A new pleiotropic bacteriophage P1 mutation, bof, affecting c1 repression activity, the expression of plasmid incompatibility and the expression of certain constitutive prophage genes.

Summary In bacteriophage P1 an amber mutation in a new gene, bof, has been isolated. The bof-1 phage mutant exhibits a pleiotropic phenotype; bof product is non-essential, and acts as a positive modulator. In P1 bac-1 mutants, in which a dnaB analog product, ban, is expressed constitutively, the bof product activates ban expression both in the prophage state and in lytic growth: P1 bof bac prophages have a reduced ban activity and in lytic growth P1 bof bac phages show a lower ban activity than P1 wild type. This effect on ban activity is observed specifically in P1 bac-1 mutants; it is not mediated by the cl repressor of the lytic functions (repressor of the ban operon) since this effect occurs even if the phage carries a heat sensitive c1 repressor. Thus we concluded that the bac mutation put the ban operon under an abnormal, unknown control, modulated by the bof product. P1 bof lysogens show an increased immunity to superinfecting P1 phage and are affected in their inducibility properties; in the presence of the altered c1-100 repressor, bof product is required for maintenance of lysogeny, as shown by the induction of P1 c1-100 bof-1 lysogens at 30°. P1 bof superinfecting phage can be established together with a resident P1 bof prophage in a recA host, unlike P1 wild type which cannot form double lysogens. P1 bof double lysogens are unstable and segregate one or the other prophage. P1 Cm bof and P1 Km bof lysogens show higher levels of antibiotic resistance than the corresponding bof ⁺ lysogens. The bof gene has been mapped, in an interval defined by P1 prophage deletion end points, far from both ban and c1. All bof phenotypes are reversed by single mutations.     

Integration defective mutants of bacteriophage P2

Summary Mutants of phage P2 unable by themselves to be integrated as prophages have been isolated. These mutants (int) are complemented by the wild type allele and may then yield stable lysogenic strains carrying an int prophage at location I in Escherichia coli C. These lysogens produce either no phage or little phage, depending on the int mutant used. All int mutants isolated appear to belong to a single complementation group.Exceptional lysogens carrying two or more int prophages may be obtained: they may produce spontaneously even more phage than normal lysogens, and they segregate out defective, singly lysogenic clones at low frequency. These exceptional lysogens carry both prophages in location I, presumably in tandem.Strains carrying two or more int prophages but defective in phage production were also isolated. One of these carries its prophages at two different, not closely linked, chromosomal locations.     

Cold-sensitive mutations in the Z gene of prophage P2 that result in increased sensitivity of the lysogens to a low molecular weight product of the host bacteria

Summary Z mutants of bacteriophage P2 form clear plaques and are unable to give rise to stable lysogens in Escherichia coli C. To study the function of the Z gene in lysogenization by P2, temperature-sensitive mutants were isolated. Those that were classified as Z mutants by complementation were all cold-sensitive (cs); they were unable to form lysogens at 30° C, but had wild type phenotype at 42° C. When lysogens carrying such mutants, prepared at 42° C, were shifted to the lower temperature, the bacteria continued to multiply at the normal rate until they reached concentrations of about 5 × 10⁷ per ml, at which point the viable titer began to decrease. Inactivation of the bacteria at even lower concentrations occurred if they were transferred to medium taken from overnight cultures of the same strain, suggesting that they were sensitive to some material that had accumulated in the culture medium.The lethal material was produced not only by csZ lysogens, but by all derivatives of Escherichia coli C tested, including non-lysogens, and at both 30° C and 42° C. Only csZ lysogens were sensitive to it, however, and only at the lower temperature. A preliminary characterization of the material indicates that it is heat-stable, of low molecular weight and does not adsorb to activated charcoal.This work was supported by Research Grant 72 from the Swedish Medical Research Council     

Behavior of bacteriophage P1 inErwinia carotovora subsp.carotovora

Bacteriophage P1KMclr100 was tranferred toErwinia carotovora subsp.carotovora. P1 was stably maintained as detected by hybridization and transfer of kanamycin resistance. Lysogens ofE. carotovora failed to produce any viable P1 phage. Although total DNA from P1 lysogens ofE. carotovora hybridized to³²P-labeled P1 probe, we were not able to detect P1 DNA as an extrachromosomal element. Attempts to use bacteriophage P1 as a vector for transposon Tn5 insertion mutagenesis inE. carotovora were not successful. Our results indicate that lytic replication of P1 DNA does not occur in P1 lysogens ofE. carotovora and that P1 DNA is probably integrated into the bacterial chromosome.Journal paper 10085 from the Purdue Agricultural Experiment Research Station.     

