evidence that the campylobacter fetus sap locus is an ... · the reca and sapd sequences were...

INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.4.2237–2244.2001

Apr. 2001, p. 2237–2244 Vol. 69, No. 4

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Evidence that the Campylobacter fetus sap Locus Is an AncientGenomic Constituent with Origins before Mammals and

Reptiles DivergedZHENG-CHAO TU,1 FLOYD E. DEWHIRST,2 AND MARTIN J. BLASER1,3*

Division of Infectious Diseases, Department of Medicine, Vanderbilt University School of Medicine,1 and Departmentof Veterans Affairs Medical Center,3 Nashville, Tennessee, and Department of Molecular Genetics,

The Forsyth Institute, Boston, Massachusetts2

Received 11 September 2000/Returned for modification 11 December 2000/Accepted 29 December 2000

Campylobacter fetus bacteria, isolated from both mammals and reptiles, may be either subsp. fetus or subsp.venerealis and either serotype A or serotype B. Surface layer proteins, expressed and secreted by genes in thesap locus, play an important role in C. fetus virulence. To assess whether the sap locus represents a pathoge-nicity island and to gain further insights into C. fetus evolution, we examined several C. fetus genes in 18isolates. All of the isolates had 5 to 9 sapA or sapB homologs. One strain (85-387) possessed both sapA and sapBhomologs, suggesting a recombinational event in the sap locus between sapA and sapB strains. When weamplified and analyzed nucleotide sequences from portions of housekeeping gene recA (501 bp) and sapD (450bp), a part of the 6-kb sap invertible element, the phylogenies of the genes were highly parallel. Among the 15isolates from mammals, serotype A and serotype B strains generally had consistent positions. The fact that theserotype A C. fetus subsp. fetus and subsp. venerealis strains were on the same branch suggests that theirdifferentiation occurred after the type A-type B split. Isolates from mammals and reptiles formed two distincttight phylogenetic clusters that were well separated. Sequence analysis of 16S rRNA showed that the reptilestrains form a distinct phylotype between mammalian C. fetus and Campylobacter hyointestinalis. The phylog-enies and sequence results showing that sapD and recA have similar G 1 C contents and substitution ratessuggest that the sap locus is not a pathogenicity island but rather is an ancient constituent of the C. fetusgenome, integral to its biology.

Members of the genus Campylobacter are microaerophilic,nonfermentative bacteria, of which Campylobacter fetus is thetype species (40). C. fetus has been isolated from a wide rangeof hosts, including cattle, sheep, other ungulates, swine, hu-mans, poultry, and reptiles (41). C. fetus causes infertility andinfectious abortion in sheep and cattle and may cause bothdiarrheal and extraintestinal infections in human hosts (3, 21,39, 40, 43). The species C. fetus is currently subdivided into C.fetus subsp. fetus and C. fetus subsp. venerealis, based on theirhabitats, biological properties, and genome sizes and the dis-eases they produce (22, 28, 38, 40, 43).

C. fetus strains are either serotype A or serotype B (13, 30,35). C. fetus subsp. venerealis strains are serotype A, whereas C.fetus subsp. fetus cells may be either serotype A or serotype B(30, 35). These serotypes are associated with differences inboth lipopolysaccharide (LPS) structure and type of surfacelayer protein (SLP) (13, 20, 30, 33–35, 48). The C. fetus SLPs,which act as capsules to resist C3b binding and undergo anti-genic variation to protect against antibody-mediated opsoniza-tion, are important virulence factors allowing both persistenceand systemic infection (5, 6, 8, 10, 11, 29, 32, 47). Our previousstudies have shown that the SLPs produced by strains isolatedfrom reptiles (see below) are antigenically cross-reactive withSLPs isolated from mammals (46), and a reptile strain was

found to be the cause of an acute diarrheal illness in a child(24). This observation suggests that reptile strains produceSLPs that also serve as important virulence factors, similar tothose for the SLPs from strains isolated from mammals. C.fetus cells possess a unique sap promoter that allows expressionof the full complement of sap homologs that encode theseSLPs (9, 45). The sap locus (including all the sap homologs, thesap promoter, and sapC, -D, -E, and -F on an invertible ele-ment) is tightly clustered on the C. fetus chromosome in aregion of ,93 kb, representing ,8% of the genome (9, 44, 45).

