Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells.

Lysogenic strains of Bacillus subtilis 168 were reduced in their level of transformation as compared to non-lysogenic strains. The level of transformation decreased even further if the competent lysogenic cells were allowed to incubate in growth media prior to selection on minimal agar. This reduction in the frequency of transformation was attributable to the selective elimination of transformed lysogenic cells from the competent population. Concurrent with the decrease in the number of transformants from a lysogenic competent population was the release of bacteriophage by these cells. The lysogenic bacteria demonstrated this dramatic release of bacteriophage only if the cells were grown to competence. Both the selective elimination of transformed lysogens and the induction of prophage was prevented by the inhibition of protein synthesis. Additionally, competent lysogenic cells released significantly higher amounts of exogenous donor transforming deoxyribonucleic acid than did competent non-lysogenic cells or competent lysogenic cells incubated with erythromycin. These data establish that the induction of the prophage from the competent lysogenic cells was responsible for the selective elmination of the lysogenic transformants. A model is presented that accounts for the induction of the prophage from competent lysogenic bacteria via the induction of a repair system. It is postulated that a repair system is induced or derepressed by the accumulation of gaps in the chromosomes of competent bacteria. This hypothetical enzyme(s) is ultimately responsible for the induction of the prophage and the selective elimination of transformants.     

Mechanism of Transfection with Deoxyribonucleic Acid from the Temperate Bacillus Bacteriophage φ105

Bacteriophage phi105 is a temperate phage for the transformable Bacillus subtilis 168. The infectivity of deoxyribonucleic acid (DNA) extracted from mature phi105 phage particles, from bacteria lysogenic for phi105 (prophage DNA), and from induced lysogenic bacteria (vegetative DNA) was examined in the B. subtilis transformation system. About one infectious center was formed per 10(8) mature DNA molecules added to competent cells, but single markers could be rescued from mature DNA by a superinfecting phage at a 10(3)- to 10(4)-fold higher frequency. Single markers in mature DNA were inactivated at an exponential rate after uptake by a competent cell. Prophage and vegetative DNA gave about one infectious center per 10(3) molecules added to competent cells. Infectious prophage DNA entered competent cells as a single molecule; it gave a majority of lytic responses. Single markers in sheared prophage DNA were inactivated at the same rate as markers in mature DNA. Prophage DNA was dependent on the bacterial rec-1 function for its infectivity, whereas vegetative DNA was not. The mechanism of transfection of B. subtilis with viral DNA is discussed, and a model for transfection with phi105 DNA is proposed.     

Transformation and Transfection in Lysogenic Strains of Bacillus subtilis 168

Strains of Bacillus subtilis lysogenic for either temperate bacteriophage phi105 or SPO2 were reduced to less than 1.0% of the level of transformation of the nonlysogenic strains. Strains lysogenic for both phi105 and SPO2 are virtually nontransformable, indicating that the effect of lysogeny is additive. Lysogenic cultures transfected at essentially wild-type levels with deoxyribonucleic acid (DNA) isolated from bacteriophages phi29 and SPO1. The residual transformation and transfection achieved by the lysogenic cultures changed dramatically during growth in SPII medium, whereas nonlysogenic strains remained competent for 5 hr in SPII medium. Despite a marked reduction in transformation, lysogenic cultures initially irreversibly bound as much DNA as nonlysogenic cultures. After 60 min in SPII medium, there was a rapid decrease in the capacity of lysogenic cells to bind DNA irreversibly. These results, as discussed, indicate that the inhibition of transformation is probably due to an alteration of the cell surface or a differential inactivation of bacterial genes after lysogenic conversion.     

Fate of transforming bacteriophage HP1 deoxyribonucleic acid in Haemophilus influenzae lysogens.

