Table 1.
C. fetus genome attributes, including the extra-chromosomal element.
Figure 1.
Genome comparisons of C. fetus subspecies.
Plots were generated using C. fetus subsp. venerealis 84-112 (Cfv) as a reference (A) or C. fetus subsp. fetus 82-40 (Cff) (B). Inside tracks represent GC-content (ring 1) and GC-skew (ring 2). Cff is shown in blue and Cfv in red. Variation regions (VR) relative to the reference genome are indicated in orange/yellow and named according to the corresponding Genomic Island (GI) or the subspecies definition region (SDR). (V) and (F) in the feature names designate the subspecies venerealis and fetus, respectively. Important genes or features are indicated in parenthesis. Positions of selected mobility genes are indicated.
Figure 2.
Comparative overview of Genomic Islands (GIs).
(A) VGI I (PAI) with the T4SS and putative prohage I, (B) VGI II with a vir-gene cluster and plasmid-related genes, (C) VGI III containing the surface array protein cluster and prohage III, (D) VGI IV containing the CRISPR-array and prophage IV and (E) FGI II with prophage-related genes (prophage II) and the CRISPR-cluster (array and cas-genes). The GI borders to genes shared between the subspecies (grey) are indicated with nucleotide position. Gene clusters are colored as follows: phage-related genes (orange), plasmid related genes (green), integrases and transposases (blue), T4SS (red), effector proteins (yellow), surface array proteins (purple), cas-genes (lavender), tRNAs (green boxes); Each x represents a hypothetical protein and their numbers in tandem are indicated above.
Figure 3.
Schematic representation and structural comparison of VGI III and FGI I (sap region).
MAUVE was used to compare the VRs of both subspecies for visualization of rearrangements and insertions. Regions free of rearrangements are indicated by colored colinear blocks. White color within these blocks indicates insertions or non-homologous regions. Important orfs are colored and labeled. S-layer genes (purple) were identified in both C. fetus strains. The sap-promoter is indicated. In C. fetus subsp. venerealis 84-112, the sap genes were disrupted by an inserted prophage (orange). White boxes are mainly hypothetical proteins. Detailed annotation information can be found in File S1. Genes are labeled with RAST-peg numbers and the inset table lists homologous sap genes of the subspecies.
Figure 4.
C. fetus promoter sequence and transcriptional organization of the sap-locus.
Promoter consensus sequence for (A) C. fetus subsp. venerealis 84-112 (Cfv) and (B) C. fetus subsp. fetus 82-40 (Cff). The promoter motif is defined by an extended Pribnow box (tgnTAtaAT) at the −10 position. The −35 motif is replaced by a periodic AT-rich signal upstream of position −14 (dotted line). (C) Transcriptional organization of Cfv VG III (top) and Cff FGI I (bottom), identical sap-promoter sequence of Cfv and Cff (middle).
Figure 5.
WcbK is important for LPS-biosynthesis, attenuates survival in blood, and promotes acid resistance.
(A) SDS-PAGE pattern of purified LPS after silver staining. Samples were isolated from C. fetus. subsp. fetus (Cff) 82-40 (lane 1), Cff ATCC 27374 (type B) (lane 2), wcbK mutant K19 (wcbK::Km) (lane 3) and K19 [pSW2] (wcbK in trans) (lane 4); C. fetus subsp. venerealis (Cfv) ATCC 19438 (lane 5) and Cfv 84-112 (lane 6). (B) Cff serum resistance assays. Strains were incubated either with EMEM (-), heat-inactivated (I) or active (A) human serum and colony forming units (CFU) were counted. Results shown are for Cff ATCC 27374, K19 and K19 [pWS2]. Cff 82-40 served as a type A comparator. (C) Same as in (B) but for better visualization, CFU/ml obtained after treatment with active serum are displayed separately. **p<0.002 (D) Acid resistance assays. Cff were incubated in PBS pH range 7.3 to 3.4, plated and CFU determined. Survival after exposure to different pH of the wild type, K19 and K19 [pSW2] was compared. (E) For better visualization, CFU/ml for the three strains after treatment with pH 3.4 were plotted separately. **p<0.003.
Figure 6.
Phylogeny, niche specificity and virulence of C. fetus subspecies.
MLST tree showing the phylogeny of C. fetus, with original scale as reported [4]. Reptile C. fetus represent a distinct clade harboring mat1 and galE. Diversification of C. fetus subsp. fetus (Cff) type B happened prior to the diversification of Cff type A and C. fetus subsp. venerealis (Cfv) type A strains. Cff type B strains harbor galE, mat1 and wcbK. The latter gene provides protection from acid, and this genotype is associated with animal hosts. Cfv type A represents the bovine clone harboring mat1 and galE which is also prone to HGT. Cff type A have lost mat1 but acquired glf correlating with serum resistance in Cff.