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In addition to the cryptic lambdoid prophage genes that are known to reside at the rac locus in Escherichia coli K12 strains, a second cryptic lambdoid prophage has been located near the gal operon. This prophage was shown to contain DNA that is homologous to the QSR genes of lambda phage.
The induction of prophage lambda by ultraviolet light has been measured in E. coli K12 lysogenic cells deficient in DNA polymerase I. The efficiency of the induction process was greater in polA1 polC(dnaE) double mutants incubated at the temperature that blocks DNA replication than in polA+ polC single mutants. Similarly, the polA1 mutation sensitized tif-promoted lysogenic induction in a polA1 tif strain at 42 degrees. In strains bearing the polA12 mutation, which growth normally at 30 degrees, induction of the prophage occurred after the shift to 42 degrees. It is concluded that dissapearance of the DNA polymerase I activity leads to changes in DNA replication that are able, per se, trigger the prophage induction process.
The demonstrated relationship between carcinogenicity of a chemical compound in mammals and its tendency to cause prophage induction in bacteria provides a method for biologically based carcinogen screening. Because of the need for this type of screening and the abundance of lysogens in the marine environment, 14 isolates were evaluated for the degree of prophage induction in exponentially growing cultures in the presence of a known mutagen (0.5 microg/ml mitomycin C). Assays were performed both in liquid culture and in microtiter plates. Virus-like particles were enumerated by epifluorescence microscopy after staining with SYBR-Gold. Two isolates designated P94-4B3 (identified as Halomonas aquamarina) and P94-4S3 (identified as Pseudomonas aeruginosa) were further evaluated for potential use. Because of the rapid growth, larger size of its virus-like particles, and linear response to increasing dose of mitomycin C, the P. aeruginosa st. P94-4S3 was determined a better candidate for the marine prophage induction assay (MPIA). The Pseudomonas isolate was then used in several experiments for the development and optimization of the MPIA procedure. Initial screenings of the MPIA were also performed with selected environmental pollutants.
11 platinum compounds with nitrogen donor ligands, previously tested for anti-tumour activity, were studied for induction of prophage lambda and for mutagenicity in the Ames assay, with various strains of Salmonella. The compounds included cis and trans isomers of Pt(II) and Pt(IV) complexes and were tested with and without metabolic activation. All the cis compounds elicited prophage induction, whereas the trans compounds were inactive. Mutagenicity was found only in strains containing the R factor, indicating that SOS-type repair processes are required for the conversion of initial DNA lesions into mutations. Mutation induction was also influenced by the excision-repair process. The 2 trans compounds were not, or only slightly, mutagenic; all other compounds were mutagenic in at least one strain, exhibiting a 2-20-fold increase over the spontaneous background level. Addition of liver homogenate had no significant effect on the number of mutants. One compound induced exclusively frameshift mutations. The other mutagenic compounds induced frameshift mutations as well as base-pair substitutions. 7 compounds were more mutagenic for the repair-proficient than for the repair-deficient strains; only one showed the opposite effect. This suggests that for mutagenicity testing of platinum compounds, repair-proficient strains are more sensitive indicators. The differences in response of the various strains are more sensitive indicators. The differences in response of the various strains toward the compounds suggest the formation of different DNA lesions and/or a selective action of repair processes on these lesions. In general, a good qualitative correlation was observed between prophage-inducing capacity, mutagenicity in bacterial and mammalian cells and anti-tumour activity.
Characteristics of lambda c/ts857 prophages that can be attributed to the ability of the temperature-sensitive phage repressor to renature at low temperature are not apparent in host cells that contain a mutation in the htpR gene. Host killing by prophages that are N- or are blocked in DNA synthesis is not prevented by the return of mutant cells to low temperature, and recovery of cells in which the phage remains derepressed is not delayed if the prophage is a mutant that cannot kill. These and other findings suggest that the phage repressor protein is unusually susceptible to inactivation in cells that are unable to respond to heat shock.
