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.     

Superinfection exclusion and changes in cellular transport processes in phage infected Salmonella typhimurium.

Summary The changes induced by bacteriophage P22 in the cellular transport process(es) of the host Salmonella typhimurium (Taneja et al., 1975; Khandekar et al., 1975; Bandyopadhyay and Chakravorty, 1976) involve interactions between the superinfection exclusion system of the resident prophage and the C immunity region of the superinfecting phage. The sieA gene of the prophage interferes with the changes in the cellular transport process induced by the superinfecting phage. However, if the superinfecting phage carries active C ₁ and C ₂ genes of the superinfecting phage seem to be expressed in the sie A⁺ lysogen.     

A new gene of bacteriophage P22 which regulates synthesis of antirepressor

Two new mutants of bacteriophage P22 are described which define a new regulatory gene, arc (for antirepressor control). The properties of the arc mutants and of 31 phenotypic revertants indicate that the arc gene codes for a trans-acting protein whose primary role is to depress synthesis of P22 antirepressor protein during the lytic cycle of infection. Failure to regulate antirepressor production apparently leads secondarily to a lethal defect (i.e. failure to produce progeny phage).Although under certain conditions the arc function can be expressed by P22 prophages and can act as a weak barrier to superinfecting homologous phage, the arc product is neither necessary nor sufficient for maintenance of the prophage state or superinfection immunity in lysogens. Instead, as shown previously by others (Levine et al., 1975; Botstein et al., 1975), the prophage mnt gene product is responsible for repressing antirepressor synthesis, both by the prophage and by superinfecting phage.     

Ant-mediated transactivation of early genes in Salmonella prophage P22 by superinfecting virulent P22 mutants

Summary The virulent mutants P22 vir B vy and P22 vy mutants, both insensitive to mnt-repressor, transactivate the early genes of a P22 prophage. The transactivation of early P22 prophage genes depends strictly on the expression of gene ant (antirepressor-protein) by the superinfecting P22 mutant and therefore occurs by derepression.     

Regulation of gene expression in Salmonella phage P22. II. Regulation of expression of late functions

Transactivation experiments were performed involving the genetically related Salmonella phages P22, L and Px1 in order to find out if more than one positively acting regulatory product is engaged in the expression of vegetative gene functions of each of these phages. The results obtained with Px1- and L-lysogenic cells superinfected with P22 suggest the following conclusions: 1. The expression of the early genes 12 and 23 and of the late gene 19 (lysozyme synthesis) is positively regulated by two different regulatory products, since P22 transactivates in prophage Px1 both early and late genes (Prell, 1973), in prophage L only late genes. 2. The transactivation by P22 of the lysozyme gene of prophage L takes place in the presence of L repressor. This conclusion is suggested, since the superinfecting P22 does not derepress early gene expression (see 1.), and is confirmed by demonstration of replication inhibition for L phage in L lysogenic cells doubly superinfected with L and P22 phages (Thomas-Bertani-experiment). 3. The late gene regulatory protein seems to be synthesized by gene 23, as transactivation experiments with both L- and Px1 prophages suggest. 4. The expression of gene 23 itself is turned on by an early regulatory product. The gene which codes for it is still unidentified. However its product seems to by highly specific, since it is active on Px1- but not on L-prophage.     

Phage and prophage decision in defective lambda-lysogens: the role of cII product

Summary The cII product of prophage plays an ambivalent role: immediately after irradiation it promotes lytic response to superinfection by the homoimmune phage, later, in some cells, it helps to reestablish immunity.     

Non-receptivity for kappa phage of kappa-lysogenic Serratia and reactions to superinfection of receptive cells with a mutant prophage

Summary l_I-lysogenic cells of Serratia marcescens as opposed to l_I ⁺-lysogenic cells, are receptive to further infection. After superinfection with wild type or several clear plaque mutants killing and lytic response were observed to a varying extent. From some of the surviving cells doubly lysogenic colonies with a l_I ⁺ and a l_I prophage could originate, l_I ⁺ being dominant and the cells therefore non-receptive. By this property, combined with a special color reaction, the colonies could be easily screened. Evidence is presented that the l_I ⁺ gene product probably does not interfere with the formation of new phage receptors but that under its influence receptors are masked.Two mutants resembling int^- mutants are described which only rarely give double lysogenization and prophage substitution following superinfection. The phage-coded function normally achieving these reactions is likely to work constitutively after superinfection. Transduction experiments performed with one of the two mutants pointed at integration of prophage into the host DNA near to the trp gene.Abbreviations moi multiplicity of infection - OD optical density - NG N-methyl-N-nitro-N-nitrosoguanidine - NB nutrient broth - BS buffered saline - EMB eosmemethylene blue - leu leucine - met methionine - pro proline - trp tryptophan     

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.     