In this study, we investigated the evolutionary relationshipsbetween the two C. fetus subspecies among strains originatingfrom different hosts. We hypothesized that the sap locus mightrepresent a pathogenicity island (23) which entered the C. fetusgenome after the species was formed. To test this hypothesis,we compared sapD, a gene that is conserved in the invertiblesap element (44), and recA, a widely conserved housekeepinggene (16). Based on differences observed in these phylogeneticanalyses, we examined the 16S rRNA genes of six C. fetusstrains to better understand the position of C. fetus in relationto other Campylobacter spp.

MATERIALS AND METHODS

Strains. The 18 C. fetus strains used in this study are listed in Table 1. Threeisolates were C. fetus subsp. venerealis. The other 15 isolates were C. fetus subsp.fetus; 9 were serogroup A (6 mammal and 3 reptile) and 6 were serogroup B. Thethree reptile strains were isolated from turtles after one had been found to be thecause of an acute diarrheal illness in a child (24). Strains 99-256 (ATCC 33561)and 99-257 (ATCC 19438) are from the American Type Culture Collection,Manassas, Va.; the other 16 strains had been collected and identified as C. fetus

* Corresponding author. Mailing address: Department of Medicine,New York University School of Medicine, 550 First Ave., New York,NY 10016. Phone: (212) 263-6394. Fax: (212) 263-7700. E-mail: [email protected].

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in the Vanderbilt University Campylobacter laboratory using standard criteria (9,18, 21, 40).

Immunoblot assay. C. fetus cells were harvested from 48-h plate cultures,protein concentrations were assayed using the Pierce bicinchoninic acid proteinreagent assay (Pierce Chemical Co., Rockford, Ill.), and 1-mg protein sampleswere assayed by electrophoresis on a 7% sodium dodecyl sulfate-polyacrylamidegel. S-layer proteins were detected with polyclonal rabbit serum (1:10,000 dilu-tion) against the C. fetus strain 82-40LP 97-kDa SLP, as described previously(33). The secondary antibody (1:2,000 dilution) was goat anti-rabbit immuno-globulin G–alkaline phosphatase (Boehringer Mannheim Biochemicals, India-napolis, Ind.).

PCR. The PCR primers used in this study are listed in Table 2. To determinewhether a strain was a sapA or sapB type, chromosomal DNA from the selectedstrains was amplified with SAF01 and SAR01 or with SBF01 and SBR01, re-spectively. To amplify recA or sapD, the primers RAF01 and RAR01 or SDF01and SD01, respectively, were used. The 16S rRNA cistrons were amplified withbacterial universal primers F24 and F25. The products of PCR amplification

were examined by electrophoresis in 1% agarose gels. DNA was stained withethidium bromide and visualized under short-wavelength UV light.

Southern hybridization. C. fetus chromosomal DNA was prepared using theWizard genomic DNA purification kit (Promega, Madison, Wisc.), digested withHindIII, electrophoresed on a 0.7% agarose gel, and transferred to a nylonmembrane (MSI, Westborough, Mass.). The membranes were hybridized withDNA probes labeled using the Renaissance nonradioactive chemiluminescencekit supplied by NEN Research Products (Boston, Mass.). The probes were thePCR products specific for either the sapA 59 conserved region, which was am-plified using primers SAF01 and SAR01, or for the sapB 59 conserved region,which was amplified using primers SBF01 and SBR01 (Table 2).

Sequencing. After the 501-bp recA fragments, the 450-bp sapD fragments, andthe 16S rRNA cistrons were amplified, the PCR products were purified using theQiaQuik PCR purification kit (Qiagen Inc., Valencia, Calif.). Purified DNA fromPCR was sequenced using an ABI prime cycle-sequencing kit (BigDye termina-tor cycle sequencing kit with AmpliTaq DNA polymerase FS; The Perkin-ElmerCorp., Norwalk, Conn.), and reactions were run on an ABI 377 DNA sequencer.

