Complementation experiments between conditional lethal mutants of bacteriophage ?X174

Summary Complementation experiments with temperature sensitive (ts) and suppressor sensitive (sus) mutants of bacteriophage X174 unambiguously revealed five cistrons on the basis of a clear bipartition of burst sizes.A new group of sus mutants (emeralds) was found, defective in a function essential for growth in Shigella sonnei V64.The complementation between ts and sus mutants was in general asymmetric in that the yield of ts particles was lower than that of the sus particles. The mutants of one cistron (defective in RF-replication) showed a completely asymmetric complementation behaviour both of ts and sus mutants. The ts mutants of this group, which show to be early, appear to be defective in two functions.The possibility is discussed that in each cell only one phage genome is replicated. This would explain both kinds of asymmetric complementation and the low burst sizes that were obtained when mutants of particular genes were complemented.     

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.     

Studies on the In Vitro Assembly of Bacteriophage φ80 and φ80-Lambda Hybrids

Suppressor-sensitive (sus) mutants of bacteriophage 80 defective in late functions were classified, by means of in vitro assembly tests, into two complementation groups: head donors and tail donors. Each group of mutants was subdivided, by means of two-factor crosses, into six cistrons. Deletion mapping revealed clustering of tail and also of head cistrons. The two clusters were located in the left arm of vegetative 80 (the tail specifying cluster being distal). In vitro cross complementation between 80 and lambda sus mutants revealed that whereas lambda heads could quite efficiently bind 80 tails to form viable phage, the union of 80 heads and lambda tails was very much less efficient. Deletion mapping of the 80 sus mutants, using both 80 and i⁸⁰h^λ deletion lysogens indicated congruent gross gene arrangement in the two related bacteriophages.     

Genetic mapping of the G101 bacteriophage (Pseudomonas aeuruginosa by temperature-sensitive mutations

Temperature-sensitive (ts) mutations of the G101 phage were isolated after mutagenesis with hydroxylamine. A complementation analysis of 61ts mutants showed that these mutants may be divided into at least 12 complementation groups. Twots mutants probably originated in genes which control lytic functions of the G101 phage. It was shown by three factor crosses that all of the 12ts mutations tested are localized on that side of the “c” region where the probablecI repressor gene is positioned. Sevents mutations is closely linked to thecI ₂₆ clear marker, three exhibit a closer linkage and two do not exhibit any linkage withcI. All mutations isolated until now can be arrange linearly. According to the present knowledge the preliminary genetic map of the G101 phage is linear.     

Virulent mutants of temperate phage Mu-1

Summary Virulent mutants of phage Mu have been isolated after mutagenesis. The virulent phenotype results from most probably 2 mutations located in the c-A region of the Mu genome.Vir mutants are trans-dominant; they induce the resident prophage upon infection in broth of any Mu lysogen. They however form plaques only on certain lysogens, that are monolysogenic for a mutant prophage. We further isolated secondary mutations in Mu Vir which suppress the virulent phenotype.     

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.     

Defective prophages of bacteriophage Mu

Summary A method is described for the isolation of thermoinducible defective Mu lysogens. Four of these defective lysogens were studied more extensively. By marker-rescue experiments it was shown that the strain harbouring the smallest defective prophage contains the immunity gene cts and the genes A and B; the strain with the largest defective prophage still contains all the known essential genes of Mu, A to S (see Fig. 1).After induction at 43° C all the defective lysogens are killed, whereas no lysis occurs.Although in all the thermoinducible defective lysogens the A and B gene products could be demonstrated by complementation, these gene products are not responsible for the killing of the host, suggesting the presence of another unknown early gene product of Mu. The level of complementation of a mutation in gene A is reduced by the presence in the cell of another defective Mu prophage containing the G part of Mu. This effect on A gene complementation is markedly enhanced when the defective prophage, containing the G part, is located on an episome instead of on the chromosome. Complementation of late genes by a defective prophage located on the chromosome, is extremely low or undetectable. A stimulation of complementation by a factor of 10 to 40 was found when the same defective prophage was situated on a F factor. A possible explanation for this episome effect will be discussed.     

