P22 Mutants Research Articles

Temperature-inducible mutants of P2 phage

Mutants of P2 with temperature-sensitive immunity ( c ts ) are not induced from the prophage state at high temperature, because the derepressed prophage cannot be excised ( L. E. Bertani, 1968). Starting from P2 c ts we have isolated a P2 mutant that is induced at high temperature. This phage carries a second mutation ( nip) which maps in the immunity region. The nip mutation alters the control of int expression.

P2 phage amber mutants: Characterization by use of a polarity supressor

Seventy amber mutants of temperate phage P2 have been characterized, using an Escherichia coli strain which suppresses polarity. Two new genes involved in tail synthesis were defined. The eighteen known P2 late genes can be divided into four transcription units. One of these is transcribed in the opposite direction from the other three. The largest transcription unit contains four head genes, a gene involved in cell lysis, and two tail genes.

Purification and Properties of Phage P22-induced Lysozyme

Abstract Phage P22 induces a lysozyme in Salmonella typhimurium cells toward the later stage of its multiplication. P22 lysozyme has been purified about 1000-fold starting from the lysate of C1 (clear plaque-forming mutant of phage P22)-infected cells. The enzyme has an optimum pH between 7 and 8 and its activity is dependent on the ionic strength of the assay medium. Salts like MgCl2, NaCl, and KCl are inhibitory to the lysozyme. Gram-negative cells act as better substrates for the lysozyme than do gram-positive cells. The enzyme has a molecular weight of about 2 x 104 and rapidly looses its activity at temperatures higher than 40°. The properties of P22 lysozyme have been compared with those of λ and T4 lysozymes. All three lysozymes have more or less the same molecular weight and have similar properties although P22 and T4 lysozymes seem to be closer than P22 and λ lysozymes.

On the Process of Cellular Division in Escherichia coli: Replication of the Bacterial Chromosome Under Control of Prophage P2

The temperature-sensitive mutant CRT46 of Escherichia coli K12 is unable to initiate new rounds of DNA replication at 42 degrees C. Mutants of bacteriophage P2 have been isolated, which, in the prophage state, allow mutant CRT46 to grow at 42 degrees C. The lysogenic bacteria that grow at 42 degrees C are apparently replicating under the control of prophage P2, which substitutes for the bacterial initiation system. The ability of prophage P2 cause this suppression phenomenon depends on the position of the prophage on the bacterial chromosome. Those lysogenic strains that are able to grow at 42 degrees C all carry the prophage close to metE. The P2 mutants that allow CRT46 to grow at 42 degrees C have insertions in the early region of the P2 genome. The suppression requires the cis-acting protein formed by gene A of P2.

Relation between Px phages and generalised transducing particles of phage P22.

P22 mutants with an altered ability to form generalised transducing particles were tested for their ability to form the hybrid Px phages during growth on Py-lysogenic strains of Salmonella typhimurium. The mutant which produces more (less) transducing particles produces also more (less) Px phages. It is suggested that Px particles might be regarded as self-replicating generalised transducing particles.

Vegetative DNA of temperate coliphage P2.

In order to study P2 DNA synthesis by incorporation of tritium-labeled thymine, mitomycin C has been used to selectively suppress host DNA synthesis. Several conditional lethal mutants of P2 (temperature-sensitive as well as amber) have been tested for their ability to synthesize DNA under non-permissive conditions. Mutants representing the two “early” genes were found unable to synthesize DNA normally.

LIpid changes in Salmonella typhimurium on infection with bacteriophage P22

On infection of Salmonella typhimurium with bacteriophage P22 mutants c 2 and Lys − c 2, changes in the phospholipid pattern of extractable phospholipids are observed, when investigated using 14C- and 32P-labeled bacterial cultures, either in unsupplemented M9 or in low phosphate M 9 medium. Thin layer chromatography resolved phospholipids into twelve areas which were qualitatively the same in both infected and uninfected cells. Moderate quantitative differences were found. In Lys − c 2 infected cells phosphatidyl glycerol rises sharply during the first 5 min after infection, and by 10 min has returned part way to its former level. In the first 10 min phosphatidyl ethanolamine and cardiolipin also increase at a faster rate than in uninfected cells, but to a lesser degree than phosphatidyl glycerol. From 10 min to the time of lysis the content of individual phospholipids scarcely changes although the cells continue to grow and divide. During the early stages of lysis induced by the addition of cysteine, phosphatidyl ethanolamine rises appreciably, but phosphatidyl glycerol and cardiolipin drop slightly. As lysis proceeds phospholipids are degraded with the liberation of free fatty acids which accumulate steadily. In c 2 infected cells, which do continue to grow but rarely divide, the lipid changes up to the time of lysis are less abrupt and more difficult to interpret. During and following lysis the changes are similar to those in Lys − c 2 infections. An attempt is made to correlate lipid changes with other changes occurring in the cell.