Analysis of the phase variation in lambda reduced immunity lysogens

Summary Two distinct phases characterized by different levels of immunity that appear in some E. coli strains lysogenic for reduced immunity mutants of bacteriophage lambda are identified as single and tandem double lysogens respectively on the basis of DNA-DNA hybridization experiments and the requirement of the phage xis function for the transition from a single to a double, and of the host recA function for the transition from a double to a single lysogen (in a xis ^- condition). Rim lysogens with a further increase in immunity, containing some 5 copies of the lambda genome per host genome, have also been observed.It is argued that the different levels of immunity are a direct reflection of the CI gene dosage effect.An unexplained finding is that rim single lysogens yield double lysogens with a frequency of near 1% per generation, whereas cured cells fail to appear even at a frequency 100 times lower.     

Temperate Coliphage HK022: Clear Plaque Mutants and Preliminary Vegetative Map

Wild type phage HK022 was mutagenized by N-methyl-N′-nitro-N-nitrosoguanidine to induce clear plaque mutants. A total of 225 clear plaque mutants were isolated and 198 of these were assignable to one or the other of the two complementation groups of the corresponding cistrons which have been designated as cI and cII, respectively. Approximately 25% of the c mutants were found to be temperature-sensitive (cts); producing turbid plaques at 32 C and clear plaques at 38 C and above. From complementation tests involving cI and cII mutants, bacteria lysogenic for cII prophage were frequently obtained. Double lysogens harboring a cI and a cII prophage were infrequently found and single lysogens harboring only a cI prophage have not been recovered. Bacterial lysogens harboring a prophage carrying a cts mutation in the cI cistron were readily obtainable. However, such lysogens show a lethal phenotype at 40 C and above, although they appear to be fully viable at 32 C. It is shown that by incubation of lysogens harboring a cts mutant of the cI cistron at 42 C, it is possible to isolate cryptic lysogens which are non-immune but harbor at least one of the phage sus⁺ alleles. Genetic data involving cI, cII, and two complementing sus mutants of essential genes are presented. From these data the following vegetative map is deduced: sus4–cII-cI-sus3.     

Genetic analysis of bacteriophage lambda strains which transduce the genes for leucine biosynthesis

Summary By means of two specific genetic tests as well as additional transduction studies, the genetic compositions of four different leucine-transducing lambda-phages and the two E. coli lysogens from which these phages originated were analyzed. Three of the phages, No. 267, 517 and 889, are of the bio-type, e.g. carry bacterial genes adjacent to prophage attachment element P. The former two contain a large portion of the E. coli leucine operon (genes leu B through leu D, see Fig. 2), the latter carries only gene leu B and part of leu C. Phage No. 518 is of the gal-type and carries at least part of the leu A gene. The two lysogens, No. 73 and 75, from which these phages arose, contain the prophage between two mutation sites in gene leu A and leu B, in an orientation that is opposite to the normal one for lambda.     

Plaque forming specialized transducing phage P1: Isolation of P1CmSmSu, a precursor of P1Cm

Summary E. coli strains lysogenic for various types of P1-R hybrids were isolated. These carry all the essential genes for vegetative phage production and lysogenization including P1 immunity and P1 incompatibility, together with drug resistance genes derived from the R plasmid NR1. In particular, P1Cm and P1CmSmSu derivatives were studied. When strains lysogenic for these phages were induced in the absence of helper phage, yields of phage particles as high as with wild type P1 were obtained. All P1Cm phages isolated were of plaque forming type and usually every plaque contained Cm^r lysogens. Lysates of P1CmSmSu lysogens transduced Cm^rSm^rSu^r at high frequency and they formed plaques with an efficiency of 10^-4 to 10^-2 per phage particle. Only a minority of these plaques contained drug resistant bacteria. Cm^rSm^rSu^r transductants isolated from bacteria infected at a high multiplicity with phage P1CmSmSu were lysogens for the original P1CmSmSu. In contrast, Cm^rSm^rSu^r transductants isolated after infection at low multiplicity appeared to carry the Cm^rSm^rSu^r markers integrated into the host chromosome. The results described suggest that P1CmSmSu prophages carry the resistance genes transposed into the P1 genome without in principle causing a loss of essential gene functions. However, since these prophages are longer than the wild type P1 genome, the DNA packaged into phage particles has a reduced redundancy which seriously affects the reproduction and lysogenization abilities.Plaque forming P1Cm can be obtained from P1CmSmSu. Thus, P1CmSmSu is a precursor of P1Cm. P1Cm is also obtainable from P1 and NR1 under the recA ^- condition. The mechanism of formation of plaque forming P1Cm is discussed.     

