Role of the cro gene in bacteriophage lambda development

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 cI protein (λ repressor) at a substantial rate; (2) the cause of this diminished synthesis of cI protein is the capacity of cro product to repress synthesis of the cII and cIII proteins, which normally activate the cI 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 cI 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.     

Mutations in the right operator of bacteriophage lambda: physiological effects

The right operator in bacteriophage lambda vs326 has one-twentieth the in vitro binding affinity for repressor as λv⁺; for comparison λv3 has one-quarter the affinity of λv⁺. In vivo, both mutants constitutively express genes in the right operon. Both λv3 and λvs326 express gene O constitutively because they complement λimm434Oam^? in a λ lysogen, vs, more efficiently than v3. The v3 allele in cis (but not in trans) to vs326 gives significantly greater phage yields in a λ lysogen than λvs326 alone, cro gene function, measured by arrest of exonuclease synthesis, suggested the following series of increasing degree of conatitutivity: v3, vs326, v3 vs326. λv2 vs326 forms plaques on lysogens that carry λcI857, but λv2 v3 does not. These results indicate that vs326, like v3, is an operator constitutive mutation but stronger in its effects. These mutants exemplify a uniform correlation between relative weakness of repressor binding and degree of constitutive gene expression.     

Modulation of Lactobacillus casei bacteriophage A2 lytic/lysogenic cycles by binding of Gp25 to the early lytic mRNA

The genetic switch of Lactobacillus casei bacteriophage A2 is regulated by the CI protein, which represses the early lytic promoter P_R and Cro that abolishes expression from the lysogenic promoter P_L. Lysogens contain equivalent cI and cro‐gp25 mRNA concentrations, i.e., CI only partially represses P_R, predicting a lytic cycle dominance. However, A2 generates stable lysogens. This may be due to Gp25 binding to the cro‐gp25 mRNA between the ribosomal binding site and the cro start codon, which abolishes its translation. Upon lytic cycle induction, CI is partially degraded, cro‐gp25 mRNA levels increase, and Cro accumulates, launching viral progeny production. The concomitant concentration increase of Gp25 restricts cro mRNA translation, which, together with the low but detectable levels of CI late during the lytic cycle, promotes reentry of part of the cell population into the lysogenic cycle, thus explaining the low proportion of L. casei lysogens that become lysed (～ 1%). A2 shares its genetic switch structure with many other Firmicutes phages. The data presented may constitute a model of how these phages make the decision for lysis versus lysogeny.     

High-level expression of genes under control of cro translation initiation signals in Escherichia coli

The expression of several genes in Escherichia coli under the control of the lambda p_R promoter and translation initiation signals of the lambda cro gene were studied. Fusions were made in frame at the initiation codon and/or with 5′ translated cro fragments. Expression fluctuated strongly when genes were fused directly at the ATG, whereas constructs, which encode hybrid genes that include at least the first nine codons of the cro gene, always directed high-level synthesis. These fusion proteins were mainly intracellularly precipitated. Our results confirm the poor reliability of ATG vectors for the expression of cloned genes. On the other hand, useful levels of expression are obtained when genes are fused to 5′ cro coding sequences, presumably due to an efficient ribosome binding site configuration.     

Mutational analysis of the operators of bacteriophage lambda

Summary O ^c mutations in the operators of bacteriophage lambda have been used to analyze the functional organization of the operators. In each operator, repressor binding sites 1 and 2, as identified biochemically, were found to be primarily responsible for the repressor affinity of the operators in vitro and for the repression of lytic functions in vivo. In addition, both sites were shown to be involved in the action of cro product at the operators. The data obtained have been used to estimate the repressor affinities of the individual binding sites. These affinities suggest that repressor bound at O _R1 and O _R2 interacts cooperatively. The results obtained support a model for repression of the early lambda operons where repressor bound at binding sites 1 and 2 interferes with RNA polymerase binding to the promoter sites.     

Kinetics of bacteriophage lambda repressor synthesis directed by the PRE promoter: influence of temperature, multiplicity of infection, and mutation of PRM or the cro gene

