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PubMed1969

Mechanism of transfection with deoxyribonucleic acid from the temperate Bacillus bacteriophage phi-105.

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.

Bacillus subtilis↗
PubMed2001

[Isolation and functional-structural characteristics of Bacillus polymyxa RP4::Mucts62 transcipients].

Conjugative-like transfer of hybrid plasmid RP4::Mucts62 from Escherichia coli to plasmid-free Bacillus polymyxa was carried out. Bacillus transcipients are detected by the markers of kanamycin and tetracyclin resistance RP4 and thermal sensitivity to growth at 40-42 degrees C, determined by prophage Mucts62 in the plasmid. The technique of transception using millipore filters on solid media has been improved. For comparison with experimental samples, restriction mapping of the native plasmid in donor E. coli GA570 strain was carried out with identification of the prophage location in point 30.5 n. p. of RP4 map with counter-clockwise orientation of the right terminal towards IS21 element. Two phenotypes of bacillus transcipients selected for restriction mapping were singled out. Phenotype KmRTcRTS retains all markers of donor strain plasmid, and by the sum of restricts electrophoretically either corresponds to intact hybrid plasmid or contains deletions in RP4 sites (1-8 n. p.) adjacent to the left arm of prophage or rarely on the right arm (up to 3 n. p.). KmRTcRTS transcipients lose kanamycin resistance and on restriction map show greater prolongation of deletions from the prophage right terminal to kan RP4 gene, shortening it to 13 n. p. without involving the prophage proper. Its left terminal is retained, but 2-7 n. p. sites are deleted in the RP4 area adjacent to it. The possibility of transception in nature and horizontal routes of drug resistance dissemination among genetically remote bacteria are discussed.

Bacillus↗
PubMed1981

[Effectiveness of plasmid RP4 mobilization of the bacterial chromosome in Escherichia coli strains lysogenic for phages Mu and lambda].

The effect of phage lambda on mobilization of Escherichia coli chromosome mediated by the Mu phage and RP4 plasmid has been studied. The efficiency of bacterial chromosome mobilization is an order of magnitude lower than that in the control strain, monolysogenic for phage Mu provided a termoinducible prophage lambda is located separately from Mu. This efficiency is an order of magnitude higher in comparison with the control strain in case prophage is incorporated in the Mu-lambda-Mu structure and does not inhibit Mu phage development. Thus the effect of bacteriophage Mu on mobilization of E. coli chromosome determined by this phage and RP4 plasmid is shown to be dependent on localization of prophage lambda, as well as on functioning of certain prophage genes. A possibility of replication of Mu genomes from the ori site of prophage lambda incorporated into the Mu-lambda-Mu structure is discussed. This replication is presumed to have a stimulating effect on the Mu-induced mobilization of the bacterial chromosome.

Bacteriophage lambda↗
PubMed2004

Identification of bacteriophage types and their carriage in Staphylococcus aureus.

Conserved genomic sequences distinctive of Staphylococcus aureus phage types 3A, 11, 77, 187 and Twort, representative of phage serogroups A, B, F, L and D, were identified and characterized. PCR primers designed for the above sequences were used for development of a multiplex PCR assay which enabled us not only to classify all phages of the International Typing Set plus 16 additional phages, but also to detect prophages in S. aureus genomes. One to four different prophages were unambiguously detected in experimentally lysogenized S. aureus strains, and substantial variation in prophage content was found in 176 S. aureus clinical strains of different provenance. In addition, by using a comparative genomics approach, all the prophages in the S. aureus genomes sequenced to date could be revealed and classified.

DNA Primers↗
PubMed2003

Biology of the temperate Streptococcus thermophilus bacteriophage TP-J34 and physical characterization of the phage genome.

