Evidence for horizontal gene transfer between Chlamydophila pneumoniae and Chlamydia phage

Abstract

Chlamydia-infecting bacteriophages, members of the Microviridae family, specifically the Gokushovirinae subfamily, are small (4.5-5 kb) single-stranded circles with 8-10 open-reading frames similar to E. coli phage ϕX174. Using sequence information found in GenBank, we examined related genes in Chlamydophila pneumoniae and Chlamydia-infecting bacteriophages. The 5 completely sequenced C. pneumoniae strains contain a gene orthologous to a phage gene annotated as the putative replication initiation protein (PRIP, also called VP4), which is not found in any other members of the Chlamydiaceae family sequenced to date. The C. pneumoniae strain infecting koalas, LPCoLN, in addition contains another region orthologous to phage sequences derived from the minor capsid protein gene, VP3. Phylogenetically, the phage PRIP sequences are more diverse than the bacterial PRIP sequences; nevertheless, the bacterial sequences and the phage sequences each cluster together in their own clade. Finally, we found evidence for another Microviridae phage-related gene, the major capsid protein gene, VP1 in a number of other bacterial species and 2 eukaryotes, the woodland strawberry and a nematode. Thus, we find considerable evidence for DNA sequences related to genes found in bacteriophages of the Microviridae family not only in a variety of prokaryotic but also eukaryotic species.

Keywords: gokushovirinae; horizontal gene transfer; microviridae; putative replication initiation protein (PRIP).

Figure 1.

Comparison among Chlamydia bacteriophages. (A) Matrix derived from MUSCLE analysis of the phage DNA sequences. The matrix shows the extent of identity between 2 phage sequences in pairwise alignment. (Chp1 shows ∼60% identity to the other phages, whereas the others show >90% identity to one another. (B) Phylogenetic analysis of the 6 phage genomes. The evolutionary history was inferred by using the maximum likelihood method based on the Hasegawa-Kishino-Yano model using 1000 bootstraps. The tree with the highest log likelihood (−11567.9705) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sites was < 100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise BIO neighbor joining method with maximum composite likelihood distance matrix was used. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 5.9605)). The analysis involved 6 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 3729 positions in the final dataset. Evolutionary analyses were conducted in MEGA5.

Figure 2.

Phylogenetic Analysis of the 11 PRIP Proteins. The evolutionary history was inferred by using the maximum likelihood method based on the Whelan and Goldman model. The tree with the highest log likelihood (−713.8725) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 11 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 88 positions in the final data set. Evolutionary analyses were conducted in MEGA5.

Figure 3.

Comparison of the 5 C. pneumoniae strains in the region containing the PRIP gene. Ten kb regions centered around the PRIP gene for each of the 5 sequenced C. pneumoniae strains were compared to each other using the WebACT (Artemis Comparison Tool). Light blue arrows represent the open-reading frames on each sequence; PRIP is outlined with a heavier black line. The red chords between sequences signify homology in those regions; blue chords show homology to the opposite strand. In LPCoLN and AR39, PRIP is found on the bottom strand, while in CWL029, J138, and TW183, PRIP is on the top strand. The sequence in LPCoLN that does not match anything in the other strains corresponds to the additional phage sequences similar to VP3 not found in the human-infecting strains.