Homologous Recombination between Genetically Divergent Campylobacter fetus Lineages Supports Host-Associated Speciation

Homologous Recombination between Genetically Divergent Campylobacter fetus Lineages Supports... Homologous recombination is a major driver of bacterial speciation. Genetic divergence and host association are important factors influencing homologous recombination. Here, we study these factors for Campylobacter fetus, which shows a distinct intraspecific host dichotomy. Campylobacter fetus subspecies fetus (Cff)and venerealis are associated with mammals, whereas C. fetus subsp. testudinum (Cft) is associated with reptiles. Recombination between these genetically divergent C. fetus lineages is extremely rare. Previously it was impossible to show whether this barrier to recombination was determined by the differential host preferences, by the genetic divergence between both lineages or by other factors influencing recombination, such as restriction-modification, CRISPR/Cas, and transformation systems. Fortuitously, a distinct C. fetus lineage (ST69) was found, which was highly related to mammal-associated C. fetus, yet isolated from a chelonian. The whole genome sequences of two C. fetus ST69 isolates were compared with those of mammal- and reptile-associated C. fetus strains for phylogenetic and recombination analysis. In total, 5.1– 5.5% of the core genome of both ST69 isolates showed signs of recombination. Of the predicted recombination regions, 80.4% were most closely related to Cft,14.3% to Cff, and 5.6% to C. iguaniorum. Recombination from C. fetus ST69 to Cft was also detected, but to a lesser extent and only in chelonian-associated Cft strains. This study shows that despite substantial genetic divergence no absolute barrier to homologous recombination exists between two distinct C. fetus lineages when occurring in the same host type, which provides valuable insights in bacterial speciation and evolution. Key words: Campylobacter fetus, homologous recombination, speciation, host association, reptile, whole genome sequencing. Introduction Campylobacter fetus is recognized as an important veteri- There are several underlying processes driving bacterial speci- nary and occasional human pathogen (van Bergen et al. 2008; ation. One of these processes is homologous recombination, Wagenaar et al. 2014). Three C. fetus subspecies are currently in which genetic material is exchanged between two identical recognized: C. fetus subspecies fetus (Cff)and venerealis (Cfv), or similar molecules of DNA. Here, the effect of homologous which are closely related and occur in mammals, primarily recombination on speciation is studied in Campylobacter fe- ungulates, and C. fetus subsp. testudinum (Cft), which is ge- tus, which shows several distinct genetically divergent host- netically divergent from Cff and Cfv, and primarily occurs in associated lineages. reptiles (Gilbert et al. 2014). These subspecies show a strict The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non- commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 716 Genome Biol. Evol. 10(3):716–722. doi:10.1093/gbe/evy048 Advance Access publication February 22, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/716/4904301 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Homologous Recombination between Genetically Divergent C. fetus Lineages GBE host dichotomy: Cff and Cfv have never been isolated from The average coverage was 216-354, the number of contigs reptiles, whereas Cft has never been isolated from mammals, was 22-30 and the number of gaps was 23-31 for both excluding occasional human cases, in which contact with genomes. The level of completeness (99.77%) and contami- reptiles is suspected (Patrick et al. 2013). The current situa- nation (1.96%) was determined for both genomes based on tion shows two major dominating coherent lineages of the Campylobacter genus using CheckM 1.0.5. The whole C. fetus in mammals (Cff and Cfv) and reptiles (Cft). genome sequences of C. fetus isolates 12S01208-4 and However, during independent studies assessing the 12S01908-5 have been deposited at GenBank under acces- Campylobacter diversity in reptiles, a genetically distinct sion numbers MTDX00000000 and MTDY00000000, respec- C. fetus lineage, comprising multilocus sequence types tively. All other C. fetus strains were sequenced as described 43 and 69, was obtained from captive-held red-footed previously (Gilbert, Miller, Yee, Zomer, et al. 2016; van der tortoises (Chelonoidis carbonaria)in Taiwan and the Graaf-van Bloois et al. 2016) and are present in GenBank. Netherlands, adding up to the genetic diversity of C. fetus (Wang et al. 2013; Gilbert et al. 2014). Surprisingly, Average Nucleotide Identity phylogenetic analysis based on 16S rRNA and multilocus sequence typing (MLST) showed that this lineage was As a measure of genomic relatedness the average nucleotide highly related to mammal-associated Cff and Cfv,al- identity (ANI) was used (Konstantinidis and Tiedje 2005; though it was isolated from reptiles (Wang et al. 2013). Konstantinidis et al. 2006). Using the OrthoANIu tool (Lee Noteworthy, recombination was virtually absent be- et al. 2016), ANI values based on whole genome sequences tween mammal-associated Cff/Cfv and reptile-associated were calculated for C. fetus isolates 12S01208-4 and Cft, which suggested allopatric speciation within C. fetus 12S01908-5, Cff strains 04/554 and 82-40, Cft strains 03- (Gilbert, Miller, Yee, Zomer, et al. 2016). A barrier to recom- 427, 13S00388-15, 85-387, and SP3, Cfv strain 97/608, bination was apparent, but it remained to be shown and C. iguaniorum strain 1485E. whether this was caused by the host dichotomy (mammal- or reptile-associated) or by intrinsic factors which inhibit Genome Analysis recombination between these lineages. The C. fetus lineage closely related to mammal-associated Cff and Cfv, yet iso- Protein-, rRNA-, and tRNA-encoding genes were identified lated from reptiles, might play a pivotal role in our under- using Prokka (Seemann 2014). An all versus all BLAST was standing of these processes. performed for all predicted proteins of the whole genomes Based on whole genome comparison, we explore the ho- (supplementary table S1, Supplementary Material