Host-Bacteriophage Interaction in Agrobacterium tumefaciens III. Phage-Coded Endolysins

Endolysins were detected in a sensitive strain of Agrobacterium tumefaciens (B6) after infection with phage LV-1 and in the lysogen A. tumefaciens V-1 after induction with mitomycin C. A similar endolysin was found in mitomycin C-induced A. tumefaciens C-58, which apparently harbors a defective prophage.     

Genetic analysis of spo0A and spo0C mutants of Bacillus subtilis with a phi 105 prophage merodiploid system.

An 8.0-kilobase chromosomal fragment of Bacillus subtilis which contained an intact spo0A gene was recloned onto temperate phage phi 105 from the rho 11dspo0A+-1 transducing phage. A specialized transducing phage, phi 105-dspo0A+-1, was constructed and used to transduce the spo0A12 mutant strain 1S9. A Spo+ transductant which was a single lysogen of the phi 105dspo0A+-1 transducing phage was isolated. From competent cells of this Spo+ transductant was isolated a Spo- (Spo0A) strain which was immune to phi 105. It was used to prepare a lysate of the phi 105dspo0A12 phage. Transduction of the spo0C9V recE4 strain with the phi 105dspo0A12 and phi 105dspo0A+-1 phages was carried out. The phi 105dspo0A+-1 phage gave rise to a large number of heat-resistant cells, but the phi 105dspo0A12 phage formed no heat-resistant cells. These results indicate that the spo0A12 and spo0C9V mutant genes do not complement each other in the ability to sporulate and that the spo0C9V mutation is located within the spo0A gene. Although the spo0C9V strain was completely asporogenous, the spo0C9V/spo0C9V diploid strain produced heat-resistant cells at a frequency of ca. 10(-3) in the sporulation medium. This result indicates that two copies of the spo0C9V mutant gene partially restore the ability of these cells to sporulate.     

Participation of hup gene product in replicative transposition of Mu phage in Escherichia coli

The closely related Escherichia coli genes hupA and hupB each encode a bacterial histone-like protein HU. We report here that mutator phage Mucts62 was unable to replicate in a hupA hupB double mutant, although it could replicate in hupA or hupB single mutant as efficiently as in the wild-type strain. Mucts62 was able to lysogenize the double mutant at 30 degrees C; cell killing occurred when the lysogen was incubated at 42 degrees C, but did not result in phage production. High-frequency non-replicative integration of Mu into host genomic DNA soon after infection could not be detected in the hupAB double mutant. These results provide the evidence that HU protein is essential for replicative transposition of Mu phage in E. coli, and also participates in high-frequency conservative integration.     

Superinfection Exclusion by P22 Prophage and the Replication Complex

Wild-type sie(+) P22 prophage converted Salmonella typhimurium lysogens to exclude deoxyribonucleic acid (DNA) injected by superinfecting phage. DNA from a P22 superinfecting virulent phage associated with the replication complex in a sie(-) lysogen but not in a sie(+) lysogen.     

Chromosomal Recombination in HAEMOPHILUS INFLUENZAE

Haemophilus influenzae cultures doubly lysogenic for defective phage HP1, with a prophage marker sequence +b+/a+c, always contained some free wild-type phage. Single ultraviolet-irradiated cells produced either no wild-type phage or large numbers of them. This suggested that the phage was not released by the original double lysogen but by internal recombinants, i.e., by double lysogens with altered prophage marker sequence such as +++/abc or +b+/++c. Thirty-one wild-type phage-producing clones have been isolated independently from cultures of this double lysogen and identified. They fell in five classes. Two classes, still possessing all three prophage markers, can be explained by Campbell's (1963) prophage recombination model. The other classes had lost one or more markers. They can be explained by interchromosomal double-strand DNA breakage and rejoining. A single-DNA-strand gene conversion model is discussed in view of the fact that genetic transformation involves single-DNA-strand exchanges. A number of potentially interesting mutants has been analyzed of which only the derivatives of rec1 mutant DB117 (obtained from Dr. J. Setlow) were incapable of internal recombination.     

New Deoxyribonuclease Activity After Bacteriophage P22 Infection

Extracts from P22-infected and uninfected cultures of Salmonella typhimurium were subjected to deoxyribonucleic acid (DNA)-cellulose and diethylaminoethyl-cellulose chromatography. Comparison of the elution patterns revealed that in infected cells there is a decrease in the amount of nuclease activity specific for denatured DNA and an increase in the amount of nuclease activity specific for native DNA. The latter activity was shown to differ from a similar host enzyme in Mg²⁺, Mn²⁺, and pH optima. This new activity is not found after infection of a lysogen with a nonvirulent phage or after infection under nonpermissive conditions with P22ts25.1 (a mutant in gene 25 that carries out no known functions other than adsorption and injection) and thus appears to be specified by the phage genome.     

