Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK1
Department of Medicine, University of California at San Francisco, Box 0868, San Francisco,CA 94143-0868, USA2
Author for correspondence: Ru-ching Hsia. Tel: +44 20 7927 2290. Fax: +44 20 7612 7871. e-mail: r.hsia{at}lshtm.ac.uk
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ABSTRACT |
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Keywords: Chlamydia, bacteriophage, capsid protein, Rep protein
Abbreviations: EB, elementary body; GPIC, Guinea Pig Inclusion Conjunctivitis; MOMP, major outer-membrane protein; RB, reticulate body; RCR, rolling circle replication; Rep, replication initiation protein
The GenBank accession number for the sequence reported in this paper is U41758.
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INTRODUCTION |
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The apparent diversity in host and associated disease pathology belies common elements that are central to the chlamydial pathogenic process at both the molecular and cellular levels. All members of the Chlamydiaceae share a common obligate intracellular life cycle, and comparative genomic analysis of two members of the family (C. trachomatis and C. pneumoniae) reveals a core set of shared genes whose putative functions encompass most activities known or presumed to be required for intracellular survival, growth and differentiation (Kalman et al., 1999 ). A counterpoint to this hypothesis is that differential properties, such as host and tissue tropism, the ability to persist, and the capacity of certain strains to cause disseminated infection, are more likely to be specified by factors that are unique to specific strains of the Chlamydiaceae, may not be readily annotated, and may ultimately be difficult to identify. Hence the wealth of genomic information currently gathered, however useful in allowing predictions, is unlikely to provide immediately satisfying answers to many questions regarding chlamydial infection and disease. This is particularly true where differential pathogenic outcomes are concerned, and this difficulty will be enhanced by the continuing lack of genetic methodologies for these bacteria. It is therefore essential that efforts to identify new virulence factors and tools to manipulate these factors genetically be developed concurrently with genomic and post-genomic advances to enable the new information to be exploited optimally.
We describe in this report a microvirus that infects Chlamydia psittaci strain Guinea pig Inclusion Conjunctivitis (GPIC). This bacteriophage, designated CPG1, is the second Chlamydia phage ever isolated and the first known to infect a member of the Chlamydiaceae infecting a mammalian species.
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METHODS |
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Cloning of the CPG1 genome.
Supernatants from CPG1-infected GPIC culture (48 h post-infection) were collected and cleared by centrifugation at 30000 g for 30 min at 4 °C and filtration through a 0·22 µm membrane. Following treatment with DNase I (20 µg ml-1) at 37 °C for 1 h,
CPG1 particles were sedimented by centrifugation at 100000 g for 3 h at 4 °C and treated with proteinase K (200 µg ml-1) at 37 °C for 1 h. Single-stranded phage genomic DNA was recovered by phenol/chloroform extraction and ethanol precipitation. Conversion to double-stranded DNA was achieved using Klenow fragment and random hexamer priming, and second-strand synthesis was confirmed by mobility shift upon agarose gel electrophoresis. The resulting double-stranded DNA template was digested with BamHI, generating a major linearized molecule migrating at approximately 4·5 kb on a TAE-agarose gel. The whole BamHI digest was shotgun cloned into Bluescript pKS+.
All transformants generated contained identical plasmid inserts of 4·5 kb. Partial nucleotide sequence analysis of a representative clone (pCPG1) revealed sequence encoding the amino terminus of the capsid protein (VP1), thus confirming the viral origin of the insert. In order to ensure that plasmid pCPG1 contained the complete phage genome, i.e. that no genomic sequence was missing as could result from more than one BamHI site, a fragment spanning 135 bp upstream and 254 bp downstream of the BamHI cloning site was amplified using purified phage DNA as template. Sequence analysis of the amplified fragment revealed no extra BamHI site and confirmed that pCPG1 contains the whole genome of CPG1.
Nucleotide sequence analysis.
For each strand, nucleotide sequence analysis was performed on overlapping segments, either by subcloning small fragments into Bluescript pSK+, and subsequently sequencing using T3 and T7 primers, or by subcloning larger fragments into pMOB, generating nested insertions and sequencing using transposon-specific primers as described by Strathmann et al. (1991)
. A combination of manual (Sanger et al., 1977
) and automated (Applied Biosystems) sequencing methods was used for the analysis of the complete
CPG1 genome.
