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The Actin-Like Mreb Cytoskeleton Organizes Viral DNA Replication in Bacteria

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The Actin-Like Mreb Cytoskeleton Organizes Viral DNA Replication in Bacteria The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria Daniel Mun˜ oz-Espína,b, Richard Danielb,c, Yoshikazu Kawaic, Rut Carballido-Lo´ pezd, Virginia Castilla-Llorentea, Jeff Erringtonb,c,1,2, Wilfried J. J. Meijera,1, and Margarita Salasa,1,2 aInstituto de Biología Molecular ‘‘Eladio Vin˜uela’’ and Centro de Biología Molecular ‘‘Severo Ochoa,’’ Consejo Superior de Investigaciones Cientificas-Universidad Auto´noma de Madrid, Canto Blanco, 28049 Madrid, Spain; bSir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom; cInstitute for Cell and Molecular Biosciences, Newcastle University, Newcastle-upon-Tyne NE2 4HH, United Kingdom; and dGe´ne´ tique Microbienne, Institut National de la Recherche Agronomique, 78352 Jouy-en Josas Cedex, France Contributed by Margarita Salas, June 12, 2009 (sent for review April 20, 2009) Little is known about the organization or proteins involved in occurs via a so-called protein-primed mechanism (22, see Fig. membrane-associated replication of prokaryotic genomes. Here S1). Phage ␾29 DNA transcription is divided into early and late we show that the actin-like MreB cytoskeleton of the distantly stages (see Fig. S1 for a genetic and a transcriptional map). related bacteria Escherichia coli and Bacillus subtilis is required for Genes encoding DNA replication proteins such as DNA poly- efficient viral DNA replication. Detailed analyses of B. subtilis merase (p2) and TP (p3) are located in the left-side early operon. phage ␾29 showed that the MreB cytoskeleton plays a crucial role The right-side early operon contains gene 16.7 that encodes a in organizing phage DNA replication at the membrane. Thus, membrane protein (p16.7) required for optimal in vivo ␾29 DNA phage double-stranded DNA and components of the ␾29 replica- replication (23, 24). Additional functional, biochemical and tion machinery localize in peripheral helix-like structures in a structural studies have provided strong evidence that p16.7 is cytoskeleton-dependent way. Importantly, we show that MreB responsible for attaching ␾29 DNA to the bacterial membrane interacts directly with the ␾29 membrane-protein p16.7, respon- (24–26). Crystallographic resolution of the p16.7 DNA binding sible for attaching viral DNA at the cell membrane. Altogether, the domain (p16.7C) in complex with dsDNA revealed that 1 results reveal another function for the MreB cytoskeleton and dsDNA binding unit is formed by 3 p16.7C dimers that are describe a mechanism by which viral DNA replication is organized arranged in such a way that they form a deep positively charged at the bacterial membrane. longitudinal cavity that interacts with the phosphate backbone of dsDNA (27). Bacillus subtilis ͉ phage ␾29 Here we have analyzed the subcellular localization of com- ponents of the phage ␾29 replication machinery and found that ␾29 DNA polymerase, protein p16.7, and replicating ␾29 enes of the mreB family encode homologues of eukaryotic dsDNA localize in a helix-like pattern near the membrane of Gactin (1, 2) that form a cytoskeleton in most non-spherical infected B. subtilis cells. In addition, this helical organization bacteria (3–6). MreB proteins form filamentous structures fol- depends on all 3 host-encoded MreB proteins. Moreover, we lowing a helical path around the inner surface of the cytoplasmic show that MreB interacts directly with p16.7 in vivo. A model membrane (1). These actin-like filaments are continuously re- integrating these results is discussed. modelled during cell-cycle progression (7–11). Evidence is ac- cumulating that the bacterial MreB cytoskeleton plays key roles Results in several important cellular processes such as cell shape deter- Efficient Phage DNA Replication Requires an Intact Bacterial MreB mination, chromosome segregation, and cell polarity (1, 3, 8, Cytoskeleton. B. subtilis mreB mutant strains can be propagated 12–16). Whereas Gram-negative bacteria have a single mreB with near wild-type growth rate and cell morphology in growth gene, Gram-positive bacteria often have multiple mreB homo- media supplemented with high concentrations of magnesium logues. Bacillus subtilis encodes 3 MreB isoforms: MreB, Mbl, (28). Therefore, when cytoskeleton mutant strains were used in and MreBH (17–19). this work, media were supplemented with 25 mM MgSO4. For decades, evidence has been provided that replication of To examine a possible role of the bacterial actin-like cytoskel- phage DNA, like that of other prokaryotic genomes, occurs at eton in viral DNA replication we determined the efficiency of the cytoplasmic membrane (for review see 20). However, little is DNA replication of phages ␾29, SPP1, and PRD1 in infected known about the proteins or their organization in membrane- wild-type and mreB mutant cells. ␾29 and SPP1 infect the associated replication of viral