Cloning of the immunity repressor determinant of bacteriophage P2 in the pBR322 plasmid

Summary Through in vitro recombination of DNA restriction fragments, we have constructed a plasmid, which expresses in vivo the immunity repressor gene (C) of bacteriophage P2. A bacterial strain carrying such a plasmid showed a high level of P2 specific immunity. It was lysogenized normally by an infecting P2. but the frequency of spontaneous phage production was reduced about 10⁴ fold as compared to a normal P2 lysogen. Satellite phage P4, known to derepress P2 lysogens, was unable to derepress the plasmid-carrying lysogenic strain so to allow growth of coinfecting P2. Phage P4 multiplied on the plasmid-carrying. P2-lysogenic strain, but due to a prolonged latent period failed to form plaques on this strain.     

The activity of ant product of the Salmonella phage P22 against the closely related but heteroimmune phage L.

Summary P22 mutants defective in the early gene 24 are complemented by phage L in mixed infection. P22 12 ^- and P22 23 ^- mutants are not complemented by phage L. Gene function 24 of an L prophage is turned on by a superinfecting P22 24 ^- mutant and complements the missing function of the defective P22 phage. Since this transactivation of prophage gene 24 depends on a functional gene ant in the superinfecting P22 mutant, it indicates derepression for leftward directed gene expression in prophage L. On the contrary neither the rightward directed expression of gene 12 nor of gene 23 in prophage L can be turned on by superinfecting P22 24 ^- 12 ^- or P22 24 ^- 23 ^- mutants (and also not by P22 12 ^- and P22 23 ^-) to a degree sufficient for complementation of simultaneously superinfecting L virB 12 ^- or L virB 23 ^- mutants. The failure to detect release of repression for rightward directed gene expression of prophage L corresponds to the earlier observation (Prell, 1975) that P22 superinfecting L lysogens cannot release replication inhibition for simultaneously infecting phage L. The results are discussed with respect to the mechanism underlying the different action of P22 antirepressor in L and in P22 lysogens.     

Synthesis of P1 ban protein in minicells infected by P1 mutants

Summary Phage P1 encodes a dnaB analog (ban) protein. Synthesis of ban protein has been studied in minicells infected by P1 mutants and has been identified as a polypeptide of 56,000 molecular weight by immunoprecipitation using antibody directed against E. coli dnaB protein. The amount of ban protein synthesized by P1 mutants increases in the order: P1 wild type, P1bac, P1crr, and P1bac crr. The relative amount of ban protein identified in P1bac- and P1bac crr-infected minicells is approximately the same as that previously found in dnaBsdban heteromultimers isolated from the corresponding P1 lysogens.     

Lysogeny in the blue-green algaNostoc muscorum

Lysogens were isolated after confluent lysis of the blue-green algaNostoc muscorum by N-1 virus and characterised. The spontaneous liberation of active virus particles in lysogens was not lost after treating them with viricidal concentration of EDTA and virus titre obtained in their cultures was >5×10³ PFU/ml. The virus adsorbed on the lysogens with a slower rate than on parent alga, although it followed the pattern of a first order reaction. The heat treatment (45°C for 14h) of the lysogens failed to induce lysis, whereas mitomycin C (1–2 g/ml) was effective in inducing lysis.List of Abbreviations EDTA ethylene diamine tetra-acetic acid - IM input multiplicity - k adsorption rate constant - k _v specific constant for virus production - MC mitomycin C - PFU plaqueforming units     

Genetic exchanges caused by ultraviolet photoproducts in phage λ DNA molecules: The role of DNA replication

