Primary structure of an EcoRI fragment of lambda imm434 DNA containing regions cI-cro of phage 434 and cII-o of phage lambda.

Digestion of phage lambda imm434 DNA with restriction endonuclease EcoRI yields 7 fragments. The shortest among them (1287 bp) contains the right part of the phage 434 immunity region and the phage DNA portion proximal to it. The complete primary structure of this fragment has been determined using the chemical method of DNA sequencing. Hypothetical amino-acid sequences of proteins coded by the cro gene of phage 434 and the cII gene of phage lambda, as well as NH2-terminal amino-acid sequences of the cI protein of phage 434 and the O protein of phage lambda, have been deduced solely on the basis of the DNA sequence. The fragment studied contains also the pR and probably prm promoters and the oR operator of phage 434. The sequence coding for them differs from the respective DNA sequence of phage lambda.     

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.     

Coliphage 434 tofprotein: NH2-terminal amino acid sequence and kinetic and equilibrium measurements of DNA binding

Summary Coliphage 434 tof protein was purified to a substantially pure state from imm ⁴³⁴ cI dv carrier cells. The minimum molecular weight is 7,500±500 as estimated by polyacrylamide gel electrophoresis. The amino acid sequence of the nine NH₂-terminal residues was determined, by manual Edman degradation of the intact protein, to be Met-Gln-Thr-Leu-Ser-Glu-Arg-Leu-(Lys)-.The purified protein at low concentrations binds specifically to imm ⁴³⁴dv DNA and at high concentrations also binds to imm ²¹dv and dv DNA. The curve of the specific binding is of Michaelis type, while that of the nonspecific binding is sigmoidal. The specific binding does not show marked temperature dependency at 4°–37°C. We have analyzed the equilibrium and kinetic data of specific binding. The equilibrium dissociation constant is 1.9x10^-11M at 0°C. The association rate constant and the dissociation rate constant are 1.1–2.9x10⁸M^-1s^-1 and 2.7x10^-3s^-1, respectively, at 0°C. The half life of the tof protein-operator DNA complex is 260s. These results suggest that the tof protein-operator interaction is much weaker than the interaction between the cI repressor and the operator reported by other workers.     

Purification and some properties of presumptive tof gene product of Coli phage 434.

Summary The presumptive tof gene product of Coli phage 434 has been purified from cells carrying imm–⁴³⁴ cIdv plasmid known to contain only some of the early genes of phage 434 and . It was detected and tentatively identified as tof protein primarily by its ability to specifically bind to phage 434 DNA. The protein has a molecular weight of about 11,000 and requires Mg²⁺ for specific DNA binding, unlike 434 cI-repressor.     

Nucleotide sequence of the cro-cII-oop region of bacteriophage 434 DNA.

The nucleotide sequence of a 869 bp segment of phage 434 DNA including the regulatory genes cro and cII is presented and compared with the corresponding part of the phage lambda DNA sequence. The 434 cro protein as deduced from the DNA sequence is a highly basic protein of 71 amino acid residues with a calculated molecular weight of 8089. While the cro gene sequences of phage 434 and lambda DNA are very different, the nuleotide sequences to the right of the lambda imm434 boundary show differences only at 11 out of 512 positions. Nucleotide substitutions in the cII gene occur with one exception in the third positions of the respective codons and only one out of several DNA regulatory signals located in this region of the phage genomes is affected by these nucleotide substitutions.     

DNA sequence of the att region of coliphage 434

Phages lambda and 434 are related phages that insert at the same site on the Escherichia coli chromosome. A 5.9-kb SalI-BamHI fragment derived from phage 434 was shown to hybridize to a 0.5-kb probe carrying attP-lambda. A 0.8-kb Bam HI-TaqI fragment subcloned into pBR327 was used for sequencing. The sequence of the 500 bp around the insertion site is given here, Comparison of the lambda and 434 sequence shows that the following regions are conserved: the coding sequence for the integrase protein (only 162 bp have been sequenced corresponding to the carboxy terminus), the 15-bp common core at the insertion site, and the three integrase-binding sites flanking the insertion site. The lambda and 434 sequences diverge radically to the left of base-197, suggesting that DNA to the left of that point plays no specific role in insertion or its regulation.     

The bacteriophage 434 repressor dimer preferentially undergoes autoproteolysis by an intramolecular mechanism

Inactivation of the lambdoid phage repressor protein is necessary to induce lytic growth of a lambdoid prophage. Activated RecA, the mediator of the host SOS response to DNA damage, causes inactivation of the repressor by stimulating the repressor's nascent autocleavage activity. The repressor of bacteriophage lambda and its homolog, LexA, preferentially undergo RecA-stimulated autocleavage as free monomers, which requires that each monomer mediates its own (intramolecular) cleavage. The cI repressor of bacteriophage 434 preferentially undergoes autocleavage as a dimer specifically bound to DNA, opening the possibility that one 434 repressor subunit may catalyze proteolysis of its partner subunit (intermolecular cleavage) in the DNA-bound dimer. Here, we first identified and mutagenized the residues at the cleavage and active sites of 434 repressor. We utilized the mutant repressors to show that the DNA-bound 434 repressor dimer overwhelmingly prefers to use an intramolecular mechanism of autocleavage. Our data suggest that the 434 repressor cannot be forced to use an intermolecular cleavage mechanism. Based on these data, we propose a model in which the cleavage-competent conformation of the repressor is stabilized by operator binding.     

