Architecture of the flexible tail tube of bacteriophage SPP1

Bacteriophage SPP1 is a double-stranded DNA virus of the Siphoviridae family that infects the bacterium Bacillus subtilis. This family of phages features a long, flexible, non-contractile tail that has been difficult to characterize structurally. Here, we present the atomic structure of the tail tube of phage SPP1. Our hybrid structure is based on the integration of structural restraints from solid-state nuclear magnetic resonance (NMR) and a density map from cryo-EM. We show that the tail tube protein gp17.1 organizes into hexameric rings that are stacked by flexible linker domains and, thus, form a hollow flexible tube with a negatively charged lumen suitable for the transport of DNA. Additionally, we assess the dynamics of the system by combining relaxation measurements with variances in density maps.

Bacteriophage SPP1 Chu Is an Alkaline Exonuclease in the SynExo Family of Viral Two-Component Recombinases

ABSTRACT Many DNA viruses concatemerize their genomes as a prerequisite to packaging into capsids. Concatemerization arises from either replication or homologous recombination. Replication is already the target of many antiviral drugs, and viral recombinases are an attractive target for drug design, particularly for combination therapy with replication inhibitors, due to their important supporting role in viral growth. To dissect the molecular mechanisms of viral recombination, we and others previously identified a family of viral nucleases that comprise one component of a conserved, two-component viral recombination system. The nuclease component is related to the exonuclease of phage λ and is common to viruses with linear double-stranded DNA genomes. To test the idea that these viruses have a common strategy for recombination and genome concatemerization, we isolated the previously uncharacterized 34.1 gene from Bacillus subtilis phage SPP1, expressed it in Escherichia coli, purified the protein, and determined its enzymatic properties. Like λ exonuclease, Chu (the product of 34.1) forms an oligomer, is a processive alkaline exonuclease that digests linear double-stranded DNA in a Mg2+-dependent reaction, and shows a preference for 5′-phosphorylated DNA ends. A model for viral recombination, based on the phage λ Red recombination system, is proposed.

Characterization of the Putative Replisome Organizer of the Lactococcal Bacteriophage r1t

ABSTRACT Analysis of the nucleotide sequence of the genome of the lactococcal bacteriophage r1t showed that it may encode at least two proteins involved in DNA replication. On the basis of its similarity with the G38P protein encoded by the Bacillus subtilis phage SPP1, the product of orf11 (Pro11) is thought to be involved in the initiation of phage DNA replication. This protein was overexpressed in Lactococcus lactis and partially purified. Gel retardation analysis using various r1t DNA fragments indicates that Pro11 specifically binds to a sequence located within its cognate gene. DNase I footprinting showed that Pro11 protects a stretch of DNA of 47 bp. This region spans four 6-bp short direct repeats, which suggests that the region contains four binding sites for Pro11. 1,10-Phenanthroline-copper footprinting confirmed the protection of the hexamers. An asymmetric protection pattern of each strand was observed, suggesting that Pro11 contacts each DNA strand separately at contiguous hexamers. We propose a model for the binding of Pro11 to its target sites that may account for the torsion strain required for strand opening at the origin of replication.

The effect of magnesium on transfection and transformation in Bacillus subtilis

The influence of Mg2+ on phage SPP1 DNA-mediated transfection as compared with chromosomal transformation was studied.A differential influence of this cation in both processes was detectable by analyzing the competence development and the kinetics of appearance of transformants and transfectants. Binding and uptake of DNA and release of acid-soluble products by competent cells in high- and low-Mg2+ media was measured for 3H-labelled SPP1 and 3H-labelled Bacillus subtilis DNAs. Phage SPP1 DNA was shown to be subjected to endonucleolytic cleavage after exposure to competent cells.