Bacteriophage P2: Replication of the chromosome requires a protein which acts only on the genome that coded for it

Some mutants of phage P2 with unusual properties are described. These mutants, which are of temperature sensitive or amber type, are located in a gene called A, at the right end of the P2 map. Gene A mutants are unable to replicate their DNA under nonpermissive conditions, even if another P2 is actively replicating in the same cell. They are also unable to complement with mutants in any other essential gene. It is concluded that the gene A protein is required for DNA replication and expression of other essential genes, and that it works only on the chromosome from which it was formed, i.e., it works only in cis. The function and mechanism of action of this protein are discussed.

Control of the replication complex of bacteriophage P22.

A replication complex for the vegetative synthesis of the deoxyribonucleic acid (DNA) of the temperate phage P22 previously has been described. This complex is an association of parental phage DNA, most of the newly synthesized phage DNA made during pulses with (3)H-thymidine, and other cell constituents, and has a sedimentation rate in neutral sucrose gradients of at least 1,000S. The complex is one of the intermediates, intermediate I, in the synthesis and maturation of phage P22 DNA after infection or induction. Evidence supporting the replicative nature of intermediate I is presented. Phage replication is repressed in lysogenic bacteria. On superinfection of P22 lysogens with nonvirulent phage, little association of the input phage DNA with a rapidly sedimenting fraction is demonstrable. However, after induction with ultraviolet light, the superinfecting parental phage DNA quickly acquires the rapid sedimentation rate characteristic of intermediate I; phage DNA synthesis follows; and progeny phages are produced. Infection with a virulent mutant of P22 produces progeny phages in lysogens. Its DNA associates with intermediate I. In mixed infection with the virulent phage, replication of nonvirulent phage P22 is still repressed, even though the virulent replicates normally. The nonvirulent input DNA does not associate with intermediate I. The repressor of the lysogenic cell prevents replication by interfering with the physical association of template material with intermediate I. A phage function is required for association of phage template with the replication machinery.

Bacteriophage lambda; abortive infection of bacteria lysogenic for phage P2.

The efficiency of plating of wild-type lambda on a host lysogenic for P2 is less than 10(-6), and only a small number of infected cells produce progeny phage. Lambda can adsorb and inject its DNA normally in such cells; the DNA can circularize and is not nicked or degraded, but replication is severely impaired. Mutants of P2, which as prophages no longer interfere with lambda, have been isolated and found to be recessive to wild type, implying that P2 prophage codes for a diffusible product involved in lambda interference. The P2 gene product responsible for preventing lambda growth also kills recombination-deficient bacteria of the recB and recC classes under conditions where P2 does not normally kill the host. Mutants of lambda that are resistant to interference are recessive to wild-type lambda. Thus lambda actively participates in its own interference. The lambda-mutants that are resistant to interference are unable to synthesize at least two nonessential proteins. In addition, they are unable to grow on recombination-deficient bacteria of the recA class, but they can grow on recA recB double mutants.

Delayed lysis in Salmonella phage P22: The appearance of free fatty acids coincident with induced cell lysis

Temporally associated with cell lysis, extractable free fatty acids are detected in phage P22 infections of Salmonella typhimurium. In infections with a P22 mutant showing delayed lysis ( Lys − c 2) free fatty acids are observed only after induction of lysis with cysteine, histidine, cyanide, or EDTA. Fatty acid appearance is detected at the same time as or later than the onset of lysis as indicated by the decrease in optical density of infected cell suspensions. Since free fatty acids accumulate during incubation of sonicated but uninfected S. typhimurium cells (and also sonicated E. coli cells), it is concluded that their release in both infected and uninfected cells is the result of activation of a bacterial lipase as a consequence of cellular disorganization rather than as a cause of it.

Isolation of UGA and UAG nonsense mutants of bacteriophage P22

Isolation of UGA and UAG nonsense mutants of bacteriophage P22

Delayed Lysis with a Mutant of Salmonella Bacteriophage P22

A mutant of bacteriophage P22 (Lys(-)) was isolated which shows a plaque morphology on mixed plates comparable to the r(+) plaques of the T-even phages. When Lys(-) and normal Lys(+) plaques are juxtaposed on a petri dish, the Lys(+) plaque exhibits a flat side adjacent to the Lys(-) plaque. The mutant is identical to P22 under an electron microscope, is inactivated at the same rate by antiserum and heat, and has the same kinetics of attachment. It does not plate on Salmonella lysogenic for phage P22 nor on strain St/22. In liquid culture, the lysis of mutant infections in M9CAA medium is delayed between 20 and 40 min. Cells mixedly infected in M9CAA with Lys(-) and Lys(+) phage lyse later than Lys(+)-infected cells and even later than Lys(-)-infected cells. In unsupplemented M9 medium, however, mixedly infected cells again lyse later than Lys(+)-infected cells, but Lys(-)-infected cells require more than 3 hr to lyse. In supplemented and unsupplemented M9 media, intracellular phage development and endolysin synthesis proceed in Lys(-) infections at least as rapidly as in Lys(+)-infected cells. In diluted infections, the latent and eclipse periods of Lys(-) and Lys(+) infections are indistinguishable. The possible mechanisms involved in the control and timing of lysis are discussed.