Analysis of the Escherichia coli proBA locus by DNA and protein sequencing

A 2.9 kb DNA fragment carrying the Escherichia coli proBA region, which encodes the first two enzymes of the proline biosynthetic pathway, was subcloned onto an expression plasmid carrying both the bacteriophage lambda PL promoter (lambda PL) and the lambda gene encoding a thermolabile cI repressor protein (cI857). Derepression of the lambda PL promoter by thermal inactivation of the cI857 repressor protein resulted in the simultaneous overproduction of the proB (gamma-glutamyl kinase) and proA (gamma-glutamyl phosphate reductase) gene products. Nucleotide sequence analysis of the proBA locus allowed gene assignments consistent with the NH2 and COOH-terminal analyses and amino acid compositions of homogeneous preparations of the proB and proA proteins. The contiguous nature of the proB and proA genes suggests that the two genes constitute an operon in which proB precedes proA.     

C1 and cro repressors of lambda phages. I. Construction of vectors for expression of cro repressor of bacteriophage lambda imm434

Eight derivatives of recombinant plasmid pBRcro434, that consists of pBR322 and fragment of immunity region of phage lambda imm434 have been constructed and characterised. These derivatives contain the deletions in the region adjacent to OR3 operator and in the structural gene of cro-repressor of lambda imm434. The deletions have been produced by the treatment of pBRcro434 with exonuclease III of Escherichia coli and S1 nuclease of Aspergillus orizae and precisely mapped. The unique EcoRI-restriction sites have been reconstructed with the aim of using this deletion plasmids as a vectors for cloning.     

Role of the tof gene in the production and perpetuation of the lambdadv plasmid.

A series of lambda derivatives carrying tof mutations were tested for their ability to give rise to plasmid lambda dv. Phages carrying tof mutations that distorted expression of the pRoR-tof-OP operon, were unable to produce lambda dv. Phages carrying an altered tof gene, having only a moderate effect on the same operon, produced unstable lambdadv's. On the other hand, those tof mutants were only the expression of the pLoL-N-exo operon, but not that of the pRoR-tof-OP operon was affected, produced stable lambdadv's.     

Construction of lambda gt103, a derivative of phage lambda gt10 that has unique EcoRI, NotI, SacI and SpeI sites and retains positive selection for recombinants.

A phage vector, lambda gt103, that has unique EcoRI, NotI, SacI and SpeI sites within the imm434 cI repressor gene, was constructed by PCR-aided site-directed mutagenesis of lambda gt10 [Huynh et al., DNA Cloning Techniques: A Practical Approach, 1985, pp. 49-78]. This vector allows directional cloning and retains positive selection for recombinants on Escherichia coli C600hfl strains (since only phages with disrupted cI genes plate on this host). Libraries made with this phage vector can be efficiently screened for clones in which a part of the insert is homologous to probe DNAs derived from a plasmid-based library, without cross-hybridization.     

Preparation of plasmids from lambdoid phages and studies on their incompatibilities.

Various lambdoid phages, including those carrying the immunity region of 434, 21, and φ80, were found to give rise to fragments of DNA that can be perpetuated in the plasmid state. The plasmids, imm434dv, imm21dv, and φ80dv, were similar to the already known plasmid λdv in size, genetic constitution, oligomeric state, copy number, and stability. Cells carrying two kinds of dv plasmids were constructed by transformation. It was shown that a pair of plasmids is compatible in a cell if they originate from phages differing in immunity region. On the other hand, a pair of plasmids is incompatible if they are derived from phages carrying the same immunity region. These observations were taken to imply that the incompatibility of a pair of plasmids is determined by the “immunity region” of the plasmid genome that contains an autorepressor gene and a promoter-operator. The region that carries initiator genes and a site for initiation of plasmid replication is not primarily important for determination of the incompatibility. Plasmids λdv and imm434dv, which are very closely related to each other, behaved in an intermediate fashion.     

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.     

Prediction of 3-D structure of the Cro protein from phage 434

A comparative model building process has been utilized to predict the three-dimensional structure of the bacteriophage 434 Cro protein. Amino acid sequence similarities between the 434 Cro protein and other bacteriophage repressor and Cro proteins have been used, in conjunction with secondary structure prediction and the known structures of other base sequence specific DNA binding proteins, to derive the model. From this model the interactions between the 434 Cro protein and its operator DNA have been deduced. These proposed interactions are consistent with the known properties of the bacteriophage 434 Cro protein.     

DNA-induced conformational changes in bacteriophage 434 repressor

Although bacteriophage 434 repressor binds to its specific DNA sites only as a dimer, formation of the dimers in solution occurs at concentrations three orders of magnitude higher than those needed to bind the 434 operator DNA. Our results suggest that both specific and non-specific DNA induce conformational changes in repressor that lead to formation of repressor dimers. The repressor conformational changes induced by DNA occur at concentrations much lower than those needed for binding of repressor, suggesting that the alternative conformations of repressor persist even if the protein is not in direct contact with DNA. Hence, DNA acts in a "catalytic" fashion to induce a steady-state amount of an alternative repressor conformation that has an enhanced affinity for its specific binding site. These findings suggest that the repressor conformer induced by non-specific DNA is the form of the repressor that is optimized for searching for DNA binding sites along non-specific DNA. Upon finding a binding site, the repressor protein undergoes an additional conformational change that allows it to "lock-on" to its specific site.     

Molecular cloning of the complementary DNA for a human folate binding protein

The complementary DNA for a human folate binding protein has been cloned from a lambda gt11-cDNA library prepared from cultured KB cells. A number of clones were selected by immunoscreening with a monospecific antiserum and by oligonucleotide probes corresponding to the NH2-terminal sequence of the folate binding protein. A partial nucleotide sequence of the cDNA was determined directly from the lambda gt11 phage and after subcloning into M13. The 18 amino acids deduced from the initial 19 codons were exactly the same as the amino acid sequence obtained by peptide analysis of the purified protein providing proof that this clone is the folate binding protein cDNA.