Saccharopolyspora hirsuta strain 367 releases JHJ-1, a bacteriophage capable of propagation on old mycelium

Bacteriophage JHJ-1 was isolated from Saccharopolyspora hirsuta strain 367 NRRL 12045 as an endogenous but virulent phage. The plaque size was not self-limiting, since a few p.f.u. could completely lyse a lawn. Electron microscopy showed that this phage belonged to group B of Bradley's morphological classification. The JHJ-1 genome is a linear DNA molecule of 41.1 kbp with cohesive ends and a G + C content of 68.8-70.0 mol%. The DNA cleavage map was established for 12 restriction endonucleases. The host range is apparently very narrow, being limited to two strains of S. hirsuta (NRRL 12045 and NRRL B-5792). However, JHJ-1 did not lytically infect S. hirsuta strain 367 UC 8106. Phage JHJ-1 was shown, by Southern blot analysis, to lysogenize both S. hirsuta NRRL 12045 and UC 8106. It thus appears to behave as a virulent mutant of a temperate phage on one, but not on the other, JHJ-1 lysogen.     

Prophage Curing in Lactobacillus casei by Isolation of a Thermoinducible Mutant

To eliminate the occurrence of virulent phage in industrial fermentation, attempts were made to obtain prophage-cured derivatives from Lactobacillus casei lysogenic strain S-1. A thermoinducible mutant lysogen was isolated from mutagenized strain S-1, since S-1 cannot be induced under laboratory conditions. The mutation responsible for thermoinducibility was located on the prophage. Prophage-cured strains were selected after heat induction of the mutant. These cured strains did not produce the virulent phage and should be valuable for industrial fermentation.     

Behavior of a temperate bacteriophage in differentiating cells of Bacillus subtilis.

During the first 6 hr of sporulation, infection of Bacillus subtilis by by phi105 wild type or the clear-plaque mutant phi105 c30 was nonproductive, but phage DNA was trapped inside developing spores. After infection with either wild-type or mutant phage at early times of sporulation (T1-T3), phage DNA entered the developing spores in a heat-stable form, which may represent integration of the phage DNA into the host chromosome. Phage DNA in carrier spores produced by infection at later times (T4-T6) was much more heat sensitive. Spore preparations containing either phi105 wild type or phi105 c30 carrier spores gave rise to a spontaneous burst of phage during outgrowth, although the fraction of carried wild-type phage that chose lysis over lysogeny at germination has not been determined. Heat induction of the thermoinducible lysogen 3610 (phi105 cts23) was also abortive during sporulation. Furthermore, induction neither prevented eventual spore formation nor resulted in the conversion of prophage DNA to the carrier state; during outgrowth, the previously induced lysogenic spores remained stable lysogens. However, if the sporulating lysogenic cells were plated immediately after induction, they did not form colonies at high efficiency, as though transfer to fresh medium allowed sufficient phage expression to kill the host.     

Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor

Wiesmeyer, Herbert (Vanderbilt University, Nashville, Tenn.). Prophage repression as a model for the study of gene regulation. I. Titration of the lambda repressor. J. Bacteriol. 91:89-94. 1966.-The concentration of lambda repressor molecules within a lambda lysogenic cell was estimated from the multiplicity of superinfecting homologous phage necessary to permit replication and release of plaque-forming units. A multiplicity of 20 superinfecting phage was found sufficient to permit replication to occur in the normal lambda lysogen. The phage released after lysis of the superinfected lysogen was composed of both prophage and superinfecting phage types. Superinfection of the lysogen at lower multiplicities resulted in the lysis of only a small percentage of infected cells and is thought to represent a possible heterogeneity of repressor concentration in the lysogenic population. Viability of the superinfecting particle was found to be unnecessary for titration of the repressor. The repressor concentration in three lysogens of the nonultraviolet-inducible mutant of lambda, lambda(ind-), was found to be greater than 20 regardless of the host bacterium. However, the number of cells yielding phage after superinfection was found to vary with the particular host. The specificity of the lambda repressor was shown to be limited to homologous phage, as determined following heterologous superinfection experiments with phages T6r, 82c, 434c, 434hy, and 424. In all instances except that of superinfection with phage 434hy, only heterologous phage replication occurred. Superinfection by phage 434hy resulted in the release of both prophage and superinfecting phage types. The latter type represented approximately 80% of the total phage released.     

