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Phage HK139 is UV inducible and lambda homoimmune and has the host range of phi80. It can recombine with lambda as well as with phi80, and in the prophage form it is found integrated between the loci his and supD.
We used electron microscopy and serum blocking power tests to determine the phenotypes of 47 phage P1 amber mutants that have defects in particle morphogenesis. Eleven mutants showed head defects, 30 showed tail defects, and 6 had a defect in particle maturation (which could be either in the head or in the tail). Consideration of previous complementation test results, genetic and physical positions of the mutations, and phenotypes of the mutants allowed assignment of most of the 47 mutations to genes. Thus, a minimum of 12 tail genes, 4 head genes, and 1 particle maturation gene are now known for P1. Of the 12 tail genes, 1 (gene 19, located within the invertible C loop) codes for tail fibers, 6 (genes 3, 5, 16, 20, 21, and 26) code for baseplate components (although one of these genes could code for the tail tube), 1 (gene 22) codes for the sheath, 1 (gene 6) affects tail length, 2 (genes 7 and 25) are involved in tail stability, and 1 (gene 24) either codes for a baseplate component or is involved in tail stability. Of the four head genes, gene 9 codes for a protein required for DNA packaging. The function of head gene 4 is unclear. Head gene 8 probably codes for a minor head protein, whereas head gene 23 could code for either a minor head protein or the major head protein. Excluding the particle maturation gene (gene 1), the 12 tail genes are clustered in three regions of the P1 physical genome. The four head genes are at four separate locations. However, some P1 head genes have not yet been detected and could be located in two regions (for which there are no known genes) adjacent to genes 4 and 8. The P1 morphogenetic gene clusters are interrupted by many genes that are expressed in the prophage.
Lysates of P1 from all hosts tested contained at least three morphological variants with respect to head size. These were termed "big" (P1B), "small" (P1S), and "minute" (P1M). Since successive clonings of plaques isolated on many different hosts failed to change the proportions of the variants, we concluded that the production of variants was a function of the P1 genome rather than that of the host. In the electron microscope, the heads appeared to be icosadeltahedra, having face-to-face head diameters of 86 +/- 2 nm, 65 +/- 2 nm, and 47 +/- 2 nm. Assuming the head capsids to be composed of the same protein subunits, these diameters were compatible with T = 16, 9, and 4 with a lattice constant (intercapsomere distance) of 12 to 13 nm. The tails of all variants were morphologically indistinguishable. Each consisted of a hollow tail tube surrounded by a contractile sheath attached to the head by means of a "head-neck connector" which could be a specialized vertex capsomere. In CsCl gradients, a number of bands were observed. One band contained the majority of P1B particles and 99% of the plaque-forming units. Two other bands contained P1S particles whose densities suggested a content of about 40 and 60% of the complete P1B genome. The less dense of these two bands also contained defective P1B particles with a calculated content of only 60% of the complete genome. The P1S particles tested injected their deoxyribonucleic acid (DNA) into host cells and killed them. Genetic markers contained in this band could be rescued by infectious P1B particles, confirming the evidence of Ikeda and Tomizawa that this fraction contains P1 DNA.
Plaque morphology indicated that the five Escherichia coli K1-specific bacteriophages (A to E) described by Gross et al. (R. J. Gross, T. Cheasty, and B. Rowe, J. Clin. Microbiol. 6:548-550, 1977) encode K1 depolymerase activity that is present in both the bound and free forms. The free form of the enzyme from bacteriophage E was purified 238-fold to apparent homogeneity and in a high yield from ammonium sulfate precipitates of cell lysates by a combination of CsCl density gradient ultracentrifugation, gel filtration, and anion-exchange chromatography. The enzyme complex had an apparent molecular weight of 208,000, as judged from its behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was dissociated by sodium dodecyl sulfate at 100 degrees C to yield two polypeptides with apparent molecular weights of 74,000 and 38,500. Optimum hydrolytic activity was observed at pH 5.5, and activity was strongly inhibited by Ca2+; the Km was 7.41 X 10(-3) M. Rapid hydrolysis of both the O-acetylated and non-O-acetylated forms of the K1 antigen, an alpha 2----8-linked homopolymer of N-acetylneuraminic acid, and of the meningococcus B antigen was observed. Limited hydrolysis of the E. coli K92 antigen, an N-acetylneuraminic acid homopolymer containing alternating alpha 2----8 and alpha 2----9 linkages, occurred, but the enzyme failed to release alpha 2----3-, alpha 2----6-, or alpha 2----9-linked sialic residues from a variety of other substrates.
H (heavy) and L (light) MS-2 particles differ in density, absorption spectrum, and infectivity. Studies on their sedimentation, ribonucleic acid (RNA) content and infectivity, appearance under the electron microscope, ribonuclease sensitivity, and A-protein content failed to demonstrate any difference between the two particle types. Studies on the size, RNA content, and density of the capsid and two smaller coat protein components were also conducted. The antigenic relatedness of five different viral and subviral particles of MS-2 were studied by using immunodiffusion and neutralization. Capsids and the H and L viral particles were shown to be antigenically related, whereas the coat protein monomers and dimers were shown to be unrelated to the higher-molecular-weight particles.