genomes in bacteria. Phages ␾29 Gram-positive bacterium B. subtilis but use different modes of and SPP1 infect the Gram-positive bacterium B. subtilis, and DNA replication [reviewed in (21)]. In contrast, PRD1 uses a phage PRD1 infects the Gram-negative bacterium Escherichia similar DNA replication mechanism as ␾29 but infects the coli. Whereas PRD1 and ␾29 use the protein-primed mechanism Gram-negative bacterium E. coli (reviewed in 21). Fig. 1 shows of DNA replication, phage SPP1 replicates its DNA initially via that the efficiency of DNA replication of these phages was the theta mode and later via a rolling circle mode [reviewed in severely affected in the absence of an intact MreB cytoskeleton. (21)]. Here we show a key role for the MreB cytoskeleton in In the case of ␾29, deletion of any of the 3 mreB-like genes phages replicating by different modes in the distantly related bacteria E. coli and B. subtilis. Thus, the efficiency of replication CELL BIOLOGY of phage PRD1, and that of phages SPP1 and ␾29, is severely Author contributions: D.M.-E., R.D., J.E., W.J.J.M., and M.S. designed research; D.M.-E. and Y.K. performed research; V.C.-L. contributed new reagents/analytic tools; D.M.-E., R.D., affected in the absence of an intact cytoskeleton. R.C.-L., J.E., W.J.J.M., and M.S. analyzed data; and D.M.-E., J.E., W.J.J.M., and M.S. wrote the The underlying mechanism by which the cytoskeleton leads to paper. efficient phage DNA replication was analyzed in detail for B. The authors declare no conflict of interest. ␾ subtilis phage 29, whose DNA replication has been well char- 1J.E., W.J.J.M., and M.S. contributed equally to this work. ␾ acterized in vitro. The 29 genome consists of a linear double- 2To whom correspondence may be addressed. E-mail: [email protected] or stranded DNA (dsDNA) with a terminal protein (TP) covalently [email protected]. Ј linked at each 5 end that is the primer for the initiation of phage This article contains supporting information online at www.pnas.org/cgi/content/full/ DNA replication. Hence, initiation of ␾29 DNA replication 0906465106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906465106 PNAS ͉ August 11, 2009 ͉ vol. 106 ͉ no. 32 ͉ 13347–13352 Downloaded by guest on September 24, 2021 Fig. 1. Efficient viral DNA replication requires an intact MreB cytoskeleton. The amount of intracellular accumulated ␾29 (Left), SPP1 (Center), or PRD1 (Right) phage DNA was quantified by real-time PCR at different times after infection of the wild-type and the indicated mreB mutant strains. In the case of B. subtilis, strains used were DM-010 (control) and the isogenic cytoskeleton mutants DM-011 (⌬mreB), DM-012 (⌬mbl), and DM-013 (⌬mreBH). For E. coli, strains were DM-040 (control) and the isogenic cytoskeleton mutant DM-041 (⌬mreB). Samples were taken at different times after infection and processed as described in the Materials and Methods. The amounts of accumulated phage DNA (␮g viral DNA per mL culture) are expressed as a function of time after infection. caused a similar deleterious effect on DNA replication. Analyses in most cases spanned the entire length of the infected cells. of phage DNA accumulation by agarose gel electrophoresis Three-dimensional reconstruction of a set of deconvolved Z showed that intracellular phage DNA was detected early after sections demonstrated that p16.7 forms helical structures at the infection of both wild-type and mutant cells (see Fig. S2), membrane of infected cells (see SI Text and Movie S1). To study indicating that the cytoskeleton mutations have no or little effect the localization pattern of p16.7 in live cells, a B. subtilis strain on phage DNA injection. The results show that the bacterial MreB cytoskeleton is required for efficient phage DNA repli- cation in Gram-positive and Gram-negative bacteria, regardless of the phage DNA replication mechanism. The role of the cytoskeleton in viral DNA replication was studied in detail for B. subtilis phage ␾29. To get further evidence that an intact cytoskeleton is required for efficient ␾29 DNA ABC J K replication, phage DNA synthesis was studied using the B. subtilis strain 2060, which contains a disrupted mreB gene and a xylose-inducible copy of c-myc-mreB. The results presented in Fig. S3 show that the amount of phage DNA increased rapidly when the cells were grown in the presence of xylose. In contrast, the efficiency of ␾29 DNA replication was low in the absence of D E F L M the inductor. Importantly, efficient phage DNA replication was restored when xylose was added to the conditional strain 30 min after infection, confirming that MreB plays a role in phage DNA replication. ␾29 DNA Polymerase Localizes in a Helix-Like Pattern at the Periphery G H I N O of Infected Cells During ␾29 DNA Replication. To determine the subcellular distribution of the ␾29 replication machinery we first studied the localization of the gene 2-encoded ␾29 DNA poly- merase fused to the green fluorescent protein (GFP) in both infected and non-infected live cells.
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