Harrison Echols

Control of phage λ development by stability and synthesis of cll protein: Role of the viral clll and host hflA, himA and himD genes

The cII protein of bacteriophage lambda has a decisive role in the regulatory switch between the lysogenic and lytic pathways of viral development. Recent work has indicated that cII may be the primary control function providing for the initial partition between the two pathways, with other host and viral regulatory genes acting to determine the levels of cII in an infected cell. We have studied the synthesis and stability of cII protein with two experimental systems, phage infection and a cII-producing plasmid. We have found that the stability of cII is controlled by the host hflA and viral cIII genes; hflA protein facilitates degradation of cII, whereas cIII protects cII. The synthesis of cII appears to be under the positive control of the host himA and himD genes. We conclude that posttranscriptional regulation of cII by host and viral genes is critical for the choice of a developmental pathway.

hflB, a new Escherichia coli locus regulating lysogeny and the level of bacteriophage lambda cII protein

The level of the viral cII protein has been proposed to be the crucial determinant in the lysis-lysogeny decision of bacteriophage lambda. A new Escherichia coli locus (hflB) has been identified in which a mutation (hflB29) leads to high frequency of lysogeny by lambda. A double mutant defective in both hflB and the previously identified hflA gene displays a more severe Hfl- phenotype than either single mutant. The hflB locus is at 69 minutes on the E. coli map, 85% co-transducible with argG. The hflB29 mutation results in increased stability of the phage cII protein (increasing its half-life twofold) and is recessive to hflB+. We conclude that the hflB+ locus is a negative regulator of cII, perhaps coding for or regulating a protease that acts on cII. In addition, we observe that the can1 mutation, an alteration of the cII gene that results in enhanced lysogenization, leads to increased stability of cII protein. These observations reinforce the view that the level of cII is a key factor in the lysis-lysogeny decision of lambda.

Role of the cro gene in bacteriophage λ development

Abstract Previous experiments have shown that the product of the cro gene of baeteriophage λ can exert an anti-repression activity, defined by the capacity of certain “ cro -constitutive” defective lysogens to channel a superinfecting λ phage toward lytic development. We have used a combination of biological and biochemical assays to draw two main conclusions concerning this anti-repression activity: (1) after infection of a cro -constitutive cell, the superinfecting phage is unable to establish repression because it is unable to commence synthesis of c I protein (λ repressor) at a substantial rate; (2) the cause of this diminished synthesis of c I protein is the capacity of cro product to repress synthesis of the c II and c III proteins, which normally activate the c I gene to establish repression in an infected cell. From our experiments and those of others, we suggest that cro product possesses a repression activity which is similar to that of the c I protein itself, but normally exerts a very different physiological role: the turnoff of synthesis of replication, recombination and regulation proteins as the virus enters the late stage of lytic development.

Role of the Xis protein of bacteriophage λ in a specific reactive complex at the attR prophage attachment site

Abstract Phage λ controls its integration and excision by differential catalysis of the forward and reverse reactions. The λ Int protein is required for both directions, but Xis for excision only. Previous electron microscopic observations have shown that Int protein forms a stable, condensed protein-DNA complex with the phage ( att P) and prophage left ( att L) substrate sites, but not with the host ( att B) or prophage right ( att R) sites. We have found that Int and Xis together produce a stable, condensed complex with att R. The att R complex involves the P region DNA to the left of the crossover point (O site). In contrast, the att P complex includes DNA on both sides of the crossover point (P and P′), and the att L structure involves the P′ DNA to the right of O. In the presence of Int and Xis, the att L and att R sites form a paired structure. We conclude that the role of Xis is to provide a distinct reactive structure at att R, allowing att L and att R to pair efficiently.

Effect of mutations in the cII and cIII genes of bacteriophage λ on macromolecular synthesis in infected cells

We have investigated the time-course of macromolecular synthesis following infection by cI−, cII− and cIII− mutants of λ in an effort to understand the role of the cII and cIII genes in the establishment of repression and lysogeny. cII− and cIII− mutants of λ begin to synthesize the “late” proteins tail antigen and lysozyme substantially before a cI− mutant; the shift to late messenger RNA production from the head and tail genes also occurs earlier after cII− or cIII− infection. In contrast, no appreciable difference among cI−, cII−, or cIII− mutants was found in synthesis of the “early” protein λ-exonuclease or in the rate of total or λ-specific DNA synthesis. These results suggest that the λ cII and cIII gene products function to delay the lytic response by inhibiting, directly or indirectly, the production of messenger RNA from the late λ genes. When lysozyme synthesis was studied after infection by cI− cII− and cI− cIII− double mutants, results were obtained which were similar to those found in the case of single cII− and cIII− mutants. The results with the double mutants suggest that the cII and cIII gene products may also participate in the regulation of cI represser synthesis or activity.