The temperate Streptococcus thermophilus bacteriophage TP-J34 was identified in the lysogenic host strain J34. The majority of phage particles produced upon induction was defective and noninfectious, consisting of DNA-filled heads lacking tails. A physical map (45.6 kb) was established. Analysis of minor restriction bands of the DNA isolated from phage particles as well as the analysis of the protein pattern indicated that phage TP-J34 is a pac-type phage. This was confirmed by immunoelectron microscopy using antisera raised against virulent cos- and pac-type S. thermophilus phages. The lysogenic host J34 but not its noninducible derivate J34-12 contained phage DNA in the nonintegrated state and exhibited autolysis at elevated temperatures. Prophage-carrying strains grew homogeneously while 16 of 20 prophage-cured derivatives aggregated and sedimented rapidly. When phage TP-J34 was propagated lytically on a prophage-cured host strain, a 2.7-kb site-specific deletion occurred in the phage genome. This deletion was also identified in the prophage DNAs of relysogenized strains.

DNA, Viral↗
PubMed2005

Host gene expression changes and DNA amplification during temperate phage induction.

Salmonella enterica serovar Typhimurium LT2 harbors four temperate prophages. The lytic cycle of these phages was induced with hydrogen peroxide or mitomycin C. Microarray analysis was used to monitor the increase in phage genome copy number and the changes in RNA expression. Phage gene transcription was classified temporally, and host genes that responded to hydrogen peroxide, mitomycin C, or phage induction were also identified. A region of the serovar Typhimurium LT2 host genome encompassing hundreds of genes, flanking the Fels-1 lambdoid prophage, was amplified manyfold during lytic induction, presumably due to Fels-1 runoff replication prior to excision, a phenomenon termed escape replication. An excisionase (xis) mutant of Fels-1 also induced escape replication but did not get packaged. Gifsy-1, a lambdoid prophage that does not normally produce escape replication, did so after deletion of either its integrase or excisionase genes. Escape replication is probably widespread; large regions of host genome amplification were also observed after phage induction in serovar Typhimurium strains SL1344 and 14028s at the suspected integration site of prophage genomes.

DNA Nucleotidyltransferases↗
PubMed2001

The protelomerase of the phage-plasmid N15 is responsible for its maintenance in linear form.

The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. Upon infection of an Escherichia coli cell, the phage DNA circularises via cohesive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). We demonstrate that this enzyme acts in vivo on specific substrates, and show that it is necessary for replication of the linear prophage. We show that protelomerase is an end-resolving enzyme responsible for processing of replicative intermediates. Removal of protelomerase activity resulted in accumulation of replicative intermediates that were found to be circular head-to-head dimers. N15 protelomerase and its target site constitute a functional unit acting on other replicons independently of other phage genes; a mini-F or mini-P1 plasmid carrying this unit replicates as a linear plasmid with covalently closed ends. Our results suggest the following model of N15 prophage DNA replication. Replication is initiated at an internal ori site located close to the left end of plasmid DNA and proceeds bidirectionally. After replication of the left telomere, protelomerase cuts this sequence and forms two hairpin loops telL. After duplication of the right telomere (telR) the same enzyme resolves this sequence producing two linear plasmids. Alternatively, full replication of the linear prophage to form a circular head-to-head dimer may precede protelomerase-mediated formation of hairpin ends.

Base Sequence↗
PubMed1998

DNA requirements in vivo for phage T4 packaging.

Phage T4 terminase, comprising the products of genes 16 and 17, packages headfuls of DNA from a concatemer but its mechanism of DNA recognition remains to be determined. Phage T4 terminase gene sequences were introduced into prophage lambda imm434 and plasmids in order to assess their effect on packaging as measured by transduction frequency and DNA content of T4-transducing particles. Multiple copy prophage lambda imm434 genes were transduced at 100-fold higher frequency, and high copy plasmids were transduced at 1000-fold higher frequency than single copy prophage or chromosomal genes T4 16 gene inserts enhanced both prophage and plasmid packaging; terminase gene-containing plasmid DNA in T4 transducing particles could exceed 10% of the total. Deletion or base change of the 24-bp gene 16 3' region which is required for sequence specific amplification of terminase gene 17 (Hp 17 mutations) depressed these elevated plasmid transduction frequencies, suggesting that this is a preferred T4 pac sequence. Moreover, a specific gene 16-containing pac fragment could be detected in mature, packaged phage T4 DNA following restriction endonuclease digestion. We conclude that both the copy number of homologous sequences and the DNA pac sequence(s) themselves are important for packaging, consistent with a synapsis model for regulation of terminase cutting and packaging in phage T4.