Summary Genetic recombination induced by structural damage in DNA molecules was investigated in E. coli K12 () lysogens infected with genetically marked phage . Photoproducts were induced in the phage DNA before infection by exposing them either to 313 nm light in the presence of acetophenone or to 254 nm light. To test the role of the replication of the damaged phage DNA on the frequency of the induced recombination, both heteroimmune and homoimmune crosses were performed.First, samples of a heteroimmune phage imm434 P80 exposed to these treatments were allowed to infect cells lysogenic for prophage cI857 P3. Phage DNA replication and maturation took place, and the resulting progeny phages were assayed for the frequency of P ⁺ recombinants. Recombination was less frequent in infected cells exposed to visible light and in wild type cells able to perform excision repair than in excision-defective lysogens. Therefore, much of the induced recombination can be atributed to the pyrimidine dimers in the phage DNA, the only photoproducts known to be dissociated by photoreactivating enzyme.Second, in homoimmune crosses, samples of similarly treated homoimmune P3 phages were allowed to infect lysogens carrying cI857 P80. Replication of the phage DNA containing ultraviolet photoproducts was repressed by immunity, and was futher blocked by the lack of the P gene product needed for replication. The lysogens were purified and scored for both colony forming ability and for P ⁺ recombinant prophages. The 254 nm photoproducts increased the frequency of recombination in these homimmune crosses, even though phage DNA replication was blocked. Irradiation with 313 nm light and acetophenone M, which produces dimers and unknown photoproducts, was not as effective per dimer as the 254 nm light.It is concluded from these results that certain unidentified 254 nm photoproducts can cause recombination even in the absence of DNA replication. They are not pyrimidine dimers, as they are not susceptible to excision repair or photoreactivation. In contrast, pyrimidine dimers appear to cause recombination only when the DNA containing them undergoes replication.     

Suppression of a thermosensitive dnaA mutation of Escherichia coli by bacteriophage P1 and P7.

Like other plasmids, the P1 and P7 prophages suppress E. coli dnaA(Ts) mutations by integrating into the host chromosome. This conclusion is supported by three lines of evidence: (1) Alkaline sucrose gradients reveal the absence of plasmid DNA in suppressed lysogens; (2) the prophage is linked to host chromosomal markers in conjugation; and (3) auxotrophs whose defect is linked to the prophage are found among suppressed colonies. No phage or bacterial mutation is required for suppression. Integrative suppression by P1 and P7, unlike suppression by F, does not require the host recA⁺ function. Among suppressed P7 lysogens are some that do not produce phage; these contain defective prophages. The genetic extent of the deletions contained by these defective prophages delineates the prophage regions which are not necessary for suppression of dnaA(Ts). The possible mechanisms of integration and deletion formation are discussed.     

Coping with inadvertent lysis of Escherichia coli cultures: Strains resistant to lysogeny and infection by the stealthy lysogenic phage Φ80

Phage Φ80 can infect Escherichia coli in a stealthy manner and persist by forming lysogens. Such Φ80 lysogens are fairly common and often go undetected unless the host is grown at temperatures below 37°C. Since low growth temperatures are required for growing temperature-sensitive mutants and often preferred for large-scale applications such as protein production, Φ80-resistant strains would be useful. We report the construction of E. coli strains that cannot be efficiently lysogenized or infected by bacteriophage Φ80. These strains contain combinations of deletions or mutations in the bacterial attachment site for Φ80 integration and/or deletions in the genes required for phage absorption to the host outer membrane. These strains should help contain and prevent Φ80 infection of E. coli cultures in a laboratory or industrial setting.     

Genetic analysis of bacteriophage P4 using P4-plasmid ColE1 hybrids

Summary A set of plasmids that contain fragments of the bacteriophage P4 genome has been constructed by deleting portions of a P4-ColE1 hybrid. A P4 genetic map has been established and related to the physical map by examining the ability of these plasmids to rescue various P4 mutations. The P4 vir1 mutation and P4 genes involved in DNA replication (), activation of P2 helper genes ( and ), polarity suppression (psu) and head size determination (sid) have been mapped, as has the region responsible for synthesis of a nonessential P4 protein.One of the deleted plasmids contains only 5900 base pairs (52%) of P4 but will form plaques if additional DNA is added to increase its total size to near that of P4. This plasmid is also unique in that it will not form stable associations with P2 lysogens of E. coli which are recA ⁺. P4 mutants can be suppressed as a result of replication under control of the ColE1 part of the hybrid.     

Prevalence of Stx Phages in Environments of a Pig Farm and Lysogenic Infection of the Field <Emphasis Type="Italic">E. coli</Emphasis> O157 Isolates with a Recombinant Converting Phage

