- Research article
- Open Access
A horizontal gene transfer supported the evolution of an early metazoan biomineralization strategy
© Jackson et al; licensee BioMed Central Ltd. 2011
Received: 16 June 2011
Accepted: 12 August 2011
Published: 12 August 2011
The synchronous and widespread adoption of the ability to biomineralize was a defining event for metazoan evolution during the late Precambrian/early Cambrian 545 million years ago. However our understanding on the molecular level of how animals first evolved this capacity is poor. Because sponges are the earliest branching phylum of biomineralizing metazoans, we have been studying how biocalcification occurs in the coralline demosponge Astrosclera willeyana.
We have isolated and characterized a novel protein directly from the calcified spherulites of A. willeyana. Using three independent lines of evidence (genomic architecture of the gene in A. willeyana, spatial expression of the gene product in A. willeyana and genomic architecture of the gene in the related demosponge Amphimedon queenslandica), we show that the gene that encodes this protein was horizontally acquired from a bacterium, and is now highly and exclusively expressed in spherulite forming cells.
Our findings highlight the ancient and close association that exists between sponges and bacteria, and provide support for the notion that horizontal gene transfer may have been an important mechanism that supported the evolution of this early metazoan biomineralisation strategy.
Results and Discussion
Isolation and evolutionary characterization of the novel biocalcification protein Spherulin
Using previously described de-mineralization, protein extraction and separation techniques , our continuing efforts to characterize the spherulitic proteome of A. willeyana identified a prominent protein of 52 kDa on SDS-PAGE gels. This band was excised, and an amino terminal fragment sequenced by Edman degradation. The resulting peptide was used to design degenerate PCR primers which amplified a fragment of DNA from a cDNA library. This was cloned, sequenced and used to design gene specific primers that allowed the full-length cDNA to be isolated (Additional file 1). Due to the association of this protein with spherulites we have named it 'Spherulin'.
Initial BLAST searches using partial RACE fragments returned hits against uncharacterized proteins with similarity to sugar transporters derived exclusively from bacteria. Conversely, exhaustive BLAST searches directed against a wide phylogenetic range of eukaryotic unicellular and multicellular draft or complete genomes (more than 270) did not return any positive hits (see Additional file 2 for a complete list of eukaryotic genomes searched). One exception to this was the identification of a clear Spherulin homolog in the draft genome of the demosponge Amphimedon queenslandica. This restricted phylogenetic distribution of Spherulin led us to the initial hypothesis that the poriferan Spherulin sequences were in fact derived from the abundant bacterial communities known to exist throughout A. willeyana and A. queenslandica, and which are particularly numerous within the cells that initiate spherulite formation in A. willeyana [8, 9]. However, once we isolated the full length mRNA from A. willeyana the presence of a putative poly-A signal and poly-A tail (Additional file 1) suggested that this may not be a bacterially derived gene product, and could be the product of a horizontal gene transfer (HGT).
Three independent lines of evidence demonstrate Spherulinis now located within poriferan genomes
To test this hypothesis we first localized Awi-Spherulin expression using whole mount in situ hybridization (WMISH). These experiments clearly show that Awi-Spherulin is expressed in the same spherulite forming cells that Astrosclerin is expressed in (Figure 1f-k), a gene we previously demonstrated to be involved in spherulite formation . Furthermore, endobiotic bacteria associated with A. willeyana are visible following WMISH, and Spherulin expression was not detected in these bacterial cells after multiple rounds of WMISH (data not shown).
While the A. queenslandica Spherulin homolog is intron-less, genomic sequence immediately flanking Aqu-Spherulin codes for metazoan specific genes (Virilizer and a lecithin:cholesterol acyltransferase), indicating that Aqu-Spherulin is located within the A. queenslandica genome (Figure 2b).
The deep ancestry of the Spherulin HGT event has removed evidence of Spherulin'sbacterial heritage
Phylogenetic incongruence, where evolutionary relationships are well established, can indicate a history of HGT activity , and is considered to be a gold standard for the demonstration of HGT ancestry . We therefore performed phylogenetic analyses using a range of Spherulin sequences. All of our analyses grouped the two poriferan Spherulins together with high support, while all other prokaryotic sequences formed distantly related clades (Figure 2c). While this result is not phylogenetically incongruent (sponge Spherulin proteins would be expected to form an outgroup to all other prokaryotic Spherulin-like sequences in the absence of HGT), it does not provide evidence against HGT. It does however, provide information regarding the origins of the poriferan Spherulin gene. Because Awi- and Aqu-Spherulin form a well separated and supported clade, it is clear that an HGT event established this gene in a common ancestor of A. willeyana and A. queenslandica, and must therefore have occurred during or prior to the Triassic 265-220 MYA when these two species of sponge shared a common ancestor . Because of the long branch that leads to the two poriferan genes, they must have since been evolving under pressures distinct from those of the prokaryotic Spherulin homologs.