Bacteriophage T4↗
PubMed1979

W reactivation is inefficient in repair of the bacterial chromosome.

UV-inducible "SOS" processes associated with W reactivation of phage lambda were studied for their effect on repair of lambda prophage integrated in the bacterial chromosome. For this purpose, lambda c1857 ind red-lysogens were used. These lysogens, although non-inducible by UV light, can be induced by raising the temperature from 30 degrees to 42 degrees. If the W reactivation processes are involved in repair of the bacterial DNA, when the lysogens are incubated at 30 degrees after UV exposure W reactivation should be fully expressed and should also exert an effect on the bacterial chromosome and the prophage inside it. When heat-induction is delayed until the time at which W reactivation reaches its maximum, a considerable increase in phage survival might then be expected. The results presented in this report show, however, that the delayed induction had only a small effect on the survival of prophage in the wild-type strain (possibly attributable to excision repair) and no detectable effect on prophage in a uvrA strain. From these results we conclude that W reactivation is largely irrelevant to the repair of UV-damaged bacterial DNA.

Bacteriophage lambda↗
PubMed1980

Restriction and modification in B. subtilis: the role of homology between donor and recipient DNA in transformation and transfection.

Non-modified DNAs from phages SPO2 and phi 105, and prophage DNAs extracted from lysogens carrying these phages, were used to transfect isogenic r+m+ B. subtilis recipients which were either non-lysogenic, or had been lysogenized with a homologous or a non-homologous phage. Restriction of transfecting phage and prophage DNA occurred in non-lysogenic recipients and in recipients lysogenic for a non-homologous phage. No effect of restriction was observed when phage or prophage DNA was used to transfect recipients carrying a homologous prophage. This is analogous to the absence of restriction in transformation and indicates that in B. subtilis the distinction between transforming and transforming and transfecting DNA is not made at the initial stages of DNA uptake and processing, but rather at later stages, where recognition of homologous regions in donor and recipient DNA plays an important role.

Bacillus subtilis↗
PubMed1981

Rac-E. coli K12 strains carry a preferential attachment site for lambda rev.

Lambda rev is a hybrid lambdoid phage formed by recombination between lambda and a defective lambdoid prophage (Rac) present in most E. coli K12 derivatives. We show here that three independently derived Rac-E. coli K12 strains are specifically deleted for the entire Rac prophage consistent with loss of Rac by excisive recombination between hybrid attachment sites that flank the prophage (c.f. excision of a lambda prophage). lambda rev, in which int and PP' of lambda have been replaced by integrative recombination genes and an attachment site derived from Rac (Gottesman et al. 1974), integrates site-specifically and in the correct orientation at the preferential attachment site generated by Rac excision.

Attachment Sites, Microbiological↗
PubMed1992

RexAB proteins of bacteriophage lambda enhance the effect of photolyase-dimer complexes on lacZ gene expression in Escherichia coli.

Expression of the lacZ gene in Escherichia coli is inactivated by exposure to ultraviolet light (UV). Inactivation is exceptionally effective when cells contain amplified levels of DNA photolyase (which forms complexes with pyrimidine dimers in the absence of light for actual photoreversal) and a lambda prophage. Without amplified photolyase, the lambda prophage or both, inactivation rates are similar and much lower. UV-inactivation of lacZ gene expression in the presence of both amplified photolyase and lambda is even more effective if lambda cI857 is used in place of the wildtype prophage but is wholly unexceptional if the prophage carries defects in the lambda genes rexA or rexB. When Rex AB proteins are provided by expression from a plasmid and the cell also contains amplified photolyase, exceptional inactivation rates again obtain; in fact inactivation is most effective under these conditions. The data are considered to reveal a role for Rex AB proteins, which mediate superinfection exclusion, in the exceptional inactivation of gene expression by photolyase bound to pyrimidine dimers in DNA. Photolyase-dimer complexes may mimic the structure of certain complexes that arise during phage development and thus influence Rex A and/or B proteins, thereby shutting down cell metabolism.

