Effect of salt shock on stability of lambdaimm434 lysogens

The affinities of the bacteriophage 434 repressor for its various binding sites depend on the type and/or concentration of monovalent cations. The ability of bacteriophage 434 repressor to govern the lysis-lysogeny decision depends on the DNA binding activities of the phage's cI repressor protein. We wished to determine whether changes in the intracellular ionic environment influence the lysis-lysogeny decision of the bacteriophage lambda(imm434). Our findings show that the ionic composition within bacterial cells varies with the cation concentration in the growth media. When lambda(imm434) lysogens were grown to mid-log or stationary phase and subsequently incubated in media with increasing monovalent salt concentrations, we observed a salt concentration-dependent increase in the frequency of bacteriophage spontaneous induction. We also found that the frequency of spontaneous induction varied with the type of monovalent cation in the medium. The salt-dependent increase in phage production was unaffected by a recA mutation. These findings indicate that the salt-dependent increase in phage production is not caused by activation of the SOS pathway. Instead, our evidence suggests that salt stress induces this lysogenic bacteriophage by interfering with 434 repressor-DNA interactions. We speculate that the salt-dependent increase in spontaneous induction is due to a direct effect on the repressor's affinity for DNA. Regardless of the precise mechanism, our findings demonstrate that salt stress can regulate the phage lysis-lysogeny switch.     

Precise excision of bacteriophage Mu DNA

The temperate bacteriophage Mu is a transposable element that can integrate randomly into bacterial DNA, thereby creating mutations. Mutants due to an integrated Mu prophage do not give rise to revertants, as if Mu, unlike other transposable elements, were unable to excise precisely. In the present work, starting with a lacZ::Muc62(Ts) strain unable to form Lac+ colonies, we cloned a lacZ+ gene in vivo on a mini-Mu plasmid, under conditions of prophage induction. In all lac+ plasmids recovered, the wild-type sequence was restored in the region where the Mu prophage had been integrated. The recovery of lacZ+ genes shows that precise excision of Mu does indeed take place; the absence of Lac+ colonies suggests that precise excision events are systematically associated with loss of colony-forming ability.     

Bacteriophage crosstalk: coordination of prophage induction by trans-acting antirepressors

Many species of bacteria harbor multiple prophages in their genomes. Prophages often carry genes that confer a selective advantage to the bacterium, typically during host colonization. Prophages can convert to infectious viruses through a process known as induction, which is relevant to the spread of bacterial virulence genes. The paradigm of prophage induction, as set by the phage Lambda model, sees the process initiated by the RecA-stimulated self-proteolysis of the phage repressor. Here we show that a large family of lambdoid prophages found in Salmonella genomes employs an alternative induction strategy. The repressors of these phages are not cleaved upon induction; rather, they are inactivated by the binding of small antirepressor proteins. Formation of the complex causes the repressor to dissociate from DNA. The antirepressor genes lie outside the immunity region and are under direct control of the LexA repressor, thus plugging prophage induction directly into the SOS response. GfoA and GfhA, the antirepressors of Salmonella prophages Gifsy-1 and Gifsy-3, each target both of these phages' repressors, GfoR and GfhR, even though the latter proteins recognize different operator sites and the two phages are heteroimmune. In contrast, the Gifsy-2 phage repressor, GtgR, is insensitive to GfoA and GfhA, but is inactivated by an antirepressor from the unrelated Fels-1 prophage (FsoA). This response is all the more surprising as FsoA is under the control of the Fels-1 repressor, not LexA, and plays no apparent role in Fels-1 induction, which occurs via a Lambda CI-like repressor cleavage mechanism. The ability of antirepressors to recognize non-cognate repressors allows coordination of induction of multiple prophages in polylysogenic strains. Identification of non-cleavable gfoR/gtgR homologues in a large variety of bacterial genomes (including most Escherichia coli genomes in the DNA database) suggests that antirepression-mediated induction is far more common than previously recognized.     

The antirepressor needed for induction of linear plasmid-prophage N15 belongs to the SOS regulon

The physiological conditions and molecular interactions that control phage production have been studied in only a few families of temperate phages. We investigated the mechanisms that regulate activation of lytic development in lysogens of coliphage N15, a prophage that is not integrated into the host chromosome but exists as a linear plasmid with covalently closed ends. We identified the N15 antirepressor gene, antC, and showed that its product binds to and acts against the main phage repressor, CB. LexA binds to and represses the promoter of antC. Mitomycin C-stimulated N15 induction required RecA-dependent autocleavage of LexA and expression of AntC protein. Thus, a cellular repressor whose activity is regulated by DNA damage controls N15 prophage induction.     

