Phage ϕ1402 has a contractile tail and thus is classified as a myovirus (). As measured on 20 particles, it has an elongated 68 × 40 nm head and a tail that is, in its extended state, 62 × 17 nm. The tail has a neck that lacks a collar and, hence, collar-associated appendages. In the contracted state, the tail measures 20 × 20 nm and shows a base plate of 17 × 2 nm that separates from the sheath. The base plate has a set of 5–7 nm long fibers. Electron microscopy of empty heads reveals capsomers of 5 nm diameter that are unusually easy to distinguish, presumably because of the absence of any supplementary head decoration proteins. The precise number and exact pattern of organization of these capsomers in the capsid has not yet been determined.
Figure 1 Electron micrographs of ϕ1402. (A and B) Complete phages with extended tails and some empty heads. Arrows in (B) indicate tail fibers. The ring structure in the upper left of (B) is a contracted tail sheath standing on its end. (C) Particles with (more ...)
The phage genome is a 23,931 bp, circularly permuted dsDNA molecule with a GC content of 50.4% (). Restriction digest profiles (data not shown) identified a fragment containing a pac
site which is diagnostic of phages using a terminally redundant, head-full packaging strategy.11
The DNA sequence encodes 42 ORFs, which are nearly all located on the positive strand and have no significant overlaps. The genome has no obvious phylogenetic relationship to any other phage in public databases. Remarkably, only two of its ORFs can clearly be considered as phage-related: ORF11, the large subunit of the phage terminase and ORF33 which is related to various fibrous phage proteins (). Both these sequences are among the most highly conserved and ubiquitous sequences in phage genomes. Five other ϕ1402 proteins had significant E-values (≤10−4
) when compared to cellular proteins: ORF01 is related to ssDNA-binding proteins; ORF10 belongs to an RNA-binding Conserved Domain Database (CDD) protein family; and three ORFs have homologies to hypothetical cellular proteins. The remaining 35 proteins (83%) are ORFans—database entries without any known homologs. Iterative PSI-BLAST, applying less stringent selection criteria, found weak hits to three additional ORFs (ORF21/24/38). These are putatively identified as dehydrogenases/reductases, potassium/proton antiporters and peptidoglycan-binding domain proteins, respectively, but the significance of such weak homologies is dubious.
Figure 2 Genome sequence of ϕ1402. The circles, from outermost to innermost, represent: the scale in kilobases; the rightward-transcribed ORF s (blue); the leftward-transcribed ORF s (red); the %GC content (black for above-average and gray for below-average); (more ...)
ϕ1402 ORF s with identifiable homologs/gene functions
The highly compact ϕ1402 genome (95% coding) contains no tRNAs and dot-plot analysis reveals no significant direct- or inverted-repeat sequences. A comparison of the ϕ1402 genome and that of its host revealed that both had similar GC contents and codon usage patterns (data not shown). The intergenic regions of the phage genome contained no obvious consensus promoter sequences; however, the host promoter sequences of Bdellovibrio spp. have not yet been defined either. The ϕ1402 genome contains a single large (~1 kb) untranslated region (UTR) located between the end of the forward-transcribed genes and the small cluster of genes transcribed leftward. Although this UTR has a substantially reduced GC ratio, there are no additional indications that it corresponds to an attP
site of a temperate phage. ϕ1402 always behaved as a standard lytic phage, with no particularities regarding plaque morphology, stock production, nor latent period (Fane B, personal communication). However, BLASTn did find an identical 16 bp sequence (GTA ACT CCT CAA GAA T) in the phage UTR and in a Bdellovibrio bacteriovorus
intergenic sequence (coordinates 3,528,980–3,528,995 on NC_005363) that is located between an asparaginase gene and a gene for a hypothetical protein, thus quite unlike the site of many temperate phage attP
sites that are often in close proximity to tRNA sequences.12
The function of this large phage UTR region, therefore, remains unknown. Although an investigation of the DNA sequence with Ori-Finder was inconclusive, there are 6 putative shifts in GC skew indicating possible origins of replication, with the strongest candidate being near the 0 coordinate.
In conclusion, phage ϕ1402 differs from other myoviruses by its extremely small size, the presence of visible capsomers, and its very small dsDNA genome. Tailed-phage capsids are generally smooth; the visualization of capsomers after negative staining is exceptional in tailed phages and many indicate structural differences separating ϕ1402 from other phages. Among the myoviruses, only the satellite coliphages P4 and ϕR73 have smaller DNAs, of 11.6 and 12.7 kb, respectively. Both of these have small isometric heads (~45 nm diameter) and tails of approximately 140 nm in length.13–15 A phylogenetic analysis of the ϕ1402 terminase protein () further reinforces the notion that this unusual Bdellovibrio phage has diverged considerably from all other currently known phage groups. In the absence of any evidence that ϕ1402 is a satellite phage, it appears to represent the smallest known autonomous myovirus and thus an interesting example of reductionism carried to the extreme. It will be interesting to identify all the functions that ϕ1402 has been able to jettison on its evolutionarily reductionist pathway and, equally important, what few genes it was constrained to retain. It seems likely that even this small “simplistic” myovirus would require at least 25 genes to encode all the essential virion components and the associated genes for correct assembly of the virion. Such a conservative estimate leaves only 17 functions unaccounted for in the ϕ1402 genome, but many of these must be involved in the phage's takeover of host macromolecular synthesis and phage transcription/replication. It will be interesting to see what the few remaining unaccounted for phage functions do and how they do it with such modest genetic resources.
Large subunit terminase phylogeny. Neighbor-joining tree of the ϕ1402 terminase with its 7 closest phage homologs. Values at the nodes indicate the results of 100 bootstrap replicates and the scale bar indicates 0.1 substitutions per site.