The packaging initiation site of phage P22

Summary P22 lysates were grown on Salmonella strains carrying P22 prophages deleted to various extents. Transducing bacterial markers at both sides of the prophage insertion site it could be shown that: (i) transduction of markers can be enhanced by the prophage pac site; (ii) the recognition signal pac is in the area of gene 3 on the phage genome and thus close to the cutting site(s); (iii) transposon Tn10 may also act as a signal for packaging initiation; (iv) (at least) Tn10 initiates packaging sequences in both directions.     

Superinfection exclusion by heteroimmune corynebacteriophages.

Superinfection of Corynebacterium diphtheriae C7(beta) by heteroimmune phage gamma is productive, whereas superinfection by gamma-bin mutants is for the most part nonproductive. Exclusion of gamma-bin phage occurred after its DNA had penetrated and was partially expressed in the heteroimmune lysogen. All of the infected cells were killed, and lysis was observed. The beta inhibitor causing exclusion was produced during the prophage state and appeared to be distinct from immune repressor. The ability of gamma-bin phage to superinfect C7(beta) productively could be restored by recombination with beta phage, indicating that both beta and gamma phages contain either indentical or similar alleles of the bin gene. The bin gene was mapped by vegetative and prophage crosses and found to be located in the region of the phage genome concerned with regulation. Both beta and gamma wild-type phages induced the resident prophage in a significant fraction of superinfeted heteroimmune lysogens. This, coupled with the fact that induction of C7(beta) abolished exclusion, suggests that the bin gene product acts as antirepressor, i.e., it reduces the level of heteroimmune repressor either directly or indirectly. The gamma-bin mutants either failed to produce antirepressor or did so with reduced efficiency. Antirepressor activity was negatively controlled by homoimmune repressor. The isolation of beta mutants that appeared bin-like suggests that beta and gamma phages contain homologous systems of exclusion and antiexclusion. Exclusion of gamm-bin by beta phage in gram-positive C. diphtheriae exhibited striking parallels to the sieB exclusion described for phages P22 and lambda in gram-negative organisms. The extended similarities of these coryngephages to lambda bacteriophage is noted.     

Specialized Transduction by Bacteriophage P22 in SALMONELLA TYPHIMURIUM: Genetic and Physical Structure of the Transducing Genomes and the Prophage Attachment Site

P22pro-1 and P22pro-3 are specialized transducing derivatives of phage P22 that carry the proA and proB genes of Salmonella typhimurium. These genes lie immediately adjacent to the prophage attachment site on the bacterial chromosome. By examining DNA heteroduplexes in the electron microscope, we found that DNA molecules from P22pro-1 and P22pro-3 each contain a substitution which adds length to the composite genome making the intracellular replicated genome too long to fit into a single phage particle. In this respect, and in many of their biological properties, the proline-transducing phages resemble P22Tc-10, another specialized transducing phage with an oversize, intracellular replicated genome which carries a tetracycline-resistance determinant from an R-factor.—Unlike P22Tc-10, however, P22pro-1 and P22pro-3 fail to integrate normally during lysogenizing infections, even when provided with all known integration functions. These results suggest that the proline substitutions have created a defect in the phage attachment site and suggest that the Campbell model for the formation of specialized transducing phages is applicable to phage P22 with the additional feature that oversize genomes can be produced and propagated.—A physical and genetic map of the P22 genome near the prophage attachment site was constructed which shows that the insertion from the R-factor in P22Tc-10 is not at the attachment site: it is therefore unlikely that P22Tc-10 was formed in an abnormal prophage excision event as envisioned in the Campbell model, but was instead the result of a direct translocation from the R-plasmid to P22.     