Integration defective mutants of bacteriophage P2

Mutants of phage P2 unable by themselves to be integrated as prophages have been isolated. These mutants (int) are complemented by the wild type allele and may then yield stable lysogenic strains carrying an int prophage at location I in Escherichia coli C. These lysogens produce either no phage or little phage, depending on the int mutant used. All int mutants isolated appear to belong to a single complementation group.

Calcium sensitive and other mutants of bacteriophage P2.

Summary Several types of mutants variously affected by the concentration of calcium ions in the medium may be isolated from phage P2 (host: Escherichia coli). As compared with the wild type, the mutants may be more or less dependent on calcium for adsorption, more rapidly inactivated in the presence of calcium, or less prone to establish lysogeny at low calcium concentrations.

Abortive induction of bacteriophage P2

Five temperature-sensitive clear-plaque mutants of bacteriophage P2 were isolated, and the properties of strains lysogenic for these mutants were studied. Upon incubation at 42°, all five strains are inactivated and concomitantly lose immunity to superinfection, although at characteristically different rates. In no case, however, is much phage produced, the proportion of productive bacteria varying from 0.1% to 0.5%. The proportion of bacteria producing phage carrying the c allele of the prophage can be increased to 100% by superinfection at 42° with ultraviolet-irradiated wild-type P2. By means of bacterial crosses, it was shown that the temperature-sensitive prophages are able to detach from the bacterial chromosome only if the bacteria are first superinfected. In contrast, it was found that λ prophages, following induction by either temperature shift or ultraviolet irradiation, detach even in the absence of superinfection. The derepression of at least one P2 prophage gene following incubation at 42° was clearly demonstrated. A strain lysogenic for a temperature-sensitive clear mutant, blocked also in this gene, was found to be inactivated much more slowly at 42°, although the immunity of the strain was lost at the rate characteristic of that particular clear mutant.

Defective phage formation by lysogens of integration deficient phage P22 mutants

Integration deficient ( L) mutants of phage P22 can be complemented with L + phage to yield L mutant lysogens. Once established the L prophage is stable in the absence of L + function. Ultraviolet or thermal induction of L lysogens leads to production of defective phage particles and low yields of infectious phage. The defective particles contain a normal amount of DNA but the DNA is mainly bacterial in origin and appears to derive from the pro C region adjacent to one end of the prophage. The defective lysates transduce pro C at high frequency relative to other bacterial markers. The representation of phage genes is strongly polar with markers distal to pro C being rarely present. It appears that L + function is required not only for efficient integration of the prophage, but for normal recovery of the prophage genome following induction.

A phage P22 gene controlling integration of prophage.

Abstract A mutually noncomplementing group of phage P22 mutants (L mutants) have been isolated which cannot accomplish a late step necessary for prophage integration. L mutants produce turbid plaques and lead to cell survival on high multiplicity infection, but the infecting phage genomes are incapable of integrating as prophage and are progressively diluted among the segregating cells. L mutants can become prophage by complementation with L+ phage. The L+ gene apparently produces a diffusible product which acts trans to accomplish integration. Experiments with temperature sensitive L mutants show that the gene begins acting late, after the infected cells have recovered and begun to divide, and does not accomplish its effect in all the cells until at least the third generation. L lysogens formed by complementation are stable. The L+ gene is thus necessary for integration but not for maintenance of the lysogenic state.

SEQUENTIAL GENE ACTION IN THE ESTABLISHMENT OF LYSOGENY.

A study of temperature sensitive clear-plaque-forming mutants of bacteriophage P22 demonstrates that the c(1) and c(2) loci must function in a temporal sequence in the establishment of lysogeny in Salmonella typhimurium. The c(1) locus acts for only a 4-minute interval between the 7th to 11th minute of the infection. The c(2) locus begins to function some minutes later, and its continued activity is necessary for perpetuation of the lysogenic state.

EFFECT OF OPSONINS ON PHAGOCYTOSIS AND KILLING OF SALMONELLAE BY AMOEBA PROTEUS, AND THE USE OF PHAGE P22 TO ELIMINATE SURPLUS EXTRACELLULAR BACTERIA.

SummaryMammalian serum opsonins have been shown to possess moderate power to promote phagocytosis of Salmonella enteritidis by Amoeba proteus, but serum opsonins had no measurable effect on the rate of intracellular killing of these bacteria or of Salmonella typhimurium. The survival time of opsonised salmonellae after phagocytosis by amoebae was considerably longer than would be expected if similar bacteria were to be ingested by mammalian phagocytes. The possible effect of temperature on intracellular bactericidal mechanisms is discussed.Also described is a technique in which a lytic mutant of phage P22 was used to eliminate residual extracellular salmonellae from washed amoebae after a standard period of phagocytosis.