online) at mologous recombination between two genetically divergent an E-value cutoff of 1E 6. To determine the orthologous bacterial lineages, which both occur naturally in a reptilian relationships of all proteins, protein sequences were clustered host, and provide novel insights in bacterial speciation and using Roary with a 75% identity cutoff (Page et al. 2015). evolution. Core genome alignment was performed using Parsnp 1.2 (Treangen et al. 2014). DNA regions present in all isolates were extracted and gaps were removed using trimAl Materials and Methods (Capella-Gutierrez et al. 2009). Based on this core genome Strains alignment, phylogenomic reconstruction and prediction of re- combination events was performed using Gubbins (Croucher Campylobacter fetus isolates 12S01208-4 and 12S01908-5 et al. 2015) with the default settings. Phylogenetic dendro- were obtained on February 23 and March 16, 2012, respec- grams were created using Fasttree (Price et al. 2009). A BLAST tively, from a red-footed tortoise (Chelonoidis carbonaria) search of the predicted recombination regions of C. fetus suffering from a Mycoplasma induced pneumonia. Isolates ST69 isolate 12S01908-5 (supplementary information S1, were grown on Columbia agar with 5% sheep blood Supplementary Material online) against the genomes of Cft (Oxoid, the Netherlands) in a microaerobic atmosphere strain 03-427 and Cff strain 82-40 and against the NCBI non (83.3% N ,7.1% CO ,3.6% H ,and 6% O )at 37 Cfor 2 2 2 2 redundant (nr) database was performed to search for partic- 48 h. All other 61 C. fetus strains (Cff,19 strains; Cfv,22 ular recombination between these reptile-associated taxa and strains; Cft, 20 strains) were identical to those used in previous other species. The same procedure was performed for all studies (Gilbert, Miller, Yee, Zomer, et al. 2016; van der Graaf- predicted recombination regions of Cft (supplementary infor- van Bloois et al. 2016). mation S2, Supplementary Material online) and the genomes of C. fetus ST69 isolate 12S01908-5 and Cff strain 82-40. Whole Genome Sequencing Percentage sequence identity of C. fetus ST69 recombination Sequencing of C. fetus isolates 12S01208-4 and 12S01908-5 regions with Cft and Cff and percentage sequence identity of was performed using Illumina MiSeq with 300 bp paired end Cft recombination regions with C. fetus ST69 and Cff were reads. The reads were assembled using SPAdes 3.1.1. plotted in an x–y graph using Microsoft Excel. Genome Biol. Evol. 10(3):716–722 doi:10.1093/gbe/evy048 Advance Access publication February 22, 2018 717 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/716/4904301 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Gilbert et al. GBE Multilocus Sequence Typing Gilbert, Miller, Yee, Zomer, et al. 2016; Gilbert et al. 2017), is absent from C. fetus ST69. The loci for the C. fetus multilocus sequence typing (MLST) A whole genome-based phylogeny of C. fetus shows that scheme (van Bergen et al. 2005) were extracted from the C. fetus ST69 is most closely related to mammal-associated Cff whole genome of C. fetus isolate 12S01208-4 and submitted and Cfv (fig. 1). Campylobacter fetus ST69, Cff and Cfv are to the Campylobacter MLST database (www.pubmlst.org/ highly divergent from Cft. The average nucleotide identity campylobacter; last accessed March 17, 2016). (ANI) was used as a measure of genomic relatedness. The The 501-nt trimmed pgm alleles of C. fetus and C. igua- ANI between both C. fetus ST69 isolates was 99.96%. niorum strain 1485E were extracted from the Campylobacter Campylobacter fetus ST69 and Cff showed 98% ANI, which MLST database and from the genome (GenBank accession is well above the 95% species delineation (supplementary number CP009043), respectively. Alignment and phyloge- table S2, Supplementary Material online). The ANI between netic analysis, based on the neighbor-joining method with C. fetus ST69 and Cft was 92%. Although this is below the bootstrap values using 500 repetitions, was performed using species delineation, these lineages are considered conspecific MEGA 6.05. based on many shared genotypic and phenotypic character- istics, including the presence of an S-layer, as examined pre- Results viously for C. fetus (Fitzgerald et al. 2014). The ANI between The genomes of Campylobacter fetus isolates 12S01208-4 C. fetus ST69 and reptile-associated C. iguaniorum was 76%. and 12S01908-5 are highly similar and show a high degree Extensive recombination was detected in C. fetus ST69. In of synteny. In total, 34 discriminatory SNPs were identified in isolates 12S01208-4 and 12S01908-5, 5.1% and 5.5% of the the core genomes of both isolates; 88.2% (30/34) were lo- gapless core genome was predicted to be recombined, re- cated inside recombination regions, 11.8% (4/34) were lo- spectively. In the Cft and Cff/Cfv genomes, on an average cated outside recombination regions. Both isolates belong 2.9% and 0.4% of the core genome was predicted to be to the same multilocus sequence type, ST69. Based on the recombined, respectively. The ratio of base substitutions pre- presence of LPS-biosynthesis gene wcbK, encoding a putative dicted to have been imported through recombination to GDP-mannose 4, 6-dehydratase, the predicted serotype of those occurring through point mutation is 0.32 for C. fetus bothisolatesisB or AB (Kienesberger et al. 2014; Gilbert, ST69, 0.06 for Cft, and 0.03 for Cff/Cfv, indicating that re- Miller, Yee, Zomer, et al. 2016). As in other C. fetus lineages, combination is a major driver of mutation in C. fetus ST69. the S-layer encoding sap genes, considered important in A total of 56 different recombination regions were identi- C. fetus virulence (Blaser et al. 2008), are present in C. fetus fiedinthe C. fetus ST69 genomes, of which two were ST69 isolate 12S01908-5. However, no sapCDEF genes were uniquely present in isolate 12S01908-5 (fig. 1). In Cft and in detected in isolate 12S01208-4. A CRISPR/Cas system is pre- Cff/Cfv, onanaverage 31.9(6 5.4) and 1.5 (6 1.1) recombi- sent in C. fetus ST69, including two CRISPR repeat regions nation regions were identified, respectively. A BLAST search (30 nt spacers; 36 and 39 repeats, respectively). However, the against the NCBI nonredundant database showed that additional locus encoding