 (, immP22). One such hybrid, immP22dis, was determined by heteroduplex analysis to contain approximately 40% of the P22 genome. Genetic studies established that immP22dis possesses the two widely separated immunity control regions of P22 (immC and immI) and that these loci are expressed in E. coli K-12 lysogenic for immP22dis. In addition, immP22dis contains the P22 a1 locus responsible for somatic 0–1 antigen conversion in Salmonella. Although the immP22dis phage particle has the head and tail, the phage genome also carries P22 tail gene 9 as evidenced by the production of free P22 tails. It also has the P22 att site as indicated by the integration of the immP22dis prophage near the proA locus on the bacterial chromosome.     

The role of ant-product of Salmonella phage P22 in the process of transactivation of prophage Px1 genes.

Summary Phage P22 replicates in Px1-lysogenic cells because the ant-product (antirepressor-protein) of P22 removes the repression exerted by the Px1-prophage. P22 ant ^- phages however are repressed in Px1-lysogens. The ant-product of P22 is also required for the transactivation of the early genes 24, 12, and 23 of the Px1-prophage. P22 virB3 ant ^- mutants, which are insensitive to c-repression and therefore replicate in Px1-lysogens, are unable to transactivate early genes of the Px1-prophage or of a coinfecting Px1 phage. Transactivation of the late gene 19 is insensitive to repression and independent of ant-activity. This suggests the presence of a separate, and o_R-independent, promotor for late gene expression. —A more rigorous proof for functional homology in early gene regulation of P22 and Px1 is presented.Reinhard W. Kaplan zum 65. Geburtstag in Dankbarkeit gewidmet     

Regulation of late functions in Salmonella bacteriophages P22 and L studied by assaying endolysin synthesis.

The glycopeptides obtained by pronase digestion of two ecotropic strains of murine leukemia virus (MuLV) were compared by gel filtration. Four different glycopeptide size classes, designated G(1), G(2), G(3), and G(4), with molecular weights of approximately 5,100, 2,900, 2,200, and 1,500, respectively, were shown to be associated with Rauscher MuLV virions grown in JLS-V9 cells. Various sugar precursors, including glucosamine, galactose, fucose, and mannose were incorporated into G(1) and G(2), suggesting that these are complex (type I) glycopeptides. The two smaller glycopeptide size classes, G(3) and G(4), were shown to be mannoserich (type II) glycopeptides. G(4) was more sensitive to digestion with endo-beta-N-acetylglucosaminidase H than G(3), suggesting that the core of G(3) may contain fewer mannose residues. Glycopeptides of the same size class as G(1) and G(2) were associated with both Rauscher MuLV and AKR-MuLV grown in III6A (mouse embryo) cells. Previous studies have shown that gp52, a proteolytic cleavage product of gp70, possessed primarily G(1) glycopeptides and that gp52 was more highly sulfated than gp70. We observed that G(1) is approximately twofold more highly sulfated than G(2), explaining the observed difference in sulfation of gp52. The unusually large size of G(1) suggested that infection with MuLV may alter the host cell glycosylation pattern. To test this possibility, glycopeptides from Sindbis virions grown in uninfected and Rauscher MuLV-infected JLS-V9 cells were compared, and no differences were observed. G(1) was not detected in Sindbis virions, indicating that acquisition of G(1) depends on properties of the virus-coded polypeptide backbone of the gp70 molecule.     