Effects of mine acid on the longevity and reproductive rate of the gastrotricha Lepidodermella squammata (Dujardin)

Effects of mine acid on longevity and reproductive rate of the parthenogenic gastrotrich Lepidodermella squammata were studied under laboratory culture conditions. Water from unpolluted and polluted streams was used, directly or mixed, to establish a series of test conditions at pH 8.1, 7.1, 6.4, 5.2, 4.6 and 3.3. Eggs, cultured individually under each test condition, were observed at 12 h intervals for hatching, daughter egg laying and death. Data representing 50 animals under each test condition were used in the construction of a series of life tables. From these were calculated maximal life expectancy (e_x), net reproductive rate per individual lifetime (R_o) and intrinsic rate of natural increase (r_max).Values of e_x, R_o and r_max were maximal at pH 7.1 and were reduced slightly at the higher pH; e_x was greatly reduced and R_o zero at pH 6.4 and 5.2; and e_x was zero at pH 4.6 and 3.3. Analysis of variance tests indicate significant differences between e_x values of L. squammata cultured at pH 8.1 and those cultured at pH 7.1, but no differences between R_o or r_max.Associated with the decrease in pH was an increase in total conductivity and a decrease in carbonate alkalinity and hence in carbonate conductivity. It appears that L. squammata is capable of living and reproducing at pH 6.0 to 6.5 under field conditions low in carbonates, providing non-carbonate ions are not abundant, or under field conditions high in non-carbonate ions, providing sufficient carbonates are present.     

Cis- and trans-activity of P22 antirepressor protein against c-repression specified by the closely related Salmonella phages L and Px1

Summary The ant product of Salmonella phage P22, synthesized by its immI region, releases when acting in cis replication inhibition for phages P22 and L. When ant product acts in trans on a coinfecting immunity sensitive phage (Thomas-Bertani-experiment as test for release of replication inhibition) full replication ensues only if both superinfecting phages are homologous in the specificities of their immC and immI regions. If these regions are heterologous, differing in immC, immI or in both, the replication of the phage expected to be complemented by ant is inhibited. This inhibition is observed in both L- and Px-lysogenic bacteria and can be released in case of ant ^- amber phages by action of ant in cis in su ⁺lysogenic bacteria.     

The role of ant-product of Salmonella phage P22 in the process of transactivation of prophage Px1 genes.

Summary Phage P22 replicates in Px1-lysogenic cells because the ant-product (antirepressor-protein) of P22 removes the repression exerted by the Px1-prophage. P22 ant ^- phages however are repressed in Px1-lysogens. The ant-product of P22 is also required for the transactivation of the early genes 24, 12, and 23 of the Px1-prophage. P22 virB3 ant ^- mutants, which are insensitive to c-repression and therefore replicate in Px1-lysogens, are unable to transactivate early genes of the Px1-prophage or of a coinfecting Px1 phage. Transactivation of the late gene 19 is insensitive to repression and independent of ant-activity. This suggests the presence of a separate, and o_R-independent, promotor for late gene expression. —A more rigorous proof for functional homology in early gene regulation of P22 and Px1 is presented.Reinhard W. Kaplan zum 65. Geburtstag in Dankbarkeit gewidmet     

The role of c3 product and anti-immunity in zygotic induction of bacteriophage lambda

Summary A nonlysogenic cell has twenty fold higher (26% versus 1.3%) probability to survive phage infection than entry of the same genome via conjugation (prophage infection). When the entering genome bears a cIII^- mutation, this difference increases to one hundred fold (6% versus 0.06%). A lysogenic im^- cell harbouring a defective prophage able to synthetize anti-immunity (product of gene tof) has ten fold higher probability to survive prophage infection than phage infection (20% versus 2%). Here, cIII^- mutation does not affect the survival. When the cell is simultaneously infected with the phage and prophage, the decision of the phage whether to enter the lytic cycle (in im^- cells) or not (in nonlysogens) is always epistatic to that of the prophage.     

