The similarity with the entire 0305w8-36 operon is highest for contraction initiates at the tail tip.8,9 The procedure developed lytic bacteriophage SPO1, which has a homolog for orfs 201 is based on centrifugation through a sucrose gradient (Materials (coupling ATPase; Table 2, row 9) and orf207 (Table 2, row 12), and Methods Section). not present in the other genomes (Fig. 2). The other bacterio- After the revised ultracentrifugation, we removed the sucrose phages, all lysogenic, also have neighboring genes for integration/ by dialysis and observed the purified bacteriophages by electron site specific recombination that 0305w8-36 does not have (Fig. 2, microscopy of a negatively stained specimen. The result was rightmost genes in the bottom three rows). that the tail sheath contraction was not observed in electron Yet, we did not find a single bacteriophage-borne member micrographs of wild type bacteriophage particles. However, most of any of the other mobilization/relaxase families (reviewed in of the wild type bacteriophage particles were in large aggregates, ref. 28). Hence, there may be something about this parti- making problematic their further characterization (not shown). cular mobilization system that adapts it for function within a Thus, we repeated the revised purification with a spontane- phage genome, but the reason for its peculiar association with ously generated, reduced-aggregation 0305w8-36 mutant that phages is not known. had been selected by serial propagation (beyond the propagation Remainder of the genes missing in the D(118,397–124,982) used for the original cloning) in the laboratory. As judged by the mutant. The genes deleted in D(118,397–124,982) also included in situ (in-plaque) fluorescence microscopy of reference 4, the orf205 (Fig. 1B), an orf that had no detected character of a mutant had less than 1% of the aggregation previously observed gene with a function in translocation. Orf205 encodes a protein for the wild type bacteriophage in reference 4 (not shown). previously identified as a capsid protein.8 In addition, orf205 is In confirmation of the results obtained with the wild type inverted relative to the other deleted orfs, suggesting that it is bacteriophage, the revised purification yielded mutant bacterio- ©the a comparatively funct2ionof 0orf205 recent 1isdiff2erent addition to the fromLthe genome afunctinonof and is in the dtheorfescosntraction class phage particles Bwas without oibseorved tail fosrthe tip-initiated cmutiant eafter sheath npurificcation contraction. eina A . of genes sometimes called morons.29 We assume, therefore, that field of several particles is in Figure 3A. Nonetheless, sheath in which it is embedded. cesium chloride density gradient (not shown), as previously seen The remaining orfs in the deleted region have no known for wild type bacteriophage. The bacteriophage particles are function and presumably represent at least one more function, monomeric in Figure 3A, even though the sample is concentrated making a total of at least three functions in an island deletable enough so that bacteriophages are close to overlapping. laboratory. Do not disasmotstcroncliusivbelyseuenin tmoreedilu.te areas of a specimen; without any detected effect on propagation of 0305w8-36 in the However, some bacteriophage particles were not monomeric, The second deletion mutant, D(181,412–189,531). The these particles were less than 10% of the total. The multimeric second deletion mutant, D(181,412–189,531), was missing a bacteriophage particles were always joined tail tip-to-tail tip second island (8.119 Kb; 3.71% of the genome) that also had and over 90% of them were dimeric; a few trimers were seen. genes of at least three different functions. This deletion mutant Dimers are shown in the higher magnification images of was mapped by the procedures used for the first deletion mutant. Figure 3B and C. The tail tip-to-tail tip, multimer-forming The deleted genes include genes for (1) two metallo-protein interaction was confirmed by the following transformation, seen chaperonins (orf063 and orf066), (2) two tRNAs and (3) six only in multimers. The tail sheaths underwent splitting into unknown functions (Fig. 1C). Bacteriophages T430 and T531 also two segments. “S” in Figure 3B and C indicates each of the two have deletable tRNA genes. Deletion of tRNA genes lowers sheath segments. The splitting left a section of the tail tube both burst size and rate of protein synthesis in the case of T4.32 exposed, as indicated by “T” in Figure 3B and C. The remainder Orf66 encodes one of the Mox-R ATPase-like metal chelatase of the tube is presumably encased within the sheath. The sheath proteins,33 which provide energy for inserting metals into other splitting is different from the previously observed8,9 sheath con- proteins and are in a diverse family of macromolecule remodeling, traction because the point of sheath breakage varies throughout P-loop ATPases called AAA+ proteins.34 Orf63 encodes a vWA- the tail in sheath splitting, whereas the point of breakage is only like protein, which are metal binding and usually found to work atthe tail tip in sheath contraction. Tail-to-tail dimerization with AAA+ proteins.33,34 apparently places the entire tail sheath under stress. We do not Both the D(118,397–124,982) and the D(181,412–189,531) know whether the sheath splitting occurs before or during deletions are in the right half of the 0305w8-36 genome (Fig. 1A). preparation for electron microscopy. The D(181,412–189,531) deletion did cause a significant growth Although some dimers appeared to consist only of bacterio- defect, as seen via significant reduction of D, but not I (Table 1). phage particles, as in Figure 3B, others appeared to have an Bacteriophage dimerization. To begin analysis of both the additional component, indicated by the arrow labeled “M” in aggregation pathway of 0305w8-36 and the possible role of Figure 3C. Although the composition of this M component is aggregation in the phenotype of 0305w8-36, we further charac- not known from direct characterization, the M component varies terized 0305w8-36 bacteriophage particles. We initially developed in size and shape among different dimers. Thus, we think likely an ultracentrifugal purification procedure that did not inactivate that this component is part the host, probably part of the cell 0305w8-36. We had to do this because centrifugation in a cesium wall or cell membrane. chloride density gradient, the procedure previously used, causes Tail-to-tail dimerization occurred before electron microscopy, inactivation accompanied by contraction of the tail sheath. The as judged by single-particle fluorescence microscopy of www.landesbioscience.com Bacteriophage 29