The high‐resolution functional map of bacteriophage SPP1 portal protein

Isidro, Anabela; Santos, Mário A.; Henriques, Adriano O.; Tavares, Paulo

doi:10.1046/j.1365-2958.2003.03880.x

Cited by 40 publications

(42 citation statements)

References 46 publications

(98 reference statements)

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“…The region from rtp23 to rtp43 contains genes for head and tail morphogenesis; this region is highly syntenic between phages Rtp and T1. The precise location of the border between the head and tail genes is difficult to predict from the (51,81). Rtp25 is a possible prohead protease.…”

Section: Resultsmentioning

confidence: 99%

The Genome of the Novel Phage Rtp, with a Rosette-Like Tail Tip, IsHomologous to the Genome of Phage T1

et al. 2006

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A new Escherichia coli phage, named Rtp, was isolated and shown to be closely related to phage T1. Electron microscopy revealed that phage Rtp has a morphologically unique tail tip consisting of four leaf-like structures arranged in a rosette, whereas phage T1 has thinner, flexible leaves that thicken toward the ends. In contrast to T1, Rtp did not require FhuA and TonB for infection. The 46.2-kb genome of phage Rtp encodes 75 open reading frames, 47 of which are homologous to phage T1 genes. Like phage T1, phage Rtp encodes a large number of small genes at the genome termini that exhibit no sequence similarity to known genes. Six predicted genes larger than 300 nucleotides in the highly homologous region of Rtp are not found in T1. Two predicted HNH endonucleases are encoded at positions different from those in phage T1. The sequence similarity of rtp37, -38, -39, -41, -42, and -43 to equally arranged genes of lambdoid phages suggests a common tail assembly initiation complex. Protein Rtp43 is homologous to the J protein, which determines host specificity. Since the two proteins differ most in the C-proximal area, where the binding site to the LamB receptor resides in the J protein, we propose that Rtp43 contributes to Rtp host specificity. Lipoproteins similar to the predicted lipoprotein Rtp45 are found in a number of phages (encoded by cor genes) in which they prevent superinfection by inactivating the receptors. We propose that, similar to the proposed function of the phage T5 lipoprotein, Rtp45 prevents inactivation of Rtp by adsorption to its receptor during cells lysis. Rtp52 is a putative transcriptional regulator, for which 10 conserved inverted repeats were identified upstream of genes in the Rtp genome. In contrast, the much larger E. coli genome has only one such repeat sequence.Phages form the largest group of "organisms" on earth and display a huge genetic diversity. To date, 228 phage genomes have been sequenced and the 252 sequenced bacterial genomes contain many prophages and phage-like elements. Horizontal gene transfer between phages and their hosts largely contributes to the genetic variety of the phages and their hosts. During evolution, phages endowed bacteria with new functions enabling them to occupy new environmental niches. Phage genomics provides data for tracing phage and bacterial evolution. It is therefore desirable to determine the sequences of as many phages as possible.Escherichia coli and a selected group of E. coli phages paved the way into molecular biology. Among the phages was T1 (1, 29), whose genome sequence was, however, not determined until 2004, over 60 years after its discovery (75). The phage T1 genome encodes 77 open reading frames (ORFs), 37 of which are homologous to described phage ORFs. Functions have been assigned to 27 ORFs. The largest functional group consists of tail proteins. Twenty-two ORFs encoding putative proteins of fewer than 100 amino acid (aa) residues lie predominantly close to the termini. These putative proteins display no sequence similarity to...

