Dynamic relocalization of phage phi 29 DNA during replication and the role of the viral protein p16.7

Abstract

We have examined the localization of DNA replication of the Bacillus subtilis phage phi 29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7.

Fig. 1. Schematic representation of the genetic and transcriptional organization of the φ29 genome and its in vitro DNA replication mechanism. (A) Genetic and transcription map of the phage φ29 genome. The direction of transcription and length of the transcripts are indicated by arrows. The positions of the various genes are indicated between the two DNA strands. The positions of the open reading frames 16.9, 16.8, 16.6 and 16.5, located at the right side of the φ29 genome, are indicated with the numbers .9, .8, .6 and .5, respectively. TD1 is the position of a bidirectional transcriptional terminator. Filled circles represent the covalently linked φ29 terminal protein. The map is adapted from Mellado et al. (1976a). (B) Mechanism of in vitro φ29 DNA replication (see text for details). Circles and triangles represent TP and DNA polymerase, respectively. Synthesized DNA strands are indicated with broken lines.

Fig. 2. Localization of DNA in non-infected (A) and φ29 sus14(1242) infected (B) B.subtilis cells using DAPI staining. At t = 0, a logarithmically growing B.subtilis culture was split into two aliquots. One of these was infected with phage φ29 sus14(1242) and the other one served as mock-infected control. Samples of both cultures, withdrawn at t = 45 min, were fixed, treated with lysozyme, and the cellular positions of the DNA were determined by fluorescence microscopy in the presence of DAPI. Overlays of phase contrast and DAPI-captured images of non-infected (A) and infected (B) cells are shown.

Fig. 3. Localization of TP during the φ29 infection cycle. At the indicated times after infection of B.subtilis 110NA cells with phage φ29 sus14(1242), aliquots were harvested, fixed and analysed by immunofluorescence using affinity-purified antibodies against TP. FITC immunofluorescence images (A, D and G) are shown in the left frames, corresponding DAPI images are shown in the middle frames (B, E and H) and combined images of both signals are shown in the right frames (C, F and I).

Fig. 4. Localization of φ29 DNA in B.subtilis cells infected with sus14(1242) (A–F) or sus14(1242)/sus16.7(48) (G–L) phage. HpUra was administered to logarithmically growing B.subtilis 110NA cultures 2 min before BrdU addition. Cells were harvested 10 min (A–C), 22 min (D–I) or 45 min (J–L) after infection. After fixation and acid treatment (15 min with 4 M HCl), samples were analysed by immunofluorescence using monoclonal antibodies against BrdU. Phase contrast images (left frames; A, D, G and J), corresponding FITC immunofluorescence images (middle frames; B, E, H and K) and overlays of both images (right frames; C, F, I and L) are shown. The black arrows in (A) indicate a nucleoid observed as a dark structure in the phase contrast image. Single BrdU foci per nucleoid in early stage infected cells in (B) and (C) are indicated by white arrows. Grey arrows in (B) and (C) indicate additional phage DNA sometimes observed in early stage infected cells. White arrows in (E) and (F) point to the ring-like BrdU signals seen in middle stage infected cells.

Fig. 5. Localization of protein p16.7 during the φ29 infection cycle. Aliquots of B.subtilis 110NA cells, harvested 10 min (A–C) or 30 min (D–F) after infection with sus14(1242) phage, were fixed and analysed by immunofluorescence using affinity-purified antibodies against p16.7. FITC immunofluorescence images (A and D) are shown in the left frames, corresponding DAPI images are shown in the middle frames (B and E) and combined images of both signals are shown in the right frames (C and F). Note that in the image shown at 30 min, the middle cell of the chain was probably not infected with φ29, so no p16.7 signal was detected.

Fig. 6. Schematic representation of φ29 DNA during its infection cycle. See text for details. Transcription foci are indicated with white circles. Phage DNA and phage transcripts are indicated in green and yellow, respectively. p16.7 is indicated in red. Phage DNA replication sites are indicated with blue circles.