Isolation and characterization of a plaque-forming lac-transducing phage phi80plac with special reference to its integration and excision

Summary From a double lysogen for 80dlac type II (Beckwith and Signer, 1966) and 80, we isolated a plaque-forming lac-transducing coliphage 80plac after selecting a strain with a suitable deletion in the 80 prophage. The lac region of the phage is i ⁺ o ⁺ z ⁺ y ⁺ a ^- and supposed to be located between genes 15 (N) and imm (CI). The phage showed feckless phenotype indicating deletion of genes of the red system. The phage is also deleted for int or att function, and integrates exclusively at the host lac region, largely dependent on the host rec system. Excision of the prophage upon UV-irradiation or by mating the male lysogen with a non-lysogenic female was efficient and largely dependent on the host rec system. But a considerable amount of rec-independent excision was observed at least in the case of zygotic induction, which was not likely to be caused by int-xis, red or ter system of the phage. 80plac/o ^e phage was also isolated by incorporation of o ^e1 mutation from strain 2000o ^e.     

The kinetics of the requirement for X gene product in bacteriophage P 22

Summary The kinetic study of the requirement for X gene product showed that the average burst size of the P22 phage depended on the length of the permissive interval in which the X function was expressed. Results of the temperature shift experiments with the clear plaque recombinants tsX c ₂ ⁵ and ts 25.1 c ₂ ⁵ gave a complicated pattern of the phage yield response.It is concluded that X gene product, besides the control function in the initiation of the phage development, is involved directly or indirectly in the control of late functions and is required throughout the entire period of the phage development.     

Mycobacteriophage Fruitloop gp52 inactivates Wag31 (DivIVA) to prevent heterotypic superinfection

Bacteriophages engage in complex dynamic interactions with their bacterial hosts and with each other. Bacteria have numerous mechanisms to resist phage infection, and phages must co‐evolve by overcoming bacterial resistance or by choosing an alternative host. Phages also compete with each other, both during lysogeny by prophage‐mediated defense against viral attack and by superinfection exclusion during lytic replication. Phages are enormously diverse genetically and are replete with small genes of unknown function, many of which are not required for lytic growth, but which may modulate these bacteria–phage and phage–phage dynamics. Using cellular toxicity of phage gene overexpression as an assay, we identified the 93‐residue protein gp52 encoded by Cluster F mycobacteriophage Fruitloop. The toxicity of Fruitloop gp52 overexpression results from interaction with and inactivation of Wag31 (DivIVA), an essential Mycobacterium smegmatis protein organizing cell wall biosynthesis at the growing cellular poles. Fruitloop gene 52 is expressed early in lytic growth and is not required for normal Fruitloop lytic replication but interferes with Subcluster B2 phages such as Hedgerow and Rosebush. We conclude that Hedgerow and Rosebush are Wag31‐dependent phages and that Fruitloop gp52 confers heterotypic superinfection exclusion by inactivating Wag31.     

Characterization of Temperate <Emphasis Type="Italic">Lactobacillus gasseri</Emphasis> Phage LgaI and Its Impact as Prophage on Autolysis of Its Lysogenic Host Strains

We show by electron microscopy that Lactobacillus gasseri phage LgaI, a temperate phage residing in the chromosome of Lactobacillus gasseri ATCC33323, belongs to the family of Myoviridae phages. The LgaI DNA is packed by the “head-full” mechanism, as demonstrated by analysis of restriction patterns of heated (74°C) or non-heated DNA. By isolating prophage-cured cells, we were able to demonstrate phage LgaI to be responsible for the strong autolytic phenotype observed for Lactobacillus gasseri ATCC33323. In addition, we show that a copy of the LgaI prophage resides in the chromosome of Lactobacillus gasseri NCK102. The LgaI prophage was not inducible in L. gasseri NCK102-adh by mitomycin C, however, it apparently contributed to the autolytic phenotype of this strain.     

Isolation of P22 Specialized Transducing Phage following F''-Episome Fusion

A P22 specialized transducing phage has been constructed which carries the structural gene for aspartate transcarbamylase (ATCase). This gene (pyrB) was first brought close to the P22 attachment site by fusing an F' pyrB⁺ episome to an F' prolac episome which carries a P22 prophage attachment site. A prophage was added to these fused F' episomes and the lysogen was UV-induced. The specialized transducing phage was isolated from the resulting lysate. The phage also carries argI, the structure gene for ornithine transcarbamylase.