Mutants in the y region of bacteriophage lambda constitutive for repressor synthesis: their isolation and the characterization of the Hyp phenotype

Bacteriophage lambdahyp mutants have been isolated as survivors of Escherichia coli K-12 bacteria lysogenic for lambda Nam7am53cI857. The hyp mutants are characterized by (i) their localization in the y region very close to the imm lambda/imm434 boundary, (ii) polarity on O gene expression, (iii) immediate recovery of lambda immunity at 30 degrees C after prolonged growth of lambda Nam7am53cI857 hyp lysogens at 42 degrees C even in the presence of an active cro gene product, (iv) ability of phage lambda v2v3vs326 but not lambda v1v2v3 to propagate on lambda cI+hyp lysogens, (v) inability to express lambda exonuclease activity after prophage induction, and (vi) inviability at any temperature of phage carrying the hyp mutation. All these properties are referred to collectively as the Hyp phenotype. We show that the Hyp phenotype is due to cII-independent constitutive cI-gene-product synthesis originating in the y region, which results in the synthesis of anti-cro RNA species, and constitutive levels of cro gene product present even in lambda cI+hyp lysogens. A model is presented which is consistent with all the experimental observations.     

Regulation of Bacteriophage P22 DNA synthesis and repressor levels in P22cly infections.

A rifampin-resistant mutant of Salmonella typhimurium carries an altered RNA polymerase. Wild-type (c+) phage P22 displays clear plaques and a reduced lysogenization frequency on this mutant host. The cly mutants of P22 were isolated on the basis of their ability to lysogenize such mutant hosts. Two classes of regulatory events, both of which are dependent on P22 gene c1 activity, are necessary for the establishment of lysogeny in P22. The positive events culminate in repressor synthesis; the negative events cause a retardation in phage DNA synthesis. Neither the positive nor the negative events are observed in P22c+ infections of the mutant host. Both effects are found in P22cly infections of the mutant host. Observable results of both the negative and the positive events are exaggerated in P22cly infections of wild-type hosts as compared to P22c+ infections. The cly mutation apparently increases the positive and negative regulatory events so that they are detectable in the mutant host and exaggerated in wild-type hosts. Possible mechanisms that result in the high frequency of lysogenization that characterizes the cly mutation and the nature of the cly mutation are discussed.     

The cleavage specificity of RNase III.

We determined sites in lambda cII mRNA that are cleaved by RNase III in the presence of lambda OOP antisense RNA, using a series of OOP RNAs with different internal deletions. In OOP RNA-cII mRNA structures containing a potential region of continuous double-stranded RNA bounded by a non-complementary unpaired region, RNase III cleaved the cII mRNA at one or more preferred sites located 10 to 14 bases from the 3'-end of the region of continuous complementarity. Cleavage patterns were almost identical when the presumptive structure was the same continuously double-stranded region followed by a single-stranded bulge and a second short region of base pairing. The sequences of the new cleavage sites show generally good agreement with a consensus sequence derived from thirty-five previously determined cleavage sequences. In contrast, four 'non-sites' at which cleavage is never observed show poor agreement with this consensus sequence. We conclude that RNase III specificity is determined both by the distance from the end of continuous pairing and by nucleotide sequence features within the region of pairing.     

Escherichia coli grpE gene codes for heat shock protein B25.3, essential for both lambda DNA replication at all temperatures and host growth at high temperature.

We have identified the grpE gene product as the B25.3 heat shock protein of Escherichia coli on the following evidence: (i) a protein similar in size and isoelectric point to B25.3 was induced after infection of UV-irradiated bacteria by lambda grpE+ transducing phage, (ii) mutant phage lambda grpE40, isolated by its inability to propagate on grpE280 bacteria, failed to induce the synthesis of the B25.3 protein, and (iii) lambda grpE+ revertants, derived from phage grpE40 as able to propagate on grpE280 bacteria, simultaneously recovered the ability to induce synthesis of the B25.3 protein. In addition, we show that E. coli bacteria carrying the grpE280 mutation are temperature sensitive for bacterial growth at 43.5 degrees C. Through transductional analysis and temperature reversion experiments, it was demonstrated that the grpE280 mutation is responsible for both the inability of lambda to replicate at any temperature tested and the lack of colony formation at high temperature. At the nonpermissive temperature the rates of synthesis of DNA and RNA were reduced in grpE280 bacteria.     

Stability of coliphage lambda DNA replication initiator, the lambda O protein.