The biological fate of temperate phage HP1 deoxyribonucleic acid (DNA) was followed after uptake by defectively lysogenic competent Haemophilus influenzae cultures. The similar inactivation kinetics of three single phage genetic markers and of their triple combination indicated a complete rather than partial destruction of about half of the adsorbed DNA molecules. Intracellular DNA breakdown products were tentatively identified by hydroxyapatite column chromatography as short single strands and extensively damaged short double strands. Integrated donor DNA (after single-strand insertion?) was still highly efficient for triple-marker co-transformation. This suggests that whole or nearly whole donor DNA molecules were integrated. Some donor DNA was never integrated but remained largely unaltered. This DNA fraction did not contain significant amounts of recipient prophage marker activity. It is concluded that it had not participated in some kind of reciprocal recombination event involving the recipient chromosome. Since very similar phage DNA marker inactivation rates were observed after adsorption by competent nonlysogenic recipients (transfection), the relationship between biological inactivation of adsorbed donor phage DNA and its integration in lysogenic recipients is not clear.     

Effect of lysogeny on transfection and transfection enhancement in Bacillus subtilis.

Strains of Bacillus subtilis 168 lysogenic for bacteriophage phi105 transfer with deoxyribonucleic acid (DNA) isolated from bacteriophage SPO2 at a higher efficiency than non-lysogenic strains. This enhancement of transfection was not the result of recombination between bacteriophages SPO2 and phi105. Superinfection marker rescue increased transfection with DNA from bacteriophage phi105 occurred simultaneously with the addition of the transfecting DNA. Again, this enhancement of transfection was not the result of recombination but rather a protection of the transfecting DNA by the superinfecting bacteriophage. The ability of the superinfecting bacteriophage to protect the transfecting DNA from inactivation was maximal when the bacteria were just becoming competent. Bacteriophage phi1 cannot replicate after the transfection of competent bacteria lacking a functional DNA replication system, whereas bacteriophage phi1 was able to replicate after infection of competent bacteria grown under comparable conditions. These observations support the hypothesis that GAPase and an inducible repair system play an important role in the development of competence.     

Relationship Between Lysogeny, Spontaneous Induction, and Transformation Efficiencies in Bacillus subtilis

The low transformation efficiency of Bacillus subtilis 168 lysogenic for phages ?105 or SPO2 is shown to result from the induction of lytic phage replication in competent cells. Lysogenic competent cells have a higher rate of spontaneous prophage induction than noncompetent cells. Mutants of ?105 and SPO2 which form lysogens resistant to spontaneous induction were isolated, and these lysogens exhibited higher transformation levels than those formed by wild-type phage. These results suggest that the physiological state of competence in B. subtilis promotes prophage derepression leading to cell death and the loss of potential transformants.     

Genetic and physical characterization of lysogeny by bacteriophage MX8 in Myxococcus xanthus

Myxophage MX8 can initiate a lysogenic cycle in Myxococcus xanthus. The lysogenic phage was gentically stable in vegetative cells and persisted in the latent state through many cell generations in the absence of extracellular phage reinfection. The latent state also was stable during the host developmental cycle, since myxospores transmitted latent MX8 genetic information to future progeny cells. DNA hybridization experiments to probe the structure of the lysogenic phage provided physical evidence that MX8 formed a prophage. During lysogenization, MX8 DNA was cut at a specific site (attP) on phage DNA, and we have concluded that genetic recombination between attP and a bacterial DNA site (attB) leads to integration of MX8 DNA and formation of stable MX8 prophage. The genetic and physical properties of MX8 that we describe should make MX8 useful in the analysis of development of M. xanthus by genetic methods.     

Chaperone-assisted excisive recombination, a solitary role for DnaJ (Hsp40) chaperone in lysogeny escape

Temperate bacteriophage lytic development is intrinsically related to the stress response in particular at the DNA replication and virion maturation steps. Alternatively, temperate phages become lysogenic and integrate their genome into the host chromosome. Under stressful conditions, the prophage resumes a lytic development program, and the phage DNA is excised before being replicated. The KplE1 defective prophage of Escherichia coli K12 constitutes a model system because it is fully competent for integrative as well as excisive recombination and presents an atypical recombination module, which is conserved in various phage genomes. In this work, we identified the host-encoded stress-responsive molecular chaperone DnaJ (Hsp40) as an active participant in KplE1 prophage excision. We first show that the recombination directionality factor TorI of KplE1 specifically interacts with DnaJ. In addition, we found that DnaJ dramatically enhances both TorI binding to its DNA target and excisive recombination in vitro. Remarkably, such stimulatory effect by DnaJ was performed independently of its DnaK chaperone partner and did not require a functional DnaJ J-domain. Taken together, our results underline a novel and unsuspected functional interaction between the generic host stress-regulated chaperone and temperate bacteriophage lysogenic development.     