CRISPR/Cas system-associated 80.4% (45/56) of the recombination regions in C. fetus RAMP superfamily proteins, which is well conserved in Cff, ST69 were most closely related to Cft, 14.3% (8/56) to Cff, Cfv,and Cft, was absent from both C. fetus ST69 isolates and 5.6% (3/56) to C. iguaniorum. (supplementary table S1, Supplementary Material online). A scatter plot of the BLAST identities between the C. fetus Of the genes specifically present in or absent from C. fetus strain 12S01908-5 recombination regions and Cff 82-40 and ST69 only, or C. fetus ST69 and either Cff/Cfv or Cft,a dis- Cft 03-427 confirmed that most recombination regions were proportionally high number encoded proteins related to DNA more homologous with Cft than with Cff (fig. 2A). On an uptake and defense, such as competence, transformation sys- average, the recombination regions showed 95.9% (6 2.3) tem, restriction-modification system, and CRISPR/Cas system homology with Cft and 93.2% (6 1.7) homology with Cff.Of proteins (supplementary table S1, Supplementary Material the recombination regions, 98.2% (55/56) showed>87% online). homology with Cff and>89% homology with Cft.The Notably, 60 genes were exclusively shared between C. fe- homology of the recombination regions with Cft gradually tus ST69 and Cft strain 13S00388-15, which was isolated increased from 89.4% to 99.5%, while this range was from Chelonoidis denticulata, a chelonian species closely re- 87.3–98.7% for Cff. Recombination regions became scarcer lated to Chelonoidis carbonaria. Most of these genes encode with decreasing BLAST identity. Of the recombination regions, hypothetical proteins, but also several phage-specific proteins 12.5% (7/56) and 1.8% (1/56) was>98% homologous with and likely represent a prophage. Cft and Cff, respectively. The tcuRABC locus, involved in catabolism of tricarbally- Two unique recombination regions were identified only in late (a citrate analog), which has been shown present in Cft C. fetus ST69 isolate 12S01908-5 (indicated in blue in fig. 1). and in many other reptile-associated Campylobacter and One of those regions was 99.8% (3,801/3,808 nt) homolo- Helicobacter taxa (Gilbert, Miller, Yee, Kik, et al. 2016; gous with C. fetus ST69 isolate 12S01208-4. All nucleotide 718 Genome Biol. Evol. 10(3):716–722 doi:10.1093/gbe/evy048 Advance Access publication February 22, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/716/4904301 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Homologous Recombination between Genetically Divergent C. fetus Lineages GBE FIG.1.—Core genome-based phylogeny for Campylobacter fetus. Identical recombination regions in two or more strains are indicated in red; unique recombination regions are indicated in blue. Campylobacter fetus ST69 is associated with reptiles (chelonians), C. fetus subsp. fetus (Cff)and venerealis (Cfv) are associated with mammals (primarily ungulates), and C. fetus subsp. testudinum (Cft) is primarily associated with reptiles. substitutions were synonymous and did not alter the amino housekeeping gene which is part of the C. fetus MLST acid sequence of the proteins affected. The other recombined scheme. A dendrogram based on all C. fetus pgm alleles pre- sequence was 100% (114/114 nt) homologous with reptile- sent in the pubMLST database (www.pubmlst.org/campylo- associated C. iguaniorum strains 1485E and 2463 D. Due to bacter; last accessed March 17, 2016) confirmed that pgm this recombination, the start site of the gene encoding from Cft strain 85-387 was identical to pgm from C. fetus threonyl-tRNA synthetase and the amino acid composition ST69, indicating recent recombination from the C. fetus ST69 of the first part of the translated protein were altered, result- lineage to Cft strain 85-387 (supplementary fig. S1, ing in a shorter open reading frame. Supplementary Material online). In Cft, recombination regions which likely originated from C. fetus ST69 were observed as well, but to a much lesser Discussion extent than from Cft to C. fetus ST69. Of the recombination regions extracted from Cft, 83.1% (108/130) showed>87% This study shows that homologous recombination between homology with Cff and C. fetus ST69. Two well-separated divergent bacterial lineages occurs despite substantial genetic clusters of recombination regions which showed 52.3– distance when present in the same host. As shown previously, 75.4% and 87.5–99.9% homology with Cff and C. fetus Cff/Cfv and Cft show no or very few recombination between ST69 were identified (fig. 2B). In total, only 2.3% (3/130) of each other (Gilbert, Miller, Yee, Zomer, et al. 2016). In con- the recombination regions likely originated from C. fetus ST69 trast, no absolute barrier to recombination exists between (>98% homology). These regions were detected in Cft strains divergent C. fetus lineages when occurring in the same host 13S00388-15 and 85-387, isolated from the chelonian spe- type, as shown in this study. Apparently, a species-level diver- cies Chelonoidis denticulata and Terrapene carolina,respec- gence of 8% between both lineages is no barrier to homol- tively. Notably, one recombination region in Cft strain 85-387, ogous recombination. Notably, most of the recombination showing high homology with C. fetus ST69, contained phos- regions detected in both C. fetus ST69 and Cft showed high- phoglucosamine mutase encoding pgm (glmM), an essential est homology with C. fetus, implicating that recombination is Genome Biol. Evol. 10(3):716–722 doi:10.1093/gbe/evy048 Advance Access publication February 22, 2018 719 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/716/4904301 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Gilbert et al. GBE FIG.2.