F-plasmid gene srnB+ complements the function of the S gene of phage lambda

Abstract The srnB ⁺ gene located on the F plasmid was assayed for its capacity to facilitate the release from infected cells of phage λ lacking the usual lytic activity. The srnB ⁺ plasmid pOY54, carrying the 1.4–2.5F fragment in the Eco RI- Bam HI fragment of pBR322, induced bacteriolysis and the release of progeny phage of the λcI 857 susS 7 lysogen in the presence of rifampin at 42°C. An srnB 1 mutant plasmid, pOY541, did not promote bacteriolysis. These results suggest that the srnB ⁺ gene of the F plasmid complements the function of the λ S gene in the nonpermissive host strain.     

Efficient excision of phage lambda from the Escherichia coli chromosome requires the Fis protein.

The Escherichia coli protein Fis has been shown to bind a single site in the recombination region of phage lambda and to stimulate excisive recombination in vitro (J. F. Thompson, L. Moitoso de Vargas, C. Koch, R. Kahmann, and A. Landy, Cell 50:901-908, 1987). We demonstrate that mutant strains deficient in fis expression show dramatically reduced rates of lambda excision in vivo. Phage yields after induction of a stable lysogen are reduced more than 200-fold in fis cells. The defect observed in phage yield is not due to inefficient phage replication or lytic growth. Direct examination of excisive recombination products reveals a severe defect in the rate of recombination in the absence of Fis. The excision defect observed in fis cells can be fully reproduced in fis+ cells by using phages that lack the Fis binding site on attR, indicating that the entire stimulatory effect of Fis on excisive recombination is due to binding at that site.     

Influence of resistance-factors on the phage types ofSalmonella Panama

The resistance to antibiotics which has been increasingly observed in naturally occurringSalmonella panama, is due to an R-factor. A relationship was found between phage pattern and the presence of this R-factor. All strains belonging to phage types A, C and E are sensitive to all antibiotics and are indicated in phage-typing by wild-type phage 47 or host-range mutants of phage 47. All strains belonging to phage types B, D and F possess an R-factor and are indicated by host-modified variants of phage 47. Phage type G, indicated by a host-range mutant, and group Z contain strains with, as well as without an R-factor. Spontaneous drug-sensitive segregants of type B, D and F strains have the phage pattern A, C and E respectively. Conversely, the phage pattern of A, C and E type strains change into B, D and F respectively after infection with the R-factor ofS. panama. The theory can be advanced that type B type A+R-factor, D — C+R-factor and F = E+R-factor. This change in phage type can be considered to be due to the fact that the R-factor exerts restriction and modification of the phage which indicates theS. panama strain without the R-factor.Many of the antibiotic-resistantEscherichia coli strains found in nature possess an R-factor which can be transferred toS. panama in vitro. Relatively few of these R-factors were found to possess also the restriction marker. Thus up to the present the number ofE. coli strains possessing an R-factor which is able to create a dependable combination of phage type and drug resistance inS. panama is relatively small.     

Infection of Competent Mycobacterium smegmatis with Deoxyribonucleic Acid Extracted from Bacteriophage B1

A relatively competent state of Mycobacterium smegmatis for infection with deoxyribonucleic acid (DNA) extracted from phage B1 was found in the late log phase of bacterial growth. This state of the culture was used in quantitative studies on the infectivity of the DNA. The buoyant density of B1 DNA was 1.728 g/cc in CsCl, and 1 mug of the DNA produced 84 infective centers, the phage equivalent of which was 1.5 x 10(-8). The infectivity was destroyed by catalytic amounts of deoxyribonuclease but not by specific B1 antiserum. Tween 80, which prevents phage adsorption, did not prevent DNA infection. The response of plaque-forming ability to DNA concentration suggested that two or more molecules are required to initiate an infective center. The low efficiency of DNA infection in mycobacteria was considered to be caused by a limiting population of competent cells in the culture employed; in this experiment less than 10(-5) of the cells were infected with DNA. A typical cycle of infection was observed, although the latent period was prolonged and the burst size reduced after DNA infection. The transition of B1 DNA infection to deoxyribonuclease insensitivity had a lag period of about 10 min, and increased linearly with a velocity of about 0.24 infective centers per min per mug of DNA. Half of the infective titer was inactivated by heating at 92 C for 15 min. The melting temperature was about 96 C. Species barriers were not crossed by B1 DNA; however, the DNA was infectious for a B1-resistant mutant of the host.     

