The first deletion mutant, Δ(118,397–124,982)
After selection for mutants stable to elevated temperature, we isolated three independent mutants, total, two of which were deletion mutants, based on the restriction endonuclease analysis described in the Materials and Methods Section. In , a pair of vertical red lines indicates each of the regions deleted, within a low-resolution map of the entire 218.948 Kb genome (6.479 Kb terminal repeat; 247 identified orfs).
Figure 1. Maps of the bacteriophage 03058-36 genome and its deletions. (A) A low-resolution map of the entire genome. (B) A high-resolution map of the Δ(118,397–124,982) deletion with distance from the left end (Kb) at the top, orf number (more ...)
The first 0305
8-36 deletion mutant was missing nucleotides 118,397–124,982 (3.01% of the genome) and will be called Δ(118,397–124,982). Orfs 200–207 were deleted, as shown at higher resolution in . We found a single deletion and its approximate position by restriction endonuclease analysis that detected a single deletion in the 110,000–130,000 area (not shown). We obtained confirmation and a more precise position by PCR amplification across the site of the deletion. Finally, we located the deletion precisely by Sanger sequencing of the PCR fragment. Details are in the Materials and Methods Section. As described below, the genes deleted included a DNA translocation operon.
The propagation phenotype of Δ(118,397–124,982)
Next, we determined whether the Δ(118,397–124,982) mutant had a propagation defect in relation to the wild type phage. The following characteristics were compared: average plaque diameter (D) and average infectious particle number per ml of a plaque (I). The value of I was assayable because the plaque-supporting, 0.1% agarose gel was weak enough so that a 0.2 ml portion of a plaque (about 25% of the total cleared region) was pipeted for titering. No significant difference between wild type and Δ(118,397–124,982) deletion mutant was observed for either D or I (). That is to say, no growth defect was detected for the Δ(118,397–124,982) mutant.
Propagation phenotypes of deletion mutants
Deletion of a DNA relaxase gene in the Δ(118,397–124,982) mutant
The genes deleted in Δ(118,397–124,982) included orf200, which was found via multi-iteration PsiBlast and reverse PsiBlast searches to be related to the DNA relaxase from the DNA translocation operon of Enterobacter cloacae
plasmid CloDF13 (mobC).20
The weakest link in this association was challenged using HHpred as described in the Materials and Methods Section. Alignments of proteins clearly related to either orf200 or CloDF13 mobC were developed using SAM with a stringent inclusion threshold of E = 1.0 × 10−9
. Each was picked out of the library with the other as queried by HHpred, with an E value of 4.4 × 10−22
(, row 1). However, both families also matched a variety of helix-turn-helix domains in their N-terminal domains (, row 2).
Table 2. Homologs of the 03058-36 relaxase, coupling ATPase and membrane binding protein
To test that orf200 and CloDF13 mobC were related beyond both simply having helix-turn-helix domains, the entire operation was repeated with their C-terminal domains only. The result was finding of significant homology (, row 3). Thus, it is clear that 0305
8-36 orf200 contains a DNA relaxase domain, with homologs that include CloDF13 mobC, the latter described in reference 21
. Conjugation relaxases are usually encoded adjacent to a DNA translocase that acts as a coupling protein for transfer of the nicked DNA into the conjugation system.21,22
Deletion of a coupling protein/translocase gene in the Δ(118,397–124,982) mutant
The candidate for coupling protein in 0305
8-36 is the adjacent orf201, originally annotated8
as a VirD4-like protein, based on the closest annotated sequences found by PsiBlast. More detailed analysis revealed that the similarity is confined to a C-terminal ATPase domain. Query with this domain hits Pfam family AAA_10, an ATPase domain frequently found in conjugation-associated ATPases (annotator's comments; PF12846).
