Temperate Bacteriophage TP901-1 Research Articles

Structural and dynamics studies of a truncated variant of CI repressor from bacteriophage TP901-1.

The CI repressor from the temperate bacteriophage TP901-1 consists of two folded domains, an N-terminal helix-turn-helix DNA-binding domain (NTD) and a C-terminal oligomerization domain (CTD), which we here suggest to be further divided into CTD1 and CTD2. Full-length CI is a hexameric protein, whereas a truncated version, CI∆58, forms dimers. We identify the dimerization region of CI∆58 as CTD1 and determine its secondary structure to be helical both within the context of CI∆58 and in isolation. To our knowledge this is the first time that a helical dimerization domain has been found in a phage repressor. We also precisely determine the length of the flexible linker connecting the NTD to the CTD. Using electrophoretic mobility shift assays and native mass spectrometry, we show that CI∆58 interacts with the OL operator site as one dimer bound to both half-sites, and with much higher affinity than the isolated NTD domain thus demonstrating cooperativity between the two DNA binding domains. Finally, using small angle X-ray scattering data and state-of-the-art ensemble selection techniques, we delineate the conformational space sampled by CI∆58 in solution, and we discuss the possible role that the dynamics play in CI-repressor function.

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

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.

The Role of MOR and the CI Operator Sites on the Genetic Switch of the Temperate Bacteriophage TP901-1

A genetic switch controls whether the temperate bacteriophage TP901-1 will enter a lytic or a lysogenic life cycle after infection of its host, Lactococcus lactis. We studied this bistable switch encoded in a small DNA fragment of 979 bp by fusing it to a reporter gene on a low-copy-number plasmid. The cloned DNA fragment contained the two divergently oriented promoters, PR and PL, transcribing the lysogenic and lytic gene clusters; the two promoter-proximal genes, cI and mor; and the three CI operator sites, OR, OL and OD. We show that mor encodes a protein and that this protein in concert with CI is required for the bistability. Furthermore, interaction of CI at OR represses transcription from the lysogenic promoter, PR. Thus, CI regulates its own transcription. Interaction of CI at OL represses transcription from the lytic promoter, PL. The presence of only OL (absence of OR and OD) is enough to maintain a bistable system. The distantly located operator site, OD, functions as a helper site by increasing binding of CI at OR and OL. In the immune state, OD increases repression of the lytic promoter, PL. Our results strongly support the model that a hexameric form of CI binds cooperatively to the three operator sites in the immune state forming a CI–DNA loop structure. Finally, we show that in the anti-immune state, repression of the lysogenic promoter is independent of the known CI operator sites but requires the presence of both CI and MOR.

Analysis of the Complete DNA Sequence of the Temperate Bacteriophage TP901-1: Evolution, Structure, and Genome Organization of Lactococcal Bacteriophages

A complete analysis of the entire genome of the temperate lactococcal bacteriophage TP901-1 has been performed and the function of 21 of 56 TP901-1-encoded ORFs has been assigned. This knowledge has been used to propose 10 functional modules each responsible for specific functions during bacteriophage TP901-1 proliferation. Short regions of microhomology in intergenic regions present in several lactococcal bacteriophages and chromosomal fragments of Lactococcus lactis are suggested to be points of exchange of genetic material through homologous recombination. Our results indicate that TP901-1 may have evolved by homologous recombination between the host chromosome and a mother phage and support the observation that phage remnants as well as prophages located in the Lactococcus chromosome contribute significantly to bacteriophage evolution. Some proteins encoded in the early transcribed region of the TP901-1 genome were more homologous to proteins encoded by phages infecting gram-positive hosts other than L. lactis. This protein homology argues for the occurrence of horizontal genetic exchange among these bacteriophages and indicates that they have access to a common gene pool.

Use of the integration elements encoded by the temperate lactococcal bacteriophage TP901-1 to obtain chromosomal single-copy transcriptional fusions in Lactococcus lactis.