A dose-dependent prophage induction by furazolidone exhibited a gradual rise to a maximum, corresponding to an exposure dose of 1.2 microgram/ml X h and a gradual fall thereafter. A 2-3-fold higher level of induction was achieved when the lysogens were treated with furazolidone in the presence of a metabolizing mixture. A maximum of about 70% efficiency of induction was achieved. Kinetics of prophage induction by any concentration of furazolidone exhibited a common pattern, viz., an initial rise for 15-20 min, then a plateau extending up to about 60 min and a faster rise thereafter. Higher concentrations of the drug (10 micrograms/ml) exhibited a toxic effect. Chloramphenicol at a concentration of 20 micrograms/ml inhibited the furazolidone-induced prophage induction, the plaque-forming units gradually decreasing from several minutes after the chloramphenicol treatment. The burst size of the lysogens was not significantly affected by treatment with 2 micrograms/ml of furazolidone up to a period of about 10 min, but thereafter, decreased faster with the duration of furazolidone treatment. The "latent period' of induction decreased linearly with the duration of furazolidone treatment.
The abilities of 4 antimalaria drugs (Daraprim, Fansidar, Nivaquine and Camoquine) on their own and in combination with aflatoxin B1 to induce prophage in tester strains E. coli D21 and D22 were studied. The 4 drugs were found to induce prophage in the tester strains; Daraprim and Fansidar without the need for activation by liver mixed function oxidases (S9 microsomal fraction), while Nivaquine and Camoquine did require activation by this system. Aflatoxin B1 was used as a positive control in all the tests. The combined effects of each of the drugs with aflatoxin B1 were lower than that of the individual drugs acting alone. From these results, it may be concluded that the drugs may, on their own, pose a carcinogenic hazard to the population in the Nigerian environment exposed to them. In addition, the depression of prophage induction observed when the drugs were combined with aflatoxin B1 may be indicative of a common target site of action in the tester strains.
A comparative survey of the mutagenic, prophage-inducing and antibacterial activities of 3 structure-related series of 5-nitro-furan derivatives including 5-nitro-2-furohydrazide imide, 5-nitro-2-furamide oxime and 5-nitro-2-furohydrazide has been undertaken. Among the compounds assayed, the 5-nitro-2-furohydrazide imide series was found to be most active with regard to mutagenic and antibacterial activities against Salmonella typhimurium TA100 and prophage-inducing activity in Escherichia coli GY5027. A clear correlation was observed between the chemical structure and the mutagenic and prophage-inducing activities which were approximately correlated to the antibacterial activity.
Integration of bacteriophage P2 into the Escherichia coli host genome involves recombination between two specific attachment sites, attP and attB, one on the phage and the other on the host genome, respectively. The reaction is controlled by the product of the phage int gene, a basic polypeptide of about 37 kDa [Ljungquist and Bertani, Mol. Gen. Genet. 192 (1983) 87-94]. The int gene appears to be expressed differently by an infecting phage, as opposed to a prophage [Bertani, Proc. Natl. Acad. Sci. USA 65 (1970) 331-336]. A 1200-bp region of P2 DNA containing the int gene and attP, the prophage hybrid ends attL and attR, and one bacterial attachment site, the preferred site locI from E. coli strain C, have all been sequenced. An open reading frame coding for a polypeptide of 337 amino acids corresponds to the int gene. The gene has no obvious promoter sequence preceding it. The int gene transcript seems to continue past the attP site downstream from it, suggesting a possible explanation for the previously observed difference in integration and excision. A comparison of the four attachment sites reveals a common 'core' sequence of 27 bp: 5'-AAAAAATAAGCCCGTGTAAGGGAGATT-3'. The P2 nip1 mutation, which increases prophage excision [Calendar et al., Virology 47 (1972) 68-75], was found to lie within the int gene itself. The P2 saf variant, which has altered site preference [Six, Virology 29 (1966) 106-125], has a bp substitution within the core sequence. Three deletion/substitution mutants, vir22, vir94 and del3, also have altered core sequences.
We describe the isolation and characterization of a new Q-independent substitution mutant of lambda, lambdaqin111, which differs from other characterized Q-independent lambda phages. This mutant defines a new lambda-like prophage in the bacterial chromosome, as seen by homologous recombination between lambdaqin111 and the host DNA and by DNA/DNA hybridization methods. Genetic and electron microscopy data show that this new prophage carries, at least, genes analogous to Q-S-R of lambda and also a cos site functionally identical to lambda cos. It is located near 34 min on the Escherichia coli K12 map, i.e. in the same region but at a different site from the defective Rac prophage.