Induction of Prophage SPO2 in Bacillus subtilis: Prophage Excision in the Absence of Bacterial or Bacteriophage DNA Synthesis

Bacillus subtilis lysogenic for SPO2 wild type was induced under conditions preventing synthesis of both bacterial and phage DNA. The infectivity of phage DNA in transfection is strongly decreased under these conditions, whereas the activity of single phage genes as measured by marker rescue with superinfecting phage is unaffected. DNA from induced cells was sedimented in neutral sucrose gradients. After induction, phage DNA was detected at a position in the gradients, which was different from the bulk of the bacterial DNA, corresponding to linear double-stranded DNA of about 25 x 10(6) daltons. Similar results were obtained with bacteria lysogenic for a SPO2 prophage carrying a DNA-negative mutation. No separation of phage and bacterial DNA activity was detected when chloramphenicol was present during the induction period. These experiments show that prophage SPO2 can excise from the bacterial chromosome without previous replication.     

Anti-SOS effects induced in Bacillus subtilis by a ϕ105 mutant prophage

The presence of the mutant prophage 105cts23 in Bacillus subtilis strains strongly affected several biological parameters including the viability of protoplasts and the establishment of plasmid pC194. A defective inducibility of the prophage after treatments that de-repress the SOS-like response were also observed. Although these alterations suggested a Rec-deficient phenotype, homologous recombination was not impaired in these lysogenic derivatives. In fact, chromosomal DNA transformation in these competent cells was more efficient than in cells carrying the wild type prophage: cell death due to prophage induction upon competence development was lower than expected. Alterations in the response to SOS-inducing agents and to osmotic stress correlated with the presence of this particular mutant prophage or the cloned thermosensitive repressor at the permissive temperature. The induction of an anti-SOS effect is discussed.     

Induction of prophage SPO2 in Bacillus subtilis: isolation of excised prophage DNA as a covently closed circle.

Bacillus subtilis tryC2, thyA, thyB, lysogenic for the phage DNA polymerase negative mutant SPO2 susL244, was induced under conditions preventing phage and bacterial DNA synthesis. The biological activity of DNA from induced cells and from uninduced controls was assayed by transformation and transfection, respectively. About 50% of the phage DNA biological activity in DNA extracted from induced cells was resistant to exposure to pH 11.8 TO 11.9. This DNA was operationally defined as alkali-resistant phage DNA. Transforming bacterial DNA from uninduced or induced cells and transfecting DNA from uninduced cells were more than 95% inactivated after exposure to high pH. The alkali-resistant phage DNA was characterized by sucrose gradient centrifugation, by centrifugation in cesium chloride-propidium iodide, and by electron microscopy. It was found to consist of a majority of covalently closed circular DNA molecules. Length measurements of a few relaxed circular molecules indicate a molecular weight of these similar to that previously found for mature SPO2DNA. Attempts to isolate similar covalently closed circular phage DNA from induced bacteria lysogenic for SPO2 phage with a functional DNA polymerase gene were unsuccessful. The gene order in mature and prophage SPO2 was determined by rescue of single and double markers from the respective type of DNA. The data obtained show that prophage DNA is (genetically) permuted relative to mature DNA. The phage attachment site is suggested to be located between genes I and J.     

W-mutagenesis in competent cells of Bacillus subtilis

Summary The relative yield (N _m/N) of fluorescent mutants Ind^- after the transformation of Bacillus subtilis cells by means of UV-irradiated DNA is much higher in an uvr ^- recipient than in an uvr ⁺ strain, when compared at equal fluence, but practically identical at equal survival. Ind^- mutations are induced by UV-irradiation of separated single strands of transforming DNA. The H-strand is much more sensitive to the mutagenic action of UV light. Preliminary irradiation of competent recipient cells by moderate UV fluences increases the survival of UV-or -irradiated transforming DNA (W-reactivation) and the frequency of Ind^- mutations (W-mutagenesis). During transfection of B. subtilis cells by UV-irradiated prophage DNA isolated from lysogenic cells B. subtilis (Ø105 c ⁺) c-mutants of the phage are obtained in high yield only in conditions of W-mutagenesis, i.e. in UV-irradiated recipient cells. These data show that there is no substantial spontaneous induction of error-prone SOS-repair system in the competent cells of B. subtilis.     