—Scatter plot based on the BLAST-based nucleotide identities of all recombination regions identified in Campylobacter fetus ST69 (Cf ST69) isolate 12S01908-5 (A)and all C. fetus subsp. testudinum (Cft)strains (B). A BLAST search was performed for each recombination region and the genomes of Cft strain 03-427 and C. fetus subsp. fetus (Cff) strain 82-40 (A)and Cff strain 82-40 and Cf ST69 isolate 12S01908-5 (B). The nucleotide identities of the recombination regions are plotted as circles; the nucleotide identities of the reference genomes are plotted as squares. The dashed line shows the98% nucleotide identity. x-and y-axis range 80–100% (A) or 50–100% (B). occurring most frequently within C. fetus or a closely related recombination regions and gene content. As the number of Campylobacter lineage. Of the C. fetus ST69 core genome, discriminatory SNPs outside the recombination regions was 5.1–5.5% showed signs of recombination and the majority of low in the core genomes of both isolates (n¼ 4), these can the recombination regions in C. fetus ST69 were most closely be considered recent events. Furthermore, as 88.2% of the related to Cft, indicating that this lineage is the most impor- discriminatory SNPs in the core genomes of both isolates tant DNA donor. could be attributed to recombination, this can be considered The majority of the recombination regions in C. fetus the main driver of the short-scale divergence in C. fetus ST69. ST69 were most closely related to Cft, yet showed relative The differences in gene content could predominantly be at- low nucleotide identity (<98%). These more divergent re- tributed to the sap genes. The absence of sapCDEF genes in combination regions likely represent ancient recombination C. fetus ST69 isolate 12S01208-4 has been observed in other events, followed by divergent evolution of those recombi- C. fetus strains as well and may represent a spontaneous nation regions based on point mutations in both donor and mutation occurring in vitro (Dworkin et al. 1995; Gilbert, recipient. The gradual increase from 89.4% to 99.5% ob- Miller, Yee, Zomer, et al. 2016). served in the BLAST sequence identities of the C. fetus ST69 Recombination also occurred in essential housekeeping recombination regions with Cft suggests that this has been genes, such as pgm in Cft strain 85-387. As pgm is part of a long-term and continuous process which may still be on- the C. fetus MLST scheme, recombination can distort the going. Indeed, parts of similar bacterial genomes can main- inferences made based on MLST (Dingle et al. 2010). In ad- tain the ability to recombine over long timespans before dition, the observation that Cft strain 85-387, isolated in genetic isolation between two lineages is complete 1984, has an identical pgm allele as C. fetus ST69 isolates (Retchless and Lawrence 2007). With sets of niche-specific 12S01208-4 and 12S01908-5, isolated in 2012, indicates genes being maintained in populations that freely recom- that both lineages occur together for at least 28 years and bine at other loci, different parts of the genome may be that no mutation has occurred in this allele during that period. genetically isolated at different times, suggesting temporal Reciprocal recombination between C. fetus ST69 and Cft fragmentation of speciation. strains from chelonian hosts suggests that recombination be- Despite the high genetic similarity between both C. fetus tween both lineages primarily occurs in chelonian hosts. In ST69 isolates, small-scale differences were observed in addition, the presence of an identical prophage in C. fetus 720 Genome Biol. Evol. 10(3):716–722 doi:10.1093/gbe/evy048 Advance Access publication February 22, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/716/4904301 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Homologous Recombination between Genetically Divergent C. fetus Lineages GBE ST69 and Cft strain 13S00388-15, which was isolated from Literature Cited Chelonoidis denticulata, further affirms that both C. fetus Blaser MJ, Newell DG, Thompson SA, Zechner EL. (2008). lineages occur in the same niche. Pathogenesis of Campylobacter fetus. In: Nachamkin I, Szymanski CM, Blaser MJ, editors. Campylobacter. Washington Interestingly, only little recombination was observed be- (DC): ASM Press. p. 401–428. tween C. fetus ST69 and reptile-associated C. iguaniorum, Capella-Gutierrez S, Silla-Martinez JM, Gabaldon T. 2009. trimAl: a tool for which showed a prevalence of 33.3% (5/15) in Chelonoidis automated alignment trimming in large-scale phylogenetic analyses. carbonaria and Chelonoidis denticulata and was isolated from Bioinformatics 25(15):1972–1973. the same animal and the same samples as both C. fetus ST69 Croucher NJ, et al. 2015. Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins. isolates (Gilbert et al. 2014). Recombination between C. fetus Nucleic Acids Res. 43(3):e15. ST69 and C. iguaniorum does occur, but the genetic diver- Dingle KE, et al. 2010. Genetic relationships among reptilian and mam- gence between both species may lead to an altered protein malian Campylobacter fetus strains determined by multilocus se- (function), and likely reduced fitness in most cases, making it quence typing. J Clin Microbiol. 48(3):977–980. less likely that these recombination regions will be fixed in the Dworkin J, Tummuru MK, Blaser MJ. 1995. A lipopolysaccharide-binding domain of the Campylobacter fetus S-layer protein resides within the genome over time. conserved N terminus of a family of silent and divergent homologs. Cft and C. fetus ST69 diverged in isolation of each other, J Bacteriol. 177(7):1734–1741. either in space or due to intrinsic barriers to recombination. Fitzgerald C, et al. 2014. Campylobacter fetus subsp. testudinum subsp. Upon contact in a shared reptilian host, recombination be- nov., isolated from humans and reptiles. Int J Syst Evol Microbiol. 64(Pt tween both lineages occurred. Recombination between 9):2944–2948. Fraser C, Hanage WP, Spratt BG. 2007. Recombination and the nature of C. fetus ST69 and Cft appears to be bidirectional. However, bacterial speciation. Science 315(5811):476–480. the number and size of recombination regions was larger Gilbert MJ, Duim B, Timmerman AJ, Zomer AL, Wagenaar JA. 2017. from Cft to C. fetus ST69 than vice versa. This asymmetry Whole genome-based phylogeny of reptile-associated Helicobacter could be the result of numerical dominance of Cft over indicates independent niche adaptation followed by diversification in C. fetus ST69 in the reptilian intestine, which is supported a poikilothermic host. Sci Rep. 7(1):8387. Gilbert MJ, et al. 2014. Occurrence, diversity, and host association of in- by the prevalence rates (Gilbert et al. 2014), and which has testinal Campylobacter, Arcobacter,and Helicobacter in reptiles. PLoS been suggested previously for C. coli showing introgression of One 9(7):e101599. C. jejuni DNA (Sheppard et al. 2008). 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Washington (DC): ASM Press. p. 213–225. Associate editor: Howard Ochman 722 Genome Biol. Evol. 10(3):716–722 doi:10.1093/gbe/evy048 Advance Access publication February 22, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/716/4904301 by Ed 'DeepDyve' Gillespie user on 16 March 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Genome Biology and Evolution Oxford University Press

