Binding Specificities of the Telomere Phage ϕKO2 Prophage Repressor CB and Lytic Repressor Cro

Abstract

Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the temperate telomere phages N15, PY54, and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor (cI or cB), the lytic repressor (cro) and a putative antiterminator (q). The roles of these products are thought to be similar to those of the lambda proteins CI (CI prophage repressor), Cro (Cro repressor), and Q (antiterminator Q), respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ϕKO2 are reminiscent of lambda-like phages. We determined binding sites of the ϕKO2 prophage repressor CB and lytic repressor Cro on the ϕKO2 genome in detail by electrophoretic mobility shift assay (EMSA) studies. Unexpectedly, ϕKO2 CB and Cro revealed different binding specificities. CB was bound to three OR operators in the intergenic region between cB and cro, two OL operators between cB and the replication gene repA and even to operators of N15. Cro bound exclusively to the 16 bp operator site OR3 upstream of the ϕKO2 prophage repressor gene. The ϕKO2 genes cB and cro are regulated by several strong promoters overlapping with the OR operators. The data suggest that Cro represses cB transcription but not its own synthesis, as already reported for PY54 Cro. Thus, not only PY54, but also phage ϕKO2 possesses a genetic switch that diverges significantly from the switch of lambda-like phages.

Extreme divergence between one-to-one orthologs: the structure of N15 Cro bound to operator DNA and its relationship to the λ Cro complex

“…We also tested binding to O R 2 and O R 1 sites and observed no binding by EMSA up to ∼1 μM protein. For comparison, ϕKO2 Cro, which has ∼90% sequence identity with N15 Cro and an identical cognate O R 3 sequence, binds O R 3 with apparent K D = 44 nM by EMSA under somewhat different conditions, and binds ϕKO2 O R 2 and O R 1 very weakly ( K D ∼ 1–2 μM) (45). The cognate affinities of N15/ϕKO2 Cro for O R 3 DNA are both weaker than that of λ Cro, which binds λ O R 3 with apparent K D = 3.6 ± 0.5 nM in under EMSA conditions comparable to those used here for N15 Cro (21).…”

“…N15 Cro cocrystallized with a 16 base-pair symmetrical consensus O R duplex that also contained a single thymine overhang at each 5′ end. Sixteen base pairs represent the minimal binding site size found for ϕKO2 Cro (45). The symmetrical site differs from O R 3 by a single base pair near the center (see Figure 1), which is not contacted directly by the protein (see below).…”

“…The symmetrical site differs from O R 3 by a single base pair near the center (see Figure 1), which is not contacted directly by the protein (see below). ϕKO2 Cro binds both O R 3 and this symmetrical site in EMSA experiments (45). The symmetrical site also represents the consensus sequence of the six O R half sites of N15 (7).…”

“…The two proteins play similar biological roles in promoting lytic growth (50–54). Both bind O R 3 and N15 Cro shares λ Cro’s preference for O R 3 over other O R sites (45). But N15 Cro and λ Cro lack significant sequence similarity, adopt different folds and form completely different homodimer interfaces (9,11).…”

The Burkholderia thailandensis Phages ΦE058 and ΦE067 Represent Distinct Prototypes of a New Subgroup of Temperate Burkholderia Myoviruses

“…After verification of the insert by Sanger sequencing (Eurofins Genomics), the lysin gene was expressed by adding IPTG (1.0 mM) at an optical density (OD 588 nm ) of 0.5 to 0.8. Gene expression and protein purification were visualized by SDS-PAGE (Hammerl et al, 2016b). The lysin was purified using the ProBond Purification System (Thermo Fisher Scientific).…”