Bacteriophage lambda↗
PubMed1983

A genetic study of Escherichia coli strains carrying Mu-lambda-Mu structures.

We have studied the interaction of bacteriophages Mu and lambda after their simultaneous induction and the influence of lambda on Mu-dependent mobilization of the E. coli chromosome by the RP4 plasmid. Heterolysogenic E. coli strains carrying Mu-lambda-Mu structures were constructed (Faelen et al. 1975). The Mu and lambda prophages are linked in such structures, and the functions of some lambda genes are disturbed depending on the integration site. A study of the inhibition of Mu growth by lambda after their simultaneous induction was performed and the region of the lambda genome (R-H) which contains the gene(s) responsible for the inhibitory effect of lambda on Mu was identified. The efficiency of Mu-dependent mobilization of the bacterial chromosome by RP4 is shown to be an order of magnitude lower in strains with unlinked Mu and lambda and an order of magnitude higher in strains with some permutations of the lambda prophage than in the control Mu-monolysogenic E. coli strain. Thus the effect of Mu on mobilization depends on the localization of the lambda prophage and on the functioning of its genome within a Mu-lambda-Mu structure. It is presumed that the mobilization of the bacterial chromosome is stimulated by effective replication of the Mu genome starting from the ori site (origin of replication) of the lambda prophage within the Mu-lambda-Mu structure. We propose a model to explain the interaction of Mu and lambda in E. coli strains carrying Mu-lambda-Mu structures.

Bacteriophage lambda↗
PubMed1983

Absence of functional RNA encoded by a silent chromosome in non-complementing diploids obtained from protoplast fusion in Bacillus subtilis.

The molecular basis for lack of phenotypic expression of one chromosome in Bacillus subtilis non-complementing diploid clones (Ncd cells) was investigated. Correlations between chromosomal inactivation and absence of functional transcripts were determined with wild-type prophage phi 105 or a thermoinducible mutant phi 105 cts23, on either the expressed or the silent chromosome. It appears that no significant amount of phage mRNA is detectable in Ncd cells carrying the prophage in the inactive chromosome. However, phi 105 mRNA represents 0.23% of total cellular mRNA in an Ncd strain with the prophage in the expressed chromosome and 0.28% in the parental lysogenic strain. The lack of an mRNA repressor of phi 105 prophage from the silent chromosome was confirmed by the absence of repressor activity in Ncd clones with a temperature sensitive mutant phi 105 located in the silent chromosome. After heat induction, no phage production was observed. As expected these clones do not exhibit phi 105 immunity when superinfected with the same phage. The combined data of the present and previous work suggest that control of phenotypic suppression of Ncd strains should, at the transcription level, involve a different DNA tertiary organisation in one of the two chromosomes.

Bacillus subtilis↗
PubMed1979

Location and characterisation of a new replication origin in the E. coli K12 chromosome.

A segment of DNA located in the region of the E. coli K12 chromosome previously identified by the Rac phenotype can function as a self-replicating plasmid. Evidence is presented that this plasmid, the oriJ plasmid, contains the origin of replication of a defective prophage postulated to be located in this chromosomal region by Low (1973). The plasmid can only be maintained in strains in which this postulated prophage has been deleted. In strains which possess the prophage selection for plasmid maintenance permits the isolation of clones containing new deletions which we postulate are the result of prophage excision.

Chromosome Mapping↗
PubMed1985

Processive action of terminase during sequential packaging of bacteriophage lambda chromosomes.