Three kinetically different reactions (Rx1, Rx2, and Rx3) have been distinguished in the photoinactivation of phage T3 in the presence of the dye proflavine. The response of these reactions to the presence of the radical trap l-cysteine has been examined. At dye concentrations equal to or less than 2.2 mug/ml, Rx1 was composed of at least two parallel first-order reactions, one cysteine-insensitive (Rx1A) and one cysteine-inhibited (Rx1B). Rx2 was completely cysteine-insensitive (Rx2A). The cysteine sensitivity of these reactions changed abruptly at dye concentrations above 2.2 mug/ml. Rx1A and Rx1B now operated in tandem, rather than simultaneously, with Rx1B being confined to the first 1 min at most. Rx2, on the other hand, was completely cysteine-inhibited (Rx2B). Rx3 was inhibited roughly 75 to 80% by saturating concentrations of cysteine regardless of the time of addition of cysteine. The dark inactivation associated with Rx3 was inhibited roughly 85% whether the radical trap was added during the light or dark regimes. Changes of initial phage titer did not alter the cysteine sensitivity of a reaction.
Ultracentrifugational studies with deoxyribonucleic acid (DNA) extracted from phage exposed to light in the presence of either 0.25 or 8.5 mug of proflavine per ml reveal that the lethal damage of Rx1 renders DNA alkali-labile, with lethality resulting from damage that occurs singly on either strand or simultaneously on both strands. Apparently nonlethal damages temporally associated with Rx1 consist of (i) heat and alkali-labile cross-links (which produce undenatured DNA that migrates at 45S) and (ii) heat-labile bonds. The formation of 45S material is a linear function of light dose, and the production of this material ceases when Rx2 appears at the higher dye concentration. No tendency to plateau is seen at 0.25 mug of dye per ml. The nature of the lethal damage of Rx2 could not be determined. Damages that were temporally associated with Rx2 at 8.5 mug of dye per ml were heat-labile, alkali-stable cross-links (undenatured DNA, 38 to 41S; alkali-denatured-reneutralized DNA, 85 to 89S) and some double-stranded breaks. No such changes were seen at 0.25 mug of dye per ml.
Hybrids of lambda and adjacent bacterial deoxyribonucleic acid carried in T1 particles were able to transduce Gal(+) with a greatly increased efficiency to strains which were not immune to lambda compared to immune strains. The enhanced transduction was dependent on a functional recA(+) gene in the recipient. Mutations of the donor's lambda prophage which abolished the function of either the cI, O, or P genes in the recipients led to a further enhancement of transduction. The rate of transduction of a nonlysogenic recipient such as W3350 by the hybrid particles may be as much as 140 times greater than transduction of the lysogenic recipient W3350(lambda). In addition to the effect of lambda immunity in blocking enhanced transduction, mutations of the N gene of the donor's lambda prophage abolished enhanced transduction. Mutations in the red, int, xis, and Q genes of the donor's prophage had no significant effect on transduction. The hybrids which mediated the enhanced transduction are called (lambda-gal)T1.
One of the crossovers leading to the transduction of Escherichia coli W3350 or one of its derivatives for the Gal(+) marker by T1 grown on donors lysogenic for lambda must occur to the right of the galactose operon. The location of this crossover determined both the rate of transduction and the lambda genes which control the transduction by (lambda-gal)T1. When the crossover occurred either to the left of gene N or the right of the cI gene, it was affected in a positive fashion by the gene product of the N(+) gene. When the crossover occurred either to the left of gene N or between N and gene Q, its efficiency was not significantly affected by the expression of Q(+). However, crossovers to the right of gene Q were greatly stimulated by the presence of a functioning Q gene on the chromosome of (lambda-gal)T1 hybrid. The repressor made by the cI(+) gene of lambda blocked efficient transduction. Therefore, the control of efficient transduction by (lambda-gal)T1 is the same as the control of transcription of lambda. It is concluded that the increased efficiency of transduction by (lambda-gal)T1 in nonimmune recipients is not caused by any particular product of a gene but rather by the process of transcription itself.
Electron microscopic studies of Escherichia coli infected with bacteriophage T5(+) have revealed that host nuclear material disappeared before 9 min after infection. This disappearance seemed to correspond to the breakdown of host deoxyribonucleic acid (DNA) into acid-soluble fragments. Little or no host DNA thymidine was reincorporated into phage DNA, except in the presence of 5-fluorodeoxyuridine (FUdR). Progeny virus particles were observed in the cytoplasm 20 min postinfection. Most of these particles were in the form of hexagonal-shaped heads or capsids, which were filled with electron-dense material (presumably T5 DNA). A small percentage (3 to 4%) of the phage heads appeared empty. On rare occasions, crystalline arrays of empty heads were observed. Nalidixic acid, hydroxyurea, and FUdR substantially inhibited replication of T5 DNA. However, these agents did not prevent virus-induced degradation of E. coli DNA. Most of the phage-specified structures seen in T5(+)-infected cells treated with FUdR or with nalidixic were in the form of empty capsids. Infected cells treated with hydroxyurea did not contain empty capsids. When E. coli F was infected with the DO mutant T5 amH18a (restrictive conditions), there was a small amount of DNA synthesis. Such cells contained only empty capsids, but their numbers were few in comparison to those in cells infected under permissive conditions or infected with T5(+). The cells also failed to lyse. These results confirm other reports which suggest that DNA replication is not required for the synthesis of late proteins. The data also indicate that DNA replication influences the quantity of viral structures being produced.
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The coprecipitate of A protein and RNA which results from acetic acid treatment of bacteriophage R17 has been shown to form two bands after equilibrium density gradient centrifugation in Cs2SO4. The band of higher density, whose position coincides with that of phage RNA prepared by phenolisation possesses neither A protein nor infectivity while the band of lower density which contains both RNA and A protein is infectious for intact E. coli cells. The nature of the bonding between the A protein and the RNA was also investigated.