TABLE 1. Characteristics of the C. fetus strain studied

Strain no.(in Fig. 1–3)

Straindesignation

C. fetussubspecies

Animal source(site) Serotypea sap typeb Major SLP

(kDa)cNo. of saphomologs

1 80-109 fetus Human (blood) A A 127 82 82-40 fetus Human (blood) A A 97 83 83-94 fetus Human (NA) NA A 97 54 84-32d fetus Bovine (vagina) A A 97 85 84-86 fetus Human (blood) A A 97 86 84-92 fetus Bovine (feces) A A 97 77 85-388 fetus Reptile (feces) A A 97 98 85-389 fetus Reptile (feces) A A 149 89 84-112 venerealis Bovine (genital) A A 149 810 99-256 venerealis Bovine (vagina) NA A 97 811 99-257 venerealis Human (blood) NA A 97 712 85-387 fetus Reptile (feces) NA A/B 97 813 84-87 fetus Human (blood) B B 97 814 84-90 fetus Bovine (feces) B B ND 715 84-91 fetus Human (blood) B B 97 816 84-94 fetus Human (blood) B B 127 817 84-104 fetus Monkey (blood) B B 97 718 84-107 fetus Human (blood) B B 97 7

a Serotype based on LPS type (35, 47). NA, not available.b Based on PCR and Southern hybridization.c By immunoblot using polyclonal rabbit serum. ND, not detected.d Originally characterized as strain 23D by McCoy et al. (29).

TABLE 2. PCR and sequencing primers

Primerdesignation Gene Position Orientation Sequence (59339)a

F24 16S rRNA 9–27 Forward AGTTTGATYMTGGCTCAGF22 16S rRNA 344–358 Reverse RCTGCTGCCTCCCGTF23 16S rRNA 344–359 Forward ACGGGAGGCAGCAGYF15 16S rRNA 519–533 Reverse TTACCGCGGCTGCTGF16 16S rRNA 789–806 Forward TAGATACCCYGGTAGTCCF17 16S rRNA 907–926 Reverse CCGTCWATTCMTTTGAGTTTF18 16S rRNA 1099–1113 Forward GCAACGAGCGCAACCF20 16S rRNA 1226–1242 Reverse CCATTGTARCACGTGTGF25 16S rRNA 1525–1541 Reverse AAGGAGGTGWTCCARCCSAF01 sapA 0–19 Forward ATGTTAAACAAAACAGATGTSAR01 sapA 513–531 Reverse ATCAAGATCACTAGCACTASBF01 sapB 19–40 Forward TTCAGAGCTATTTATAGTTCSBR01 sapB 504–524 Reverse TCAACACTACTACTATTACTARAF01 recA 12–41 Forward ATAAGAAAAAAAGCCTAGACCRAR01 recA 607–625 Reverse TAGTTTCAGGAGTGCCATASDF01 sapD 825–844 Forward AGCTGGATCAATACTATTAGSDR01 sapD 1348–1367 Reverse CCGTCCGGAAGTCTTAG

a R 5 A or G; M 5 A or C; W 5 A or T; Y 5 C or T.

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The recA and sapD sequences were determined on both strands using the sameprimers used for their PCR amplification. For 16S rRNA sequencing, primersF15-F18, F20, and F22-F25 (Table 2) were used.

Data analysis. Multiple nucleotide alignments were created using the GeneticsComputer Group (GCG) program (Wisconsin Package version 9.1; GCG, Mad-ison, Wisc.). Phylograms based on recA and sapD nucleotide sequences weregenerated using both parsimony and distance matrix methods, using PAUP4.0b2, and the phylograms were displayed using Treeview and PAUP 3.1 (D. L.Swofford. 1993. PAUP: phylogenetic analysis using parsimony, version 3.1. Illi-nois Natural Survey, Champaign, Ill.) 16S rRNA sequence data were enteredinto RNA, a program for data entry, editing, sequence alignment, secondarystructure comparison, similarity matrix generation, and dendrogram constructionfor 16S rRNA, written in Microsoft QuickBasic for use with PC computers, andwere aligned as previously described (31). The 16S database contains over 1,000sequences obtained at the Forsythe laboratory and over 500 obtained fromGenBank (27). Similarity matrices were constructed from the aligned sequencesby using only those sequence positions for which 90% of the strains had data. Thesimilarity matrices were corrected for multiple base changes at single positions bythe method of Jukes and Cantor (26). Phylogenetic trees were constructed usingthe neighbor-joining method of Saitou and Nei (37).