Comparison of two serologically distinct ribonucleic acid bacteriophages. II. Properties of the nucleic acids and coat proteins

Overby, L. R. (University of Illinois, Urbana), G. H. Barlow, R. H. Doi, Monique Jacob, and S. Spiegelman. Comparison of two serologically distinct ribonucleic acid bacteriophages. II. Properties of the nucleic acids and coat proteins. J. Bacteriol. 92:739-745. 1966.-The ribonucleic acid (RNA) molecules and coat proteins of two RNA coliphages, MS-2 and Qbeta, have been characterized. MS-2 RNA shows an S(20,w) of 25.8 and a molecular weight by light scattering of 10(6). The corresponding parameters for Qbeta-RNA were 28.9 and 0.9 x 10(6). A difference in base composition was reflected in the adenine-uracil ratio, which was 0.95 for MS-2 and 0.75 for Qbeta. The two RNA preparations are readily separated by chromatography on columns of methylated albumin. Both gave identical bouyant densities in cesium sulfate of 1.64 g/ml. The coat protein subunits were of similar molecular weights: 15,500 (Qbeta) and 14,000 (MS-2). They differed, however, in that the Qbeta-protein lacked tryptophan and histidine, whereas the MS-2 protein lacked only histidine.     

Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic pathway

We have identified and characterized structural intermediates in phage P22 assembly. Three classes of particles can be isolated from P22-infected cells: 500 S full heads or phage, 170 S empty heads, and 240 S “proheads”. One or more of these classes are missing from cells infected with mutants defective in the genes for phage head assembly. By determining the protein composition of all classes of particles from wild type and mutant-infected cells, and examining the time-course of particle assembly, we have been able to define many steps in the pathway of P22 morphogenesis.In pulse-chase experiments, the earliest structural intermediate we find is a 240 S prohead; it contains two major protein species, the products of genes 5 and 8. Gene 5 protein (p5) is the major phage coat protein. Gene 8 protein is not found in mature phage. The proheads contain, in addition, four minor protein species, PI, P16, P20 and PX. Similar prohead structures accumulate in lysates made with mutants of three genes, 1, 2 and 3, which accumulate uncut DNA. The second intermediate, which we identify indirectly, is a newly filled (with DNA) head that breaks down on isolation to 170 S empty heads. This form contains no P8, but does contain five of the six protein species of complete heads. Such structures accumulate in lysates made with mutants of two genes, 10 and 26.Experiments with a temperature-sensitive mutant in gene 3 show that proheads from such 3^? infected cells are convertible to mature phage in vivo, with concomitant loss of P8. The molecules of P8 are not cleaved during this process and the data suggest that they may be re-used to form further proheads.Detailed examination of 8^? lysates revealed aberrant aggregates of P5. Since P8 is required for phage morphogenesis, but is removed from proheads during DNA encapsulation, we have termed it a scaffolding protein, though it may have DNA encapsulation functions as well.All the experimental observations of this and the accompanying paper can be accounted for by an assembly pathway, in which the scaffolding protein P8 complexes with the major coat protein P5 to form a properly dimensioned prohead. With the function of the products of genes 1, 2 and 3, the prohead encapsulates and cuts a headful of DNA from the concatemer. Coupled with this process is the exit of the P8 molecules, which may then recycle to form further proheads. The newly filled heads are then stabilized by the action of P26 and gene 10 product to give complete phage heads.     

Morphing molecular specificities between Arm-peptide and NUT-RNA in the antitermination complexes of bacteriophages lambda and P22

Bacteriophage lambda's N-protein includes a 17-amino-acid segment, Arm, rich in arginine and having specific affinity for a 15-nucleotide RNA stem-loop called BOX-B. Parallel but different Arm/BOX-B sequences in lambda's cousin, phage P22, account for some of the type specificity that distinguishes lambda from P22: the N of each works only with its cognate BOX-B in vivo. We find that the specificity of N(lambda) can be shifted gradually to that of N(22) by substituting sets of particular amino acids from Arm(22) into Arm of N(lambda). The determinative amino acids are generally those shown by nuclear magnetic resonance to contact BOX-B RNA; gain or loss of these contact amino acids is reasonably expected to contribute to the affinity of each amino acid sequence. Intermediate sequences may show no function with either BOX-B, or weak function with both BOX-B(lambda) and BOX-B(22), the latter suggesting possible evolutionary paths for specificity shifts.     