Bioinformatics.
Sequence analysis was performed using software of the Wisconsin Package Version 10.0, Genetics Computer Group, Madison, WI, through the UK Human Genome Mapping Project Resource Centre in Cambridge (PEPTIDEMAP, GAP, BLASTN, STEMLOOP), software from Le Centre de Ressources Infobiogen, Université dEvry-Val dEssone (CLUSTALW 1.8), and from the National Center for Biotechnology Information, National Library of Medicine, US National Institute of Health (PSI-BLAST).
Electron microscopy.
Samples were fixed in 2% (v/v) glutaraldehyde and 0·5% (w/v) paraformaldehyde for processing and embedding in Epon-Araldite resin as outlined by Wyrick et al. (1989) . Ultrathin sections were stained with uranyl acetate and lead citrate and examined using a Zeiss EM900 transmission electron microscope operated at 60 kV. Negatively stained [2% (w/v) uranyl acetate] specimens were observed on a Philips CM12 electron microscope.
Other methods.
SDS-PAGE was performed according to Laemmli (1970) .
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RESULTS |
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Isolation and structural analysis of CPG1
Confirmation of the presence of a contaminating phage in GPIC cultures was initially obtained by direct visualization. GPIC-infected HeLa lysates were examined by transmission electron microscopy of ultra-thin sections. Particles similar in size and shape to Chp1 virions were observed in abundance in GPIC-infected HeLa lysates, in particular in association with membranes of lysed chlamydiae (Fig. 2a). GPIC-infected HeLa culture supernatants were fractionated according to Storey et al. (1989b)
and phage-enriched fractions were observed by electron microscopy of negatively stained samples (Fig. 2b
) and by SDS-PAGE. The particles observed were consistent with an isometric phage of approximately 25 nm diameter, superficially similar to that of the Escherichia coli bacteriophage
X174 (Hayashi et al., 1988
). Some of the particles had a central electron lucency and may represent viral particles empty of their nucleic acid complement. SDS-PAGE of purified phage fractions (Fig. 3
) revealed three major polypeptide bands of apparent molecular masses 62, 26 and 13 kDa, similar to the SDS-PAGE profile of purified Chp1 (Storey et al., 1989b
).
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Analysis of the CPG1 genome
The CPG1 genome was isolated from culture supernatant fractions and cloned in Bluescript, generating plasmid pCPG1. Analysis of the 4529 bp included within the pCPG1 insert revealed five major non-overlapping ORFs, in one transcriptional orientation (Fig. 4
). These were similar to those in Chp1, with the sequence identity of the predicted products ranging from 25·6% to 54·3% (Table 1
). As the five predicted gene products of
CPG1 were highly homologous to their Chp1 counterparts, we adopted the nomenclature already in place for Chp1 for the three proposed structural proteins (i.e. VP13) and further propose to name the last two VG4 and VG5 for virus genes 4 and 5 (corresponding to ORF4 and ORF5 in Chp1, respectively).
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A search of the databases revealed similarities between CPG1, Chp1 and SpV4, a bacteriophage infecting Spiroplasma melliferum, a helical mollicute pathogenic for honey bees (Renaudin et al., 1987
). However, the similarity is restricted to VP1, VP2 and VG4 and the genetic organization is different in SpV4 (Fig. 4
). Moreover, while Chp1 and
CPG1 share similar G+C contents and codon usage, the G+C (32 mol%) and codon usage of SpV4 closely reflect its Spiroplasma host (Renaudin et al., 1987
).
Sequence analysis of VP1, the major capsid protein
Analysis of the nucleotide sequence of the CPG1 genome revealed a hypothetical gene downstream of the BamHI site, the translated amino terminal sequence of which was identical to the amino-terminal sequence determined for the 62 kDa band, confirming the high homology to the sequence of VP1 of Chp1 (Table 1
). VP1 displayed the highest sequence conservation between
CPG1, Chp1 and SpV4, reflecting its important role as the major structural component of the capsid (Chipman et al., 1998
; Renaudin et al., 1987
; Storey et al., 1989a
).
