A 979-bp fragment from the TP901-1 genome (positions 2622 to 3600) is known to function as a bistable genetic switch. (A) The gray boxes indicate the positions of the cI and mor genes. The numbers below the line correspond to the location in the TP901-1 genome. The positions and directions of the promoters, P_L and P_R, are indicated as arrows above and below the line. The CI binding sites, O_R, O_L, and O_D, are shown as black boxes. (B) Schematic representations of the cloned DNA fragments used in this study. Plasmid pAJ80 was used for the transposon mutagenesis, and pAJ149 and pAJ155 were used as reporter plasmids for measuring activity of P_L or P_R in the presence of wild-type or mutant CI proteins expressed in trans. Plasmid pMAP50 and its derivatives (pMAP131 to pMAP133, pMAP140, and pMAP141) contain a functional mor gene and were used to determine the activity of P_L-expressing wild-type or mutant CI protein in cis. The gray boxes indicate the position of the reporter genes lacLM. (C) In the immune state, CI is suggested to bind to the three operator sites, O_R, O_L, and O_D, forming a CI-DNA loop structure. Momentary release of CI from O_R, which has the lowest affinity for CI, allows transcription from P_R. (D) In the anti-immune state, a CI:MOR complex is suggested to bind DNA and prevent transcription from P_R, whereas transcription from P_L is allowed. Repression of P_R in this state requires the presence of both functional mor and cI genes, whereas the O_R site is not required (22). The nature of the protein complex and its location on the DNA are unknown. The head-to-head arrows represent the palindromic sequences of the CI operator sites.

Limited proteolysis of CI using subtilisin. (A) SDS-PAGE of purified CI protein treated with the protease subtilisin. First lane, molecular weight standard. The numbers to the left of the gel show the sizes of the molecular weight markers. Second lane, CI before addition of subtilisin. Third to eighth lanes, samples of CI treated with subtilisin for 2, 5, 10, 15, 30, and 60 min. The arrows at the left indicate the positions of the full-length CI protein. The lines to the right show polypeptides appearing following digestion with subtilisin. Each polypeptide band was analyzed by N-terminal sequencing and mass spectrometry. (B) The individual peptide bands were purified from the gel, treated with trypsin or Asp-N, and analyzed by mass spectrometry. The black lines indicate the sequences identified following trypsin digestion and mass spectrometry analysis, and the gray lines indicate the sequences identified following Asp-N digestion and mass spectrometry analysis. The amino acid numbers at the right of the figure indicate the minimal lengths of the peptides produced after subtilisin digestion.

Amino acid sequences of the TP901-1 CI repressor protein. The gray boxes represent regions involved in DNA binding (helix-turn-helix motif) and oligomerization (residues 138 to 180) (23). The positions where five amino acids were inserted by transposon mutagenesis are shown above the sequences as triangles, and the numbers identify the positions of the inserts in CI. At two positions (78 and 89) marked with asterisks, five amino acids were inserted by site-specific mutagenesis. Three lines to the left of the sequence are marked with P_L, P_R, or P_Lmor, and the triangles in each line represent the state of P_L, P_R, or P_L in the presence of a functional mor gene, resulting from the given CI mutant, respectively. ▾, positions where linker insertion in CI abolish repression of P_L or P_R; ▿, positions where linker insertion still allows repression of P_L or P_R. Wild-type CI protein represses transcription from both P_L and P_R (white triangles) in the absence of mor. In the presence of a functional mor gene and wild-type CI, each transformant has a choice between two states, i.e., P_L repressed or P_L open (hatched triangle). Insertion of linkers at positions 98 and 135 showed wild-type phenotypes, but the repressed state of P_L was not inducible.

Western blot analysis using specific antibodies against TP901-1 CI. (A) Protein extracts were obtained from cells expressing cI, cI3, cI6, cI11, cI14, cI15, cI20, cI41, cI75, cI98, cI101, cI113, cI120, cI121, cI122, or cI135 from the lysogenic promoter, P_R, cloned in the pCI372 plasmid. Cells containing plasmid pCI372 without a cI insert was used as a negative control. The relative amount of CI was estimated using the ImageQuant TL software. (B) Protein extracts were obtained from cells containing a functional mor gene in concert with cI, cI78, cI89, cI98, cI120, or cI135. The state of the lytic P_L promoter (open or repressed) is shown below each lane.

No cleavage product of CI is observed in vivo following P_L induction or following incubation at alkaline pH in vitro. (A) In vivo induction of the repressed P_L promoter analyzed by Western blotting using CI-specific antibodies. The cloned bistable switch from phage TP901-1 carrying P_L in the repressed state was treated with mitomycin C, which activates the host SOS response, thereby allowing increased transcription from P_L. Lane 1, protein sample from the host strain Lactococcus lactis subsp. cremoris MG1363 without the genetic switch from phage TP901-1. Lanes 2 and 3, protein samples collected at 10 and 2 min, respectively, before treatment with mitomycin C. At time zero the culture was divided into two, and one of the cultures was treated with mitomycin C. Lanes 4 and 5, are protein samples collected at 15 and 60 min, respectively, after time zero (no mitomycin C was added). Lanes 6 and 7, protein samples collected 15 and 60 min, respectively, after addition of mitomycin C. (B) Purified CI protein incubated at alkaline pH. First lane, protein marker. Numbers to the left of the gel represent molecular weights in thousands. Second lane, CI protein at pH 7.5 without incubation. Lanes 1a and b, CI incubated at pH 7.5 for 1 and 15 h, respectively. Lanes 2a and b, CI incubated at pH 8 for 1 and 15 h, respectively. Lanes 3a and b, CI incubated at pH 8.8 for 1 and 15 h, respectively. Lanes 4a and b, CI incubated at pH 9.5 for 1 and 15 h, respectively. The arrows indicate the position of CI.

Amino acid sequences of the CI repressor protein. Eighty-six amino acids from TP901-1 CI (residues 49 to 133) were searched for sequence identity to other repressor proteins. Phages encoding proteins with sequence similarity to TP901-1 CI and the corresponding host bacteria are shown to the left of the sequences. Bacteriophages that encode MOR homologous are marked with an asterisk. CI encoded by phages ul36, φ31, and 4268 are 98% identical, whereas CI proteins encoded by phages 80α and φPVL180 are 94% identical. Amino acids shown in gray are identical to amino acids in CI of phage TP901-1. Numbers to the right of the sequences indicate the amino acid numbers in the CI proteins. ▿, positions where linker insertion in CI allows repression of P_L or P_R; ▾, positions where linker insertion abolishes repression of P_L or P_R; hatched triangle, positions where linker insertion in CI in the presence of MOR allows the choice between the two different states. Two conserved regions are marked with black lines. The gray box below the sequences at the left represents a region conserved only in phages encoding MOR-homologous proteins. Insertion of five amino acids at positions 78 and 89 in this region abolished repression of P_L in the presence of MOR, suggesting that this region is important for CI:MOR interactions. The second and the fourth gray boxes represent regions where insertion of five amino acids produced CI mutant proteins that can no longer be derepressed following activation of the host SOS response, suggesting that interaction with RecA may be abolished or that the multimeric form of the protein may be changed. The third gray box represents a conserved region in phage infection in Lactococcus. Insertion of five amino acids in this region abolishes repression of P_R, suggesting that the cooperative binding of CI at O_L-O_R is reduced and hence this region is involved in oligomerization of CI.