Abstract. 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 ¼ 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.

A number of bacteriophages belonging to the Microviridae have been described infecting chlamydiae. Phylogenetic studies divide the Chlamydiaceae into two distinct genera, Chlamydia and Chlamydophila, containing three and six different species, respectively. In this work we investigated the biological properties and host range of the recently described bacteriophage Chp2 that was originally discovered in Chlamydophila abortus. The obligate intracellular development cycle of chlamydiae has precluded the development of quantitative approaches to assay bacteriophage infectivity. Thus, we prepared hybridomas secreting monoclonal antibodies (monoclonal antibodies 40 and 55) that were specific for Chp2. We demonstrated that Chp2 binds both C. abortus elementary bodies and reticulate bodies in an enzyme-linked immunosorbent assay. Monoclonal antibodies 40 and 55 also detected bacteriophage Chp2 antigens in chlamydia-infected eukaryotic cells. We used these monoclonal antibodies to monitor the ability of Chp2 to infect all nine species of chlamydiae. Chp2 does not infect members of the genus Chlamydia (C. trachomatis, C. suis, or C. muridarum). Chp2 can infect C. abortus, C. felis, and C. pecorum but is unable to infect other members of this genus, including C. caviae and C. pneumoniae, despite the fact that these chlamydial species support the replication of very closely related bacteriophages.

The host range of CPAR39 is limited to four Chlamydophila species: C. abortus, C. caviae, C. pecorum, and C. pneumoniae. Chp3 (a newly discovered bacteriophage isolated from C. pecorum) shares three of these hosts (C. abortus, C. caviae, and C. pecorum) but can additionally infect Chlamydophila felis. The ability to support replication was directly correlated with the binding properties of the respective bacteriophages with their host species. Binding studies also show that CPAR39 and Chp3 use different host receptors to infect the same host cells: cell binding is sensitive to proteinase K treatment, confirming that the chlamydiaphage receptors are proteinaceous in nature.Chlamydiae have an obligate intracellular developmental cycle that alternates between the infectious elementary body and the replicative form, the reticulate body (RB). Five bacteriophages have been isolated from the chlamydiae. The first chlamydial bacteriophage to be characterized (13) was found by thin-section transmission electron microscopy (EM) of avian Chlamydophila psittaci RBs (14). Although samples containing Chp1 were later lost, a second bacteriophage (CPG1) (7) was found to infect Chlamydophila caviae (8) and a third bacteriophage, Chp2, was isolated from Chlamydophila abortus (10). The Chlamydophila pneumoniae strain AR39 genomesequencing project revealed the presence of a double-stranded DNA extrachromosomal element (11), which was subsequently shown to be the replicative form of bacteriophage CPAR39 (4). Recently Chp3 was isolated from Chlamydophila pecorum (6) and was found to share 97.1% nucleotide sequence identity with Chp2. Chlamydiaphages CPAR39 and CPG1 also show 97.1% identity, whereas the two groups (Chp2-Chp3 and CPAR39-CPG1) share 93.3% identity. All the chlamydiaphages share similar features; they are small icosahedral T ϭ 1 particles containing circular, single-stranded DNA genomes, and molecular characterization showed that they belong to the virus family Microviridae (9-11, 17).Recently we described the host range of Chp2 and showed that Chp2 was unable to infect C. caviae or C. pneumoniae. It was therefore of interest to investigate the host range of the very closely related Chp3 and the slightly more diverse bacteriophages CPAR39 and CPG1, which were isolated from host chlamydiae resistant to Chp2. In our work, monolayers of BGMK (Buffalo green monkey kidney) or HEp2 cells in 25-cm 2 flasks were infected by centrifugation at 1,000 ϫ g for 1 h in medium containing cycloheximide (1 g/ml) and gentamicin (25 g/ml) with C. pecorum bearing Chp3 or C. pneumoniae (11) bearing CPAR39. At 72 h postinfection the culture medium was replaced with a small volume of phosphate-buffered saline (PBS), and the flasks were frozen at Ϫ70°C. One hundred flasks of phage-infected chlamydiae were prepared, stored frozen, and then processed as a single batch. The flasks were frozen and thawed three times to lyse the chlamydial RBs and release phage. Any remaining monolayer that had not detached after this procedure was scraped off. The s...

Chlamydiaphage Chp2 is a member of the family Microviridae, of which bacteriophage X174 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.Although chlamydiae are widespread bacterial pathogens causing a wide range of illnesses, such as blindness, respiratory infections, and possibly coronary artery disease (23, 24, 26), detailed molecular mechanisms of pathogenesis will remain obscure as long as significant barriers hinder laboratory manipulation. One of these barriers is the lack of a stable gene transfer system. In recent years, five chlamydiaphages belonging to the family Microviridae, of which the prototype is bacteriophage X174, have been isolated (11,14,22,25). The mechanisms and techniques to package X174 in vitro and in vivo are well defined (1, 12). Thus, the microviruses of chlamydiae have the potential of becoming the basis of a genetic transfer system.Critical to this goal is the identification of a chlamydiaphage procapsid, the assembly intermediate into which DNA is packaged. Although the chlamydiaphages are within the same family as X174, the relationship is distant. The family has a deep evolutionary divide between the X174-like phages (coliphages) that infect free-living enterobacteria and the Chp-like phages infecting obligate intracellular parasites and mollicutes, such as the chlamydiae, Bdellovibrio bacteriovorus, and spiroplasma (3). The most striking differences are structural and morphogenetic (3, 5). Structurally, the X174-like phages contain a large pentameric spike protein complex at each vertex of the T ϭ 1 virion. These spikes and their requisite genes are not present in the Chp-like phages, which have elaborate viral coat protein protrusions on the three fold axes of symmetry not seen in the coliphages. Accordingly, structural differences have led to different morphogenetic requirements vis-à-vis scaffolding proteins. The X174-like phages utilize two scaffolding proteins during assembly: an internal and external species, proteins B and D, respectively (10). While it is evident that the Chp-like phages do not encode an external scaffolding pr...

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