Interaction of the ΦHSIC Virus with Its Host: Lysogeny or Pseudolysogeny?

The marine phage ΦHSIC has been previously reported to enter into a lysogenic relationship with its host, HSIC, identified as Listonella pelagia. This phage produces a variety of plaques on its host, including turbid and haloed plaques, from which lysogens were previously isolated. These lysogens were unstable during long-term storage at −80^° C and were lost. When HSIC was reinfected with phage ΦHSIC, pseudolysogen-like interactions between the phage and its host were observed. The cells (termed HSIC-2 or HSIC-2e) produced high viral titers (10¹¹ ml⁻¹) in the absence of inoculating phage and yet reached culture densities of nearly 10⁹ ml⁻¹. Prophages were not induced by mitomycin C or the polyaromatic hydrocarbon naphthalene in cells harboring such infections. However, such cells were homoimmune to superinfection. Colonies hybridized strongly with a gene probe from a 100-bp fragment of the ΦHSIC genome, while the host did not. Analysis of chromosomal DNA preparations suggested the presence of a chromosomally integrated prophage. Phage adsorption experiments suggested that HSIC-2 was adsorption impaired. Because of the chromosomal prophage integration and homoimmunity, we interpret these results to indicate that ΦHSIC establishes a lysogenic relationship with its host that involves an extremely high level of spontaneous induction. This could be caused by a weak repressor of phage production. Additionally, poor phage adsorption of HSIC-2 compared to the wild type probably helped maintain this pseudolysogen-like relationship. In many ways, pseudolysogenic phage-host interactions may provide a paradigm for phage-host interactions in the marine environment.     

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     

Observations on the formation of clones containing araB-lacZ cistron fusions

Summary Casadaban (1976) developed a technique for isolating E. coli clones containing fusions of the amino terminal-encoding portion of any cistron with the carboxy terminal-encoding portion of lacZ. The technique utilizes prophage Mu homology to bring the two cistrons into proximity. I have followed the appearance over time of colonies containing araB-lacZ fusions from a strain where the begining of the araB cistron is connected to lacZ by an intact Mucts62 prophage. Cultures of the starting strain grown on a variety of media have fewer than 2 in 10¹⁰ cells capable of forming colonies within three days after plating on selective arabinose-lactose medium. At 32°C, there is a delay of between 4 and 19 days before the first colony appears. The kinetics of colony appearance over the next two to four weeks then shows a rapid increase in the number of new colonies emerging per day followed by a decline. The pattern of colonial emergence and the final numbers of fusion colonies obtained are not grossly affected by reducing the number of cells plated over five orders of magnitude. Fusion colonies sometimes show a clustered pattern when they first emerge. Innoculation of pre-existing fusion clones at specific locations on the arabinose-lacteredselection plates seeded with the starting strain leads to the formation of inhibitory zones where no fusion colonies appear. Selection plates contain many microcolonies and papillae which do not proliferate into scoreable colonies but nonetheless contain cells capable of growth when replated on the same selective medium. Up to 39% of all plated cells are capable of producing fusion clones. The kinetics of fusion colony appearance can be altered by environmental and genetic manipulations. Partial derepression of the Mucts prophage at 37° accelerates the appearance of colonies but also reduces the final yield. Addition of limiting concentrations of glucose to the selective medium also accelerates the appearance of colonies in a specific fashion: enrichments below the level required for maximum acceleration produce a biphasic kinetics with two waves of fusion clone emergence separated by an eight-day interval. Infection with Muc ⁺ pAp phage produces dilysogens that have almost completely lost the ability to produce fusions. Infection with MuctsAampAP phage produces strains that are reduced in phage production and have delayed kinetics of fusion clone emergence. The implications of these observations for theories of hereditary change in bacteria are discussed.     

Interaction of the PhiHSIC virus with its host: lysogeny or pseudolysogeny?

