Dynamic relocalization of phage φ29 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 f29 by immuno¯uores-cence. 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. Ef®cient 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. Keywords: Bacillus subtilis/bacteriophage f29/ immuno¯uorescence/in vivo DNA replication IntroductionThe Bacillus subtilis phage f29 is one of the best characterized phages and serves as a paradigm for the study of double-stranded DNA viruses in Gram-positive bacteria (Salas, 1991;Salas and Rojo, 1993;Salas et al., 1996). The genome of f29 is a linear double-stranded DNA molecule of 19 285 bp, whose sequence is completely known (Yoshikawa and Ito, 1982;Garvey et al., 1985;Vlcek and Paces, 1986). Regulation of f29 DNA transcription, divided into an early and a late stage, has been studied extensively in vivo as well as in vitro (for reviews see Salas and Rojo, 1993;Rojo et al., 1998). The late expressed genes, all transcribed from a single operon present in the central part of the phage genome, encode the phage structural proteins, proteins involved in phage morphogenesis and those required for lysis of the infected cell ( Figure 1A). The early expressed genes are present in two operons that¯ank the late operon ( Figure 1A). The operon located at the right side of the f29 genome encodes, in addition to protein p17 (Crucitti et al., 1998) and p16.7 (W.J.J.Meijer, A.Serna-Rico and M.Salas, submitted), four proteins of unknown function. The early operon located at the left side of the f29 genome encodes the transcriptional regulator protein p4 and various proteins that are directly involved in phage DNA replication, such as the DNA polymerase, terminal protein (TP), single-stranded DNA-binding protein (SSB), doublestranded DNA-binding protein (DBP) and protein p1 (see Discussion).A schematic overview of the in vitro f29 DNA replication mechanism is shown in Figure 1B. Initiation of f29 DNA replication occurs via a so-called proteinprimed mechanism (reviewed in Salas, 1991;Salas et al., 1996). The linear f29 genome contains a TP molecule covalently linked at each of its 5¢ DNA ends, which constitute the origins of replication. Initiation of DNA replication starts by recognition of the origin by a heterodimer formed by the f29 DNA polymerase and the primer TP. The DNA polymerase then catalyses the ad...

The Bacillus subtilis Phage φ29 Protein p16.7, Involved in φ29 DNA Replication, Is a Membrane-localized Single-stranded DNA-binding Protein

“…that the N-terminal region of the protein, constituting a transmembrane-spanning domain, is responsible for its membrane localization (2,10). Together with the results obtained in this work, we conclude that the native protein p16.7 is a membranelocalized, dimeric, ssDNA-binding protein.…”

“…By immunofluorescence microscopy we have indeed confirmed that the majority of the replicated doublestranded 29 DNA molecules present at late infection times were no longer localized at the membrane. Rather, these molecules were found within the bulk of the host nucleoid (2). Finally, although the possibility, proposed by Ivarie and Pène (8), that the 29 gene 2 product, the DNA polymerase, could be directly involved in attachment of replicating 29 DNA to the membrane still holds, another perhaps more logic explanation can be considered.…”

“…These authors showed (i) that it took at least 10 min before the parental infected 29 DNA molecules became attached to the membrane, (ii) that early expressed viral protein(s) was required for membrane association, (iii) that replicating 29 DNA molecules were attached to the membrane and that the fully replicated doublestranded 29 DNA molecules were first released from the membrane before they were packaged into the phage particles, and (iv) that phage DNA of a 29 mutant containing a temperature-sensitive mutation in gene 2 (later shown to encode the DNA polymerase) did not become associated to the membrane when infected at the non-permissive temperature. Replication of 29 DNA does not start until about 10 min after infection (2,8), implying that until this time no ssDNA-containing replication intermediates are present in the infected cell. This explains why the infecting 29 DNA molecules, present in its double-stranded form, are not associated to the membrane at the very early infection times even though p16.7 is already detected as soon as 6 min after infection (this work and Ref.…”

“…Despite this, little is known about the in vivo organization of DNA replication. To gain a better insight in this fundamental process, we studied the in vivo DNA replication of the Bacillus subtilis bacteriophage 29 (2). The detailed knowledge of its in vitro mechanism of DNA replication (for reviews, see Refs.…”

“…7). Also, replication of 29 DNA takes place at the membrane of the infected cell (2,8,9). Gene 16.7, present in the early expressed operon located at the right side of the 29 genome (see Fig.…”