8-36 orf201 differed from both VirD4 and mobB in not having a N-terminal domain composed primarily of transmembrane helix. The N-terminal domain of 0305
8-36 orf201 had no detectable transmembrane helical segment. To determine whether this absence of a transmembrane helical segment occurs in other, similar genes, we searched for genes similar to 0305
8-36 orf201 and found them among genes hit by queries from family models of both 0305
8-36 orf201 and a homolog, ING1 (, rows 10 and 11), the latter also a homolog of the traG translocase. Although traG has a N-terminal transmembrane helix domain,23,24
about 75% of hits from queries with both ING1 and 0305
8-36 orf201 models had no transmembrane helices (not shown). Thus, (1) the absence of a transmembrane domain is not unusual among translocases and (2) orf200 and 201 appear to have a relatively standard organization to be the core of a conjugation relaxase/translocase operon.
Completion of the operon: Membrane attachment sites
Mobilization complexes have to make an association with the membrane-bound conjugation system in order to function. Hence, the absence of a transmembrane domain in orf201 might mandate that a separate protein(s) provides for membrane association. If so, the genes in the 0305
8-36 version of this operon should include at least one orf that encodes a protein with a membrane attachment site. For the following reasons, we conclude that orf203 and orf207 are orfs of this type. Orf203 is homologous to the virB6 locus, which encodes a membrane-associated protein that interacts with the DNA mobilization complex and the sex pilus during Ti plasmid-mediated conjugation.25
Orf207 is homologous to mreB, which encodes a bacterial protein that is structurally homologous to actin and that associates with the cell membrane where it influences cell shape and is an anchor for another arm of the DNA translocase family involved in chromosome segregation.26,27
Thus, the translocation operon includes orfs 200, 201, 203 and 207, no two of which have homologs from a single known bacterial operon.
However, we found that a partial version of a similar 0305
8-36 DNA translocation operon does exist in other bacteriophages, based initially on hits from both entire relaxase genes and their C-terminal segments. The data for the following bacteriophages are in (bacteriophage, followed in succession by gi number, orf number and the row(s) in that have data for either the entire gene or the C-terminal domain): (1) Bacillus subtilis
bacteriophage SP01 (209972955; 27.3; rows 4–5), (2) Geobacillus
bacteriophage GBSV1 (158267614; 43; row 6), (3) Bacillus
virus 1 (155042415; 40; row 7), (4) Bacillus anthracis
bacteriophage Gamma (77020183; orf not numbered; row 8) and (5) close relatives of Gamma, including Cherry, Fah22 and WBeta (data not shown).
Figure 2. Homologs of genes from the proposed DNA translocation operon of bacteriophage 03058-36. The plasmid or bacteriophage name is at the left. The NCBI accession number is above an arrow. The orf number is within an arrow, when given at the NCBI site. (more ...)
The similarity with the entire 0305
8-36 operon is highest for lytic bacteriophage SPO1, which has a homolog for orfs 201 (coupling ATPase; , row 9) and orf207 (, row 12), not present in the other genomes (). The other bacteriophages, all lysogenic, also have neighboring genes for integration/site specific recombination that 0305
8-36 does not have (, rightmost genes in the bottom three rows).
Yet, we did not find a single bacteriophage-borne member of any of the other mobilization/relaxase families (reviewed in ref. 28
). Hence, there may be something about this particular mobilization system that adapts it for function within a phage genome, but the reason for its peculiar association with phages is not known.
Remainder of the genes missing in the Δ(118,397–124,982) mutant
The genes deleted in Δ(118,397–124,982) also included orf205 (), an orf that had no detected character of a gene with a function in translocation. Orf205 encodes a protein previously identified as a capsid protein.8
In addition, orf205 is inverted relative to the other deleted orfs, suggesting that it is a comparatively recent addition to the genome and is in the class of genes sometimes called morons.29
We assume, therefore, that the function of orf 205 is different from the function of the orfs in which it is embedded.
The remaining orfs in the deleted region have no known function and presumably represent at least one more function, making a total of at least three functions in an island deletable without any detected effect on propagation of 0305
8-36 in the laboratory.