The marine phage PhiHSIC has been previously reported to enter into a lysogenic relationship with its host, HSIC, identified as Listonella pelagia. This phage produces a variety of plaques on its host, including turbid and haloed plaques, from which lysogens were previously isolated. These lysogens were unstable during long-term storage at -80( degrees ) C and were lost. When HSIC was reinfected with phage PhiHSIC, pseudolysogen-like interactions between the phage and its host were observed. The cells (termed HSIC-2 or HSIC-2e) produced high viral titers (10(11) ml(-1)) in the absence of inoculating phage and yet reached culture densities of nearly 10(9) ml(-1). Prophages were not induced by mitomycin C or the polyaromatic hydrocarbon naphthalene in cells harboring such infections. However, such cells were homoimmune to superinfection. Colonies hybridized strongly with a gene probe from a 100-bp fragment of the PhiHSIC genome, while the host did not. Analysis of chromosomal DNA preparations suggested the presence of a chromosomally integrated prophage. Phage adsorption experiments suggested that HSIC-2 was adsorption impaired. Because of the chromosomal prophage integration and homoimmunity, we interpret these results to indicate that PhiHSIC establishes a lysogenic relationship with its host that involves an extremely high level of spontaneous induction. This could be caused by a weak repressor of phage production. Additionally, poor phage adsorption of HSIC-2 compared to the wild type probably helped maintain this pseudolysogen-like relationship. In many ways, pseudolysogenic phage-host interactions may provide a paradigm for phage-host interactions in the marine environment.     

Bacteriophage Mu DNA replication is stimulated by non-essential early functions

Summary The replication of a spontaneous Kil^– mutant of bacteriophage Mu has been investigated. The Kil^– mutation (Mucts62-13/4), which was introduced into a defective prophage, is pleiotrophic, leading to the loss of also the Gam, Cim and Sot functions. The mutation is caused by an insertion with the characteristics of IS1, located just outside the B gene.Mucts62-13/4 phages form extremely small plaques on wildtype indicator strains. The replication of the insertion mutant as compared to Mucts62 is strongly reduced. Normal replication could be restored by relieving the polarity of the insertion or by complementation with defective prophages which express all early functions. Apparently, early genes other than A and B are involved in normal Mu DNA replication.     

Heterozygosis of phage 2-3 of Rhizobium meliloti: Moderate level of mismatch repair or gene conversion

Summary Analysis of clear/turbid mottled (heterozygotic plaques) of Rhizobium meliloti temperate phage 16-3 indicates that the efficiency of repair at three sites (ti3, ti4, and ti5) in the C cistron is 2 to 20-fold less than that observed in E. coli phage . In agreement with this conclusion, heterozygotic plaques were observed at similar frequency in crosses where point and small delection mutants were combined, suggesting that in Rhizobium, DNA molecules with short single-stranded loops can escape from repair as efficiently as the simple mismatches.     

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.     

Methods for Analysis of the C Cistron of Temperate Phage 16-3 of RHIZOBIUM MELILOTI

A series of clear mutants of the temperate phage 16–3 of Rhizobium meliloti were isolated that included various point and deletion mutants of the C cistron, coding for the phage repressor. It was observed that recombinant genotypes, such as c⁺ and ti (temperature-sensitive allele), which form turbid plaques, can be detected quantitatively as lysogenic colonies and scored even at frequencies as low as 10^-6. Point mutations, deletions and the autonomy of intracistronic second-site mutations were characterized by this method. Further analysis has shown that each possible pair from three ti mutants gave nonconditional clear recombinants. It was shown that these latter bear the two initial ti mutations, suggesting a cumulative effect of two conditional mutations on the structure of the repressor protein. The double mutants were utilized in fine-structure mapping of the C cistron.     

Correlation between Map Position and Phenotype of CTI Mutants in the C Cistron of RHIZOBIUM MELILOTI Phage 16–3

Nine temperature-sensitive clear mutations (Cti) in the C cistron (coding for the repressor protein) of Rhizobium meliloti temperate phage 16–3 were characterized according to the inductive temperature, the immunity of cells lysogenic for these mutant phages to superinfection by homoimmune weak virulent mutants, the phenotype of double-ti mutants and interallelic complementation. The results indicate that mutations of similar phenotypic expression are clustered on the genetic map. Furthermore, it seems probable that the C cistron of the original phage 16–3 is identical to that of the independently isolated phage strain 36.