Supplementary Material
The role of OR2 in Cro repression of PRM
We have shown that Cro binding to OR2 can repress PRM basal activity. Previously, it was concluded that Cro at OR2 does not repress PRM on the basis that a PRM reporter carrying a mutation in OR3 was not repressed by Cro (Meyer et al. 1980). However, Cro levels may have been insufficient in this experiment to give significant occupation of OR2. In addition, the PRM promoter carried a mutation, PRMup-1, that strongly increases promoter activity. The stronger RNAP binding to the PRMup-1 promoter (Hwang et al. 1988) may cause RNAP to compete better with Cro binding and thus weaken Cro repression compared to wt PRM.
In vitro, Cro binds to OR2 some 2.6 kcal/mol more weakly than it binds to OR3 (Darling et al. 2000). This is consistent with the weaker repression we saw with plasmid-supplied Cro for the OR3-KO mutant compared with the OR2-KO mutant (Figure 2B). However, OR2 alone was nevertheless able to confer ~1.8-fold repression of PRM by Cro produced from PR in cis in single copy (OR3-KO mutant Figure 2C). Since we were not able to mutate OR2 to remove this repression without causing other effects, the impact on phage development of Cro repression of PRM through OR2 is unclear. It also means that it is possible that the effects of our OR3 mutants underestimate the importance of Cro repression of PRM.
Phage constructions
Plasmid pJWL464 (Michalowski et al. 2004) contains a portion of the b region of λ, with DNA in which position 23901 to 26818 has been replaced with the kan gene from Tn5 followed by the strong trp a terminator. l.imm434 was propagated on E4300 carrying pJWL464. To select for those phages that had acquired the b::kan insertion by in vivo recombination, the progeny phage were used to infect C600. After infection, the culture was plated on LB + 12.5 mg/ml kanamycin plates to select for those bacteria lysogenic for l.imm434 carrying the b::kan insertion.
Our method for transferring mutations in the cI-OR-cro-cII region to l relies on in vivo recombination in which the imm434 region of l.imm434 is replaced by imml sequences (+ desired mutations) carried on the plasmid pAP831 (Dodd et al. 2001). Two crossovers within l sequences flanking imm434 are required. The right junction of l.imm434 (at l sequence 38427) is well defined and homologous recombination between phage and plasmid can only occur between this site and the EcoRV site at 39355. However, we found that the left junction of imml and imm434 sequences is not clearly defined. The junction is usually taken to be at the +1 position of PL (position 35582) but we found eight sequence differences between l and l.imm434 between positions 35421 and 35557 (within the N leader). Thus, the left-side crossover between pAP831 and l.imm434 (between the NheI site at 34680 and PL) may generate l phages with mutations in the N leader. We therefore checked the left (and right) junction sequences of our l phages constructed in this way.
Reporters
The PRM.lacZ transcriptional fusions (Figures 1B, 2B, 2C and 3A and 6) contain l DNA from position 37880 (LAMCG; +62 of PRM, within cI) to either position 38064 (+43 of PR, within cro) or position 38241 (at end of cro). The PR.lacZ transcriptional fusions of Figures 3B and 3C contain l DNA from position 38064 to position 37880. These DNA fragments were inserted into the polycloning site of pTL61T. The construction of the 37880 and 38064 junctions has been described previously (Dodd et al. 2001). The 38241 junction was created by PCR with a primer carrying a Hind III site located just after the cro stop codon, allowing cloning into the Hind III site of pTL61T. The fusions were transferred to lRS45DYAOL. The generic structure of the final reporter phages is shown in Figure S1A.
In the cI::lacZ translational fusions (Figures 1C, 3D), codon 20 of cI is fused to codon 10 of lacZ with two codons between. The l.lac junction is CGCCTTAAGGATCCCGTC. The underlined AAG is codon 20 of cI and the underlined GTC is codon 10 of lacZ. In the clone of Figure 1C the l DNA extends to position 38241 (end of cro) and was inserted into the EcoRI site of pRS414 and then transferred to lRS45DYAOL.
In the fusions of Figure 3D, the l DNA extends from the cI::lacZ fusion to l position 38443 (+1 of PRE) and carries the croHTH– mutation, the PR– mutation and the different OR mutations (Figure 1). l position 38443 is joined to DNA from positions 20375-20580 of coliphage 186 (NC_001317), enoding the pB promoter (Kalionis et al. 1986). The 186.l junction sequence is TATCATTTATCTAGA. The pB -10 sequence and the PRE +1 sequence are underlined and italics indicate sequence common with 186 pB –12 to +1. The 186.l fragment was inserted between the EcoRI and BamHI sites of pRS414 (Simons et al. 1987) and the fusion was transferred to lRS45.
The structure of pIT-SLlacZY is shown in Figure S1B. The sequence components (with database accession numbers) are as follows.
Bases 1–57 = M77789: 289-233 (comp); pUC19 polylinker EcoRI–HindIII.
Bases 58–61 = GGGC. Bases 62–3366 = M29896: 88–3392; pTL61T RNase III site and lacZ gene (Linn and St. Pierre 1990)(NB. Change 3253 C to A removes EcoRI site).
Bases 3367–4659 = J01636: 4421–5713; lacY gene.
Bases 4660–4665 = CTCGAG; XhoI site.
Bases 4666–4731 = X05629: 67–132; deot terminator (Larsen et al. 1987)
Bases 4732–6654 = U66308: 4078–6000; pZS4int1 spectinomycin resistance gene and lambda toop terminator (Lutz and Bujard 1997) (obtained by Pfu PCR and not sequenced).
Bases 6655–6678 = U66308: 1–24; pZS4int1 (Lutz and Bujard 1997)
Bases 6679–7806 = AY048739: 5350–4253 (comp); pCAH56 oriR.lattP.l tL3 terminator (Haldimann and Wanner 2001).
Bases 7807–8019 = AY048739: 214–2 (comp); pCAH56 (Haldimann and Wanner 2001).
Bases 8020–8815 = M29896: 7770–8565; 4 copies of rrnBT1T2 from pTL61T (Linn and St. Pierre 1990).
The l CI-Cro switch reporters (Figure 5) carry l sequence 35542-38460 (extending from +42 position of PL to within the cII gene), obtained by PCR and cloned into pTL61T. The DNA sequence was confirmed. Various combinations of the cI857, OR3-x3 and croHTH– mutations were introduced and the fragments were cloned into pIT-SLlacZY such that the l sequence (with an SpeI site added at the left end) replaces the DNA between the EcoRI and XbaI sites, and PR is transcribing towards lacZ.
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