Relationship Between Competence for Transfection and for Transformation

Deoxyribonucleic acid (DNA) extracted from phage SPP1 is highly infectious on Bacillus subtilis competent cells; the efficiency of infection is 5 x 10(3) to 6 x 10(3) phage equivalents per plaque-forming unit. This DNA was used to study the relationship between competence for transfection and for transformation. The experiments were concerned with the frequency of infection and transformation in mutants exhibiting different levels of competence, the effect of periodate on competence for infection and for transformation, the competition between phage and bacterial DNA, the transformation of cells preinfected with phage DNA, and the infection of cells pretreated with bacterial DNA. The data show that B. subtilis cells competent for transformation are also competent for transfection and vice versa; transfection with phage DNA represents, therefore, a simple way to measure the total number of competent cells in a culture. The fraction of competent cells, determined by SPP1 DNA infection, varied from 10(-2) to 7 x 10(-2).     

Number of Deoxyribonucleic Acid Uptake Sites in Competent Cells of Bacillus subtilis

Two direct methods are presented for estimating the average number of deoxyribonucleic acid (DNA) uptake sites in competent cells of Bacillus subtilis from measurement of (14)C- or (3)H-thymine-labeled DNA uptake by competent culture. Advantage is taken of two facts: (i) effective contact between competent cells and transforming DNA molecules is established within a short time after mixing them together, and (ii) DNA molecules enter the competent B. subtilis cells in a linear fashion at a finite speed. From the number of DNA molecules initially attached to competent cells by brief exposure to transforming DNA in the first method or from the rate of DNA uptake by competent culture in the second method, the average number of DNA uptake sites is calculated to be 20 to 53 per competent cell.     

The linear double-stranded DNA of phage Bam35 enters lysogenic host cells, but the late phage functions are suppressed

Bam35, a temperate double-stranded DNA bacteriophage with a 15-kb linear genome, infects gram-positive Bacillus thuringiensis cells. Bam35 morphology and genome organization resemble those of PRD1, a lytic phage infecting gram-negative bacteria. Bam35 and PRD1 have an outer protein coat surrounding a membrane that encloses the viral DNA. We used electrochemical methods to investigate physiological changes of the lysogenic and nonlysogenic hosts during Bam35 DNA entry and host cell lysis. During viral DNA entry, there was an early temporal decrease of membrane voltage associated with K+ efflux that took place when either lysogenic or nonlysogenic hosts were infected. Approximately 40 min postinfection, a second strong K+ efflux was registered that was proposed to be associated with the insertion of holin molecules into the plasma membrane. This phenomenon occurred only when nonlysogenic cells were infected. Lysogenic hosts rarely were observed entering the lytic cycle as demonstrated by thin-section electron microscopy.     

Binding of Deoxyribonucleic Acid by Cell Walls of Transformable and Nontransformable Streptococci

Cell walls isolated from competent streptococci (group H strain Challis) were shown to bind more homologous and heterologous deoxyribonucleic acid (DNA) than noncompetent walls. Heat- and alkali-denatured DNA was not bound by either wall preparation. Pretreatment of cell walls with cetyltrimethylammonium bromide sharply increased the binding of DNA but did not increase transformation of whole cells. Pretreatment of the walls with either sodium dodecylsulfate, deoxyribonuclease and ribonuclease, or with crude competence-provoking factor did not affect the binding of DNA. Antiserum prepared against whole competent cells completely blocked transformation and also inhibited DNA binding to competent cell walls. Adsorption of this antiserum with competent Challis cells removed its blocking action for both binding and transformation. Pretreatment of walls with trypsin and Pronase destroyed their ability to bind DNA. Trypsin treatment also blocked transformation in whole cells. The transforming activity of DNA bound to cell walls was found to be protected from deoxyribonuclease action. Significant differences were observed in the arginine, proline, and phenylalanine content of competent and noncompetent walls. With few exceptions, the amino acids released from competent cell walls by trypsin were several-fold greater than from noncompetent walls. The results indicate that (i) two binding sites exist, one in competent cells only and essential for subsequent transformation, and a second, present in all cells, which is not involved in transformation; (ii) both sites are protein in nature; (iii) the transformation site is blocked by antibody; and (iv) the competent cell wall possesses tryptic-sensitive protein not present in the noncompetent wall.     

