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Electron microscopy in combination with image processing is a powerful method for obtaining structural information on non‐crystallized biological macromolecules at the 10–50 A resolution level. The processing of noisy microscopical images requires advanced data processing methodologies in which one must carefully avoid the introduction of any form of bias into the data set. Using a novel multivariate statistical approach to the analysis of symmetry, we studied the structure of the bacteriophage SPP1 portal protein oligomer. This portal structure, ubiquitous in icosahedral bacteriophages which package dsDNA, is located at the site of symmetry mismatch between a 5‐fold vertex of the icosahedral shell and the 6‐fold symmetric (helical) tail. From previous studies such ‘head‐to‐tail connector’ structures were generally accepted to be homododecamers assembled in a 12‐fold symmetric ring around a central channel. Using a new analysis methodology we have found that the phage SPP1 portal structure exhibits 13‐fold cyclical symmetry: a new point group organization for oligomeric proteins. A model for the DNA packaging mechanism by 13‐fold symmetric portal protein assemblies is presented which attributes a coherent functional meaning to their unusual symmetry.

We constructed a transfer system consisting of two compatible multicopy plasmids carrying the transfer regions Tral and Tra2 of the broad-host-range IncP plasmid RP4. In this system, the plasmid containing the Tral region with the origin of transfer (oriT) was transferred, whereas additional functions essential for the conjugative process were provided from the Tra2 plasmid in trans. The Tra2 region, as determined for matings between Escherichia coli cells, maps between coordinates 18.03 and 29.26 kb of the RP4 standard map. The section of Tra2 required for mobilization of the plasmid RSF1010 (IncQ) and the propagation of bacteriophages Pf3 and PRD1 appears to be the same as that needed for RP4 transfer. Tra2 regions of RP4 (IncPa) and R751 (IncPj3) are interchangeable, facilitating mobilization of the plasmid carrying the RP4 Tral region. The transfer frequencies of both systems are similar. Transcription of Tra2 proceeds clockwise relative to the standard map of RP4 and is probably initiated at a promoter region located upstream of trbB (kilB). From this promoter region the trfA operon and the Tra2 operon are likely to be transcribed divergently. A second potential promoter has been located immediately upstream of trbB (kiIB). Plasmids encoding the functional Tra2 region can only be maintained stably in host cells in the presence of the RP4 regulation region carrying the korA-korB operon or part of it. This indicates the involvement of RP4 key regulatory functions that apparently are active not only in the control of replication but also in conjugation.The conjugative transfer system of the promiscuous plasmid RP4 consists of two distinct regions, Tral and Tra2, separated by the Par (partitioning)/Mrs (multimer resolution system) region, the fiwA locus, IS8, and the kanamycin resistance gene (aphA). Tral, containing the origin of transfer (oriT), encodes functions involved in generating the single strand to be transferred and also includes the primase genes (59; for reviews see references 19 and 60). However, functions encoded by Tra2 have not yet been characterized extensively. Genetic approaches by transposon mutagenesis and complementation studies located the Tra2 region between the genes trbB (kiIB) andfiwA (3,4,37,51 acting replication function), encoding the replication protein and the Par/Mrs region, specifying a multimer resolution system (17,41). Plasmids containing the minimal functional Tra2 region could only be maintained stably when the korA-korB operon was present in trans, implying that KorA and KorB are involved in regulating expression of the transfer loci of Tra2. MATERUILS AND METHODSStrains, phages, and plasmids. E. coli HB101 (8) and S17-1 (47) were used as hosts for plasmids, and the nalidixic acid-resistant strain HB101 Nxr was used as a recipient for filter matings. Cells were grown in YT medium (33) buffered with 25 mM 3-(N-morpholino)propanesulfonic acid (sodium salt, pH 8.0) and supplemented with 0.1% glucose and 25 ,ug of thiamine-hydrochloride per ml. When appropriate, antibiotics...

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Many icosahedral viruses use a specialized portal vertex to control genome encapsidation and release from the viral capsid. In tailed bacteriophages, the portal system is connected to a tail structure that provides the pipeline for genome delivery to the host cell. We report the first, to our knowledge, subnanometer structures of the complete portal-phage tail interface that mimic the states before and after DNA release during phage infection. They uncover structural rearrangements associated with intimate protein-DNA interactions. The portal protein gp6 of bacteriophage SPP1 undergoes a concerted reorganization of the structural elements of its central channel during interaction with DNA. A network of protein-protein interactions primes consecutive binding of proteins gp15 and gp16 to extend and close the channel. This critical step that prevents genome leakage from the capsid is achieved by a previously unidentified allosteric mechanism: gp16 binding to two different regions of gp15 drives correct positioning and folding of an inner gp16 loop to interact with equivalent loops of the other gp16 subunits. Together, these loops build a plug that closes the channel. Gp16 then fastens the tail to yield the infectious virion. The gatekeeper system opens for viral genome exit at the beginning of infection but recloses afterward, suggesting a molecular diaphragm-like mechanism to control DNA efflux. The mechanisms described here, controlling the essential steps of phage genome movements during virus assembly and infection, are likely to be conserved among long-tailed phages, the largest group of viruses in the Biosphere.DNA gatekeeper | viral infection | bacteriophage | allosteric mechanism | hybrid methods