Commitment to Lysogeny Is Preceded by a Prolonged Period of Sensitivity to the Late Lytic Regulator Q in Bacteriophage λ

A key event in development is the irreversible commitment to a particular cell fate, which may be concurrent with or delayed with respect to the initial cell fate decision. In this work, we use the paradigmatic bacteriophage λ lysis-lysogeny decision circuit to study the timing of commitment. The lysis-lysogeny decision is made based on the expression trajectory of CII. The chosen developmental strategy is manifested by repression of the pR and pL promoters by CI (lysogeny) or by antitermination of late gene expression by Q (lysis). We found that expression of Q in trans from a plasmid at the time of infection resulted in a uniform lytic decision. Furthermore, expression of Q up to 50 min after infection results in lysis of the majority of cells which initially chose lysogenic development. In contrast, expression of Q in cells containing a single chromosomal prophage had no effect on cell growth, indicating commitment to lysogeny. Notably, if the prophage was present in 10 plasmid-borne copies, Q expression resulted in lytic development, suggesting that the cellular phage chromosome number is the critical determinant of the timing of lysogenic commitment. Based on our results, we conclude that (i) the lysogenic decision made by the CI-Cro switch soon after infection can be overruled by ectopic Q expression at least for a time equivalent to one phage life cycle, (ii) the presence of multiple λ chromosomes is a prerequisite for a successful Q-mediated switch from lysogenic to lytic development, and (iii) phage chromosomes within the same cell can reach different decisions.     

Induction of prophage lambda does not require full induction of RecA protein synthesis

In mitomycin C-treated lambda lysogens, even though the rate of synthesis of RecA protein was greatly reduced by a low concentration of rifampicin (4 microgram/ml), induction of prophage lambda occurred readily as assessed by (i) cell lysis of the lysogens, (ii) production of progeny phage, and (iii) extensive cleavage of lambda repressor. The extent and the rate of cleavage of lambda repressor were not significantly affected by the low rate of synthesis of RecA protein resulting from rifampicin action. However, the yield of phage progeny was reduced and lysis of the cells was slightly delayed. We conclude that in RecA+ bacteria, induction of prophage lambda does not require full induction of RecA protein synthesis.     

Effect of mutations of Escherichia coli K-12 chromosome on the integration of bacteriophage Mu introduced into cells by infection or conjugation

We present the detailed research on the previously described Escherichia coli K-12 Mud- mutants with impaired development of bacteriophage Mu. The ability of Mu phage DNA to penetrate into mutant cells on infection was shown. If introduced into the cells or combined with mud mutation by recombination, the prophage may be induced, which results in phage Mu lythic development and phage burst from mutant cells. In the course of conjugative transfer into the mutant cells, within a DNA fragment of the lysogenic donor chromosome, MupAp1 prophage is not inherited by recombinants. At the same time, Mu prophage deficient in genes A and B, whose products are required for transposition, is inherited by the mutant with the usual frequency. These data enable us to conclude that the mud mutations disturb the stage of conservative transposition which is connected with the insertion of the Mu prophage into the chromosome, after excision from the linear DNA introduced into the cells via infection or conjugation.     

Inactivation of prophage lambda repressor in vivo.

Jacob &; Monod (1961) postulated that prophage A induction results from the inactivation of the λ repressor by a cellular inducer. Although it has been shown that the phage A repressor is inactivated by the recA gene product in vitro (Roberts et al., 1978), we wanted to determine the action of the “cellular inducer” in vivo. Our results have led to a new model, which defines the relationship between the “cellular inducer” and the recA gene product.In order to quantitate the action of the cellular inducer on the λ repressor, we made use of bacteria with elevated cellular levels of the λ repressor (hyperimmune lysogens). We determined the kinetics of repressor inactivation promoted by three representative inducing treatments: ultraviolet light irradiation, thymine deprivation and temperature shift-up of tif-1 mutants.The kinetics of repressor decay in wild-type monolysogens indicate that repressor inactivation is a relatively slow cellular process that takes a generation time to reach completion. Incomplete inactivation of the repressor without subsequent prophage development may occur in a cell. We call this phenomenon detected at the biochemical level “subinduction”. In hyperimmune lysogens. subinduction is always the case.A high cellular level of A repressor that prevents prophage λ induction does not prevent induction of a heteroimmune prophage such as 434 or 80. Although the cellular inducer does not seem specific for any inducible prophage, it does not inactivate two prophage repressors present in a cell in a random manner. We have called this finding “preferential repressor inactivation”. Preferential repressor inactivation may be accounted for by considering that the intracellular concentration of a repressor determines its susceptibility to the action of the inducer.In bacteria with varying repressor levels, a fixed amount of repressor molecules is inactivated per unit of time irrespective of the initial repressor concentration. The rate of repressor inactivation depends on the catalytic capacity of the cellular inducer that behaves as a saturated enzyme. In wild-type bacteria the cellular inducer seems to be produced in a limited amount, to have a weak catalytic capacity and a relatively short half-life. The amount of the inducer formed after tif-1 expression is increased in STS bacteria overproducing a tif-1-modified RecA protein. This result is an indication that a modified form of the RecA protein causes repressor inactivation in vivo.From the results obtained we propose a model concerning the formation of the cellular inducer. We postulate that the cellular inducer is formed in a two-step reaction. The is model visualises how the RecA protein can be induced to high cellular concentrations, even though the RecAp protease molecules remain at a low concentration. The latter accounts for the limited proteolytic activity found in vivo.     

