The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria

Muñoz‐Espín, Daniel; Daniel, Richard A.; Kawai, Yoshikazu; Carballido-Lopez, Rut; Castilla-Llorente, Virginia; Errington, Jeff; Meijer, Wilfried J. J.; Salas, Margarita

doi:10.1073/pnas.0906465106

Cited by 51 publications

(56 citation statements)

References 43 publications

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“…S3B). ϕ29 TP Recruits the Phage DNA Polymerase to the Bacterial Nucleoid, and Both Proteins Subsequently Are Redistributed in a Helix-Like Manner. We previously showed that ϕ29 DNA polymerase localizes uniformly in non-infected cells, and that at middle stages of the infection cycle, it localizes into clear helix-like structures in B. subtilis (11). Nevertheless, at 10 min postinfection, the ϕ29 DNA polymerase fused to YFP (YFP-p2) colocalizes with the bacterial nucleoid (Fig.…”

Section: N-terminal Domain Of the Tp Is Responsible For Its Nucleoidmentioning

confidence: 99%

“…We previously demonstrated that the helical localization of the ϕ29 DNA polymerase at middle stages of infection is lost in mreB cytoskeleton mutant strains (11). To investigate whether the helical-like redistribution of ϕ29 TP also depends on MreB, we examined the subcellular localization of YFP-TP in an mreB deletion mutant.…”

Section: N-terminal Domain Of the Tp Is Responsible For Its Nucleoidmentioning

confidence: 99%

“…Recently, we provided insights into the in vivo organization of proteins involved in ϕ29 DNA replication by showing that the bacterial actin-like MreB cytoskeleton, playing important roles in several cellular processes (reviewed in 10), is required for efficient DNA replication of the distantly related phages ϕ29, SPP1, and PRD1 (11). Components of the ϕ29 DNA replication machinery, such as the DNA polymerase, are redistributed in peripheral helix-like structures in a cytoskeleton-dependent way.…”

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

Muñoz‐Espín

Holguera

Ballesteros-Plaza

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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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.Bacillus subtilis | phage ø29 | DNA polymerase | DNA-binding | bacterial cytoskeleton P rotein-primed DNA replication is a mechanism used to initiate DNA synthesis in a variety of prokaryotic and eukaryotic organisms (1). Phages such as ϕ29 (infecting Bacillus subtilis) and PRD1 (infecting Escherichia coli) (1, 2), animal viruses such as adenoviruses (1, 3), bacterial species from the Streptomyces genus (1, 4), and viruses infecting Archaea (5-7) possess replication origins at their linear chromosomes constituted by inverted terminal repetitions with a terminal protein (TP) linked to both 5′ genome ends. TPs also have been reported in linear plasmids isolated from bacteria, yeast, fungi, and higher plants (1) and in animal and plant RNA viruses (1).The development of an in vitro replication system with purified proteins and DNA from the B. subtilis phage ϕ29 laid the foundations for investigating the protein-primed mechanism of DNA replication and makes ϕ29 a paradigm for studying this process (1, 2). A schematic overview of the in vitro ϕ29 DNA replication mechanism is shown in Fig. S1. The ϕ29 genome consists of a 19,285-bp linear dsDNA, with a TP of 31 kDa (the parental TP) covalently linked to each 5′ end. DNA replication starts with the recognition of the TP-containing DNA ends by a heterodimer formed by the ϕ29 DNA polymerase and a free TP molecule (the primer TP) (8). The DNA polymerase then catalyzes the formation of a covalent bond between deoxyAMP and the hydroxyl group Ser 232 of the primer TP. Replication is coupled to strand displacement, and continuous elongation of the DNA polymerase from both DNA ends generates replication intermediates that finally converge in the complete duplication of the parental strands (reviewed in ref.2). Phage ϕ29 DNA transcription is divided into early and late stages (9). Fig. S1 shows a genetic and a transcriptional map. Genes 2 and 3, encoding phage DNA polymerase and TP, respectively, are located i...

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Section: N-terminal Domain Of the Tp Is Responsible For Its Nucleoidmentioning

confidence: 99%

Section: N-terminal Domain Of the Tp Is Responsible For Its Nucleoidmentioning

confidence: 99%

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confidence: 99%

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

Muñoz‐Espín

Holguera

Ballesteros-Plaza

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…They are responsible for proper chromosome segregation and movement, as well as for cell-wall integrity during elongation and division (22,(26)(27)(28)(29). Interestingly, two proteins of Bacillus Φ29 phage interact with FtsZ and MreB, but in contrast to the T7 proteins, they do not inhibit them but rather exploit them for phage replication (30). The fact that FtsZ has been shown to confer a competitive advantage to the phage merely via inhibition of FtsZ suggests that this is also the case for MreB.…”

