Fitness benefits of low infectivity in a spatially structured population of bacteriophages.

For a parasite evolving in a spatially structured environment, an evolutionarily advantageous strategy may be to reduce its transmission rate or infectivity. We demonstrate this empirically using bacteriophage (phage) from an evolution experiment where spatial structure was maintained over 550 phage generations on agar plates. We found that a single substitution in the major capsid protein led to slower adsorption of phage to host cells with no change in lysis time or burst size. Plaques formed by phage isolates containing this mutation were not only larger but also contained more phage per unit area. Using a spatially explicit, individual-based model, we showed that when there is a trade-off between adsorption and diffusion (i.e. less 'sticky' phage diffuse further), slow adsorption can maximize plaque size, plaque density and overall productivity. These findings suggest that less infective pathogens may have an advantage in spatially structured populations, even when well-mixed models predict that they will not.

Intracellular parasitism of chlamydiae: specific infectivity of chlamydiaphage Chp2 in Chlamydophila abortus.

The obligate intracellular nature of chlamydiae presents challenges to the characterization of its phages, which are potential tools for a genetic transfer system. An assay for phage infectivity is described, and the infectious properties of phage Chp2 were determined.

Salmonella phages examined in the electron microscope.

Out of 177 surveyed bacteriophages, 161 (91%) are tailed and belong to the Myoviridae, Siphoviridae, and Podoviridae families (43, 55, and 59 viruses, respectively). Sixteen filamentous or isometric phages are members of the Inoviridae, Leviviridae, Microviridae, and Tectiviridae families (9%). Many tailed phages belong to established phage genera (P22, T1, T5, and T7), which are widespread in enterobacteria and other Gram-negatives of the Proteobacteria phylum.

Chlamydiaphage Chp2, a skeleton in the phiX174 closet: scaffolding protein and procapsid identification.

Chlamydiaphage Chp2 is a member of the family Microviridae, of which bacteriophage phiX174 is the type species. Although grouped in the same family, the relationship between the Microviridae coliphages and the Chp2-like viruses, which infect obligate intracellular parasitic bacteria, is quite distant, with major differences in structural protein content and scaffolding protein dependence. To investigate the morphogenesis of Chp2, large particles were isolated from infected Chlamydophila abortus by equilibrium and rate zonal sedimentation. A monoclonal antibody that recognizes only assembled viral coat proteins was used in these detection assays. Thus, the detected particles represent virions and/or postcapsid formation assembly intermediates. Two distinct particle types were detected, differing in both protein and DNA content. Filled particles lacked VP3, the putative internal scaffolding protein, whereas empty particles contained this protein. These results indicate that VP3 is a scaffolding protein and that the isolated VP3-containing particles most likely represent Chp2 procapsids.

Isolation, molecular characterisation and genome sequence of a bacteriophage (Chp3) from Chlamydophila pecorum.

Chlamydiae are obligate intracellular pathogens that have a unique developmental cycle. Thirty nine viable isolates representing all nine currently recognised chlamydial species were screened by immunofluorescence with a cross-reacting chlamydiaphage monoclonal antibody. A novel chlamydiaphage (Chp3) was detected in C. pecorum, a chlamydial species not previously known to carry bacteriophages. Chp3 belongs to the Microviridae, members of this virus family are characterised by circular, single-stranded DNA genomes and small T = 1 icosahedral capsids. Double-stranded replicative form Chp3 DNA was purified from elementary bodies and used as a template to determine the complete genome sequence. The genome of Chp3 is 4,554 base pairs and encodes eight open reading frames organised in the same genome structure as other chlamydiaphages. An unrooted phylogenetic tree was constructed based on the major coat proteins of 11 members of the Microviridae and Chp3. This showed that the Microviridae are clearly divided into two discrete sub-families; those that infect the Enterobacteriaceae e.g. ØX174 and the bacteriophages that infect obligate intracellular bacteria or mollicutes including SpV4 (Spiroplasma melliferum), ØMH2K (Bdellovibrio bacteriovorus) and the chlamydiaphages. Comparative analyses demonstrate that the chlamydiaphages can be further subdivided into two groupings, one represented by Chp2/Chp3 and the other by ØCPG1/ØCPAR39.

Bacteriophage observations and evolution.

