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New Insights into the Structure and Assembly of Bacteriophage P1.

ABSTRACT: Bacteriophage P1 is the premier transducing phage of E. coli. Despite its prominence in advancing E. coli genetics, modern molecular techniques have not been applied to thoroughly understand P1 structure. Here, we report the proteome of the P1 virion as determined by liquid chromatography tandem mass-spectrometry. Additionally, a library of single-gene knockouts identified the following five previously unknown essential genes: pmgA, pmgB, pmgC, pmgG, and pmgR. In addition, proteolytic processing of the major capsid protein is a known feature of P1 morphogenesis, and we identified the processing site by N-terminal sequencing to be between E120 and S121, producing a 448-residue, 49.3 kDa mature peptide. Furthermore, the P1 defense against restriction (Dar) system consists of six known proteins that are incorporated into the virion during morphogenesis. The largest of these, DarB, is a 250 kDa protein that is believed to translocate into the cell during infection. DarB deletions indicated the presence of an N-terminal packaging signal, and the N-terminal 30 residues of DarB are shown to be sufficient for directing a heterologous reporter protein to the capsid. Taken together, the data expand on essential structural P1 proteins as well as introduces P1 as a nanomachine for cellular delivery.

SUBMITTER: Gonzales MF

PROVIDER: S-EPMC9024508 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The tailed double-stranded DNA (dsDNA) bacteriophage 29 packages its 19.3-kbp genome into a preassembled procapsid structure by using a transiently assembled phage-encoded molecular motor. This process is remarkable considering that compaction of DNA to near-crystalline densities within the confined space of the capsid requires that the packaging motor work against significant entropic, enthalpic, and DNA-bending energies. The motor consists of three phage-encoded components: the dodecameric connector protein gp10, an oligomeric RNA molecule known as the prohead RNA (pRNA), and the homomeric ring ATPase gp16. Although atomic resolution structures of the connector and different pRNA subdomains have been determined, the mechanism of self-assembly and the resulting stoichiometry of the various motor components on the phage capsid have been the subject of considerable controversy. Here a subnanometer asymmetric cryoelectron microscopy (cryo-EM) reconstruction of a connector-pRNA complex at a unique vertex of the procapsid conclusively demonstrates the pentameric symmetry of the pRNA and illuminates the relative arrangement of the connector and the pRNA. Additionally, a combination of biochemical and cryo-EM analyses of motor assembly intermediates suggests a sequence of molecular events that constitute the pathway by which the motor assembles on the head, thereby reconciling conflicting data regarding pRNA assembly and stoichiometry. Taken together, these data provide new insight into the assembly, structure, and mechanism of a complex molecular machine.Viruses consist of a protein shell, or capsid, that protects and surrounds their genetic material. Thus, genome encapsidation is a fundamental and essential step in the life cycle of any virus. In dsDNA viruses, powerful molecular motors essentially pump the viral DNA into a preformed protein shell. This article describes how a viral dsDNA packaging motor self-assembles on the viral capsid and provides insight into its mechanism of action.

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New Insights into the Structure and Assembly of Bacteriophage P1.

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