DNA sequence of the control region of phage D108: the N-terminal amino acid sequences of repressor and transposase are similar both in phage D108 and in its relative, phage Mu.

We have determined the DNA sequence of the control region of phage D108 up to position 1419 at the left end of the phage genome. Open reading frames for the repressor gene, ner gene, and the 5' part of the A gene (which codes for transposase) are found in the sequence. The genetic organization of this region of phage D108 is quite similar to that of phage Mu in spite of considerable divergence, both in the nucleotide sequence and in the amino acid sequences of the regulatory proteins of the two phages. The N-terminal amino acid sequences of the transposases of the two phages also share only limited homology. On the other hand, a significant amino acid sequence homology was found within each phage between the N-terminal parts of the repressor and transposase. We propose that the N-terminal domains of the repressor and transposase of each phage interact functionally in the process of making the decision between the lytic and the lysogenic mode of growth.     

The Escherichia coli protein, Fis: specific binding to the ends of phage Mu DNA and modulation of phage growth

We show, using gel retardation, that crude Escherichia coli cell extracts contain a protein which binds specifically to DNA fragments carrying either end of the phage Mu genome. We have identified this protein as Fis, a factor involved in several site-specific recombinational switches. Furthermore, we show that induction of a Mucts62 prophage in a fis lysogen occurs at a lower temperature than that of a wild-type strain, and that spontaneous induction of Mucts62 is increased in the fis mutant. DNasel footprinting using either crude extracts or purified Fis indicate that binding on the left end of Mu occurs at a site which overlaps a weak transposase binding site. Thus, Fis may modulate Mu growth by influencing the binding of transposase, or other proteins, to the transposase binding site(s), in a way similar to its influence on Xis binding in phage lambda.     

Genetic characterization of Mu-like bacteriophage D108.

Infection of Escherichia coli by bacteriophage D108 was shown to result in the generation of apparently random chromosomal mutations. Approximately 1% of the cells lysogenized by D108, as with Mu, acquired new auxotrophic mutations. D108-induced mutations were nonreverting and were most probably the result of insertion of the D108 genome into regions of genetic function. D108 and Mu shared many similar properties but were heteroimmune and had different host ranges. Lytic infections of Mu lysogens with D108 and D108 lysogens with Mu resulted in 100-fold increases in release of phage with prophage markers over those due to spontaneous induction. Phenotypic mixing was common, with most phage carrying the prophage immunity being packaged in particles with the host range of the superinfecting phage. A fraction of the superinfecting phage genomes were, however, packaged in particles with the prophage-specified host range. Although 10% of the prophage progeny were D108-Mu genetic hybrids, superinfecting phage-induced release of the prophage with reciprocal phenotypic mixing occurred in recA hosts, in which the frequency of D108-Mu genetic hybrids was reduced 100-fold.     

Effect of the polymerase activity of DNA polymerase I on the development of moderate bacteriophages Mu, lambda red- and lambda red-gam-. II. The effect of the polymerase activity of DNA polymerase I on the development of bacteriophage Mu

The paper reports on the influence of polymerizing activity of DNA-polymerase I on different developmental stages of temperate bacteriophage Mu in Escherichia coli K-12 cells. This activity is shown to be necessary for optimization of phage Mu primary integration into cell chromosomes. The relative frequency of Mu integration into bacterial chromosomes is 5-6 times lower in polA cells than in isogenic polA+ control strains, the phage yield from cells being delayed during the phage infectious development, but not in the course of induction from the prophage state. Data have been obtained that show the process of phage Mu DNA integration into the plasmid pRP1 .2 and the process of Mu transposition from the cell chromosome into the plasmid to be independent of the polymerizing activity of DNA-polymerase I.