As reported by Storey et al. (1989a) , the major structural protein, VP1, is similar to the bacteriophage capsid F protein of
X174. Alignment of VP1 from Chp1, VG1 from SpV4 and F protein sequences from coliphages
X174,
K,
3 and G4 allowed the identification of seven insertion loops, IN17, in VP1/VG1 proteins, which are absent in the F protein family (Chipman et al., 1998
). Overlay of the VG1 structure on the three-dimensional structure of the F protein of
X174 further suggested that the larger insertion loop (IN5) lies on the exposed surface of the capsid protein and potentially trimerizes at each of the twenty three-fold axes of symmetry of the capsid, forming protrusions at the phage surface. Chipman et al. (1998)
demonstrated such protrusions on purified SpV4 particles using cryo-electron microscopy and three-dimensional image reconstruction. Sequence alignment with VP1 of
CPG1 likewise identified an IN5 insertion sequence (Fig. 5
). The IN5 insertion of
CPG1 consisted of 78 residues, with a predicted molecular mass of 8209 Da and a pI of 9·3. These gross properties are similar to those of IN5 of the Spiroplasma phage (71 residues, 7422 Da and pI of 10·5), but are relatively dissimilar to those of IN5 of Chp1 (104 residues, 10836 Da and pI of 4·25). Alignment of the IN5 sequences revealed comparable similarity in the three phages (29·5% and 31·3% identity for
CPG1 vs Chp1 and SpV4, respectively), with positionally conserved gaps in
CPG1 and SpV4 (Fig. 5
).
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Position-specific iterated BLAST (PSI-BLAST) analysis revealed weak similarities of VG4 and ORF4 with the gene A protein family of coliphages (E values ranging from 0·003 for phage 3 to 0·55 for phage G4, and from 1x10-5 for phage G4 to 0·002 for phage
3, respectively). However, this analysis indicated that an internal segment conserved in VG4 and ORF4 displays high homology (E values 2x10-28 and 2x10-29, respectively) with a segment of the plasmid-encoded replication initiation protein (Rep) protein of Brevibacillus borstelensis, which is involved in rolling circle replication (RCR) (Ebisu et al., 1995
). Consensus motifs characteristic of the superfamily I of proteins mediating RCR initiation, including two (putative) DNA-linking tyrosine residues, are conserved in both VG4 and ORF4 of the Chlamydia phages, in VG2 of SpV4 and in the gene A/A* protein family (Fig. 6
) (Ilyina & Koonin, 1992
). Moreover, these motifs are conserved in a number of bacteriophage replication initiation proteins and related cyanobacterial and archaeal plasmid Rep proteins (Ilyina & Koonin, 1992
).
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The presence of phage-derived sequence in C. pneumoniae strongly suggests that variants of a single phage or members of a group of highly related phages were recently able to infect both C. psittaci GPIC, which is isolated from guinea pigs, and C. pneumoniae, which is isolated from humans. Moreover, these findings indicate that phage genomic sequence has the capacity to integrate into the chlamydial genome.
Other predicted genes
Apart from VP1 and VG4, for which function could be assigned tentatively based on sequence similarity, there were three other major non-overlapping ORFs on the genome of CPG1 that could not be annotated based on sequence similarity alone. The predicted products, VP2, VP3 and VG5, were homologous to VP2, VP3 and ORF5 of Chp1 (Storey et al., 1989a
), while only the VP2 protein was conserved in SpV4 (Renaudin et al., 1987
) (Table 1
). VP2 and VP3 correspond to abundant polypeptides of apparent molecular mass 30 and 16·5 kDa in Chp1, as determined by SDS-PAGE (Storey et al., 1989b
), in close agreement with the calculated values (28·5 and 16·7 kDa). VP2 and VP3 of
CPG1 may likewise correspond to dominant bands of 26 and 13 kDa observed by SDS-PAGE (Fig. 3
), although their calculated molecular masses differ somewhat (Table 1
). Discrepancies in molecular mass may correspond to post-translational modifications or alternative starts in
CPG1.