By application of prophage integration and subsequent intended excision, a method to maintain an introduced DNA sequence stably onto a bacterial chromosome has been proposed. Recently-constructed integration plasmids using Campbell-type prophage integration system in Lactobacillus casei strain Shirota and its temperate phage phi FSW was modified for this purpose and a chloramphenicol (Cm)-resistance gene was used as a model passenger DNA. On the integration plasmid having an erythromycin (Em)-resistance gene as a selection marker, N- and C-terminally-truncated Cm-resistance genes were inserted into both sides of the attP of phi FSW, within which the site-specific recombination took place with the attB of phi FSW on the recipient chromosome through the phi FSW integrase. Primary integrants of the modified plasmid (integration-excision vector) exhibiting Em-resistant and Cm-sensitive phenotype generated Em-sensitive and Cm-resistant derivatives under the nonselective conditions. Sequence analyses showed that one copy of the complete Cm-resistance gene resided at the attachment site on the host chromosome and the other vector-derived sequences were excised probably by endogenous homologous recombination in the host cells to derive final integrants. The Cm-resistant phenotype of the final integrants was stable for more than 50 generations under non-selective conditions. Frequency of the homologous recombination suggests that negative selection is also adoptable. Thus, this method using the integration-excision vector gives a stable and safe derivatives of the strain and is likely to be applicable to various bacteria, since Campbell-type prophage integration system and homologous recombination are prevalent among bacteria.
We present evidence for the existence of two large (approximately 50 kb) excisable segments in the chromosome of Salmonella typhimurium. The two elements--designated Gifsy-1 and Gifsy-2--cover, respectively, the 57 units and the 24 units of the genetic map where they contribute indicative rare restriction sites. The two elements are closely interrelated and both contain a region of sequence similarity to the recE locus of the Rac prophage of Escherichia coli. Mutations within this region of Gifsy-1 yield the classical 'Sbc' phenotype: they suppress the recombination defect of recB mutants, apparently by activating a normally silent recE-like gene. At the same time, these 'sbcE' mutations activate a Xis-type function that promotes excision of one or other of the two elements. Predictably, curing of Gifsy-1 results in the loss of recB mutant suppression. Surprisingly, the suppressor phenotype is also lost in cells cured for Gifsy-2 even though the Gifsy-1-associated sbcE mutation is still present. Moreover, the excision frequency of Gifsy-1 drops dramatically in Gifsy-2-cured cells. Thus, both elements must co-operate in the activation of recombination and excision functions. Overall, the data presented here suggest that Gifsy-1 and Gifsy-2 are cryptic prophages. They are distinct from previously described Fels prophages. Unlike Fels, they are not specific to S. typhimurium strain LT2 since they are both also found in a virulent S. typhimurium isolate (ATCC 14028s).
Coliphage N15 is a temperate bacteriophage whose prophage is a linear plasmid molecule with covalently closed ends (telomeres). The N15 prophage provided the first example of such DNA in prokaryotes and, up to now, it is the only known example of a linear plasmid in Escherichia coli. The linear N15 mature phage DNA has single-stranded cohesive ends. The phage and plasmid prophage DNAs are circularly permuted. The nucleotide structure of the telomere-forming site tel RL in phage DNA corresponds to the structures of the terminal hairpin loops. It suggests a unique mechanism for conversion of the circular phage DNA to the linear plasmid form, which is performed by the prokaryotic telomerase (protelomerase). The results of a comparison of the protelomerase with integrases lead us to suggest that these proteins may have evolved from a common ancestor. The mechanism of plasmid N15 replication is unknown. We propose that the protelomerase participates in linear plasmid replication, acting as a resolvase of replicative intermediates that are tail-to-tail linear dimers. The sequence analysis of the N15 DNA showed that it represents an evolutionary 'link' between plasmids F, P1, P4 and lambdoid bacteriophages.
Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy-2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEPhi. Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.
N15 is a bacteriophage of Escherichia coli that resembles lambda, but, unlike lambda, it lysogenizes as a linear plasmid. We show that stable maintenance of this unusual plasmid-prophage depends on the parA and parB genes, relatives of the partition genes of F and P1 plasmids. ParB of N15, like its F- and P1-encoded homologues, destabilizes plasmids carrying its target centromere, when present in excess. Within the genome of N15, we identified four unlinked, palindromic sequences that can promote the ParB-mediated destabilization of a moderate-copy vector in cis. They are distant from the parAB operon, unlike the centromeric sites of F and P1. Each of these palindromes could interact in vivo and in vitro with ParB. Each, when cloned separately, had properties characteristic of centromeric sites: exerted incompatibility against the N15 prophage and mini-N15 plasmids, and stabilized a mini-P1 plasmid depleted of its own partition genes when ParA and ParB of N15 were supplied. A pair of sites was more effective than a single site. Two of the centromeric sites are located in the proximity of promoters of phage genes, suggesting that, in addition to their function in partitioning of N15 prophage, they may control expression of N15 lytic functions.