While there are many examples of HGT between prokaryotes , well established examples of HGT from prokaryotic into metazoan organisms are relatively few, with some examples revealed to be artifacts of improper analysis [13, 21–23]. When they are detected, prokaryotic to eukaryotic HGTs often reflect relatively recent events because commonly employed methods to detect them (phylogenetic incongruence and composition analyses) require the maintenance of a phylogenetic signal . Indeed the only method that would detect the Spherulin HGT is a phyletic pattern analysis [24, 25]. In this type of analysis the phyletic distribution of the gene of interest does not reflect the known evolutionary relationships of the species it is found in. The number of independent gene losses that must be invoked in order to account for such a distribution suggests that another explanation (HGT) should be considered. Our fortuitous identification of the HGT that established Spherulin in an ancestral poriferan genome highlights the fact that phylogenetic incongruence and composition based analyses may miss HGTs between clades of organisms that either have poor genomic resources, and/or represent HGTs that occurred in the distant past.
While the HGT we describe here may have been the result of a transfer of a sponge gene into a bacterial endobiont, this is extremely unlikely due to the phyletic distribution of Spherulin homologs throughout the Proteobacteria; Spherulin homologs are present in delta- and gamma-proteobacteria (Figure 2), and can also be detected in alpha-proteobacteria and enterobacteria. Such a distribution could only be possible if the sponge Spherulin gene had been horizontally transferred from an ancestral sponge into each of these distinct bacterial lineages independently. Alternatively, sponges would need to have been present at a time when the last common ancestor of these bacterial lineages existed, a scenario not supported by fossil or molecular data.
The Spherulin gene likely supported the evolution of the Astrosclerabiomineralization strategy
HGTs are thought to often involve the transfer of enzymes involved in metabolic processes [26, however see, 27 for an alternative view]. Given its similarity with sugar transporters, the modern and/or ancestral function of Awi-Spherulin suggests that the Spherulin-HGT falls within this category. It is tempting to speculate that Awi-Spherulin is functionally required to supply the energy necessary for the metabolically demanding process of biocalcification, however such a hypothesis awaits support by functional characterization of Awi-Spherulin. Unfortunately, in vivo manipulation of Awi-Spherulin expression or translation is currently not possible as methods to culture A. willeyana in the laboratory have not been established. Additionally there is no functional data available for any of the prokaryotic Spherulin homologs. Nonetheless, the presence of Awi-Spherulin protein in purified spherulites, and its high level of expression exclusively in spherulite forming cells is strong evidence that it plays a direct role in biocalcification in A. willeyana.
Sponges are well known for the abundant bacterial communities they harbor , a condition that significantly increases the likelihood of HGTs . We provide here the first example of a HGT event into a sponge genome from a prokaryote, which serves to highlight the intimacy of the sponge-bacteria relationship. The fact that Awi-Spherulin is most likely involved in skeletogenesis suggests that this HGT event may have contributed to the evolution of A. willeyana's bodyplan. With the growing availability of metazoan genomes, and our understanding of the mechanisms that support the mobilization of genetic material, more instances of HGT into and between eukaryotic genomes will certainly be revealed. We predict that such events will often be associated with fundamental evolutionary changes to gene regulation, gene repertoire and morphological diversity.
Spherulites were isolated from snap frozen A. willeyana individuals collected around Lizard Island (Great Barrier Reef, Australia) and during the Deep DownUnder Expedition (http://www.deepdownunder.de) at various reefs on the Queensland Plateau (Australia). A prominent band at 52 kDa was excised from a standard 12% SDS-PAGE gel and sequenced by Edman degradation. From this sequence a degenerate primer (5'-TGICCDATRTCIGGDATISWRTTRTADATICCIAC-3') was designed and used to isolate a 3' fragment from a cDNA library cloned into a λ TripleX vector (Clontech). 3' and 5' RACE fragments were subsequently isolated and assembled into a putative full length mRNA. The significant BLAST hit of Awi-Spherulin against S. aurantiaca was statistically tested and confirmed using PRSS3 . WMISH was performed as previously described  using 1,234 bp DIG labeled sense and anti-sense probes. Comparative quantitative RT-PCR was performed using a Qiagen Rotor-Gene Q machine and SYBR green chemistry. Primer sequences to all genes are available in Additional file 4. For each A. willeyana individual Spherulin was set as the calibrator. A set of Spherulin protein homologs was downloaded from GenBank and aligned using ClustalW2 with gap opening and gap extension penalties set to 3 and 1.8 respectively (see Additional file 5 for the alignment). Phylogenetic analyses were conducted using MrBayes v. 3.1.2. Four runs each with 8 chains were run for 1.1 million generations at a temperature of 0.2. The first 25% of these trees were discarded as burnin. A correspondence analysis of relative synonymous codon usage was conducted using CodonW (http://sourceforge.net/projects/codonw/). A. willeyana CDSs derived from an EST collection were assembled and searched against NCBI's nr database in order to unambiguously identify correct reading frames and start and stop codons from highly conserved genes (minimum BLASTx e value 10-6). Sequences with high similarity to bacterial sequences were removed leaving a total of 181 A. willeyana CDSs (Additional file 3).
