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Extrachromosomal element pSOG3 (52,162 nucleotides) in the genome of Sodalis glossinidius contains redundant phage-related gene pairs, indicating that it may have been formed by the fusion of two ancestral phage genomes followed by gene degradation. We suggest that pSOG3 is a prophage that has undergone genome degeneration accompanying host adaptation to symbiosis.
Induction of a heat-inducible abnormal lambda prophage inserted between lysA and thyA in Escherichia coli resulted in a number of auxotrophic mutants in the surviving cured-cell populations. These mutants could not be accounted for by deletions arising on formation of lambda hybrid particles carrying regions adjacent to the insertion site. The properties of these mutants, which were almost all spontaneously revertable, have been described and mapped by F' episome complementation. Tentatively, it was suggested that induction of the lambda lysogen leads to a mutagenic state.
Single and tandem insertions of prophage lambda into R100 have been isolated. Insertions into the transfer genes, insertions into the transfer control gene finO, and insertions into regions that result in no detectable phenotypic change were found. From the last type, deletion mutants were isolated which established the sequence of antibiotic resistance genes as tet-cml-fus-str-sul-mer in R100. High frequency transducing phage preparations lambdamer, lambdasul str, and lambdasul str cml were also isolated from this type.
The recombination-defective phenotype associated with the recC genetic locus in Bacillus subtilis is not due to a chromosomal mutation at this site but rather to the presence of an integrated SPO2 prophage.
Methicillin resistance (mec) is not transduced into Staphylococcus aureus 8325-4, but is transduced into this host after it has been lysogenized with phage phi11 and has acquired the penicillinase plasmid pI524 by a separate transduction (Cohen and Sweeney, 1970, 1973). Strain 8325-4 is competent for transformation of typical plasmid or chromosomal markers and for mec only if it is lysogenic for phi11 or a related prophage (Sjöström et al., 1974, 1975). A mutant strain of phi11 that was temperature sensitive (Ts) for vegetative multiplication did not mediate competence for transformation of its 8325-4 lysogen if the lysogen had been grown at a nonpermissive temperature (Sjöström and Philipson, 1974). We isolated four Ts mutants of phi11 that did not mediate transducibility of their 8325-4(pI524) lysogens for mec after growth at nonpermissive temperatures (40 to 42 degrees C). Transduction of typical plasmid or chromosomal markers was not affected. These phi11-Ts mutants mediated normal competence of their lysogens for transformation of a tetracycline resistance plasmid. Similarly, phi11-Ts mutants that rendered their lysogens temperature sensitive for transformation did not depress the frequency of transduction of mec. These two types of phi11-Ts mutants fell into two different genetic complementation groups that differed in the physiology of deoxyribonucleic acid synthesis and in the time of expression of the mutations during a single-burst growth cycle at a nonpermissive temperature. A virulent mutant of phi11, which plaqued with 100% efficiency on 8325(phi11), also failed to condition strain 8325-4 for transducibility of mec but retained the ability to confer competence for transformation of a tetracycline resistance plasmid. Different genetic loci and physiological functions are involved in phi11 mutations that affect transducibility of mec and those that affect competence for transformation of markers generally in S. aureus 8325-4.
The attachment site for the prophage of SPbeta lies between ilvA and kauA on the chromosome of Bacillus subtilis strain 168. Specialized transduction of citK and kauA can be carried out by certain lysates of SPbeta.
Hydroxyurea inhibited synthesis of certain deoxyribonucleic acid (DNA) precursors and causes the cessation of DNA synthesis. It did not cause induction of lambda. Superinfection of an irradiated lysogen with lambdaind- could prevent induction, but the percentage of cells protected decreased as the time between irradiation and superinfection increased. The presence of hydroxyurea did not increase the time during which cells could be rescued by superinfection. The accumulation of DNA precursors after ultraviolet or ionizing radiation was not necessary for the induction of lambda prophage to occur.