RESULTS

Characterization of the C. fetus S-layer proteins. To charac-terize the 18 C. fetus strains in terms of their SLP expression,we performed immunoblotting with polyclonal antiserumraised to the 97-kDa S-layer protein from type A strain 82-40LP (46). These immunoblots indicated that the wild-type C.fetus strains contain different high-mass SLPs ranging from 97to 149 kDa, as expected (Table 1 and Fig. 1). One type B strain(84-90) is SLP2, which may reflect in vitro deletion of thesecretion apparatus and unique promoter, as has been de-scribed for type A strains (45).

Typing of strains. All C. fetus strains possess either sapA orsapB homologs (13), and each of the two families (sapA andsapB) of homologs share a unique 552-bp sequence that makesup their 59-conserved regions (13). PCR, using primers basedon the sapA and sapB 59-conserved regions (7, 13), showed that

FIG. 2. PCR to detect the sap type of the 18 C. fetus strains by using 59-conserved-region primers from sapA (panel A) or sapB (panel B). Lrepresents the 1-kb ladder. See Table 1 for strain characteristics. (The lane numbers correspond to the strain numbers in Table 1.)

FIG. 1. Identification of SLP in whole-cell preparations of 18 C. fetus strains by immunoblotting with polyclonal rabbit serum against the 97-kDaSLP from type A C. fetus strain 82-40. See Table 1 for strain characteristics. The lane numbers representing the strains correspond to the “strainnumbers” in Table 1.

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6 of the 18 strains are sapA types, 11 are sapB types, and strain85-387, isolated from a turtle, has both types (Table 1; Fig. 2).Southern hybridization, using the sapA and sapB 59-conservedregions as the type-specific probes (Fig. 2), confirmed the re-sults of PCR, and it also showed that there are 5 to 9 saphomologs in each of the 18 strains studied (Table 1 and Fig. 3).Strain 85-387 possesses seven sapB homologs and one sapAhomolog, thus representing an A/B chimera.

Patterns of divergence between strains. The polymorphicsites within recA and sapD of the 18 C. fetus strains and the 16SrRNA of the six C. fetus strains studied are shown in Fig. 4.There were 36 polymorphic sites within the 501 bp of recA thatwere sequenced in all 18 strains (Fig. 4A). Only 36 sites werepolymorphic, and the overall substitution rate was 0.10. Syn-onymous substitutions predominated, with Ka/Ks 5 0.01,where Ka is the nonsynonymous substitution rate and Ks is thesynonymous substitution rate. Within the 450 bp of sapD (Fig.4B), there were 46 sites showing any polymorphism, and theoverall substitution rate was 0.17. Synonymous substitutionsalso predominated, with Ka/Ks 5 0.03. The mean (plus orminus the standard deviation) G 1 C content of the 18 recAsequences was 40.8% 6 0.3%, similar to the 40.6% 6 0.3%content of the 18 sapD sequences.

In six C. fetus strains studied, there were 28 polymorphicsites in essentially complete 16S rRNA sequences (bases 28 to1524 using the Escherichia coli numbering) compared withCampylobacter hyointestinalis, a species similar to C. fetus (Fig.4C). The 16S rRNA sequences from sapA strain 84-32, sapB

strain 84-107, and C. fetus subsp. venerealis 99-256 were iden-tical. The three reptile strains showed identical 16S rRNAsequences, but they differed from the other C. fetus strains by10 bases and from C. hyointestinalis by 19 bases.

Phylogenetic relationships inferred from recA, sapD, and16S rRNA. The phylogeny of recA (Fig. 5A) showed that theisolates from mammals and reptiles formed two tight clustersthat were far removed from each other. Among the isolatesfrom mammals, the major distinction was between the serotypeA and serotype B strains, but there were only two nucleotidedifferences (Fig. 4). The only exception, strain 84-87, a type Bstrain, was identical to the type A consensus sequence. Thetype A C. fetus subsp. fetus and subsp. venerealis strains are onthe same branch, suggesting that their differentiation occurredafter the type A-type B split. The phylogeny of sapD is almostidentical to that for recA, with only one or two nucleotidedifferences between type A and type B mammalian strains.Strain 83-94 (type A) was the exception, with a type B sapDsequence (Fig. 5B). The dendrograms for 16S rRNA (Fig. 6)indicated that the reptile strains (as exemplified by strain 85-388) appear to form a distinct phylotype between C. fetus andC. hyointestinalis.