The second deletion mutant, Δ(181,412–189,531)
The second deletion mutant, Δ(181,412–189,531), was missing a second island (8.119 Kb; 3.71% of the genome) that also had genes of at least three different functions. This deletion mutant was mapped by the procedures used for the first deletion mutant. The deleted genes include genes for (1) two metallo-protein chaperonins (orf063 and orf066), (2) two tRNAs and (3) six unknown functions (). Bacteriophages T430
also have deletable tRNA genes. Deletion of tRNA genes lowers both burst size and rate of protein synthesis in the case of T4.32
Orf66 encodes one of the Mox-R ATPase-like metal chelatase proteins,33
which provide energy for inserting metals into other proteins and are in a diverse family of macromolecule remodeling, P-loop ATPases called AAA+ proteins.34
Orf63 encodes a vWA-like protein, which are metal binding and usually found to work with AAA+ proteins.33,34
Both the Δ(118,397–124,982) and the Δ(181,412–189,531) deletions are in the right half of the 0305
8-36 genome (). The Δ(181,412–189,531) deletion did cause a significant growth defect, as seen via significant reduction of D
, but not I
To begin analysis of both the aggregation pathway of 0305
8-36 and the possible role of aggregation in the phenotype of 0305
8-36, we further characterized 0305
8-36 bacteriophage particles. We initially developed an ultracentrifugal purification procedure that did not inactivate 0305
8-36. We had to do this because centrifugation in a cesium chloride density gradient, the procedure previously used, causes inactivation accompanied by contraction of the tail sheath. The contraction initiates at the tail tip.8,9
The procedure developed is based on centrifugation through a sucrose gradient (Materials and Methods Section).
After the revised ultracentrifugation, we removed the sucrose by dialysis and observed the purified bacteriophages by electron microscopy of a negatively stained specimen. The result was that the tail sheath contraction was not observed in electron micrographs of wild type bacteriophage particles. However, most of the wild type bacteriophage particles were in large aggregates, making problematic their further characterization (not shown).
Thus, we repeated the revised purification with a spontaneously generated, reduced-aggregation 0305
8-36 mutant that had been selected by serial propagation (beyond the propagation used for the original cloning) in the laboratory. As judged by the in situ (in-plaque) fluorescence microscopy of reference 4
, the mutant had less than 1% of the aggregation previously observed for the wild type bacteriophage in reference 4
In confirmation of the results obtained with the wild type bacteriophage, the revised purification yielded mutant bacteriophage particles without tail tip-initiated sheath contraction. A field of several particles is in . Nonetheless, sheath contraction was observed for the mutant after purification in a cesium chloride density gradient (not shown), as previously seen for wild type bacteriophage. The bacteriophage particles are monomeric in , even though the sample is concentrated enough so that bacteriophages are close to overlapping.
Figure 3. Electron microscopy of bacteriophage 03058-36 after improved fractionation. After fractionation of reduced aggregation bacteriophage particles in a sucrose step gradient, fluorescence microscopy was performed with particles from the phage band-region (more ...)
However, some bacteriophage particles were not monomeric, as most conclusively seen in more dilute areas of a specimen; these particles were less than 10% of the total. The multimeric bacteriophage particles were always joined tail tip-to-tail tip and over 90% of them were dimeric; a few trimers were seen. Dimers are shown in the higher magnification images of . The tail tip-to-tail tip, multimer-forming interaction was confirmed by the following transformation, seen only in multimers. The tail sheaths underwent splitting into two segments. “S” in indicates each of the two sheath segments. The splitting left a section of the tail tube exposed, as indicated by “T” in . The remainder of the tube is presumably encased within the sheath. The sheath splitting is different from the previously observed8,9
sheath contraction because the point of sheath breakage varies throughout the tail in sheath splitting, whereas the point of breakage is only atthe tail tip in sheath contraction. Tail-to-tail dimerization apparently places the entire tail sheath under stress. We do not know whether the sheath splitting occurs before or during preparation for electron microscopy.