The molecular structure of the transducing particles of Salmonella phage P22. II. Density gradient analysis of DNA

Summary CsCl density gradient analysis showed that the DNA of plaque forming particles ofSalmonella phageP22 is lighter than the host DNA. The DNA of transducing phages exhibits an intermediate density, but close to host DNA. BU labelling of DNA synthesized in the cells after phage infection resulted in a density increase of transducing DNA of about 0.004 gxcm^-3, whereas infectious DNA increased by about 0.045 gxcm^-3. Shearing of isolated DNA molecules from unlabelledP22 lysates demonstrated that transducing DNA consists of two pieces of DNA of different density: 90% stem from the bacterial host whereas 10% are phage DNA and therefore responsible for the BU lable in transducing phages.     

Identification and characterization of mutations in Escherichia coli that selectively influence the growth of hybrid lambda bacteriophages carrying the immunity region of bacteriophage P22.

Mutations in two Escherichia coli genes, sipA and sipB, result in a specific inhibition of the growth of certain hybrid lambdoid bacteriophages, lambda immP22, that have the early regulatory regions and adjacent genes from bacteriophage P22. The sipB391 mutation maps near minute 56 and exerts the strongest inhibitory effect on the growth of the hybrid phages. The sipA1 mutation maps near minute 72 and plays an auxiliary role: enhancing the action of sipB391. Such a role is not limited to sipA1, since there is a similar enhancement by the nusA1 and nusE71 mutations. The Sip-imposed restriction on the growth of lambda immP22 phages is not observed if the phage carries a mutation in the c1 gene. Perhaps this reflects the fact that the c1 product regulates phage DNA replication and is a major determinant in the decision governing whether the phage takes the lytic or lysogenic pathway. Consistent with this idea is the observation that lambda immP22 DNA replication is severely inhibited in bacteria carrying the sipB391 mutation. It is suggested that sip mutations exaggerate the normal role of c1 in limiting lytic growth. This causes a failure in the expression of sufficient amounts of some or all of the lytic gene products required for phage growth.     

Bacteriophage P22 is not a likely probe for zones of adhesion between the inner and outer membranes of Salmonella typhimurium.

Thin-section electron micrographs of plasmolyzed Salmonella typhimurium infected with bacteriophage P22 demonstrated that phage adsorbed to cells over sites of inner- and outer-membrane contact. Efforts were made to isolate such adsorption sites by infection of cells with 35S- and 32P-labeled phage and by separation of the membranes on sucrose gradients. At 37 degrees C, about 75% of the 35S radioactivity could be recovered in a region of intermediate density between the inner and outer membranes. This region (phi band) did not contain 32P. The gradient profile was independent of the multiplicity of infection (between 0.2 and 50) and of the presence or absence of chloramphenicol, dinitrophenol, or cyanide. However, ethylenediaminetetraacetate, when present during the infection step, prevented the formation of phi band. The density of phi band was at least 1.30 g/cm3, as demonstrated by prolonged centrifugation on a D2O-sucrose gradient. phi Band was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy to contain empty phage heads and contaminating cellular debris. In purified preparations, phage heads were the only structures, visible by negative staining, and very little cellular phospholipid or protein was associated with the phage proteins (less than 2% and 30% by weight, respectively, as determined by using [3H]glycerol or [3H]leucine). The residual cellular protein included all of the major outer-membrane proteins rather than any one specific protein. These results are interpreted as indicating that phi band probably does not contain adhesion site material stably associated with phage heads.     

Adsorption of bacteriophages phi 29 and 22a to protoplasts of Bacillus subtilis 168.

Adsorption of bacteriophages phi 29 and 22a to protoplasts of Bacillus subtilis 168 is described. The number of binding sites on bacilli and protoplasts is determined for each phage. Bacilli and protoplasts possess roughly the same number of sites per unit area for phi 29, i.e., approximately 700 sites per bacillus. There are also approximately 700 sites per bacillus for 22a, but only about one-third as many sites per unit area on the protoplast surface. A model for phi 29 adsorption is proposed.