Summary Expression of the P _RE (establishment) pathway for repressor synthesis is regulated both by phage-specific genetic elements and by physiological conditions. Here we describe the effects of temperature, multiplicity of infection, mutations in the cro gene, and a mutation in P _RM on P _RE-directed repressor synthesis. As Reichardt (1975a) has shown, repressor synthesis begins 5–15 min after infection by wildtype phage, and is shut off at 20–30 min after infection, depending on the temperature. At 43°, synthesis starts sooner, shuts off earlier, and leads to lower repressor levels than are attained at lower temperatures. Experiments with the temperature sensitive mutant crots20 demonstrate that, as had been shown previously in experiments at 30° and 37° C, cro protein is responsible for the shut-off of repressor synthesis at 43°. In addition to the effects of temperature, the kinetics of repressor synthesis are strongly affected by multiplicity of infection (moi). At mois greater than 10, repressor synthesis after infection by wildtype at 30° is dramatically inhibited. Unexpectedly, the P _RM mutation prm116, under certain conditions, can alleviate both cro-mediated shutoff and the inhibition of P _RE-directed repressor synthesis at high moi. These effects of prm116 are observed only at low temperature (30°–32° C) and at mois of about 6–10 or greater; they also appear to be cis-specific. Possible mechanisms for the effects of the prm116 mutation are discussed. Finally, these studies demonstrate that crots20, which was isolated as a temperature-sensitive lethal mutation in the cro gene (Herskowitz, unpublished), is temperature-sensitive with respect to the ability to shutoff P _RE-directed repressor synthesis; however, even at low temperature (30° C), the crots20 gene product is only partially active.     

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.     

Lambda phage cro repressor: Non-specific DNA binding

The interaction of lambda phage cro repressor with double-stranded non-specific DNA has been investigated by monitoring the quenching of its intrinsic tyrosyl fluorescence. The McGhee & von Hippel (1974) analysis of the binding of cro repressor to DNA showed that cro repressor undergoes structural variations in the ionic strength range from 0.04 to 0.18m-KCl. Under these salt conditions, the excluded binding site size of cro repressor on the DNA lattice changes from three to four base-pairs (6 to 8 nucleotides) at the lower ionic strengths, to seven to eight base-pairs (14 to 16 nucleotides) at the higher ionic strength. Quaternary structure variation, which does not cause the excluded site size variation, was also noted at low ionic strengths. Evidence is presented to indicate that cro repressor binds only one side of the DNA helix, such that cro repressor covers a stretch of 14 to 16 nucleotides along one side of the helix in the presence of 0.2 m-salt. Under conditions where the cro repressor structure is constant, approximately nine ion-pairs are formed in the cro repressor-non-specific DNA complex. These results are in agreement with the model proposed by Anderson et al. (1981).     

Changes in the Functional Activity of Phi11 Cro Protein is Mediated by Various Ions

Phi11, a temperate bacteriophage of Staphylococcus aureus, has been found to harbor a cro repressor gene which facilitates Phi11 to adopt the lytic mode of development. The Cro protein has been found to bind very specifically to a 15-bp operator DNA, located in the Phi11 cI–cro intergenic region [1]. To investigate the effects exerted by different ions upon the interaction between Cro and its cognate operator DNA, we have employed gel shift assays as well as circular dichroism spectral analysis. In this communication, we have shown that NH₄ ⁺ and acetate^? ions better facilitated the binding of Cro with its cognate operator as compared to Na⁺, K⁺ and Li⁺. Interestingly, Mg²⁺, carbonate^2? and Citrate^3? have an inhibitory effect upon the binding. The effect of the said ions upon the structure of Cro was also investigated by circular dichroism and it was found that other than Citrate^3? ions, none of the other ions destabilised the protein. On the other hand, Mg²⁺ and carbonate^2? ions maintained the structure of the protein but severely hampered its functional activity. Citrate^3? ions severely unfolded Cro and also inhibited its function. Considering all the data, NH₄ ⁺ and acetate^? ions appeared to be more suitable in maintaining the biological activity of Cro.     

A ‘phase-shift’ fusion system for the regulation of foreign gene expression by lambda represser in Gram-negative bacteria

A ‘phase-shift’ translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BelI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the λ cro gene. The λ cro gene is expressed from promoter p_R and controlled by a thermosensitive (cI857) λ repressor. The usefulness of the expression vector was demonstrated using a gal gene lacking the ATG start codon and fusing this to the p_R promoter and ATG start codon of the λ cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5′-GATC-3′) at the N terminus (provided, for example, by a BamHI linker). The A λ cI857-p_R-cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful λ p_R promoter and the efficient A repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.     

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.     

Synthesis of Bacteriophage λ Proteins <Emphasis Type="Italic">in vitro</Emphasis>

λ DNA stimulates a low level of protein synthesis in extracts of E. coli supplemented with RNA polymerase. The synthesis is efficiently and specifically repressed by addition of purified λ repressor. About 90% of the protein product is from the rightwards, or tof, promoter (P _R ) and 10% from the leftwards, or N, promoter (P _L ). The main polypeptide product is a low molecular species from the tof operon.     

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.