Although some dimers appeared to consist only of bacteriophage particles, as in , others appeared to have an additional component, indicated by the arrow labeled “M” in . Although the composition of this M component is not known from direct characterization, the M component varies in size and shape among different dimers. Thus, we think likely that this component is part the host, probably part of the cell wall or cell membrane.
Tail-to-tail dimerization occurred before electron microscopy, as judged by single-particle fluorescence microscopy of pre-dialyzed versions of the same specimens. The procedures are described in the Materials and Methods Section. A presumed dimer was revealed via two separate, resolution-limited bright regions that, during motion, were tethered together and separated by about two tail lengths (about 1,000 nm or about 4× the resolution limit of the fluorescence microscope) in some projections. These particles were undergoing tumbling and translational motion in the specimen. A series of video images, framed to include the same particle (0.5 sec between frames), is in . The two bright regions are seen separately from each other in frames indicated with an arrow; rotation brings these regions into coincidence in the other frames. The dimer was translating throughout this video series. In the second frame from the left, the leftmost bright region is from a second particle that drifted into apparent contact with the dimer; the framed particle is distinct in the other images.
By this criterion, ~10% of the particles observed were dimers. If, in analogy with the behavior of other myoviruses,35
8-36 attaches to a host cell via its tail, then dimerization will inhibit the infection of host cells.
Bacteriophage adsorption to a host cell
The tail of bacteriophage 0305
8-36 is long enough so that we determined the orientation of host-attached bacteriophage particles by in situ (in-plaque) fluorescence microscopy of DAPI-stained (Materials and Methods Section) samples of 0305
8-36 plaques. If the bacteriophage adsorbs tail tip-to-cell surface, then this orientation will be revealed by a DAPI-stained, resolution-limited spot (packaged DNA) at a distance from the cell surface that is maintained during translational and tumbling motion. Given the 486 nm rigid tail and the potentially flexible, wavy tail fibers of 0305
8-36, this distance depends on the extent to which the tail fibers are extended, but would be between 486 and ~700 nm in any case.
shows a single host cell with at least eight bacteriophages attached, the latter as revealed by resolution-limited spots derived from DNA-bound dye fluorescence. One spot is at the upper right and five distinct spots are at the lower left of the bacterial cell. Two of these latter spots are produced by fluorescence from two bacteriophages. The additional bacteriophages (1) were obscured by superposition in the frame in , (2) were seen in other frames as distinct, after tumbling generated a change in orientation (not shown) and (3) were revealed for the view in via asymmetry of the lower two spots; asymmetry can also be generated by limited time resolution during motion. Seven of the eight spots (packaged DNAs) are at a distance from the cell surface consistent with attachment of a bacteriophage particle via the tail tip or tail fibers.
Figure 4. In situ fluorescence microscopy of host-bacteriophage attachment. A portion of the clear region of a 0.1% agarose-supported plaque was pipeted, mixed with dye and examined by fluorescence microscopy, as described in the Materials and Methods Section. (more ...)
The uppermost spot at the lower left, on the other hand, appeared to be attached to the cell surface at a distance too small to be tail tip-to-cell surface attachment. This condition was maintained during tumbling, although this resolution-limited spot was slightly separated from the surface in one orientation.
We conclude that bacteriophage 0305
8-36 is selected to adsorb to its host by the tip of its tail, but that alternative patterns of adsorption can occur. We further conclude that the observed tail tip-tail tip dimerization () inhibits initiation of infection by 0305
8-36. The significance of this latter conclusion is discussed in the next section.
Parenthetically, we note that seven of the bacteriophages of had adsorbed to the host cell before the microscopy began. But, the bacteriophage at the upper right appeared to adsorb during microscopy. This bacteriophage particle was not observed before stable adsorption and appeared to have diffused into focus while it was attaching. Thus, we did not obtain information about the events that occurred before stable attachment. This limitation exists, in general, unless the thermal motion of the bacteriophage particles is restricted to a thin enough zone of solution so that the particles are tracked before and during adsorption.