Storey et al. (1989a) described several additional overlapping minor ORFs in Chp1 that do not have detectable similarity to other bacteriophage sequences. A comparison of all Chp1 minor ORFs with all minor ORFs of
CPG1 revealed only limited homology of two small ORFs, one of which lacks an AUG start, while the other is internal and antisense to VP1, and resides in a segment which is comparatively more conserved in
CPG1 and Chp1 (not shown). Whether these ORFs are genes or artefacts due to high local sequence identity is unclear. Hence, there is currently no firm evidence to support the existence of overlapping genes in
CPG1 or Chp1, although these do occur in
X174 and related phages.
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DISCUSSION |
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Coliphages related to X174 possess a circular DNA genome that undergoes replication by means of a double-stranded replicative form and is packaged as a single-stranded molecule into virions inside the cytoplasm of infected bacteria. Phage progeny is then released in abundance upon lysis of the infected host bacterium. We took advantage of these presumed properties and of consistent previous findings with Chp1 (Storey et al., 1989b
) to isolate and clone the entire genome of
CPG1. Sequence analysis of the cloned insert confirmed the relationship of
CPG1 with Chp1, in terms of general structure and predicted genetic architecture, including five conserved predicted genes (Storey et al., 1989a
) (Fig. 4
), encoding the major capsid protein VP1, corresponding to the 62 kDa protein observed by SDS-PAGE, VP2, VP3, VG4 and VG5.
Similarity was also observed between the CPG1 genome and that of SpV4, a phage of the mollicute Spiroplasma melliferum (Renaudin et al., 1987
) (Fig. 4
). Two of the three Chlamydia phage predicted proteins that are conserved in SpV4 (VP1 and VG4/ORF4) display limited sequence similarity to proteins of other bacteriophages.
VP1 of CPG1 (like its counterparts in Chp1 and SpV4) was weakly related to the capsid gene F protein family of enteric microphages including
X174,
3, S13, G4 and
K. Full-length alignment of the gene F protein family with the VP1 family defines a large internal loop, IN5, in VP1 proteins, which is absent in gene F proteins (Chipman et al., 1998
). Chipman et al. (1998)
speculate that IN5 capsid protrusions represent evolutionary substitutes for spike-forming protein G pentamers of the coliphages, and that IN5 sequence divergence may represent adaptation to different bacterial host ranges. In this context, it is surprising that the IN5 sequences of the two Chlamydia phages are relatively dissimilar. The relative divergence of capsid surface structures in the two Chlamydia phages may reflect different receptor structures in avian and guinea pig Chlamydia, and/or different lineages of the two Chlamydia phage capsid genes, a notion consistent with the observation of the narrow (restricted to avian C. psittaci) host-range of Chp1 (Richmond et al., 1990
).
Storey et al. (1989a) also reported the homology of ORF4 of Chp1 to the gene A protein of phage
X174, in which it is involved in phage DNA replication (Hayashi et al., 1988
). However, the full-length VG4 protein of
CPG1 and its SpV4 counterpart (VG2) share little homology with this gene product from either member of the
X174 group. Moreover, the primary sequence of
CPG1 VG4 is marginally more similar to its counterpart in SpV4 than to that in Chp1 (Table 1
). This is somewhat surprising in view of the predicted housekeeping function of these proteins. We therefore used PSI-BLAST to investigate whether smaller conserved motifs could be identified in VG4. This analysis led to two significant results: 1, a previously described gene A/A* protein zinc finger motif (Kodaira et al., 1992
) was not conserved in
CPG1, Chp1 and SpV4; 2, a larger segment encompassing several conserved motifs of DNA-binding Rep proteins (superfamily I) involved in RCR initiation of plasmids, phages and phagemids (Ilyina & Koonin, 1992
) was conserved across
CPG1, Chp1, SpV4 and the coliphages (Fig. 6
). Alignment of the full-length amino acid sequences based on the Rep homology also revealed that VG4 of
CPG1 lacked the amino-terminal extension of gene A protein and, as such, was similar to gene A* protein (Fig. 6
), which results from an alternative translational start within gene A of the coliphages.
