The Phage φ29 Membrane Protein p16.7, Involved in DNA Replication, Is Required for Efficient Ejection of the Viral Genome

Abstract

It is becoming clear that in vivo phage DNA ejection is not a mere passive process. In most cases, both phage and host proteins seem to be involved in pulling at least part of the viral DNA inside the cell. The DNA ejection mechanism of Bacillus subtilis bacteriophage 29 is a two-step process where the linear DNA penetrates the cell with a right-left polarity. In the first step ϳ65% of the DNA is pushed into the cell. In the second step, the remaining DNA is actively pulled into the cytoplasm. This step requires protein p17, which is encoded by the right-side early operon that is ejected during the first push step. The membrane protein p16.7, also encoded by the right-side early operon, is known to play an important role in membrane-associated phage DNA replication. In this work we show that, in addition, p16.7 is required for efficient execution of the second pull step of DNA ejection.Despite being a key step in the phage life cycle, the process whereby phages eject their genome into the cell cytoplasm during the early steps of infection has been one of the major unsolved problems. The current understanding of the phage DNA ejection process has recently been reviewed by Molineux (29). The ejection process of phages that infect gram-negative bacteria is now beginning to be understood at a detailed molecular level (24,30,33). This process is far from being a passive mechanism, just driven by the release of the pressure built inside the capsid. The cell cytoplasm has a pressure of several atmospheres with respect to the external medium. Therefore, the pressure-based mechanism would be responsible for ejection of only part of the phage genome; i.e., ejection of phage DNA by pressure will stop when the decreasing packing forces in the mature virion equal those inside the cytoplasm. Thus, internalization of the remaining part of the phage genome requires additional mechanisms, and diverse strategies have been described for different phages. For instance, some Escherichia coli phages eject their genomes into the cell largely through enzyme-catalyzed, energy-requiring processes. Phage T5 DNA ejection takes place in two steps (23). In the first step, 8% of the genome enters the cytoplasm. Then, there is a pause of about 4 min during which two proteins encoded by this DNA fragment, A1 and A2, are synthesized. The transfer of the remaining DNA (92%) takes place in a second step only if these proteins are synthesized. A2 is a DNA binding protein, thought to pull DNA into the cell (39). Phage T7 is the only one for which it has been clearly demonstrated that DNA internalization is coupled to transcription (reviewed in reference 29). About 850 bp of the left end of the T7 genome are ejected into the host (11). A molecular motor formed by viral proteins gp16 and gp15 has been proposed to control the amount of DNA that enters the cell (21). Transcription from promoters located on this short DNA sequence by the host RNA polymerase provides the force to internalize about 20% of the genome into the cell. T7 RNA polymerase is the...

The Presence of Two Receptor-Binding Proteins Contributes to the Wide Host Range of Staphylococcal Twort-Like Phages

“…Insertion of one nucleotide in orf58 caused a frameshift and generated a premature stop codon, leading to partial deletion of the gene product (amino acids [aa] 85 to 108). orf58 encodes a protein homologous to the membrane protein MbpC of staphylococcal Twort-like phage A5W; it is predicted to play a role in attachment of the complex of replicating phage DNA to a cell membrane, analogously to membrane protein p16.7 of Bacillus phage 29 (7,48). Although substitutions of amino acids in ORF33 were observed only in ⌽SA012M2, they did not accumulate among later mutant phages.…”

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

“…The left-to-right orientation of DNA movement in packaging for 29 is later reversed upon ejection, where the right end of the DNA enters the host cell first (33). Studies have shown that DNA ejection in 29 is only partially driven by the internal pressure inside the packaged head (10,11). After the right end enters the cell, genes 16.7 and 17 at the extreme right end of the genome are transcribed and the resulting proteins serve to pull the remainder of the 29 DNA into the host cell.…”

“…Covalently bound to each 5= end of the 29 DNA is the terminal protein gp3, which is essential for DNA packaging, as well as DNA replication. Relative to the genetic map, packaging initiates at the left terminal protein and proceeds in a left-to-right direction (9); consequently, the right end is the first end ejected and contains the genes needed to successfully complete the ejection process (10,11). While there is no exact equivalent of terS in 29, the DNA-binding function of terS suggests a function analogous to that of the gp3 terminal proteins (12).…”

Induction of Loci Mutation duringLactococcus lactisSpontaneous Conversion to Bacteriophage-Insensitive Phenotype

“…B. subtilis phage ø29 transfers its DNA to the cell cytoplasm in two steps as well. The second step requires the presence of viral p16.7 and p17 proteins that are involved in phage genome replication (Alcorlo et al, 2007).…”