Bacteriophages are classified into one order and 13 families. Over 5100 phages have been examined in the electron microscope since 1959. At least 4950 phages (96%) are tailed. They constitute the order Caudovirales and three families. Siphoviridae or phages with long, noncontractile tails predominate (61% of tailed phages). Polyhedral, filamentous, and pleomorphic phages comprise less than 4% of bacterial viruses. Bacteriophages occur in over 140 bacterial or archaeal genera. Their distribution reflects their origin and bacterial phylogeny. Bacteriophages are polyphyletic, arose repeatedly in different hosts, and constitute 11 lines of descent. Tailed phages appear as monophyletic and as the oldest known virus group.

Structural studies of bacteriophage alpha3 assembly.

Bacteriophage alpha3 is a member of the Microviridae, a family of small, single-stranded, icosahedral phages that include phiX174. These viruses have an ssDNA genome associated with approximately 12 copies of an H pilot protein and 60 copies of a small J DNA-binding protein. The surrounding capsid consists of 60 F coat proteins decorated with 12 pentameric spikes of G protein. Assembly proceeds via a 108S empty procapsid that requires the external D and internal B scaffolding proteins for its formation. The alpha3 "open" procapsid structural intermediate was determined to 15A resolution by cryo-electron microscopy (cryo-EM). Unlike the phiX174 "closed" procapsid and the infectious virion, the alpha3 open procapsid has 30A wide pores at the 3-fold vertices and 20A wide gaps between F pentamers as a result of the disordering of two helices in the F capsid protein. The large pores are probably used for DNA entry and internal scaffolding protein exit during DNA packaging. Portions of the B scaffolding protein are located at the 5-fold axes under the spike and in the hydrophobic pocket on the inner surface of the capsid. Protein B appears to have autoproteolytic activity that cleaves at an Arg-Phe motif and probably facilitates the removal of the protein through the 30A wide pores. The structure of the alpha3 mature virion was solved to 3.5A resolution by X-ray crystallography and was used to interpret the open procapsid cryo-EM structure. The main differences between the alpha3 and phiX174 virion structures are in the spike and the DNA-binding proteins. The alpha3 pentameric spikes have a rotation of 3.5 degrees compared to those of phiX174. The alpha3 DNA-binding protein, which is shorter by 13 amino acid residues at its amino end when compared to the phiX174 J protein, retains its carboxy-terminal-binding site on the internal surface of the capsid protein. The icosahedrally ordered structural component of the ssDNA appears to be substantially increased in alpha3 compared to phiX174, allowing the building of about 10% of the ribose-phosphate backbone.

Structural analysis of the Spiroplasma virus, SpV4: implications for evolutionary variation to obtain host diversity among the Microviridae.

BACKGROUND: Spiroplasma virus, SpV4, is a small, non-enveloped virus that infects the helical mollicute Spiroplasma melliferum. SpV4 exhibits several similarities to the Chlamydia phage, Chp1, and the Coliphages alpha 3, phi K, G4 and phi X174. All of these viruses are members of the Microviridae. These viruses have isometric capsids with T = 1 icosahedral symmetry, cause lytic infections and are the only icosahedral phages that contain single-stranded circular DNA genomes. The aim of this comparative study on these phages was to understand the role of their capsid proteins during host receptor recognition. RESULTS: The three-dimensional structure of SpV4 was determined to 27 A resolution from images of frozen-hydrated particles. Cryo-electron microscopy (cryo-EM) revealed 20, approximately 54 A long, 'mushroom-like' protrusions on the surface of the capsid. Each protrusion comprises a trimeric structure that extends radially along the threefold icosahedral axes of the capsid. A 71 amino acid portion of VP1 (the SpV4 capsid protein) was shown, by structural alignment with the atomic structure of the F capsid protein of phi X174, to represent an insertion sequence between the E and F strands of the eight-stranded antiparallel beta-barrel. Secondary structure prediction of this insertion sequence provided the basis for a probable structural motif, consisting of a six-stranded antiparallel beta sheet connected by small turns. Three such motifs form the rigid stable trimeric structures (mushroom-like protrusions) at the threefold axes, with hydrophobic depressions at their distal surface. CONCLUSIONS: Sequence alignment and structural analysis indicate that distinct genera of the Microviridae might have evolved from a common primordial ancestor, with capsid surface variations, such as the SpV4 protrusions, resulting from gene fusion events that have enabled diverse host ranges. The hydrophobic nature of the cavity at the distal surface of the SpV4 protrusions suggests that this region may function as the receptor-recognition site during host infection.