In vitro assembly of the Bacillus subtilis bacteriophage phi 29.

Bjornsti, Mary-Ann; Reilly, Bernard E.; Anderson, Dwight L.

doi:10.1073/pnas.78.9.5861

Cited by 29 publications

(41 citation statements)

References 26 publications

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“…Although phage morphogenesis and DNA packaging have not yet been analyzed for B103 or GA-1, they have been studied extensively for 29 (for a review, see reference 9), and a highly efficient in vitro 29 DNA-packaging system has been established (23,96). Moreover, the threedimensional structures of the 29 virion and its empty prohead precursor, obtained by reconstruction of cryoelectron microscopy (cryo-EM) images, have been determined recently (201).…”

Section: Structural Phage Proteins and 29 Phage Morphogenesismentioning

confidence: 99%

φ29 Family of Phages

Meijer

Horcajadas

Salas

2001

Microbiol Mol Biol Rev

187

223

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SUMMARY Continuous research spanning more than three decades has made the Bacillus bacteriophage φ29 a paradigm for several molecular mechanisms of general biological processes, such as DNA replication, regulation of transcription, phage morphogenesis, and phage DNA packaging. The genome of bacteriophage φ29 consists of a linear double-stranded DNA (dsDNA), which has a terminal protein (TP) covalently linked to its 5′ ends. Initiation of DNA replication, carried out by a protein-primed mechanism, has been studied in detail and is considered to be a model system for the protein-primed DNA replication that is also used by most other linear genomes with a TP linked to their DNA ends, such as other phages, linear plasmids, and adenoviruses. In addition to a continuing progress in unraveling the initiation of DNA replication mechanism and the role of various proteins involved in this process, major advances have been made during the last few years, especially in our understanding of transcription regulation, the head-tail connector protein, and DNA packaging. Recent progress in all these topics is reviewed. In addition to φ29, the genomes of several other Bacillus phages consist of a linear dsDNA with a TP molecule attached to their 5′ ends. These φ29-like phages can be divided into three groups. The first group includes, in addition to φ29, phages PZA, φ15, and BS32. The second group comprises B103, Nf, and M2Y, and the third group contains GA-1 as its sole member. Whereas the DNA sequences of the complete genomes of φ29 (group I) and B103 (group II) are known, only parts of the genome of GA-1 (group III) were sequenced. We have determined the complete DNA sequence of the GA-1 genome, which allowed analysis of differences and homologies between the three groups of φ29-like phages, which is included in this review.

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Section: Structural Phage Proteins and 29 Phage Morphogenesismentioning

confidence: 99%

φ29 Family of Phages

Meijer

Horcajadas

Salas

2001

Microbiol Mol Biol Rev

187

223

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“…The gpl6 was eluted in two peaks (data not shown). The gpl6 in the first peak had no biological activity in an in vitro complementation assay with a 16-14i extract (1) and was contaminated with most of the residual host proteins (data not shown). The second peak contained pure (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…The nonpermissive host B. subtilis SpoA12 (6) and the permissive hosts B. subtilis sup+4 (7) and L15 (6) were used. The 429 recombinants sus16(300)-susl4(1241) (1), sus7(614)-sus8(769)-susl4(1241), sus4(369)-sus8(22), and sus7(614)-sus8(22)-susl6(300)-susl4(1241) (4) have been described. Genes (8,9).…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Bacteriophage 429 of Bacillus subtilis is an attractive model for studying the mechanism of viral DNA packaging because of its relative simplicity and the high efficiency of in vitro assembly (1)(2)(3)(4)(5). The 12-MDa 429 DNA has the product of gene 3 (gp3) covalently bound at its 5' termini, and this protein has an essential role in DNA packaging (2).…”

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confidence: 99%

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A defined system for in vitro packaging of DNA-gp3 of the Bacillus subtilis bacteriophage phi 29.

Guo

Grimes

Anderson

1986

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

142

150

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The bacteriophage 429 DNA-gene product 3 complex (DNA-gp3) has been efficiently packaged into proheads in a completely defined in vito system. The 429 DNA packaging protein gpl6, the product of gene 16, was overproduced in Escherichia coli and purified to near homogeneity.The purified gpl6 packaged 23% of the DNA-gp3 added to purified proheads in the dermed mixture, while gpl6 in an extract of phage-infected cells packaged 26% of the DNA-gp3.No host proteins were required in the defined system. ATPdependent packaging of DNA-gp3 in the defined system was optimal with %100 copies of gpl6 per DNA-gp3, an amount similar to the production of gpl6 per DNA-gp3 in +29-infected cells.Bacteriophage 429 of Bacillus subtilis is an attractive model for studying the mechanism of viral DNA packaging because of its relative simplicity and the high efficiency of in vitro assembly (1)(2)(3)(4)(5) MATERIALS AND METHODSBacteria, Phage, and Plasmids. The nonpermissive host B. subtilis SpoA12 (6) and the permissive hosts B. subtilis sup+4 (7) and L15 (6) gpl6 accumulated as large lumps in the cells (Fig. lb). The cells (200 ml) were sedimented, resuspended in 100 ml of buffer for forming spheroplasts (0.01 M Tris-HCl, pH 8.0/0.01 M EDTA/0.125 M NaCl/500 ,ug of lysozyme per ml), and incubated for 5 min at ambient temperature. All subsequent manipulations were performed at 4°C except as noted. Spheroplasts were centrifuged at 3000 X g for 5 min, and the pellet was resuspended in 100 ml of 0.01 M Tris HCl, pH 7.5/0.001 M dithiothreitol. The spheroplasts were lysed in the French pressure cell at 69 MPa. Urea (Schwarz/Mann; 8 M, adjusted to pH 5.2 with glacial acetic acid) was added to the lysate to a final concentration of 0.7 M. After a clarifying centrifugation at 750 x g for 10 min, the supernatant was centrifuged at 7000 x g for 5 min to sediment the gpl6 lumps. The pellet was dissolved in 10 ml of solubilizing buffer [0.01 M Tris HCl, pH 7.5/0.005 M dithiothreitol/6 M guanidinium chloride (Serva, Heidelberg; or BDH)], and the solution was centrifuged at 100,000 x g for 1 hr. One milliliter of the supernatant containing gpl6 was applied to a 1 x 55 cm Sephadex G-150 column equilibrated with 7.5 M urea/0.001 M dithiothreitol adjusted to pH 5.2 as above. The gpl6 was eluted in two peaks with the same solution; fractions of the second peak were pooled, and 0.2 ml of saturated ammonium sulfate was added for each 1.0 ml of eluant. After 3 min at ambient temperature, the precipitate was collected by cenAbbreviations: gp3 and gpl6, products of phage 429 genes 3 and 16;pfu, plaque-forming units. *Permanent address: South China Agricultural University, Guangzhou (Canton), The People's Republic of China. 3505The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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