Characterization of the CI Repressor Protein Encoded by the Temperate Lactococcal Phage TP901-1

Abstract

The gene regulatory mechanism determining the developmental pathway of the temperate bacteriophage TP901-1 is regulated by two phage-encoded proteins, CI and MOR. Functional domains of the CI repressor were investigated by introducing linkers of 15 bp at various positions in cI and by limited proteolysis of purified CI protein. We show that insertions of five amino acids at positions in the N-terminal half of CI resulted in mutant proteins that could no longer repress transcription from the lytic promoter, P L . We confirmed that the N-terminal domain of CI contains the DNA binding site, and we showed that this part of the protein is tightly folded, whereas the central part and the C-terminal part of CI seem to contain more flexible structures. Furthermore, insertions at several different positions in the central part of the CI protein reduced the cooperative binding of CI to the operator sites and possibly altered the interaction with MOR.Temperate bacteriophages provide a classical example for studies of the choice between two alternative modes of development. Following infection of a sensitive host, a temperate bacteriophage may choose (i) to enter the lysogenic state, in which the viral genome is silenced and integrated into the host genome to be replicated along with the host DNA when the cell divides, or (ii) to enter the lytic state, in which phage replication in the cell leads to production of new phages, cell lysis, and release of phage progeny (4, 21, 25). The choice made by a temperate phage regarding which life cycle to enter is based on a gene regulatory system named the genetic switch (25).A DNA fragment obtained from the temperate lactococcal phage TP901-1 cloned into a low-copy-number plasmid was shown to exhibit bistable gene expression mimicking the lysislysogeny choice when introduced into Lactococcus lactis. The cloned DNA fragment contains the two divergently oriented early promoters P R and P L and the two promoter proximal genes cI and mor (Fig. 1A). P L is the lytic promoter from which MOR, the modulator of repression, is the first gene to be transcribed, and P R is the promoter for CI repressor synthesis. Furthermore, three CI binding sites, O R , O L , and O D , are present on the DNA fragment, which was fused to the reporter genes lacLM in order to measure the activity of either P R or P L (22). Following transformation of such plasmids (pMAP50 in Fig. 1B) into a lactococcal host, two different stable states are obtained; either P L is repressed by CI (immune state, shown in Fig. 1C) or P L is open (anti-immune state, shown in Fig. 1D). The open state of P L requires a large amount of MOR, which apparently counteracts CI binding to the operator sites (17). Knockout mutations introduced in either cI or mor showed that tight repression of P R in the anti-immune state requires the presence of both MOR and CI, and hence this repression of P R was suggested to occur by binding of both proteins to the DNA (Fig. 1D) (22). The nature and the location of the protein complex on the DNA are unkno...

Clear Plaque Mutants of Lactococcal Phage TP901-1

“…The localization of the mutations might indicate that the last part of CI is not necessary for DNA binding. This is in accordance with previous results, that 5 amino acid insertions in the CI protein from amino acid 78 and further downstream do not abolish repression of the lytic P L promoter[ 6 ]. The purified CI protein has been shown to be a hexamer in solution.…”

“…However, the region where the C8 mutation is located has previously been proposed to be involved in binding to the small regulatory Mor protein, which has been suggested to function as an antirepressor for CI repression at the P L promoter [ 3 ]. The proposed Mor binding region in CI is based upon homology to other CI-Mor partners and the non repressible phenotype in the presence of Mor of two 5 amino acid insertion mutations in CI [ 5 , 6 ]. It might be possible that a cI mutation could result in a clear plaque phenotype if it resulted in increased binding between CI and Mor without disrupting CI repression in the absence of Mor.…”

“…Dilutions were prepared to ensure exponential growth for at least 8 generation, and each culture was harvested after approximately 8, 11, and 14 generations. The resulting nine samples for each strain were harvested and assayed for β-galactosidase activity as described earlier[ 6 ] except that the specific activity was based upon OD 450 instead of OD 600 .…”

“…Using transcriptional fusions it has been shown that a surplus of Mor counteracts repression by CI at P L , but also increases repression by CI at P R [ 3 ]. CI-Mor protein-protein interactions have been proposed to be responsible for this behaviour [ 5 , 6 ].…”

A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria

“…The immunity repressors of temperate phages of these bacteria have been experimentally analysed in just a few cases, including B. subtilis phage ϕ105 (24), S. aureus phage ϕ11 (25) and Lactococcus lactis phage TP901-1 (26). Analysis of the lysogeny modules of a collection of temperate Siphoviridae from low GC content Gram-positive bacteria revealed a common genetic organization that differs clearly from established genera of temperate Siphoviridae, as well as a novel genomic location for this module (27).…”