Section: Discussionmentioning

confidence: 99%

Revealing bacterial targets of growth inhibitors encoded by bacteriophage T7

Molshanski-Mor

Yosef

Kiro

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Today's arsenal of antibiotics is ineffective against some emerging strains of antibiotic-resistant pathogens. Novel inhibitors of bacterial growth therefore need to be found. The target of such bacterialgrowth inhibitors must be identified, and one way to achieve this is by locating mutations that suppress their inhibitory effect. Here, we identified five growth inhibitors encoded by T7 bacteriophage. High-throughput sequencing of genomic DNA of resistant bacterial mutants evolving against three of these inhibitors revealed unique mutations in three specific genes. We found that a nonessential host gene, ppiB, is required for growth inhibition by one bacteriophage inhibitor and another nonessential gene, pcnB, is required for growth inhibition by a different inhibitor. Notably, we found a previously unidentified growth inhibitor, gene product (Gp) 0.6, that interacts with the essential cytoskeleton protein MreB and inhibits its function. We further identified mutations in two distinct regions in the mreB gene that overcome this inhibition. Bacterial two-hybrid assay and accumulation of Gp0.6 only in MreB-expressing bacteria confirmed interaction of MreB and Gp0.6. Expression of Gp0.6 resulted in lemon-shaped bacteria followed by cell lysis, as previously reported for MreB inhibitors. The described approach may be extended for the identification of new growth inhibitors and their targets across bacterial species and in higher organisms. in some bacteria, the resistance mechanisms against most conventional antibiotics have been identified (1, 2). This increasing threat is spurring the identification of novel antimicrobials against novel molecular targets in the pathogens (e.g., refs. 3-6). There are currently only a few host molecules targeted by antibiotics. These targets (and examples of the antibiotics against them) are host RNA polymerase (rifampicin), topoisomerase (quinolones), cell wall (penicillin), membranes (polymyxin), ribosome (tetracyclines, aminoglycosides, macrolids), and synthesis of nucleic-acid precursors (sulfonamides, trimethoprim). Increasing the arsenal of bacterial targets and antimicrobial drugs against them is valuable, and novel strategies to increase this repertoire are therefore of great importance.One strategy for the identification of novel antibacterial targets is to determine how bacteriophages shut down their host's biosynthetic pathways and enslave its machinery during infection. Phages have coevolved with bacteria for over 3 billion years and have thus developed molecules to specifically and optimally inhibit or divert key metabolic functions. Examples of bacterial targets inhibited by phage-derived products include the δ subunit of the DNA polymerase III clamp loader, inhibited by gene product (Gp) 8 of the coliphage N4 (7); the Staphylococcus aureus putative helicase loader, DnaI, inhibited by ORF104 of bacteriophage 77 (5); a key enzyme of folate metabolism, FolD, inhibited by Gp55.1 of the coliphage T4 (8); and the essential cell-division protein, filamenting temperature-sen...

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“…The interaction between actin and hnRNPs, which bridges transcription, chromatin remodeling and pre-mRNA processing, is an excellent example of how actin acts to bring different macromolecular assemblies together [Sjolinder et al, 2005;Obrdlik et al, 2008]. A prokaryotic example of this could be MreB, which organizes bacteriophage replication by anchoring the viral DNA to membrane proteins [Munoz-Espin et al, 2009]. As actin has a limited surface area, the competition between different factors for binding to actin may therefore act to regulate different nuclear and prokaryotic processes.…”

Section: Conclusion and Further Perspectivesmentioning

confidence: 99%

Actin on DNA—An ancient and dynamic relationship

Skarp

Vartiainen

2010

Cytoskeleton

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In the cytoplasm of eukaryotic cells the coordinated assembly of actin filaments drives essential cell biological processes, such as cell migration. The discovery of prokaryotic actin homologues, as well as the appreciation of the existence of nuclear actin, have expanded the scope by which the actin family is utilized in different cell types. In bacteria, actin has been implicated in DNA movement tasks, while the connection with the RNA polymerase machinery appears to exist in both prokaryotes and eukaryotes. Within the nucleus, actin has further been shown to play a role in chromatin remodeling and RNA processing, possibly acting to link these to transcription, thereby facilitating the gene expression process. The molecular mechanism by which actin exerts these newly discovered functions is still unclear, because while polymer formation seems to be required in bacteria, these species lack conventional actin-binding proteins to regulate the process. Furthermore, although the nucleus contains a plethora of actin-regulating factors, the polymerization status of actin within this compartment still remains unclear. General theme, however, seems to be actin's ability to interact with numerous binding partners. A common feature to the novel modes of actin utilization is the connection between actin and DNA, and here we aim to review the recent literature to explore how this connection is exploited in different contexts. V C 2010 Wiley-Liss, Inc.Key Words: actin, myosin, DNA, nucleus, transcription Introduction S ince the discovery of actin, a great number of actin-binding proteins (ABPs) have been found and implicated in various cellular processes. Traditionally, the reign of actin and ABPs has been limited to the cytoplasm, where they cooperate to form helical actin filaments, which can be bundled and cross-linked to different structural ends. The coordinated assembly of these filaments then drives for example cell migration, cytokinesis and membrane dynamics [reviewed in Pollard and Borisy, 2003]. However, during the past decade, actin has been transformed from an exclusively cytoskeletal component in eukaryotic cells to a multipurpose tool that most cells use in a number of processes. Especially the discovery of a function for actin in the nucleus [reviewed in Vartiainen, 2008] and identification of prokaryotic actin homologues [van den Ent et al., 2001] has yielded surprising data, which has considerably expanded the modes by which the actin family is utilized in different functional contexts. In the nucleus, actin has been shown to be involved all the way in the gene expression process from chromatin remodeling complexes through transcription to spliceosome function and mRNA export [reviewed in Percipalle, 2009]. In addition, nuclear actin also seems to affect the expression of subsets of genes by regulating the activity of specific transcription factors [Vartiainen et al., 2007]. However, the biochemical details of most of these actin-involving processes are lacking, and the nature and mechanics of the interact...

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The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria

Cited by 51 publications

References 43 publications

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

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

Revealing bacterial targets of growth inhibitors encoded by bacteriophage T7

Actin on DNA—An ancient and dynamic relationship

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