Homologous Recombination between Genetically Divergent Campylobacter fetus Lineages Supports Host-Associated Speciation

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Abstract

Homologous recombination is a major driver of bacterial speciation. Genetic divergence and host association are important factors influencing homologous recombination. Here, we study these factors for Campylobacter fetus, which shows a distinct intraspecific host dichotomy. Campylobacter fetus subspecies fetus (Cff)and venerealis are associated with mammals, whereas C. fetus subsp. testudinum (Cft) is associated with reptiles. Recombination between these genetically divergent C. fetus lineages is extremely rare. Previously it was impossible to show whether this barrier to recombination was determined by the differential host preferences, by the genetic divergence between both lineages or by other factors influencing recombination, such as restriction-modification, CRISPR/Cas, and transformation systems. Fortuitously, a distinct C. fetus lineage (ST69) was found, which was highly related to mammal-associated C. fetus, yet isolated from a chelonian. The whole genome sequences of two C. fetus ST69 isolates were compared with those of mammal- and reptile-associated C. fetus strains for phylogenetic and recombination analysis. In total, 5.1– 5.5% of the core genome of both ST69 isolates showed signs of recombination. Of the predicted recombination regions, 80.4% were most closely related to Cft,14.3% to Cff, and 5.6% to C. iguaniorum. Recombination from C. fetus ST69 to Cft was also detected, but to a lesser extent and only in chelonian-associated Cft strains. This study shows that despite substantial genetic divergence no absolute barrier to homologous recombination exists between two distinct C. fetus lineages when occurring in the same host type, which provides valuable insights in bacterial speciation and evolution. Key words: Campylobacter fetus, homologous recombination, speciation, host association, reptile, whole genome sequencing. Introduction Campylobacter fetus is recognized as an important veteri- There are several underlying processes driving bacterial speci- nary and occasional human pathogen (van Bergen et al. 2008; ation. One of these processes is homologous recombination, Wagenaar et al. 2014). Three C. fetus subspecies are currently in which genetic material is exchanged between two identical recognized: C. fetus subspecies fetus (Cff)and venerealis (Cfv), or similar molecules of DNA. Here, the effect of homologous which are closely related and occur in mammals, primarily recombination on speciation is studied in Campylobacter fe- ungulates, and C. fetus subsp. testudinum (Cft), which is ge- tus, which shows several distinct genetically divergent host- netically divergent from Cff and Cfv, and primarily occurs in associated lineages. reptiles (Gilbert et al. 2014). These subspecies show a strict The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non- commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 716 Genome Biol. Evol. 10(3):716–722. doi:10.1093/gbe/evy048 Advance Access publication February 22, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/716/4904301 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Homologous Recombination between Genetically Divergent C. fetus Lineages GBE host dichotomy: Cff and Cfv have never been isolated from The average coverage was 216-354, the number of contigs reptiles, whereas Cft has never been isolated from mammals, was 22-30 and the number of gaps was 23-31 for both excluding occasional human cases, in which contact with genomes. The level of completeness (99.77%) and contami- reptiles is suspected (Patrick et al. 2013). The current situa- nation (1.96%) was determined for both genomes based on tion shows two major dominating coherent lineages of the Campylobacter genus using CheckM 1.0.5. The whole C. fetus in mammals (Cff and Cfv) and reptiles (Cft). genome sequences of C. fetus isolates 12S01208-4 and However, during independent studies assessing the 12S01908-5 have been deposited at GenBank under acces- Campylobacter diversity in reptiles, a genetically distinct sion numbers MTDX00000000 and MTDY00000000, respec- C. fetus lineage, comprising multilocus sequence types tively. All other C. fetus strains were sequenced as described 43 and 69, was obtained from captive-held red-footed previously (Gilbert, Miller, Yee, Zomer, et al. 2016; van der tortoises (Chelonoidis carbonaria)in Taiwan and the Graaf-van Bloois et al. 2016) and are present in GenBank. Netherlands, adding up to the genetic diversity of C. fetus (Wang et al. 2013; Gilbert et al. 2014). Surprisingly, Average Nucleotide Identity phylogenetic analysis based on 16S rRNA and multilocus sequence typing (MLST) showed that this lineage was As a measure of genomic relatedness the average nucleotide highly related to mammal-associated Cff and Cfv,al- identity (ANI) was used (Konstantinidis and Tiedje 2005; though it was isolated from reptiles (Wang et al. 2013). Konstantinidis et al. 2006). Using the OrthoANIu tool (Lee Noteworthy, recombination was virtually absent be- et al. 2016), ANI values based on whole genome sequences tween mammal-associated Cff/Cfv and reptile-associated were calculated for C. fetus isolates 12S01208-4 and Cft, which suggested allopatric speciation within C. fetus 12S01908-5, Cff strains 04/554 and 82-40, Cft strains 03- (Gilbert, Miller, Yee, Zomer, et al. 2016). A barrier to recom- 427, 13S00388-15, 85-387, and SP3, Cfv strain 97/608, bination was apparent, but it remained to be shown and C. iguaniorum strain 1485E. whether this was caused by the host dichotomy (mammal- or reptile-associated) or by intrinsic factors which inhibit Genome