The prevalence and nature of Shiga toxin (Stx)-producing Escherichia coli (STEC) and Stx phage were investigated in 720 swine fecal samples randomly collected from a commercial breeding pig farm in China over a 1-year surveillance period. Eight STEC O157 (1.1%), 33 STEC non-O157 (4.6%), and two stx-negative O157 (0.3%) isolates were identified. Fecal filtrates were screened directly for Stx phages using E. coli K-12 derivative strains MC1061 as indicator, yielding 15 Stx1 and 57 Stx2 phages. One Stx1 and eight Stx2 phages were obtained following norfloxacin induction of the eight field STEC O157 isolates. All Stx1 phages had hexagonal heads with long tails, while Stx2 phages had three different morphologies. Notably, most of field STEC O157 isolates released more free phages and Stx toxin after induction with ciprofloxacin. Furthermore, upon infection with the recombinant phage ΦMin27(Δstx::cat), E. coli laboratory strains produced both lysogenic and lytic phage, whereas two of the eight O157 STEC isolates produced only lysogens. The lysogens from laboratory strains produced infectious particles similar to ΦMin27. Similarly, the lysogens from the STEC O157 isolates released Stx phage too, although free ΦMin27(Δstx::cat) particles were not detected. Collectively, our results reveal that breeding pig farms could be important reservoirs for Stx phages and that residual antibacterial agents may enhance the release of Stx phages and the expression of Stx.     

Analysis of Twin-Arginine Translocation Pathway Homologue in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

We investigated the relationship between expression of the O side chain of outer membrane lipopolysaccharide (LPS) and infection by a Shiga toxin 2 (Stx2)-converting phage in normal and benign strains of Escherichia coli. Of 19 wild-type E. coli strains isolated from the feces of healthy subjects, those with low-molecular-weight LPS showed markedly higher susceptibility to lytic and lysogenic infection by Stx2 phages than those with high-molecular-weight LPS. All lysogens produced infectious phage particles and Stx2. The Stx-negative E. coli O157:H7 strain ATCC43888 with an intact O side chain was found to be resistant to lysis by an Stx2 phage and lysogenic infection by a recombinant Stx2 phage, whereas a rfbE mutant deficient in the expression of the O side chain was readily infected by the phage and yielded stable lysogens. The evidence suggests that an O side chain deficiency leads to the creation of new pathotypes of Shiga toxin-producing E. coli (STEC) within the intestinal microflora.     

Role of antirepressor in the bipartite control of repression and immunity by bacteriophage P22

The bipartite immunity and repression system of the temperate Salmonella bacteriophage P22 has been analyzed by genetic means. Both parts of the immunity system, immI and immC, are necessary to confer upon lysogens immunity to superinfection with P22. The product of the c2 gene (which lies in immC) is a repressor which apparently regulates directly the expression of phage genes in a manner analogous (if not identical) with that found for coliphage λ.The immI region contains three genetic elements. One of these (mnt; Gough, 1968) appears to specify another repressor whose specific activity is continuously required for the maintenance of lysogeny. We have identified two new regulatory elements in immI through the isolation of mutants. Virulent mutations (virA) in the Vy element confer the ability to grow in immune P22 lysogens by destroying or inactivating the repression functions of the lysogen (possibly the c2-repressor itself). The third element in immI is a structural gene (ant) for a protein (antirepressor) which is regulated by mnt (repressor) and Vy (promoter/operator).We have shown that the ability of P22 to grow on immI-deletion lysogens, the dominant virulence of virA virulents, and the requirement for mnt for the maintenance of lysogeny, all depend on an intact ant⁺ gene. It is proposed that P22 antirepressor represents a new type of regulatory protein which acts by controlling other regulatory proteins.     

Carbohydrate metabolism and transport in Bacillus subtilis. A study of ctr mutations

Summary Indirect ultraviolet induction of prophage occurs when lysogenic E. coli K12 cells are mated with ultraviolet-irradiated donor strains carrying a transmissible episome such as F lac ⁺. Indirect induction occurs in wild type, uvrA, or recB recipient lysogens, but not in recA lysogens. When nonpermissive lysogens carrying prophages susO or susP are similarly mated, the defective prophages are induced and indirect curing takes place.Although indirect induction is independent of the capacity of the lysogen for repair by pyrimidine dimer excision, indirect curing (and hence indirect induction) is subject to photoreactivation when the recipient lysogen is exposed to visible light after mating. This confirms that the structure initiating indirect ultraviolet induction is a damaged transferred episome consisting of one DNA strand containing ultraviolet photoproducts and a newly synthesized discontinuous DNA strand such that pyrimidine dimers remain in single-stranded regions.F^- lac ⁺ recombinants are formed in either nonlysogenic or lysogenic Lac^- cells receiving damaged F lac ⁺ episomes from ultraviolet irradiated F lac ⁺ donors. prophage induction occurs more frequently in zygotes that form Lac⁺ recombinants than in zygotes that remain Lac^-. In contrast, cells receiving intact (undamaged) episomes are converted to F lac ⁺ secondary donors, but are rarely induced or cured.