The essential role of the cro gene in lytic development by bacteriophage λ

Abstract In an effort to understand the physiological role of the Cro repressor protein of bacteriophage λ we have studied the influence of cro− mutation on lytic and lysogenic development. Effective lytic growth is blocked by cro− mutation under conditions in which the cI repressor protein is fully active or in which active cI protein is not made; however, a partially active cI protein can substitute for Cro. The blocked lytic development under cro− cI− conditions is characterized by a severe inhibition of λ DNA synthesis, and the newly replicated DNA has an aberrant structure. Under cro−cI+ conditions, we have studied the frequency of lysogenization to determine whether the failure of lytic development in this situation results from a complete channeling into the lysogenic pathway. However, the major alteration in the lysogenization pattern for cro− phage is the appearance of a large fraction of surviving, nonlysogenic cells; we interpret this as a failure of normal lytic growth without a concomitant switch to stable lysogeny. From these results and from previous work on Cro function, we conclude that the repression exerted by Cro is essential for normal lytic development, that Cro probably functions as a “weak” repressor compared to cI, and that at least one of the essential regulatory activities of Cro concerns viral DNA replication.

Positive and negative regulation by the cII and cIII gene products of bacteriophage λ

Abstract Previous experiments have indicated that the cII and cIII gene products of bacteriophage λ serve to establish repression in an infected cell through a bifunctional regulatory activity: an activation of synthesis of cI protein (the maintenance repressor) and an inhibition of production of late lytic proteins. We have investigated further the relationship between the two regulatory activities of c II c III by measurements of genetic and environmental influences on synthesis of endolysin and cI protein. A cy− mutation which exhibits a cis dominant defect in positive regulation of the cI gene also is defective in negative regulation of endolysin. These data are consistent with a mechanism in which the cII and cIII proteins act at a single site in the y-region of λ DNA to provide for both positive and negative regulation. Multiplicity of infection exerts a strong influence on endolysin production, dependent on active cII and cIII genes; higher multiplicity favors repression. However, positive regulation of the cI gene is not substantially more effective at higher multiplicity. Thus there exists a capacity for discoordinate expression of the two regulatory activities of c II c III .

RecA protein and SOS: Correlation of mutagenesis phenotype with binding of mutant RecA proteins to duplex DNA and LexA cleavage

The RecA protein of Escherichia coli is required for SOS-induced mutagenesis in addition to its recombinational and regulatory roles. We have suggested that RecA might participate directly in targeted mutagenesis by binding preferentially to the site of the DNA damage (e.g. pyrimidine dimer) because of its partially unwound nature; DNA polymerase III will then encounter RecA-coated DNA at the lesion and might replicate across the damaged site more often but with reduced fidelity. In support of this proposal, we have found that the phenotype of wild-type and mutant RecA for mutagenesis correlates with capacity to bind to double-stranded DNA. Wild-type RecA binds more efficiently to ultraviolet (u.v.)-irradiated, duplex DNA than to non-irradiated DNA. The RecA441 (Tif) protein that is constitutive for mutagenesis binds extremely well to double-stranded DNA with no lesions, whereas the RecA430 protein that is defective in mutagenesis binds poorly even to u.v.-irradiated DNA. The RecA phenotype also correlates with capacity to use duplex DNA as a cofactor for cleavage of the LexA repressor protein for SOS-controlled operons. Wild-type RecA provides efficient cleavage of LexA only with u.v.-irradiated duplex DNA; RecA441 cleaves well with non-irradiated DNA; RecA430 gives very poor cleavage even with u.v.-irradiated DNA. We conclude that the interaction of RecA with damaged double-stranded DNA is likely to be a critical component of SOS mutagenesis and to define a pathway for the LexA cleavage reaction as well.

Positive regulation of integrative recombination by the cII and cIII genes of bacteriophage λ

Abstract We have studied the influence of the c II and c III regulatory genes of bacteriophage λ on the site-specific recombination responsible for integration and excision of the viral DNA. We find that c II/ c III exert positive regulation on integrative but not excisive recombination, and that this regulatory function involves a separate site from that used by c II/ c III to regulate the establishment of repression. Thus the two events responsible for the formation of stable lysogeny, repression and integration, are subject to coordinate positive regulation. From our experiments and those of others, we suggest that the c II/ c III proteins stimulate integrative recombination by promoting transcription of the int but not xis genes.

Repression by the cI protein of phage λ: in vitro inhibition of RNA synthesis☆

We have studied the in vitro repression of RNA synthesis by the cI protein of phage λ. We find that highly purified cI protein is an effective and specific repressor of RNA synthesis from the early gene region of λ DNA. Under optimal conditions at least 95% of the early gene RNA synthesis is repressed and this repression is eliminated or severely impaired by the use of λ DNA-carrying operator-type mutations which reduce the binding affinity of the cI protein. Highly effective repression can be demonstrated only through the use of the initiation-inhibitor rifampicin, which presumably, selects “properly” initiated RNA chains; thus we can by-pass in vitro but not yet solve the problem of how the host polymerase initiates specifically in vivo from the immediate-early promoter sites.