A dual promoter system regulating λ DNA replication initiation

Transcription and DNA replication are tightly regulated to ensure coordination of gene expression with growth conditions and faithful transmission of genetic material to progeny. A large body of evidence has accumulated, indicating that encounters between protein machineries carrying out DNA and RNA synthesis occur in vivo and may have important regulatory consequences. This feature may be exacerbated in the case of compact genomes, like the one of bacteriophage λ, used in our study. Transcription that starts at the rightward pR promoter and proceeds through the λ origin of replication and downstream of it was proven to stimulate the initiation of λ DNA replication. Here, we demonstrate that the activity of a convergently oriented pO promoter decreases the efficiency of transcription starting from pR. Our results show, however, that a lack of the functional pO promoter negatively influences λ phage and λ-derived plasmid replication. We present data, suggesting that this effect is evoked by the enhanced level of the pR-driven transcription, occurring in the presence of the defective pO, which may result in the impeded formation of the replication initiation complex. Our data suggest that the cross talk between the two promoters regulates λ DNA replication and coordinates transcription and replication processes.


I. Supplemental Materials and methods
and plasmids are described in Table S2.
Bacteriophages λpapa (from our collection) and λp O -(30) were used in density shift experiments. Phage P1vir (from our collection) was used for transduction.
MGO strain was constructed by integration of the plasmid pCAHlamO (Table S2) into the lambda attachment site using lambda site-specific recombination (79).
Plasmids pLamberA and pLamberB were constructed by deletion of fragments, containing kan or bla gene, respectively, from pLamber plasmid (73), as described in the Table 2. Both those plasmids contain λ phage region encompassing p R promoter and genes cro, cII, O and P, as well as ColE1-type origin of replication.
Plasmid pdelλO was constructed by PCR mutagenesis. Primers 1 and 2 (Table S3) were used in the reaction leading to deletion of the 291 bp fragment from pLamberB, coding for the C-terminal domain of the λO protein.
Plasmid pdelλOpowas constructed by ligation of the 5.8 kb HindIII-EcoRI fragment of pdelλO and the 1.584 bp HindIII-EcoRI fragment of pKB2po-.
A series of pdelλO-derived plasmids bearing insertions of 6, 10, 50, 100 or 500 bp, called pdelλOins6, pdelλOins10, pdelλOins50, pdelλOins100 and pdelλOins500, respectively, were constructed as follows. 6 bp and 10 bp insertions were introduced into pdelλO plasmid by PCR using following pairs of primers: 3 and 4 or 5 and 6 (Table S3). Plasmids bearing 50 bp and 100 bp insertions were constructed in two steps. First, a unique XhoI site was introduced into a pdelλO plasmid by PCR mutagenesis, using primers 7 and 8 (Table S3). Then, hybridizations of two pairs of the 50 bp-long and 100 bp-long oligonucleotides, whose sequences corresponded to fragments of the spectinomycin-resistance gene, were performed to obtain dsDNAs. Following phosphorylation of the 5'-ends by T4 polynucleotide kinase (1 unit, Promega), the double stranded oligonucleotides were ligated with pdelλO, previously linearized with XhoI. The 500 bp fragment for construction of pdelλOins500 was PCRamplified using a template containing spectinomycin-resistance gene and primers 9 an 10 (

β-galactosidase assay
Overnight cultures were diluted in the LB medium with or without indicated concentrations of the inducer and grown until OD578=0.2. After reaching desired density, cultures were chilled on ice. 100µl of culture were added to 1.5ml of the Z buffer and 100 µl of chloroform and vortexed. Subsequently, solution was incubated for 10 minutes. Following incubation, 400 µl of the ONPG solution was added and the reaction was carried out for 5 minutes. The reaction was stopped by addition of 1ml of 1M sodium carbonate. The absorbance of the solution was measured at 420nm wavelength. β-galactosidase activity was calculated as described (82).

Preparation of Fraction II
E. coli C600 strain was grown in LB medium at 37˚ to OD 600 = 1.0. The cells were collected by centrifugation, washed once in buffer A (25 mM Hepes-KOH pH 7.6, 5 mM EDTA) and then resuspended in buffer A (2 ml buffer A per 1 ml of cell paste). The cell paste was transferred to a polypropylene tube, quickly frozen in liquid nitrogen and stored at -70˚.
After thawing at 0-4˚, the liquid was adjusted to 150 mM KCl and 2mM DTT, and lysozyme was added to 0.5 mg/ml. Following incubation at 0˚ for 30 min, the cells were frozen again in liquid nitrogen, thawed at 0-4˚ and centrifuged in Beckman 50.