Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae

Abstract

The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.

Figure 1

Plot of BSRs of C.caviae proteins against C.pneumoniae and C.muridarum. For each predicted C.caviae protein, BLASTP P-values against itself and for the most similar proteins in C.pneumoniae and C.muridarum are obtained. The P-values are converted using a –ln function and the BSR calculated using the C.caviae self-alignment value. Each ratio pair (C.pneumoniae:C.caviae and C.muridarum:C.caviae) is then plotted for each C.caviae gene. Certain classes of similarity are highlighted—regions of the plot with highly conserved proteins (BSR ≥ 0.8) in red, conserved (0.5 < BSR < 0.8) in yellow, poorly conserved in green (0.1 < BSR < 0.5) and non-conserved (unique) in blue (BSR <0.1). Where the ratio of the score for a gene in one genome was at least 1.5-fold greater than the other (as delineated by the dotted lines), proteins have been marked as red squares (closer to C.pneumoniae) or blue circles (closer to C.muridarum). The pmp proteins are also highlighted (black diamond), showing their wide distribution of similarity and particularly pmpB/C, unusual for its high conservation in other chlamydial genomes. The positions of members of the guaBA-add gene cluster, found in the C.caviae replication termination region, are also indicated.

Figure 2

Breakdown of orthologous C.caviae genes in other Chlamydiaceae genomes. Numbers represent a count of the most similar genes in particular genomes to C.caviae.

Figure 3

Comparison of chromosomal synteny in the Chlamydiaceae. (A) Chlamydia trachomatis versus C.muridarum; (B) C.pneumoniae versus C.caviae; (C) C.muridarum versus C.caviae. Each panel is the plot of the most similar proteins (using BLASTP P-values), ordered by chromosomal position for each genome pair. Axes are marked with 200 kb graduations. R and T are the locations of the origin of replication and the termination region, respectively. Genomes have been rotated so that the replication origin is at ∼275 kb and the termination region at ∼850 kb.

Figure 4

Circular representation of the C.caviae chromosome. Data are from outermost circle to innermost. Circles 1 and 2: tick marks represent predicted coding sequences on the plus strand (circle 1) and minus strand (circle 2) colored by cellular role. Role categories and colors are as follows: amino acid biosynthesis, violet; biosynthesis of cofactors, prosthetic groups and carriers, light blue; cell envelope, light green; cellular processes, red; central intermediary metabolism, brown; DNA metabolism, gold; energy metabolism, light gray; fatty acid and phospholipid metabolism, magenta; protein synthesis and fate, pink; biosynthesis of purines, pyrimidines, nucleosides and nucleotides, orange; regulatory functions and signal transduction, olive; transcription, dark green; transport and binding proteins, blue–green; other categories, salmon; unknown function, gray; conserved hypothetical proteins, blue; hypothetical proteins, black. Circles 3 and 4: genes in C.caviae that are homologous to genes in C.pneumoniae (circle 3) and C.muridarum (circle 4) colored by normalized BLAST score: 0.8 < score, red; 0.5 < score < 0.8, yellow; 0.1 < score < 0.5, green; score < 0.1, blue. Circle 5: chi-square of trinucleotide skew. Circle 6: codon adaptation index (CAI) score for each gene. Circle 7: predicted tRNAs. Circle 8: predicted rRNAs.

Figure 5

Schematic comparing chlamydial replication termination regions with lymphostatin, tryptophan and purine biosynthesis determinants. View depicts gene order in the replication termination regions of C.pneumoniae AR39 (top; locus prefix CP0), C.caviae (middle; locus prefix CCA00) and C.muridarum (bottom; locus prefix TC0). Genes are colored according to role-category assignments (see legend to Fig. 4) and labeled either with the appropriate gene symbol or with the published locus numbers. Lines connect orthologs of predicted proteins between genomes. Elements of the C.caviae and C.trachomatis tryptophan operon are highlighted with gray shading. The guaA gene of C.pneumoniae and the MAC/perforin gene of C.caviae are marked with an asterisk (*) to denote the internal deletion (see text) and a frameshift, respectively. The truncation of the C.pneumoniae guaB gene is denoted with a diagonal line.

Figure 6

Variable genes found between the conserved aspartate and aromatic amino acid biosynthesis operons. View depicts the region around these genes in the replication termination region of C.pneumoniae, C.caviae, C.muridarum and C.trachomatis (descending order). Lines connect orthologs between the genomes. Chlamydophila caviae genes with homology to truncated chlamydiaphage proteins are highlighted with gray shading. Genes are colored according to role-category assignments (see legend to Fig. 4) and labeled either with the appropriate gene symbol or with the published locus numbers [C.pneumoniae AR39 (locus prefix CP0), C.caviae (locus prefix CCA00), C.muridarum (locus prefix TC0) and C.trachomatis (locus prefix CT)]. The conserved genes flanking the variable segments are dihydropicolinic reductase (dapB; CCA00715) and 3-phosphoshikimate 1-carboxyvinyl transferase (aroA; CCA00723). CP0806 is a biopterin-dependent amino acid hydroxlase. CP0808-0811 form a portion of a biotin biosynthesis operon, encoding biotin synthase (bioB), 8-amino 7-oxononanoate synthase (bioF), dethiobiotin synthase (CP0810) and adenosylmethionine 8-amino 7-oxononanoate aminotransferase (bioA), respectively.

Figure 7

Schematic depicting alignment of known and putative toxin genes with key domains highlighted. The relative locations of the four truncated C.trachomatis genes with homology to the N- and C-terminal regions of the C.caviae (CCA00558) and one of the three C.muridarum (TC0438) tox genes are shown. Escherichia coli LifA and Efa1, and Clostridium difficile ToxA are also shown. Blue: UDP-glucose binding domain; black: glycosyltransferase domain; red: YopT domain; green: transmembrane domain. Genes shaded gray indicate known or putative cytotoxic function.

Figure 8

Detail of the novel C.caviae invasin-like gene. (A) Schematic indicating relative location if the key domains within the invasin predicted protein sequence. The N-terminal half is most similar to Yersinia pseudotuberculosis invasin (SP:P11922) (72). A bacterial immunoglobulin-like domain also found in invasins and intimins is indicated. The C-terminal half shows no similarity to other invasins or intimins, lacking particularly several described sequences motifs and a disulfide loop required for binding to eukaryotic β1 chain integrins (73). Nonetheless, the presence of six repeats of approximately 90 amino acids (D1–D6) in this half suggests a similar structure and function in projecting a functional domain (D7, putative) away from the chlamydial outer membrane and into the milieu. The sequence encompassing the tandem repeats D2–D6 and some of the putative C-terminal functional domain D7 display sequence homology to the tandem repeat region of Bhp of Staphylococcus epidermidis, a paralog of Bap (74), involved in biofilm formation and virulence. The positions of the frameshifts in the nucleotide sequence and the location of the subsequences with similarity to C.muridarum TC0115 (see text) are also indicated. (B) Alignment of the bacterial immunoglobulin-like domain to the Pfam consensus sequence (75). Identical and conserved residues are indicated by red and blue, respectively. (C) Alignment of the intragenic repeats D1–D6 to each other. Identical and conserved residues are indicated by red and blue, respectively.