Spo0A, the key transcriptional regulator for entrance into sporulation, is an inhibitor of DNA replication

Castilla-Llorente, Virginia; Muñoz‐Espín, Daniel; Villar, Laurentino; Salas, Margarita; Meijer, Wilfried J. J.

doi:10.1038/sj.emboj.7601266

Cited by 55 publications

(95 citation statements)

References 51 publications

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“…Phage ϕ29 DNA replication is inhibited by the host protein Spo0A (28). Because the B. subtilis spo0A promoter switch is repressed by an excess of glucose (29), medium was supplemented with 2% glucose.…”

Section: Methodsmentioning

confidence: 99%

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: Methodsmentioning

confidence: 99%

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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“…Since phage 29 DNA replication is inhibited by Spo0A (33,34), spo0A deletion strains were used when indicated. Unless stated otherwise, the lysis-delayed mutant phage 29 sus14(1242) (35) was used.…”

Section: Methodsmentioning

confidence: 99%

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

Muñoz‐Espín

Daniel

Kawai

et al. 2009

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

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Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage 29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the 29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the 29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.Bacillus subtilis ͉ phage 29 G enes of the mreB family encode homologues of eukaryotic actin (1, 2) that form a cytoskeleton in most non-spherical bacteria (3-6). MreB proteins form filamentous structures following a helical path around the inner surface of the cytoplasmic membrane (1). These actin-like filaments are continuously remodelled during cell-cycle progression (7-11). Evidence is accumulating that the bacterial MreB cytoskeleton plays key roles in several important cellular processes such as cell shape determination, chromosome segregation, and cell polarity (1,3,8,(12)(13)(14)(15)(16). Whereas Gram-negative bacteria have a single mreB gene, Gram-positive bacteria often have multiple mreB homologues. Bacillus subtilis encodes 3 MreB isoforms: MreB, Mbl,.For decades, evidence has been provided that replication of phage DNA, like that of other prokaryotic genomes, occurs at the cytoplasmic membrane (for review see 20). However, little is known about the proteins or their organization in membraneassociated replication of viral genomes in bacteria. Phages 29 and SPP1 infect the Gram-positive bacterium B. subtilis, and phage PRD1 infects the Gram-negative bacterium Escherichia coli. Whereas PRD1 and 29 use the protein-primed mechanism of DNA replication, phage SPP1 replicates its DNA initially via the theta mode and later via a rolling circle mode [reviewed in (21)]. Here we show a key role for the MreB cytoskeleton in phages replicating by different modes in the distantly related bacteria E. coli and B. subtilis. Thus, the efficiency of replication of phage PRD1, and that of phages SPP1 and 29, is severely affected in the absence of an intact cytoskeleton.The underlying mechanism by which the cytoskeleton leads to efficient phage DNA replication was analyzed in detail for B. subtilis phage 29, whose DNA replication has been well characterized in vitro. The 29 genome consists of a linear doublestranded DNA (dsDNA) with a terminal protein (TP) covalently linked at each 5Ј end that is the primer for the initiation of phage DNA replication. Hence, initiation of 29 DNA replication occurs via a so-called protein-prim...

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“…To confirm the insertion sites, amyE region was PCR amplified using amyE_5 and amyE_3 primers (Table 2) and sequenced. Since Spo0A protein encoded by host genome inhibits ϕ29 DNA replication (Castilla-Llorente et al, 2006), the spo0A gene was disrupted by inserting neomycin resistant gene.…”

Section: Construction Of B Subtilis Strains Expressing ϕ29mentioning

confidence: 99%

Functional linkages between replication proteins of genes 1, 3 and 5 of <i>Bacillus subtilis</i> phage <b>φ</b>29

2012

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Gene 1 product (gp1) of Bacillus subtilis phage ϕ29 has been shown to be involved in viral DNA replication in vivo, but the essential role is still unknown. As part of an ongoing effort to understand the role of gp1 in viral DNA replication, we investigated genetic interaction between gene 1 and other viral genes. Because ϕ29 mutants which do not produce functional gp1 show temperaturesensitive growth, we isolated temperature-resistant phages from the ϕ29 gene 1 mutants, and eventually, obtained nine extragenic suppressors. These suppressor mutations were located in two essential genes for ϕ29 DNA replication in vivo: gene 3 encoding terminal/primer protein (gp3) or gene 5 encoding viral singlestranded DNA binding protein (gp5). Most of these mutations resulted in single amino acid substitutions in the products. By trans-complementation assay, we confirmed that the absence of gp1 at non-permissive temperature can be compensated by the suppressors which have the single amino acid substitution in either gp5 or gp3. These results indicate that gp1 has functional relationship to gp5 and gp3. From the positions of amino acid substitutions in gp3, we propose its new regulatory subdomain at which other molecules including gp1 would interact with and regulate functions of gp3.

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Spo0A, the key transcriptional regulator for entrance into sporulation, is an inhibitor of DNA replication

Cited by 55 publications

References 51 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

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

Functional linkages between replication proteins of genes 1, 3 and 5 of <i>Bacillus subtilis</i> phage <b>φ</b>29

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