The initiator of coliphage lambda DNA replication, lambda O protein, may be detected among other 35S-labeled phage and bacterial proteins by a method based on immunoprecipitation. This method makes it possible to study lambda O proteolytic degradation in lambda plasmid-harboring or lambda phage-infected cells; it avoids ultraviolet (u.v.)-irradiation of bacteria, used for depression of host protein synthesis, prior to lambda phage infection. We confirm the rapid decay of lambda O protein (half-time of 80 s), but we demonstrate the existence of a stable lambda O fraction. In the standard five minute pulse-chase experiments, 20% of synthesized lambda O is stable. The extension of the [35S]methionine pulse, possible in lambda plasmid-harboring cells, leads to a linear increase of this fraction, as if a part of the synthesized lambda O was constantly made resistant to proteolysis. Less than 5% of lambda O protein synthesized during one minute is transformed into a stable form. We presume that the stable lambda O is identical with lambda O present in the normal replication complex and thus protected from proteases. We cannot find any stable lambda O in Escherichia coli recA+ cells that were irradiated with u.v. light prior to lambda phage infection, but their recA- counterparts behave normally, suggesting that recA function interferes in the assembly of a normal replication complex in u.v.-irradiated bacteria. The stable lambda O found in lambda plasmid-harboring, amino acid-starved relA cells is responsible for the lambda O-dependent lambda plasmid replication that occurs in this system in the absence of lambda O synthesis. The existence of stable lambda O raises doubt concerning its role as the limiting initiator protein in the control of replication. Another significance of lambda O rapid degradation is proposed.     

Mutagenesis of ultraviolet-irradiated lambda phage by host cell irradiation: induction of Weigle mutagenesis is not an all-or-none process

Summary Ultraviolet mutagenesis of lambda phage to clear plaque formers is the same in the total phage population and in subpopulations of phage which have also mutated to gam ^- or at an amber codon. This is true for phage assayed in host cells in which Weigle mutagenesis has been either partially induced by low levels of ultraviolet irradiation, or fully induced by higher levels. If induction of Weigle mutagenesis were all-or-none, clear plaque formers in phage subpopulations selected for another mutation elsewhere would come mainly from induced cells; then the clear plaque mutation rate would always be that for fully induced host cells. Therefore, induction requires more than one lesion in host cell DNA.Although thymine starvation of cells induces synthesis of recA protein, it does not induce Weigle mutagenesis; in fact starvation inhibits induction of this process on subsequent ultraviolet irradiation of the cells.     

Organization of the early region of bacteriophage phi 80. Genes and proteins

An EcoRI segment containing the early region of bacteriophage phi 80 DNA that controls immunity and lytic growth was identified as a segment whose presence on a plasmid prevented growth of infecting phi 80cI phage. The nucleotide sequence of the segment (EcoRI-F) and adjacent regions was determined. Based on the positions of amber mutations and the sizes of some gene products, the reading frames for five genes were identified. From the relative locations of these genes in the genome, the properties of some isolated gene products, and the analysis of the structures of predicted proteins, the following phi 80 to lambda analogies are deduced: genes cI and cII to their lambda namesakes; gene 30 to cro; gene 15 to O; and gene 14 to P. An amber mutation by which gene 16 was defined is a nonsense mutation in the frame for gene 15 protein, excluding the presence of gene 16. An amber mutation in gene 14 or 15 inhibits phage DNA synthesis, as is the case with their lambda analogues, gene O or P. Some characteristics of proteins from the early region predicted from their primary structures and their possible functions are discussed.     

Conditionally lethal amber mutations in the dnaA region of the Escherichia coli chromosome that affect chromosome replication.

Three amber mutations, dna-801, dna-803, and dna-806, were isolated by localized mutagenesis of the dnaA-oriC region of the chromosome from an Escherichia coli strain carrying temperature-sensitive amber suppressors. When the mutations were not suppressed at 42 degrees C, the cells did not grow and DNA synthesis was arrested. They were very closely linked to each other and to the dnaA46 mutation. The mutant phenotype of each strain was converted to the wild type by infecting the mutants with specialized transducing phase lambda i21 dnaA-2 but not with lambda i21 tna. Derivatives of lambda i21 dnaA-2, each of which carried the amber mutation dna-801 dna-803, or dna-806, converted the dnaA mutant phenotype to Dna+ but did not convert rhe amber mutants to the wild-type phenotype. E. coli uvrB cells were irradiated with ultraviolet light and infected with each of these phage strains. An analysis of proteins synthesized in the cells revealed that two proteins with molecular weights of 50,000 and 43,000 were specified by lambda i21 dnaA-2 but not by lambda i21 tna. When the ultraviolet-irradiated cells did not carry an amber suppressor, the derivative phage with the amber mutation invariably failed to produce the 43,000-dalton protein, but when the host cell carried supF (tyrT), the protein was produced. The 50,000-dalton protein was unaffected.     

tif-Stimulated deoxyribonucleic acid repair in Escherichia coli K-12

Bacterial survival is significantly increased after ultraviolet irradiation in tif sfi cells, provided that the thermosensitive tif mutation has been expressed at 41 degrees C before irradiation. This tif-mediated "reactivation of ultraviolet irradiated bacteria" needs de novo protein synthesis, as is the case for the tif-mediated reactivation of ultraviolet-irradiated phage lambda. However, in striking contrast to the phage reactivation process, this tif-mediated reactivation is no longer associated with mutagenesis. It also requires the presence of the uvrA+ excision function. These results strongly suggest the existence in Escherichia coli K-12 of a repair pathway acting on bacterial deoxyribonucleic acid which is inducible, error free, and uvr dependent.     