DISCUSSION

C. fetus strains have been characterized on the basis of thesource from which they were isolated, the biochemical prop-erties defining the subspecies, and their serotype, but the workreported here indicated that the most fundamental difference

FIG. 3. Southern hybridization of HindIII digestions of chromosomal DNA from 18 C. fetus strains with probes to the 59-sapA (panel A) or-sapB (panel B)-conserved regions. See Table 1 for strain characteristics. (The lane numbers correspond to the strain numbers shown in Table 1.)The positions of molecular size markers (in kilobases) are indicated to the left of each panel.


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among C. fetus strains is reflected by whether they were iso-lated from a mammal or a reptile. Using PCR, Southern hy-bridization, and LPS typing, the three reptile strains wereshown to be type A, and earlier studies showed that reptilestrains produced SLPs that are antigenically cross-reactive withSLPs in mammals (46). However, based on the recA, sapD, and16S rRNA sequences, the reptile isolates are highly differentfrom the C. fetus strains isolated from mammals. The reptilestrains appear to form a distinct phylotype of C. fetus, but foreach of the three genes studied, sequence differences of themagnitude found could indicate that they represent different

species or different subspecies. The taxonomic relatedness ofthe reptilian, and mammalian C. fetus isolates may be resolvedby additional studies, although the decision about whether toseparate them into different species may be arbitrary.

The sequence data in this study were consistent with thehypothesis that C. fetus is an ancient organism once carried byan ancestral vertebrate host. According to this hypothesis, theancestral host carried an ancestral C. fetus strain and, as thathost evolved to differentiate into reptiles or mammals, the C.fetus strains carried by the host continued to evolve but inisolation from each other. The deep differences within com-mon loci between C. fetus strains isolated from mammals andreptiles suggest that their last common ancestor may have livedbefore these animals diverged, approximately 200 million yearsago. However, the data do not completely rule out the alter-native possibility that C. fetus was acquired after mammals andreptiles diverged and that its presence in the two types of hostsreflects interspecies (horizontal) transmission.

The source of isolates, genome size, and the degree of tol-erance to glycine have been the major differentiating featuresbetween C. fetus subsp. fetus and C. fetus subsp. venerealis (21,38, 39, 41). C. fetus subsp. fetus causes sporadic epizootic abor-tion in cattle and sheep and is involved in human infections,producing both acute intestinal illness and systemic diseases(5). C. fetus subsp. fetus strains have been isolated from manyanimal species, including cattle, sheep, other ungulates, poul-try, swine, and reptiles (40). In contrast, strains of C. fetussubsp. venerealis cause enzootic infertility in cattle and rarelyhave been associated with human infections (38). The genomesizes of C. fetus subsp. fetus and C. fetus subsp. venerealis are 1.1Mb and 1.3 Mb, respectively (38). Although classically C. fetussubsp. fetus strains but not C. fetus subsp. venerealis strainstolerate more than 1% glycine, such results are not easilyreproducible (21, 41). The two subspecies cannot be differen-tiated on the basis of serotype (35), SLP type (13, 48), fluo-rescent-antibody assay (28), fatty acid content (4), or DNA-DNA homology studies (1). All subsp. venerealis isolates areLPS type A, whereas subsp. fetus may be type A or B (30, 35).The observation that subsp. venerealis strains could not bedistinguished from type A mammalian subsp. fetus strains onthe basis of sapD, recA, and 16S rRNA sequences indicates thatthese organisms are very closely related. If they are not iden-tical, their differentiation must have been relatively recent.