Previously we showed that only one phage-expressed protein (Orf1), a 425-bp region upstream of the orf1 gene (presumably encoding a promoter), and the attP region are necessary and also sufficient for integration of the bacteriophage TP901-1 genome into the chromosome of Lactococcus lactis subsp. cremoris (B. Christiansen, L. Brondsted, F. K. Vogensen, and K. Hammer, J. Bacteriol. 178:5164-5173, 1996). In this work, a further analysis of the phage-encoded elements involved in integration was performed. Here we demonstrate that even when the orf1 gene is separated from the attP region, the Orf1 protein is able to promote site-specific integration of an attP-carrying plasmid into the attB site on the L. lactis subsp. cremoris chromosome. Furthermore, the first detailed deletion analysis of an attP region of a phage infecting lactic acid bacteria was carried out. We show that a fragment containing 56 bp of the attP region, including the core, is sufficient for the site-specific integration of a nonreplicating plasmid into the chromosome of L. lactis subsp. cremoris when the orf1 gene is donated in trans. The functional 56-bp attP region of TP901-1 is substantially smaller than minimal attP regions identified for other phages. Based on the deletion analysis, several repeats located within the attP region seem to be necessary for site-specific integration of the temperate bacteriophage TP901-1. By use of the integrative elements (attP and orf1) expressed by the temperate lactococcal bacteriophage TP901-1, a system for obtaining stable chromosomal single-copy transcriptional fusions in L. lactis was constructed. Two promoter-reporter integration vectors containing the reporter gene gusA or lacLM, encoding beta-glucuronidase or beta-galactosidase, respectively, were constructed. Immediately upstream of both genes are found translational stop codons in all three reading frames as well as multiple restriction enzyme sites suitable for cloning of the promoter of interest. By transformation of L. lactis subsp. cremoris MG1363 containing the integrase gene on a replicating plasmid, the promoter-reporter integration vectors integrated with a high frequency site specifically into the chromosomal attachment site attB used by bacteriophage TP901-1.

A Genomic Region of Lactococcal Temperate Bacteriophage TP901-1 Encoding Major Virion Proteins

Two major structural proteins, MHP (major head protein) and MTP (major tail protein), from the lactococcal temperate phage TP901-1 were sequenced at their amino acid termini, and derived degenerate oligonucleotides were used to locate the corresponding genes in the phage genome. This genomic region was sequenced. The sequence characterized includes a total of 11 open reading frames (ORFs) showing an operon structure. Upstream of each ORF, except ORFb2and ORFx,potential ribosome-binding sites were found, suggesting independent translation. However, coupled translation is suggested for ORFxand as a possibility for ORFb3and ORFc2,which have ribosome-binding sites located more distant from their start codons. ORFb2may be translationally fused withmhpat a low frequency. Themhpandmtpgenes are transcribed as a 3.7-kb mRNA with at least six additional ORFs. The organization of the genomic region analyzed resembles that of other distantly related phages, providing possible roles for the uncharacterized ORFs.

Virion Positions and Relationships of Lactococcal Temperate Bacteriophage TP901-1 Proteins

The major proteins of phage TP901-1 virion were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and structural relations were determined using specific antibodies, obtained by affinity purification from a polyclonal serum. A 23-kDa protein was identified as the major tail protein, and a 31-kDa molecule as the major head protein, respectively. Labeling experiments with antibodies against two proteins, with molecular masses of 20 and 19 kDa, indicated that they were baseplate-related components. A 72-kDa protein was found to be part of a neck passage structure, which includes a collar. Evidence for the presence of attached whiskers was also obtained. T7 RNA polymerase-mediated expression of the two major proteins confirmed the gene location of the previously sequenced region of the phage genome. The relation to other lactococcal phages was determined by DNA hybridization and antibody probing, showing that despite low DNA similarity, TP901-1 NPS epitopes were detected in both related and unrelated small isometric-headed phages.