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Section: Resultsmentioning

confidence: 99%

The Genome of the Novel Phage Rtp, with a Rosette-Like Tail Tip, IsHomologous to the Genome of Phage T1

et al. 2006

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“…Expression was controlled by the LacI repressor coded by plasmid pGB3 in E. coli or by pEB104 in B. subtilis, respectively (31). Missense mutations in gene 6 were transferred from pFiFAcc (24) to the vector by subcloning PflMI-Asp718 (coordinates 3040 -3244 of the SPP1 sequence) (mutations S279G, N290T, and E294K) or Asp718-PstI fragments (coordinates 3244 -3917 of the SPP1 sequence; the PstI site is from the vector polylinker) (mutations T319A and E352G). The replacement strategy is as described (25).…”

Section: Methodsmentioning

confidence: 99%

“…Analysis of gp6 Production-The production of gp6 in B. subtilis strains expressing different gene 6 alleles was determined by immunoblot analysis of crude extracts developed with an anti-gp6 antibody (24).…”

Section: Methodsmentioning

confidence: 99%

“…At the end of genome encapsidation, gp6 is a central component of the sensor system that triggers headful cleavage of substrate DNA after a threshold amount of DNA is reached inside the capsid (8,23). Single amino acid substitutions in gp6 can block the initiation of DNA packaging, its efficiency, or termination of the process that yields the ϳ45.9-kbp long mature viral chromosome (24,25).…”

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confidence: 99%

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Modulation of the Viral ATPase Activity by the Portal Protein Correlates with DNA Packaging Efficiency

Oliveira

Henriques²,

Tavares³

2006

Journal of Biological Chemistry

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DNA packaging in tailed bacteriophages and herpesviruses requires assembly of a complex molecular machine at a specific vertex of a preformed procapsid. As in all these viruses, the DNA translocation motor of bacteriophage SPP1 is composed of the portal protein (gp6) that provides a tunnel for DNA entry into the procapsid and of the viral ATPase (gp1-gp2 complex) that fuels DNA translocation. Here we studied the cross-talk between the components of the motor to control its ATP consumption and DNA encapsidation. We showed that gp6 embedded in the procapsid structure stimulated more than 10-fold the gp2 ATPase activity. This stimulation, which was significantly higher than the one conferred by isolated gp6, depended on the presence of gp1. Mutations in different regions of gp6 abolished or decreased the gp6-induced stimulation of the ATPase. This effect on gp2 activity was observed both in the presence and in the absence of DNA and showed a strict correlation with the efficiency of DNA packaging into procapsids containing the mutant portals. Our results demonstrated that the portal protein has an active control over the viral ATPase activity that correlates with the performance of the DNA packaging motor.The mechanism by which viral DNA is packaged into a preformed procapsid is a fascinating but still unsolved biological problem. Tailed phages and herpesviruses package DNA to densities as high as 500 mg/ml in the preformed procapsid structure (1-3). The DNA translocating machine that accomplishes this task is the most powerful biomolecular motor presently described (4, 5). Like many other molecular motors, this machine converts the chemical energy generated by ATP hydrolysis into mechanical movement of DNA. Intensive research over the past years enabled the identification and characterization of the components of the DNA translocating motor in several bacteriophages, as well as in herpesviruses (reviewed in Ref. 6). Despite some differences that may subsist between individual virus species, numerous biochemical and structural similarities have been found among the motor components, suggesting a common mechanism for DNA translocation. The motor is composed of an ATPase (terminase) and of the portal protein, a turbine-like oligomer localized at the unique vertex of the icosahedral capsid through which DNA movements occur (7-11). Packaging is initiated by docking of the DNA-terminase complex at the portal vertex. DNA is then translocated through the central tunnel of the oligomeric portal protein to be packed at high density inside the viral capsid. Translocation of the double helix is fueled by the terminase ATPase activity (12-16). Packaging is normally terminated by an endonucleolytic cleavage of the concatemeric DNA used as substrate for encapsidation by most phages (3). Previous work defined the components of the motor but elucidated neither how the DNA packaging motor assembles nor the role of the portal protein in DNA translocation.The Bacillus subtilis bacteriophage SPP1 is a well established model system for...

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“…The titers were expressed as a percentage of the titer obtained by infection of the permissive B. subtilis strain HA101B to determine the level of complementation (33). Extracts of B. subtilis were prepared and analyzed by Western blot as described previously (34).…”

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confidence: 99%