Analysis recombination between these lineages. The C. fetus lineage closely related to mammal-associated Cff and Cfv, yet iso- Protein-, rRNA-, and tRNA-encoding genes were identified lated from reptiles, might play a pivotal role in our under- using Prokka (Seemann 2014). An all versus all BLAST was standing of these processes. performed for all predicted proteins of the whole genomes Based on whole genome comparison, we explore the ho- (supplementary table S1, Supplementary Material online) at mologous recombination between two genetically divergent an E-value cutoff of 1E 6. To determine the orthologous bacterial lineages, which both occur naturally in a reptilian relationships of all proteins, protein sequences were clustered host, and provide novel insights in bacterial speciation and using Roary with a 75% identity cutoff (Page et al. 2015). evolution. Core genome alignment was performed using Parsnp 1.2 (Treangen et al. 2014). DNA regions present in all isolates were extracted and gaps were removed using trimAl Materials and Methods (Capella-Gutierrez et al. 2009). Based on this core genome Strains alignment, phylogenomic reconstruction and prediction of re- combination events was performed using Gubbins (Croucher Campylobacter fetus isolates 12S01208-4 and 12S01908-5 et al. 2015) with the default settings. Phylogenetic dendro- were obtained on February 23 and March 16, 2012, respec- grams were created using Fasttree (Price et al. 2009). A BLAST tively, from a red-footed tortoise (Chelonoidis carbonaria) search of the predicted recombination regions of C. fetus suffering from a Mycoplasma induced pneumonia. Isolates ST69 isolate 12S01908-5 (supplementary information S1, were grown on Columbia agar with 5% sheep blood Supplementary Material online) against the genomes of Cft (Oxoid, the Netherlands) in a microaerobic atmosphere strain 03-427 and Cff strain 82-40 and against the NCBI non (83.3% N ,7.1% CO ,3.6% H ,and 6% O )at 37 Cfor 2 2 2 2 redundant (nr) database was performed to search for partic- 48 h. All other 61 C. fetus strains (Cff,19 strains; Cfv,22 ular recombination between these reptile-associated taxa and strains; Cft, 20 strains) were identical to those used in previous other species. The same procedure was performed for all studies (Gilbert, Miller, Yee, Zomer, et al. 2016; van der Graaf- predicted recombination regions of Cft (supplementary infor- van Bloois et al. 2016). mation S2, Supplementary Material online) and the genomes of C. fetus ST69 isolate 12S01908-5 and Cff strain 82-40. Whole Genome Sequencing Percentage sequence identity of C. fetus ST69 recombination Sequencing of C. fetus isolates 12S01208-4 and 12S01908-5 regions with Cft and Cff and percentage sequence identity of was performed using Illumina MiSeq with 300 bp paired end Cft recombination regions with C. fetus ST69 and Cff were reads. The reads were assembled using SPAdes 3.1.1. plotted in an x–y graph using Microsoft Excel. Genome Biol. Evol. 10(3):716–722 doi:10.1093/gbe/evy048 Advance Access publication February 22, 2018 717 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/716/4904301 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Gilbert et al. GBE Multilocus Sequence Typing Gilbert, Miller, Yee, Zomer, et al. 2016; Gilbert et al. 2017), is absent from C. fetus ST69. The loci for the C. fetus multilocus sequence typing (MLST) A whole genome-based phylogeny of C. fetus shows that scheme (van Bergen et al. 2005) were extracted from the C. fetus ST69 is most closely related to mammal-associated Cff whole genome of C. fetus isolate 12S01208-4 and submitted and Cfv (fig. 1). Campylobacter fetus ST69, Cff and Cfv are to the Campylobacter MLST database (www.pubmlst.org/ highly divergent from Cft. The average nucleotide identity campylobacter; last accessed March 17, 2016). (ANI) was used as a measure of genomic relatedness. The The 501-nt trimmed pgm alleles of C. fetus and C. igua- ANI between both C. fetus ST69 isolates was 99.96%. niorum strain 1485E were extracted from the Campylobacter Campylobacter fetus ST69 and Cff showed 98% ANI, which MLST database and from the genome (GenBank accession is well above the 95% species delineation (supplementary number CP009043), respectively. Alignment and phyloge- table S2, Supplementary Material online). The ANI between netic analysis, based on the neighbor-joining method with C. fetus ST69 and Cft was 92%. Although this is below the bootstrap values using 500 repetitions, was performed using species delineation, these lineages are considered conspecific MEGA 6.05. based on many shared genotypic and phenotypic character- istics, including the presence of an S-layer, as examined pre- Results viously for C. fetus (Fitzgerald et al. 2014). The ANI between The genomes of Campylobacter fetus isolates 12S01208-4 C. fetus ST69 and reptile-associated C. iguaniorum was 76%. and 12S01908-5 are highly similar and show a high degree Extensive recombination was detected in C. fetus ST69. In of synteny. In total, 34 discriminatory SNPs were identified in isolates 12S01208-4 and 12S01908-5, 5.1% and 5.5% of the the core genomes of both isolates; 88.2% (30/34) were lo- gapless core genome was predicted to be recombined, re- cated inside recombination regions, 11.8% (4/34) were lo- spectively. In the Cft and Cff/Cfv genomes, on an average cated outside recombination regions. Both isolates belong 2.9% and 0.4% of the core genome was predicted to be to the same multilocus sequence type, ST69. Based on the recombined, respectively. The ratio of base substitutions pre- presence of LPS-biosynthesis gene wcbK, encoding a putative dicted to have been imported through recombination to GDP-mannose 4, 6-dehydratase, the predicted serotype of those occurring through point mutation is 0.32 for C. fetus bothisolatesisB or AB (Kienesberger et al. 2014; Gilbert, ST69, 0.06 for Cft, and 0.03 for Cff/Cfv, indicating that re- Miller, Yee, Zomer, et al. 2016). As in other C. fetus lineages, combination is a major driver of mutation in C. fetus ST69. the S-layer encoding sap genes, considered important in A total of 56 different recombination regions were identi- C. fetus virulence (Blaser et al. 2008), are present in C. fetus fiedinthe C. fetus ST69 genomes, of which two were ST69 