Bacteriophage lambda chromosomes are packaged in a polarized, sequential fashion from a multimeric DNA substrate. Mature chromosomes are generated when terminase introduces staggered nicks in the cohesive end sites (cos sites) bounding a chromosome. Packaging is polarized, to the initial and terminal cos sites for packaging a chromosome can be defined. To initiate packaging, terminase binds to cos at cosB, and subsequently cuts at cosN. To terminate packaging of a chromosome, a functional cosB is not required at the terminal cos. To explain this finding, it was proposed earlier that terminase scans for the terminal cosN, rather than any subsequent cosB, during packaging. In the work described here we performed helper packaging experiments to see whether processive action of terminase occurs during sequential packaging of lambda chromosomes. The helper packaging experiments involve trilysogens; strains carrying three prophages in tandem. Infection by a hetero-immune helper phage results in packaging of the repressed prophage chromosomes, since the prophage structure is analogous to the normal DNA substrate. Two chromosomes can be packaged from between the three cos sites of the prophages of a trilysogen. Both chromosomes are packaged even when the central cos is cosB-. Our interpretation of these data is that terminase is brought to the central cos by packaging; following cleavage of the central cos, the terminase remains bound to the distal chromosome; and terminase acts to begin packaging of the distal chromosome. The frequency at which terminase reads across the central cos to initiate packaging of the distal chromosome is in the range from 0.3 to 0.5 in our experiments. Reading across cos was found not to be greatly dependent on the state of cosB, indicating that cosB binding is only needed for packaging the first chromosome in a packaging series. A multilysogen was constructed in which the initial cos was cos+ and the distal cos sites were all cosB-. The initial and downstream chromosomes were found to be packaged. This result indicates that terminase that is brought to the central cos by packaging is not only able to initiate packaging of a downstream chromosome, but can also scan and terminate packaging of the downstream chromosome. A model is presented in which processive action of terminase is the basis for sequential packaging of lambda chromosomes.

Bacteriophage lambda↗
PubMed1983

Induction of SOS functions in Escherichia coli by lesions resulting from incorporation of 5-bromouracil into DNA.

Lesions induced by 5-bromouracil (BU), after its incorporation into DNA, led to effective induction of prophage lambda and W reactivation (or BU reactivation). Prophage induction due to incorporated BU occurred only with the wild-type prophage, and not for the lambda c1857 mutant with a thermosensitive repressor. Antipain, a protease inhibitor, inhibited wild-type prophage induction 70-90%. This indicates that BU-induced lesions may induce the SOS repair system. The finding that such lesions provoke BU reactivation permits the inference that BU-induced mutagenesis also proceeds via involvement of the error-prone repair system, and not directly as a result of base-pairing errors. Genetic evidence suggests that induction of the SOS repair system as a result of incorporation of BU into DNA is linked to the subsequent appearance of uracil residues and apyrimidinic sites, resulting from dehalogenation of incorporated BU. Apyrimidinic sites appear to be more effective than uracil residues in induction of the SOS system.

Bacteriophage lambda↗
PubMed1990

Stimulation of mutations suppressing the loss of replication control by small alcohols.

Transient exposure of lysogenic Escherichia coli cells to small alcohols stimulated the frequency of mutations suppressing the lethal loss of replication control from a prophage fragment of bacteriophage lambda. The stimulation in mutation frequency paralleled the effect of mutagenic agents, and in this sense the alcohols behaved as mutagens. 10-min treatments above distinct threshold concentrations at 23%, 18%, 10% and 4% (v/v) were required in order for methanol, ethanol, isopropanol and propanol to evoke mutagenic effects. The selected mutant cells were, in general, equally or more sensitive to ethanol than the starting cells. The mutagenicity of methanol and ethanol was detected only with E. coli strains with lambda fragments that included the site-specific and general recombination genes found within the phage int-kil gene interval; whereas, stimulation of the frequency of phenotypically identical mutations by nitrosoguanidine or ionizing radiation did not require that the lambda fragment encode these genes. Treatments of lysogenic cells with mutagenic concentrations of ethanol did not trigger prophage induction and were concluded not to induce a cellular SOS response nor to denature the prophage repressor, or to disrupt repressor-operator binding. The toxicity of ethanol was pH-dependent. Cellular sensitivity to ethanol toxicity was unaffected by the integrated lambda fragment(s) or by an intact lambda prophage; but, it was increased by deletions of the E. coli chromosome extending rightward from bio into uvrB, and rightward from chlA.

1-Propanol↗