Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid

Abstract

The mechanism leading to protein-primed DNA replication has been studied extensively in vitro. However, little is known about the in vivo organization of the proteins involved in this fundamental process. Here we show that the terminal proteins (TPs) of phages ϕ29 and PRD1, infecting the distantly related bacteria Bacillus subtilis and Escherichia coli, respectively, associate with the host bacterial nucleoid independently of other viral-encoded proteins. Analyses of phage ϕ29 revealed that the TP N-terminal domain (residues 1-73) possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association. Importantly, we show that in the absence of the TP N-terminal domain the efficiency of ϕ29 DNA replication is severely affected. Moreover, the TP recruits the phage DNA polymerase to the bacterial nucleoid, and both proteins later are redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent way. These data disclose a key function for the TP in vivo: organizing the early viral DNA replication machinery at the cell nucleoid.

Fig. 1.

Subcellular localization of ϕ29 and PRD1 TPs. (A) YFP, DAPI staining, phase-contrast, and merged images of typical B. subtilis cells expressing a xylose-induced YFP-ϕ29 TP fusion protein (strain DM-021) analyzed 30 min after xylose addition. (B) CFP, TO-PRO-3 staining, phase-contrast, and merged images of typical E. coli cells expressing an IPTG-induced CFP-PRD1 TP fusion protein (strain DM-051) analyzed 30 min after IPTG addition. (C) Phase-contrast overlay of B. subtilis cells expressing a xylose-induced YFP (strain DM-022) and E. coli cells expressing an IPTG-induced CFP (strain DM-049) 30 min after the addition of the inductor. (D) Phase-contrast overlay of E. coli cells expressing an IPTG-induced CFP-ϕ29 TP (strain DM-050) and B. subtilis cells expressing a xylose-induced CFP-PRD1 TP (strain DM-060) 30 min after the addition of the inductor. (E) Phase-contrast, YFP fluorescence, and merged images of typical ϕ29 sus14(1242)-infected cells expressing a xylose-induced YFP-TP fusion protein (B. subtilis strain DM-021) 10 and 30 min after infection. Fluorescence signals are shown after deconvolution of an image stack, as a max projection. DM-021 cells were grown at 37 °C in LB medium supplemented with 2% glucose to an OD₆₀₀ of 0.4. Next, xylose was added to a final concentration of 0.5%, and the culture was infected with ϕ29 mutant sus14(1242) at an MOI of 5. (F) Immunofluorescence microscopy assay. B. subtilis 110NA cells were grown at 37 °C in LB medium containing 5 mM MgSO₄. At an OD₆₀₀ of 0.4 the culture was split, and half the culture was infected with phage sus3(91) at a MOI of 25. Samples were harvested 15 min later and processed for immunodetection. Shown are typical unprocessed localization patterns of immunodetected parental TP, DAPI staining, and overlay. For clarity, in A–F YFP fluorescent signals and DAPI or TO-PRO-3 staining are false-colored red and green, respectively.

Fig. 2.

The TP N-terminal domain localizes at the bacterial nucleoid and is important for efficient ϕ29 DNA replication. (A) Three-dimensional structure of the DNA polymerase/priming TP heterodimer (12). (B) B. subtilis cells expressing CFP (strain DM-024), CFP/TP (strain DM-025), CFP/TP-Nt (strain DM-026), CFP/TP-NtI (strain DM-027), CFP/TP-I (strain DM-028), CFP/TP-ΔNt (strain DM-029), and CFP/TP-Ct (strain DM-030) IPTG-inducible fusions were grown to midexponential phase in LB medium at 37 °C. At an OD₆₀₀ of 0.4, the cultures were supplemented with 1 mM IPTG. Samples were harvested and analyzed by fluorescence microscopy 30 min after IPTG addition. For clarity, CFP fluorescent signals are false-colored red in merged images. (C) The amount of intracellular accumulated phage ϕ29 DNA was quantified by real-time PCR at different times postinfection with a sus3(91) mutant phage of the following B. subtilis strains: DM-024 (expressing CFP), DM-025 (expressing CFP-TP), DM-029 (expressing CFP-TPΔNt), and DM-032 (expressing wild-type TP). Samples were infected at a MOI of 1 and at different times postinfection were processed as described in Materials and Methods. The amounts of accumulated phage DNA (nanograms of viral DNA per milliliter of culture) are expressed as a function of time after infection. The experiment was carried out in triplicate to calculate SDs.

Fig. 3.

The N-terminal domain of the TP has sequence-independent dsDNA-binding capacity. Gel mobility shift assays using an end-labeled 297-bp DNA fragment corresponding to the right end of the ϕ29 genome (A) and an end-labeled 216-bp DNA fragment corresponding to the B. subtilis yshC gene (B). The labeled probes were incubated either in the absence (−) or presence of increasing amounts (75, 150, and 300 nM) of the indicated purified protein in a buffer containing 50 mM NaCl. After nondenaturing PAGE, the mobility of the nucleoprotein complexes was detected by autoradiography. (C) DNase I treatment of nucleoprotein complexes formed by ϕ29 wild-type TP and TP-NtI or TP-Nt truncated variants. The 297-bp right end of the ϕ29 DNA fragment was end labeled and used in DNase I footprinting assays in the absence (−) or presence of increasing amounts (0.75, 1.5, and 3 μM) of the indicated proteins. The bottom of the footprints corresponds to the right end of the ϕ29 genome. c, control lane with the dsDNA probe not subjected to DNase I treatment and protein preincubation.

Fig. 4.

The ϕ29 TP recruits the DNA polymerase to the bacterial nucleoid, and both proteins are redistributed in an MreB-dependent way. (A and B) B. subtilis strain DM-023, expressing YFP-p2 and CFP-TP in a xylose- and IPTG-dependent way, respectively, was grown in LB medium supplemented with 5 mM MgSO₄ and 2% glucose. At an OD₆₀₀ of 0.4, cultures were supplemented with 1 mM IPTG and/or 0.5% xylose, as indicated (A) and simultaneously were infected with a sus14(1242) phage at a MOI of 5 (B). Shown are phase-contrast, YFP and CFP fluorescence, and merged images. For clarity, YFP and CFP fluorescent signals are false-colored red and green, respectively. (C) B. subtilis strains expressing YFP-TP in a xylose-dependent way under wild-type (DM-021) and ΔmreB (DM-031) backgrounds were grown in LB medium supplemented with 25 mM MgSO₄ and 2% glucose. At an OD₆₀₀ of 0.4, cultures were supplemented with 0.5% xylose and infected with a sus14(1242) phage, as indicated. Samples were harvested 25 and 50 min postinfection. In A–C fluorescence signals are shown after deconvolution of an image stack, as a max projection.

Fig. 5.

Model of nucleoid-associated early ϕ29 DNA replication organized by the TP. (A) A complete ϕ29 TP-DNA molecule (linear dsDNA shown as a double helix) is shown attached to the bacterial nucleoid surface (gray mass at bottom) by the N-terminal domain (red) of the two parental TPs (red and green). (B) The priming TP interacts with the phage DNA polymerase (cyan), forming a heterodimer that associates with the nucleoid via the TP N-terminal domain and recognizes the origins of replication. (C) After a transition step, the DNA polymerases dissociate and continue processive elongation of the nascent DNA strands (red lines) coupled to strand displacement. (D and E) Once DNA replication is completed, two ϕ29 TP-DNA molecules are ready for another round of replication. For simplicity, other viral proteins involved in DNA replication are not drawn.