Loss of the biological activity of deoxyribonucleic acid in gamma-irradiated Escherichia coli cells was studied. The study is based on two sets of experimental data: (i) post-irradiation heat inducibility of the cells whose chromosomes were "labeled" with the thermoinducible lambdacI857ind prophage, and (ii) post-irradiation capacity of nonlysogenic cells to promote growth of the unirradiated lambdacI857ind phage. The results show that, at the beginning of incubation after irradiation, the number of plaques formed upon heat induction of lysogenic cells was much higher than the viable cell count of the nonheated culture. This high resistance of the heat inducibility gradually decreased during post-irradiation incubation. Finally, after a period of 4 h, there was no difference in sensitivity between the heat inducibility and the colony-forming ability of gamma-irradiated cells. The capacity of gamma-irradiated bacteria to support growth of unirradiated lambdacI857ind is radioresistant; this resistance, in contrast to that of heat inducibility, is much less affected during post-irradiation incubation. A continuous decrease in radioresistance of heat inducibility without a corresponding decrease in radioresistance of the capacity suggests that functional failure of initially undamaged and/or repaired parts of the chromosome gradually develops after irradiation. From the fact that after 4 h all colony formers are capable of being induced by heat, whereas no chromosomal activity can be detected in nonviable cells, two conclusions may be drawn: (i) gamma-irradiated E. coli cells destined to die reach their biological end point within 4 h of post-irradiation incubation; (ii) in most cells, functional failure of the whole chromosome is the immediate cause of death.
Transient induction of lambda prophage increases the ultraviolet light resistance of most exponentially growing Escherichia coli lysogens. Resistance is increased in wild-type, recB, recB recC, recB recC recF, and recB recC recL hosts. No enhancement in recA lysogens was found, nor was there enhancement in stationary cultures. Enhancement was dependent upon the lambdared recombination system. Transient induction also increases the genetic recombination rate in recB lysogens as measured in Hfr X F- matings.
Wavelength dependency of photo-inactivation and photoinduced reverse mutation of Escherichia coli sensitized with 8-methoxypsoralen, and wavelength dependency of photoinduction of lambda prophage from the sensitized lysogen were measured in a range of 298 to 400 nm. The most efficient sensitization for these biological effects was observed between 320 and 340 nm. In the presence of 8-methyoxypsoralen, the induced mutation frequency per lethal hit was highest of 298 nm in the range examined and was gradually decreased with increasing wavelength to a minimum frequency at 345 nm. This finding may be a reflection of the production of more than one type of lesions which have different efficiencies for mutation compared with the killing efficiency.
Deletion of bacterial DNA fragments is stimulated in induced Mucts62 lysogens. The host genes located proximally to the prophage are more frequently lost than those which are unlinked to the Mu genome. Genes located on either side of a Mu genome are deleted in the same manner. Like the other Mu-induced rearrangements, this process is recA independent and requires the participation of Mu DNA, as indicated by the fact that a phage genome always replaces the deleted genes. Data are presented which strongly suggest that both ends of the Mu genome are involved in deletion formation.
In previous studies of bacterial protoplast fusion, only the frequencies of cell wall regeneration and of bacterial recombination were determined. In this work the frequency of the heterozygous fusion products is measured by prophage complementation. Two multiply marked nonsuppressing strains of Bacillus subtilis, each lysogenic for a different Sus mutant of the phage phi 105, were induced by mitomycin C, protoplasted, fused, and, after dilution in hypertonic broth, incubated until plating with phi 105-sensitive indicator bacteria. When cell lysis was avoided, the frequency of the heterozygous fused cells could be determined from the number of infectious centers produced. The very high frequencies observed are in good agreement with those determined directly, with nonlysogenic strains, by electron microscopic examination of the fused protoplasts (C. Frehel, A. M. Lheritier, C. Sanchez-Rivas, and P. Schaeffer, J. Bacteriol. 137:1354--1361, 1979). Evidence is presented that fusion occurs in two steps, one polyethylene glycol dependent, the other energy requiring. The bacterial growth medium affects the ability of the protoplasts to fuse and to regenerate a cell wall. When experiments using different growth media were compared, an inverse relationship between these abilities was observed, and a direct relationship appeared between the heterozygotes (corrected for wall regeneration) and the recombinant bacteria that were found.
Escherichia coli strains containing mutations (ssbA1 and lexC113) which affect single-strand deoxyribonucleic acid binding protein have been examined. Among the properties studied were: sensitivity to ultraviolet irradiation and methyl methane sulfonate, temperature sensitivity, induction of prophage lambda by ultraviolet light, temperature, and mitomycin C, and deoxyribonucleic acid synthesis. Strains containing the ssbA1 and lexC113 mutations differ significantly in several of these properties.