Rec-Mediated Recombinational Hot Spot Activity in Bacteriophage Lambda. I. Hot Spot Activity Associated with Spi- Deletions and bio Substitutions

In order to survey the distribution along the bacteriophage lambda chromosome of Rec-mediated recombination events, crosses are performed using conditions which block essentially all DNA synthesis. One parent is density-labeled and carries a genetic marker in the left terminal lambda gene (A), while the other parent is unlabeled and carries a genetic marker in the right terminal lambda gene (R). Both parents are deleted for the lambda recombination genes int and red, together with other recombination-associated genes, by virtue of either (1) a pure deletion or (2) a bio insertion-deletion. The distribution in a cesium density gradient of the resulting A+R+ recombinant phage reflects the chromosomal distribution of the recombination events which gave rise to those phage.Crosses employing either of two different pure deletion phage strains exhibit recombinational hot spot activity located near the right end of the lambda chromosome, between the cI and R genes. This hot spot activity persists when unlimited DNA synthesis is allowed. Crosses employing bio1-substituted phage strains exhibit recombinational hot spot activity located to the right of the middle of the chromosome and to the left of the cI gene. Crosses employing either bio1 or bio69-substituted phage strains indicate that the bio-associated hot spot activity occurs in the presence of DNA synthesis, but is dependent on a functional host recB gene.     

Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli host cells irradiated with ultraviolet light

Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr+ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one to two mutant phage per mutant burst. From this and the pathways of lambda DNA synthesis, it can be argued that non-targeted mutagenesis involves a loss of fidelity in semiconservative DNA replication. A series of experiments with various mutant host cells showed a major pathway of non-targeted mutagenesis by ultraviolet light, which acts in addition to "SOS induction" (where cleavage of the LexA repressor by RecA protease leads to din gene induction): (1) the induction of mutants has the same dependence on irradiation for wild-type and for umuC host cells; (2) a strain in which the SOS pathway is constitutively induced requires irradiation to the same level as wild-type cells in order to fully activate non-targeted mutagenesis; (3) non-targeted mutagenesis occurs to some extent in irradiated recA recB cells. In cells with very low levels of PolI, the induction of non-targeted mutagenesis by ultraviolet light is enhanced. We propose that the major pathway for non-targeted mutagenesis in irradiated host cells involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and that the low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.     

Cleavage by RNase P of gene N mRNA reduces bacteriophage lambda burst size.

RNase P, an enzyme essential for tRNA biosynthesis, can be directed to cleave any RNA when the target RNA is in a complex with a short, complementary oligonucleotide called an external guide sequence (EGS). RNase P from Escherichia coli can cleave phage lambda N mRNA in vitro or in vivo when the mRNA is in a complex with an EGS. The EGS can either be separate from or covalently linked to M1 RNA, the catalytic RNA subunit of RNase P. The requirement for Mg2+ in the reaction in vitro is lower when the EGS is covalently linked to M1 RNA. Substrates made of DNA can also be cleaved by RNase P in vitro in complexes with RNA EGSs. When either kind of EGS construct is used in vivo, burst size of phage lambda is reduced by > or = 40%. Reduction in burst size depends on efficient expression of the EGS constructs. The product of phage lambda gene N appears to function in a stoichiometric fashion.     

Isolation and analysis of Escherichia coli mutants that allow increased replication of bacteriophage lambda.

Escherichia coli mutants were isolated that supported the growth of a lambda Ots and, in at least one case, a lambda Bts phage at the normally nonpermissive temperature of 39 degrees C. In one such strain, Ots and Bts suppression ability appeared to be a function of the guaB gene. Ots suppression by the mutant guaB strain was prevented if high levels of guanine or xanthine were present in the medium. No other base had any effect on Ots suppression in this strain. Other strains carrying spontaneous mutations resulting in guanine or xanthine auxotrophy (guaA or guaB lesions, respectively) all allowed lambda Ots replication at 39 degrees C; Ots suppression in these strains was also abolished by addition of guanine to the medium. Thus, reduced intracellular guanine levels resulting from guaA or guaB mutations appeared to suppress the inability of lambda Ots and, at least in some cases, Bts bacteriophage to form plaques at 39 degrees C. In burst size experiments, a guaB mutant produced a larger phage yield per infected cell of both lambda Ots and lambda O+ phage at 39 degrees C than did a similar guaB+ strain. It appeared that a lower-than-normal level of guanine (or a guanine derivative) in these cells may permit unusually efficient lambda replication. The fact that O+ and lambda Ots bursts in the guaB mutant were reduced significantly by addition of exogenous guanine to the medium is consistent with this suggestion. Another strain that suppresses the Ots allele has no known auxotrophic requirements, and suppression in this strain was unaffected by addition of guanine to the medium; however, addition of cytidine to the medium specifically eliminated Ots suppression in this strain. The mutation responsible for allowing Ots replication in this strain is unknown.