C. fetus cells may exist as either of two defined serogroups(type A or type B) based on their LPS composition (13, 30, 35).The LPS types, defined structurally and antigenically (30, 35),are consistent with the C. fetus serotyping scheme developedmore than 30 years ago (2). Reattachment of native SLP andthe recombinant sapA and sapB products to cells of the ho-mologous LPS type, but not to the heterologous LPS type, hasindicated that the conserved sapA- and sapB-encoded N ter-mini are critical for LPS-binding specificity (13, 48). Thus, theserotype (A or B), LPS type (A or B), and SLP type (A or B)of a C. fetus strain are consistent with one another. Among C.fetus isolates from mammals, the most significant phylogeneticdichotomy is between type A and B strains, as indicated byboth sapD and recA analyses.

Using PCR and Southern hybridizations based on the sapAand sapB 59-conserved regions, we found 5 to 9 sap homologsin each strain. Each strain shows a different sap profile, which

FIG. 4. Polymorphic sites within the recA (panel A), sapD (panelB), and 16S rRNA (panel C) gene sequences. Numbering (verticalformat) of the polymorphic sites of recA and sapD is from the first baseposition of each gene. The numbering of 16S rRNA sequences corre-sponds to the base positions of E. coli. For panels A and B, the positionof the site within the codon is shown below. Nearly all (90.2%) of the82 polymorphic sites for recA and sapD are in the third codon position.


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is due to DNA rearrangements in the sap locus (15, 16, 36;Z.-C. Tu, K. C. Ray, S. A. Thompson, and M. J. Blaser, sub-mitted for publication). Surprisingly, we found one (reptile)strain (85-387) with one sapA and seven sapB homologs. Thecoexistence of sapA and sapB homologs in the same strainprovides, for the first time, evidence of intraspecies recombi-nation involving the C. fetus sap locus. Although considerablehorizontal interspecies and intraspecies gene transfer has oc-curred in prokaryotes (17, 19, 25, 42), how this might occur in

C. fetus cells that have an S layer and are not naturally com-petent is not immediately apparent. The dichotomy betweentype A and type B strains involves both LPS structure (30, 35)and SLP sequence and structure (33, 48) and thus involvesdifferences in at least two different genetic loci. The occurrenceof recombination might explain why the type A-B dichotomy isnot perfectly reproduced in the sapD and recA phylogenies(Fig. 5).

The finding that the sap genes are critical for C. fetus viru-

FIG. 5. Phylogenetic trees constructed from the nucleotide sequences of recA (panel A) and sapD (panel B) using the PAUP 4.0b2 neighbor-joining method, based on Kimura’s two-parameter model distance matrices. Bootstrap values (based on 500 replicates) are represented at eachnode, and the branch length index is represented below each tree.


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lence and that pulse-field gel electrophoresis studies indicatethat these genes are clustered on the chromosome (12) sug-gests the possibility that the genes exist as a single locus rep-resenting a “pathogenicity island” (23). Mapping studies indi-cate that most if not all of the genes are contiguous (14; Z.-C.Tu and M. J. Blaser, unpublished data). Pathogenicity islandshave entered bacterial genomes after their development asparticular species, and consequently markers of their evolutionand phylogeny differ from those of “housekeeping” genes (23).However, the conservation of sapD and the sapA homologs inall C. fetus strains and the similar G 1 C content, substitutionrate, and phylogeny in relation to recA suggest that the saplocus is not a pathogenicity island but represents an ancientand highly conserved constituent of the C. fetus genome. Thestriking sequence identity between sapA and sapB (13) furthersupports a highly conserved function in the sap locus. Ourprevious studies have shown that sap DNA inversion plays animportant role in C. fetus virulence via high-frequency RecA-dependent (16) and low-frequency RecA-independent mecha-nisms (36); this redundancy of mechanisms indicates the im-portance of sap inversion to C. fetus. In total, both the earlierand present data suggest that C. fetus is an ancient organismwhose highly conserved features permit maintenance of itsniche(s) on mucosal surfaces of vertebrate hosts.

ACKNOWLEDGMENT

This research was supported in part by grant R01 A124145 from theNational Institutes of Health.

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FIG. 6. Phylogenetic tree showing the placement of strains isolated from reptiles (based on strain 85-388) on the basis of 16S rRNA sequencedata analysis. The scale bar represents a 5% difference in nucleotide sequence, as determined by measuring the lengths of the horizontal linesconnecting any two species. The positions of mammalian C. fetus are all identical whether subsp. fetus or venerealis or type A or type B, but thethree strains isolated from reptiles occupy a different position.


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