To help understand how the tif-1 mutation of the recA gene of Escherichia coli confers adenine activability on the recA protein, we used the fact that cytidine plus guanosine inhibits induction of prophage lambda and cell filamentation in a tif-1 mutant, and that adenine reverses this inhibition. We varied the amount of adenine in agar plates containing a fixed amount of cytidine and scored for survivors of three different tif-dependent lethal induction processes. Much more adenine was required for cell killing when cytidine was present than when it was absent. Therefore adenine does not override cytidine inhibition, but instead appears to compete with it for a site of action which may be on the recA protein. The competition is not at the cell transport level. Our results lead to a model in which the tif form of the recA protein is an allosteric enzyme that binds both negative and positive modulators. By varying the adenine-cytidine ratio of the medium it is possible to control the degree of induction in a tif-1 cell. For the three different tif-dependent inductions studied here, least adenine was required for lambda induction and most for lethal filamentation, presumably reflecting requirements for different amounts of activated recA protein in each process. Varying the adenine-cytidine ratio revealed two stable intermediate stages in lambda induction, as well as a stage of colicin E1 induction in which the cells produced colicin without cell death. The rate of filament formation could be similarly controlled. Experiments with tif (ColE1, lambda) gave evidence of a competition between colicin repressor and lambda repressor for activated recA protein.
A series of mutations of the P1 plasmid prophage that lead to increased copy number was isolated and analyzed. The copy number of the mutants was elevated at least five- to eightfold relative to wild-type P1, as determined by single-cell resistance to antibiotics, activity of enzymes, content of superhelical DNA, and reassociation kinetics. The copy number of two of the mutants was temperature dependent. Based on dominance tests, the mutants fell into two classes, cis specific and recessive. The latter class included a temperature-sensitive copy mutant. The existence of a class of recessive mutants suggests that the replication of the P1 plasmid is negatively regulated.
Fourteen mitomycin-resistant revertants of a recB21 recC22 strain were isolated after Tn5 mutagenesis. Eight of the mutations (type I) were essentially inseparable from aphA+ (Kanr) of Tn5; six (type II) were not. We hypothesize that the former are Tn5 and that the latter are IS50 insertions. Because of their phenotypic similarity to sbcA and sbcB mutations, which also suppress recB21 recC22, we have called them sbc mutations. sbc-lll::Tn5 was cotransducible with nirR and has thereby been located at position 29.8 on the Escherichia coli map in the vicinity of the Rac prophage and sbcA mutations. A recB21 recC22 sbc-lll::Tn5 strain was subjected to Tn10 mutagenesis, and a mitomycin- and UV-sensitive mutant was isolated. tet+ of Tn10 was 85% cotransducible with aphA+ of Tn5, locating these two transposons 0.1 map unit apart. A three-point cross located the Tn10 mutation at position 29.7. We hypothesize that the Tn10 insertion is located in recE and that the Tn5 and IS50 insertions activate expression of this gene. sbc-lll::Tn5 was found to be cis acting and dominant to its wild-type allele as were two sbcA mutations (sbcA1 and sbcA6). Five other type I and type II insertion mutations were dominant to their wild-type alleles. We hypothesize that the sbc insertion and sbcA mutations affect transcription regulation of recE and discuss the possibility that they do so differently.
We constructed a physical map of the 50-kilobase-pair (kb) DNA of the temperate Rhodobacter sphaeroides bacteriophage phi RsG1, with the relative positions of the cleavage sites for the nine restriction endonucleases KpnI, HindIII, XbaI, ClaI, BclI, EcoRV, EcoRI, BglII, and BamHI indicated. Using biotinylated phi RsG1 DNA as a probe in hybridization studies, we detected homologies with virus DNA and fragments of restriction endonuclease-digested host chromosomal DNA but not with plasmid DNA. This indicates that the prophage is integrated into the host chromosome. In addition, the use of specific probes such as the 10.4-kb BglII A fragment and the 2.65-kb BamHI H fragment allowed the determination of the position of phage attachment site (attP).
The Escherichia coli plasmids pBR322 and pBR327 can be taken up by Haemophilus influenzae but do not replicate in this organism; however, integration of pBR into the H. influenzae chromosome was achieved by ligation to a fragment of the Haemophilus phage S2 that carried a phage attachment site (attP). Once these sequences were integrated, they could serve as sites of recombination and amplification for homologous (pBR or phage) DNA. Amplification appeared to occur in one of two prophage sites (attB) present in the H. influenzae chromosome. The extent of amplification was different in different cells and reflected the ability of these sequences to undergo rearrangement leading to the formation of a DNA ladder. The ladder was obtained by treatment of DNA with restriction enzymes that cut outside of the inserted DNA, i.e., did not cut in the repeat sequence, and represented different numbers of repeat elements. Reversed-field gel electrophoresis was instrumental in resolving amplified structures. Inasmuch as single-cell isolates gave rise to the same ladder structure, it was assumed that amplification was under regulatory control and that it reproduced the same equilibrium of repeat structures. Transformation of E. coli with the amplified H. influenzae DNA resulted in precise excision and replication of the original monomeric plasmids. This excision was independent of the recA and recBC genes.