isolate 12S01908-5. However, no sapCDEF genes were uniquely present in isolate 12S01908-5 (fig. 1). In Cft and in detected in isolate 12S01208-4. A CRISPR/Cas system is pre- Cff/Cfv, onanaverage 31.9(6 5.4) and 1.5 (6 1.1) recombi- sent in C. fetus ST69, including two CRISPR repeat regions nation regions were identified, respectively. A BLAST search (30 nt spacers; 36 and 39 repeats, respectively). However, the against the NCBI nonredundant database showed that additional locus encoding CRISPR/Cas system-associated 80.4% (45/56) of the recombination regions in C. fetus RAMP superfamily proteins, which is well conserved in Cff, ST69 were most closely related to Cft, 14.3% (8/56) to Cff, Cfv,and Cft, was absent from both C. fetus ST69 isolates and 5.6% (3/56) to C. iguaniorum. (supplementary table S1, Supplementary Material online). A scatter plot of the BLAST identities between the C. fetus Of the genes specifically present in or absent from C. fetus strain 12S01908-5 recombination regions and Cff 82-40 and ST69 only, or C. fetus ST69 and either Cff/Cfv or Cft,a dis- Cft 03-427 confirmed that most recombination regions were proportionally high number encoded proteins related to DNA more homologous with Cft than with Cff (fig. 2A). On an uptake and defense, such as competence, transformation sys- average, the recombination regions showed 95.9% (6 2.3) tem, restriction-modification system, and CRISPR/Cas system homology with Cft and 93.2% (6 1.7) homology with Cff.Of proteins (supplementary table S1, Supplementary Material the recombination regions, 98.2% (55/56) showed>87% online). homology with Cff and>89% homology with Cft.The Notably, 60 genes were exclusively shared between C. fe- homology of the recombination regions with Cft gradually tus ST69 and Cft strain 13S00388-15, which was isolated increased from 89.4% to 99.5%, while this range was from Chelonoidis denticulata, a chelonian species closely re- 87.3–98.7% for Cff. Recombination regions became scarcer lated to Chelonoidis carbonaria. Most of these genes encode with decreasing BLAST identity. Of the recombination regions, hypothetical proteins, but also several phage-specific proteins 12.5% (7/56) and 1.8% (1/56) was>98% homologous with and likely represent a prophage. Cft and Cff, respectively. The tcuRABC locus, involved in catabolism of tricarbally- Two unique recombination regions were identified only in late (a citrate analog), which has been shown present in Cft C. fetus ST69 isolate 12S01908-5 (indicated in blue in fig. 1). and in many other reptile-associated Campylobacter and One of those regions was 99.8% (3,801/3,808 nt) homolo- Helicobacter taxa (Gilbert, Miller, Yee, Kik, et al. 2016; gous with C. fetus ST69 isolate 12S01208-4. All nucleotide 718 Genome Biol. Evol. 10(3):716–722 doi:10.1093/gbe/evy048 Advance Access publication February 22, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/716/4904301 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Homologous Recombination between Genetically Divergent C. fetus Lineages GBE FIG.1.—Core genome-based phylogeny for Campylobacter fetus. Identical recombination regions in two or more strains are indicated in red; unique recombination regions are indicated in blue. Campylobacter fetus ST69 is associated with reptiles (chelonians), C. fetus subsp. fetus (Cff)and venerealis (Cfv) are associated with mammals (primarily ungulates), and C. fetus subsp. testudinum (Cft) is primarily associated with reptiles. substitutions were synonymous and did not alter the amino housekeeping gene which is part of the C. fetus MLST acid sequence of the proteins affected. The other recombined scheme. A dendrogram based on all C. fetus pgm alleles pre- sequence was 100% (114/114 nt) homologous with reptile- sent in the pubMLST database (www.pubmlst.org/campylo- associated C. iguaniorum strains 1485E and 2463 D. Due to bacter; last accessed March 17, 2016) confirmed that pgm this recombination, the start site of the gene encoding from Cft strain 85-387 was identical to pgm from C. fetus threonyl-tRNA synthetase and the amino acid composition ST69, indicating recent recombination from the C. fetus ST69 of the first part of the translated protein were altered, result- lineage to Cft strain 85-387 (supplementary fig. S1, ing in a shorter open reading frame. Supplementary Material online). In Cft, recombination regions which likely originated from C. fetus ST69 were observed as well, but to a much lesser Discussion extent than from Cft to C. fetus ST69. Of the recombination regions extracted from Cft, 83.1% (108/130) showed>87% This study shows that homologous recombination between homology with Cff and C. fetus ST69. Two well-separated divergent bacterial lineages occurs despite substantial genetic clusters of recombination regions which showed 52.3– distance when present in the same host. As shown previously, 75.4% and 87.5–99.9% homology with Cff and C. fetus Cff/Cfv and Cft show no or very few recombination between ST69 were identified (fig. 2B). In total, only 2.3% (3/130) of each other (Gilbert, Miller, Yee, Zomer, et al. 2016). In con- the recombination regions likely originated from C. fetus ST69 trast, no absolute barrier to recombination exists between (>98% homology). These regions were detected in Cft strains divergent C. fetus lineages when occurring in the same host 13S00388-15 and 85-387, isolated from the chelonian spe- type, as shown in this study. Apparently, a species-level diver- cies Chelonoidis denticulata and Terrapene carolina,respec- gence of 8% between both lineages is no barrier to homol- tively. Notably, one recombination region in Cft strain 85-387, ogous recombination. Notably, most of the recombination showing high homology with C. fetus ST69, contained phos- regions detected in both C. fetus ST69 and Cft showed high- phoglucosamine mutase encoding pgm (glmM), an essential est homology with C. fetus, implicating that recombination is Genome Biol. Evol. 10(3):716–722 doi:10.1093/gbe/evy048 Advance Access publication February 22, 2018 719 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/716/4904301 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Gilbert et al. GBE FIG.2.