Moulder (1988) suggested that phages perhaps infected ancestral Chlamydia forms, prior to their irreversible commitment to an obligate intracellular lifestyle. However, our related studies (Hsia et al., 2000
) also suggest that
CPG1 may gain access to RBs by efficiently attaching to EBs prior to internalization. This de facto eliminates the presumed physical barriers on phage infection of intracellular chlamydiae and implies that phages have had and continue to have the opportunity to infect Chlamydia throughout their evolution. A multiple entry point hypothesis is consistent both with the highly conserved genetic architecture of the two Chlamydia phages, and with the contrasting finding of relative dissimilarity implying differential lineages between two of their gene products, VP1 and VG4/ORF4. A plausible interpretation of these results is that, as has been observed with double-stranded-DNA tailed bacteriophages (Hendrix et al., 1999
), Chlamydia phage evolution has been subject to frequent horizontal transfer exchanges with a large, as yet unidentified, heterologous genetic pool. This is supported by the existence of the closely related Spiroplasma phage and implies that close relatives of the Chlamydia phages may infect other free-living bacteria.
The high homology of a 375 bp segment of the C. pneumoniae genome to a segment of CPG1 genome has several important implications. First, the high homology leaves no doubt as to the relationship between the
CPG1 and C. pneumoniae sequences: either one acquired it recently from the other, or both recently acquired it from a common source. In comparison, homology of the C. pneumoniae genomic segment with the corresponding Chp1 sequence does not rise above background noise, highlighting the uniqueness of the
CPG1C. pneumoniae relationship. Given the biology of chlamydiae and bacteriophages, it is most likely that C. pneumoniae acquired this sequence from a bacteriophage closely related to
CPG1.
Identical 375 bp sequences have also been found in two other C. pneumoniae genomes. While this reflects the clonality of these strains, it also suggests that the inserted sequence may play an important role in C. pneumoniae biology. Indeed, if this insert were not essential, it would likely have been lost or inactivated by mutation in at least some of the strains. If the phage insert is essential to C. pneumoniae, it possibly is so by virtue of the product it potentially encodes, i.e. a 113 residue carboxy-terminal truncate of VG4. Homology of the integrated phage genome segment starts approximately 100 nt upstream of the start codon and stops immediately after the stop codon, suggesting that the VG4 truncate was likely generated by integration/deletion en bloc of phage sequence and did not involve further internal rearrangements/mutations in its genesis. This suggests that expression is possible by virtue of conserved promoter elements in the upstream sequence. However, the function of the expressed peptide is unclear, as only two of the three Rep motifs are found on the C. pneumoniae VG4 peptide, with the proposed tyrosine-containing DNA-binding domain missing entirely (Fig. 7).
In summary, we have reported the isolation, cloning and partial characterization of CPG1, the second phage known to infect Chlamydiaceae and the first known to infect a member of the Chlamydiaceae with a mammalian host. The phylogenetic relationship of
CPG1 with a previously described phage of avian Chlamydia has been established, but it is unclear based on sequence analysis alone whether these phages have co-evolved with their host bacteria from a single ancestor or whether they, or specific phage genes, have been independently acquired at later stages of evolution. The finding that a 375 nt sequence present in the genome of C. pneumoniae is highly homologous to a segment of the
CPG1 genome is consistent with C. pneumoniae having been recently infected by a bacteriophage closely related to
CPG1. Moreover, the previous observation of phage crystalline arrays in Chlamydia-like forms infecting Chesapeake Bay bivalves (Harshbarger et al., 1977
), the isolation of phages from avian and guinea pig C. psittaci (Richmond et al., 1982
; this work), and the likely existence of a phage infecting C. pneumoniae (Kalman et al., 1999
), suggest that phage infection is more widely spread in the genus than previously thought, and implies that phage infection may play an important role in the development of chlamydial infection and disease. Phage
CPG1 will provide the opportunity for the first time to evaluate the role of phage infection in chlamydial disease using the ocular and genital guinea pig model systems.
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ACKNOWLEDGEMENTS |
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Received 20 December 1999;
revised 21 March 2000;
accepted 7 April 2000.