Lysogeny in Leuconostoc oenos.

Thirty strains of Leuconostoc oenos were exposed to mitomycin C to induce lysogenic bacteriophages. Lysis curves typical for lysogenic strains were obtained with 19 strains. Indicator strans were found for 17 of these phages. Five were characterized by electron microscopy, lytic spectrum, molecular masses of the proteins, sequencing of five N-terminal amino acids of the two major proteins and DNA analysis (restriction patterns, cross hybridization). The results revealed a very close relationship between the phages. Hybridization experiments between the DNAs of the temperate phages and the appropriate lysogenic strains revealed phage-related sequences in the DNA of the lysogenic strain.     

Heat sensitivity of Azotobacter vinelandii genetic transformation

Heating competent Azotobacter vinelandii at 37 or 42 degrees C resulted in a total loss of competence with no loss of viability. The transformation process was relatively insensitive to heating at either temperature once DNase-resistant DNA binding was nearly complete. Although competent and 42 degrees C-treated cells bound equivalent amounts of [32P]DNA in a DNase-resistant state, no donor DNA marker (nif) or radioactivity was detected in the envelope-free cell lysate of heated cells, suggesting that DNA transport across the cell envelope was a heat-sensitive event. Competence was reacquired in a 42 degrees C-treated culture after 2 h of incubation at 30 degrees C by a process which required RNA and protein syntheses. The release of a surface glycoprotein, required for competence, from cells treated at 42 degrees C occurred in an insufficient amount to account for the total loss of competence. Recovery of competence in 42 degrees C-treated cells and further transformation of competent cells were prevented by the exposure of cells to saturating amounts of transforming DNA. Further DNase-resistant DNA binding, however, still occurred, suggesting that there were two types of receptors for DNase-resistant DNA binding to competent A. vinelandii. DNase-resistant DNA binding was dependent on magnesium ions, and at least one receptor type did not discriminate against heterologous DNA.     

EXTENSIVE VARIATION IN NATURAL COMPETENCE IN HAEMOPHILUS INFLUENZAE

The ability of some bacteria to take up and recombine DNA from the environment is an important evolutionary problem because its function is controversial; although populations may benefit in the long-term from the introduction of new alleles, cells also reap immediate benefits from the contribution of DNA to metabolism. To clarify how selection has acted, we have characterized competence in natural isolates of H. influenzae by measuring DNA uptake and transformation. Most of the 34 strains we tested became competent, but the amounts of DNA they took up and recombined varied more than 1000-fold. Differences in recombination were not due to sequence divergence and were only partly explained by differences in the amounts of DNA taken up. One strain was highly competent during log phase growth, unlike the reference strain Rd, but several strains did not develop competence under any of the tested conditions. Analysis of competence genes identified genetic defects in two poorly transformable strains. These results show that strains can differ considerably in the amount of DNA they take up and recombine, indicating that the benefit associated with competence is likely to vary in space and/or time.     

Effects of Mitomycin C and Thymidine Deprivation on Lysogenic and Sensitive Strains of Bacillus megaterium

A triple auxotroph of Bacillus megaterium strain KM was lysogenized with a phage suspension from B. megaterium 899a. The lysogenic and phage-sensitive derivatives of KM were found to die at the same exponential rate during thymineless incubation, despite the fact that the lysogenic strain became induced. The lysogenic strain was also induced by mitomycin C, and died at an exponential rate which was approximately twice that of the sensitive strain. With both strains, the lethality of mitomycin C was the same in the presence and absence of thymidine; thymidine was required for maximal phage production. Mitomycin C preferentially inhibited deoxyribonucleic acid (DNA) synthesis of both strains for the first 60 min. The (DNA) synthetic ability of the lysogenic strain was subsequently restored, due to phage production. Since there was no evidence that sensitive strains of KM contained other inducible elements (prophage or probacteriocins), it is concluded that both thymineless death and mitomycin C death can occur via mechanisms not involving induction.