PY54 is a temperate phage isolated from Yersinia enterocolitica. Lysogenic Yersinia strains harbour the PY54 prophage as a plasmid (pY54). The plasmid has the same size (46 kb) as the PY54 genome isolated from phage particles. By electron microscopy, restriction analysis and DNA sequencing, it was demonstrated that the phage and the plasmid DNAs are linear, circularly permuted molecules. Unusually for phages of Gram-negative bacteria, the phage genome has 3'-protruding ends. The linear plasmid pY54 has covalently closed ends forming telomere-like hairpins. The equivalent DNA sequence of the phage genome is a 42 bp perfect palindrome. Downstream from the palindrome, an open reading frame (ORF) was identified that revealed strong DNA homology to the telN gene of Escherichia coli phage N15 encoding a protelomerase. Similar to PY54, the N15 prophage is a linear plasmid with telomeres. The N15 protelomerase has cleaving/joining activity generating the telomeres by processing a 56 bp palindrome (telomere resolution site tel RL). To study the activity of the PY54 protein, the telN-like gene was cloned and expressed in E. coli. A 77 kDa protein was obtained and partially purified. The protein was found to process recombinant plasmids containing the 42 bp palindrome. Telomere resolution of plasmids under in vivo conditions was also investigated in Yersinia infected with PY54. Processing required a plasmid containing the palindrome as well as adjacent DNA sequences from the phage including an additional inverted repeat. Regions on the phage genome important for plasmid maintenance were defined by the construction of linear and circular miniplasmid derivatives of pY54, of which the smallest miniplasmid comprises a 4.5 kb DNA fragment of the plasmid prophage.
A simple, inexpensive, and sensitive test for potential carcinogens based upon the property of carcinogens to induce prophage lambda is described. By using chemicals activated with microsomal enzymes and E. coli K12 permeable (envA) tester bacteria also deficient in DNA repair (uvrB), the range of carcinogens detected in a lysogenic induction test (inductest) has been extended. We have provided the evidence that, after activation, carcinogenic polycyclic hydrocarbons such as benzo[a5pyrene and 7,12-dimethylbenz[a]anthracene induce prophage lambda. Three variants of the test have been developed (inductests I, II, and III), which are as sensitive as the mutagenicity test of Ames et al. [Ames, B. N., McCann, J. and Yamasaki, E. (1975) Mutat. Res. 31, 347-364]. Inductests II and III provide a quantitative estimation of the inducing activity of a carcinogen. With the latter test, one can determine: (i) the cellular toxic effect of a carcinogen and (ii) the kinetics of appearance and disappearance of active metabolites. For two series of chemicals, aflatoxins and benz[a]anthracenes, there is a good correlation between their carcinogenic activity in rodents and their prophage inducing activity in bacteria. The fact that the majority of the cell population is induced makes it possible to test the inducing activity of carcinogens at the biochemical level, e.g., by measuring lambda repressor inactivation.
To determine the structure of a prophage-containing plasmid during Mu transposition, we have monitored the physical state of pSC101[unk]Mucts after thermoinduction. We have also examined the fate of a mini Mu plasmid constructed in vitro by deleting 27 kilobases from the center of the Mu prophage in pSC101[unk]Mucts. At various times after prophage induction, DNA was extracted from Mu or mini Mu plasmid-containing strains and subjected to electrophoresis in low concentration agarose gels followed by transfer of the DNA to nitrocellulose paper. Separate hybridization with (32)P-labeled pSC101 and Mu DNA revealed the position of the plasmids and the replication of Mu DNA. At times after induction when Mu replication was clearly visible, Mu and mini Mu plasmids were found to migrate with Escherchia coli DNA. This Mu-specific association requires the phage coded A and B proteins. Electron microscopy has shown that some of the associated DNA is comprised of circular plasmid molecules which appear to be in contact with the chromosomal DNA. These structures may represent intermediates or end products of the replication-integration process. The finding that Mu and mini Mu plasmids do not give rise to any detectable excision products and apparently remain intact during Mu transposition supports our proposal that the predominant event after Mu induction is the replication of Mu DNA in situ to generate integrative intermediates.