Lysogenization of Escherichia coli him+, himA, and himD hosts by bacteriophage Mu.

The possible outcomes of infection of Escherichia coli by bacteriophage Mu include lytic growth, lysogen formation, nonlysogenic surviving cells, and perhaps simple killing of the host. The influence of various parameters, including host himA and himD mutations, on lysogeny and cell survival is described. Mu does not grow lytically in or kill him bacteria but can lysogenize such hosts. Mu c+ lysogenizes about 8% of him+ bacteria infected at low multiplicity at 37 degrees C. The frequency of lysogens per infected him+ cell diminishes with increasing multiplicity of infection or with increasing temperature over the range from 30 to 42 degrees C. In him bacteria, the Mu lysogenization frequency increases from about 7% at low multiplicity of infection to approach a maximum where most but not all cells are lysogens at high multiplicity of infection. Lysogenization of him hosts by an assay phage marked with antibiotic resistance is enhanced by infection with unmarked auxiliary phage. This helping effect is possible for at least 1 h, suggesting that Mu infection results in formation of a stable intermediate. Mu immunity is not required for lysogenization of him hosts. We argue that in him bacteria, all Mu genomes which integrate into the host chromosome form lysogens.     

Integration and Induction of Phage P22 in a Recombination-deficient Mutant of Salmonella typhimurium

Phage P22 can integrate as prophage into a recombination-deficient (Rec(-)) strain of Salmonella typhimurium. At 37 C, the integration efficiency is only 10% that in Rec(+) infection, but at 25 C the efficiencies in Rec(-) and Rec(+) hosts are similar. Rec(-) lysogens cannot be induced by ultraviolet irradiation or by treatments with the chemical inducing agents streptonigrin or mitomycin C. Heat induction of Rec(-) cells lysogenic for a temperature-sensitive c(2) mutant (ts c(2)) is normal, showing that the Rec(-) cell has the machinery necessary for prophage excision. Ultraviolet irradiation of Rec(-) (ts c(2)) lysogens prior to heat induction does not prevent the formation of infective centers after temperature shift. Thus, the noninducibility of Rec(-) lysogens is not due to destruction of the prophage as a result of ultraviolet irradiation. Deoxyribonucleic acid-ribonucleic acid (RNA) hybridization experiments demonstrate that no increase in phage-specific RNA synthesis occurs after ultraviolet irradiation of a Rec(-) (c(+)) lysogen. The Rec(-) mutant appears to lack part of the mechanism required to destroy the phage repressor and allow the initiation of early phage functions such as messenger RNA synthesis. A similar conclusion was reached previously for an Escherichia coli Rec(-) strain.     

Transfection of Escherichia coli spheroplasts. VI. Transfection of nonpermissive spheroplasts by T5 and BF23 bacteriophage DNA carrying amber mutations in DNA transfer genes.

DNA was extracted from T5 and BF23 phage carrying amber mutations in genes A2, A1, or D9 and tested for its ability to transfect su minus spheroplasts. DNA from T5 am231, defective in gene A2, transfects Escherichia coli su minus recB minus spheroplasts with an efficiency of 16% of that of wild-type T5 DNA, whereas DNA from T5 am16d or BF23 am57, both defective in gene A1 or its equivalent, transfects E. coli su minus recB minus spheroplasts with an efficiency of 1.4% of that of wild-type T5 DNA, provided E. coli su+ bacteria is used as the indicator in all cases. More than 95% of the progeny from the am231, am16d, and am57 DNA that transfects su minus recB minus spheroplasts is still amber mutant. From these efficiencies of transfection we conclude that the product of gene A2 functions mainly in the mechanism of transfer of phage DNA to intact host cells, and that this function is not essential for transfection of spheroplasts. We also conclude that gene A1 controls functions in addition to DNA transfer, in agreement with previous studies which show that mutations in gene A1 have a pleiotropic effect. Apparently, the absence of these additional functions controlled by gene A1 leads to a high frequency of abortive infection. DNA from amber mutants defective in either gene A1 or A2 does not appreciably transfect su minus rec+ spheroplasts, indicating that the products of these two genes may both be needed to protect T5 DNA from the very active rec BC nuclease in spheroplasts.