—Scatter plot based on the BLAST-based nucleotide identities of all recombination regions identified in Campylobacter fetus ST69 (Cf ST69) isolate 12S01908-5 (A)and all C. fetus subsp. testudinum (Cft)strains (B). A BLAST search was performed for each recombination region and the genomes of Cft strain 03-427 and C. fetus subsp. fetus (Cff) strain 82-40 (A)and Cff strain 82-40 and Cf ST69 isolate 12S01908-5 (B). The nucleotide identities of the recombination regions are plotted as circles; the nucleotide identities of the reference genomes are plotted as squares. The dashed line shows the98% nucleotide identity. x-and y-axis range 80–100% (A) or 50–100% (B). occurring most frequently within C. fetus or a closely related recombination regions and gene content. As the number of Campylobacter lineage. Of the C. fetus ST69 core genome, discriminatory SNPs outside the recombination regions was 5.1–5.5% showed signs of recombination and the majority of low in the core genomes of both isolates (n¼ 4), these can the recombination regions in C. fetus ST69 were most closely be considered recent events. Furthermore, as 88.2% of the related to Cft, indicating that this lineage is the most impor- discriminatory SNPs in the core genomes of both isolates tant DNA donor. could be attributed to recombination, this can be considered The majority of the recombination regions in C. fetus the main driver of the short-scale divergence in C. fetus ST69. ST69 were most closely related to Cft, yet showed relative The differences in gene content could predominantly be at- low nucleotide identity (<98%). These more divergent re- tributed to the sap genes. The absence of sapCDEF genes in combination regions likely represent ancient recombination C. fetus ST69 isolate 12S01208-4 has been observed in other events, followed by divergent evolution of those recombi- C. fetus strains as well and may represent a spontaneous nation regions based on point mutations in both donor and mutation occurring in vitro (Dworkin et al. 1995; Gilbert, recipient. The gradual increase from 89.4% to 99.5% ob- Miller, Yee, Zomer, et al. 2016). served in the BLAST sequence identities of the C. fetus ST69 Recombination also occurred in essential housekeeping recombination regions with Cft suggests that this has been genes, such as pgm in Cft strain 85-387. As pgm is part of a long-term and continuous process which may still be on- the C. fetus MLST scheme, recombination can distort the going. Indeed, parts of similar bacterial genomes can main- inferences made based on MLST (Dingle et al. 2010). In ad- tain the ability to recombine over long timespans before dition, the observation that Cft strain 85-387, isolated in genetic isolation between two lineages is complete 1984, has an identical pgm allele as C. fetus ST69 isolates (Retchless and Lawrence 2007). With sets of niche-specific 12S01208-4 and 12S01908-5, isolated in 2012, indicates genes being maintained in populations that freely recom- that both lineages occur together for at least 28 years and bine at other loci, different parts of the genome may be that no mutation has occurred in this allele during that period. genetically isolated at different times, suggesting temporal Reciprocal recombination between C. fetus ST69 and Cft fragmentation of speciation. strains from chelonian hosts suggests that recombination be- Despite the high genetic similarity between both C. fetus tween both lineages primarily occurs in chelonian hosts. In ST69 isolates, small-scale differences were observed in addition, the presence of an identical prophage in C. fetus 720 Genome Biol. Evol. 10(3):716–722 doi:10.1093/gbe/evy048 Advance Access publication February 22, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/716/4904301 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Homologous Recombination between Genetically Divergent C. fetus Lineages GBE ST69 and Cft strain 13S00388-15, which was isolated from Literature Cited Chelonoidis denticulata, further affirms that both C. fetus Blaser MJ, Newell DG, Thompson SA, Zechner EL. (2008). lineages occur in the same niche. Pathogenesis of Campylobacter fetus. In: Nachamkin I, Szymanski CM, Blaser MJ, editors. Campylobacter. Washington Interestingly, only little recombination was observed be- (DC): ASM Press. p. 401–428. tween C. fetus ST69 and reptile-associated C. iguaniorum, Capella-Gutierrez S, Silla-Martinez JM, Gabaldon T. 2009. trimAl: a tool for which showed a prevalence of 33.3% (5/15) in Chelonoidis automated alignment trimming in large-scale phylogenetic analyses. carbonaria and Chelonoidis denticulata and was isolated from Bioinformatics 25(15):1972–1973. the same animal and the same samples as both C. fetus ST69 Croucher NJ, et al. 2015. Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins. isolates (Gilbert et al. 2014). Recombination between C. fetus Nucleic Acids Res. 43(3):e15. ST69 and C. iguaniorum does occur, but the genetic diver- Dingle KE, et al. 2010. Genetic relationships among reptilian and mam- gence between both species may lead to an altered protein malian Campylobacter fetus strains determined by multilocus se- (function), and likely reduced fitness in most cases, making it quence typing. J Clin Microbiol. 48(3):977–980. less likely that these recombination regions will be fixed in the Dworkin J, Tummuru MK, Blaser MJ. 1995. A lipopolysaccharide-binding domain of the Campylobacter fetus S-layer protein resides within the genome over time. conserved N terminus of a family of silent and divergent homologs. Cft and C. fetus ST69 diverged in isolation of each other, J Bacteriol. 177(7):1734–1741. either in space or due to intrinsic barriers to recombination. Fitzgerald C, et al. 2014. Campylobacter fetus subsp. testudinum subsp. Upon contact in a shared reptilian host, recombination be- nov., isolated from humans and reptiles. Int J Syst Evol Microbiol. 64(Pt tween both lineages occurred. Recombination between 9):2944–2948. 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Washington (DC): ASM Press. p. 213–225. Associate editor: Howard Ochman 722 Genome Biol. Evol. 10(3):716–722 doi:10.1093/gbe/evy048 Advance Access publication February 22, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/3/716/4904301 by Ed 'DeepDyve' Gillespie user on 16 March 2018

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