Acknowledgements and Funding
This work was supported by Deutsche Forschungsgemeinschaft (DFG) funding to DJJ through the CRC for Geobiology and the German Excellence Initiative, and to GW and JR (Projects Wo896/4, Wo896/6 [SPP1174 Deep Metazoan Phylogeny], Wo896/7 [Deep DownUnder]). We thank Birgit Roring for initial technical assistance. The Awi-Spherulin sequence has been deposited in GenBank under accession number AY916059.
- Knoll AHK: Biomineralization and Evolutionary History. Rev Min Geo. 2003, 229-356.Google Scholar
- Morris SC: The Cambrian ''explosion'': Slow-fuse or megatonnage?. Proc Natl Acad Sci USA. 2000, 97: 4426-4429. 10.1073/pnas.97.9.4426.View ArticleGoogle Scholar
- Marshall CR: Explaining the Cambrian "Explosion" of animals. Ann Rev Earth Plan Sci. 2006, 34: 355-384. 10.1146/annurev.earth.33.031504.103001.View ArticleGoogle Scholar
- Valentine J: 2004, Chicago: University Of Chicago PressGoogle Scholar
- Philippe HP, Derelle R, Lopez P, Pick K, Borchiellini C, Boury-Esnault N, Vacelet J, Renard E, Houliston E, Quéinnec E, Da Silva C, Wincker P, Le Guyader H, Leys S, Jackson DJJ, Schreiber F, Erpenbeck D, Morgenstern B, Wörheide GW, Manuel M: Phylogenomics revives traditional views on deep animal relationships. Curr Biol. 2009, 19: 706-712. 10.1016/j.cub.2009.02.052.View ArticlePubMedGoogle Scholar
- Pick K, Hervé P, Fabian S, Erpenbeck D, Jackson DJ, Wrede P, Matthias W, Alie A, Burkhard M, Manuel M, Wörheide G: Improved phylogenomic taxon sampling noticeably affects non-bilaterian relationships. Mol Biol Evol. 2010, 27: 1983-1987. 10.1093/molbev/msq089.View ArticlePubMedPubMed CentralGoogle Scholar
- Jackson DJ, Macis L, Reitner J, Degnan BM, Wörheide G: Sponge paleogenomics reveals an ancient role for carbonic anhydrase in skeletogenesis. Science. 2007, 316: 1893-1895. 10.1126/science.1141560.View ArticlePubMedGoogle Scholar
- Jackson DJ, Thiel V, Wörheide G: An evolutionary fast-track to biocalcification. Geobiology. 2010, 8: 191-196. 10.1111/j.1472-4669.2010.00236.x.View ArticlePubMedGoogle Scholar
- Wörheide G: The reef cave dwelling ultraconservative coralline demosponge Astrosclera willeyana LISTER 1900 from the Indo-Pacific - Micromorphology, Ultrastructure, Biocalcification, Isotope Record, Taxonomy, Biogeography, Phylogeny. Facies. 1998, 38: 1-88. 10.1007/BF02537358.View ArticleGoogle Scholar
- Sheth N, Roca X, Hastings ML, Roeder T, Krainer AR, Sachidanandam R: Comprehensive splice-site analysis using comparative genomics. Nuc Acid Res. 2006, 34: 3955-3967. 10.1093/nar/gkl556.View ArticleGoogle Scholar
- Coolidge CJ, Seely RJ, Patton JG: Functional analysis of the polypyrimidine tract in pre-mRNA splicing. Nuc Acid Res. 1997, 25: 888-896. 10.1093/nar/25.4.888.View ArticleGoogle Scholar
- Newby M, Greenbaum N: Sculpting of the spliceosomal branch site recognition motif by a conserved pseudouridine. Nat Struct Biol. 2002, 9: 958-965. 10.1038/nsb873.View ArticlePubMedGoogle Scholar
- Nakashima K, Yamada L, Satou Y, Azuma J, Satoh N: The evolutionary origin of animal cellulose synthase. Dev Gen Evol. 2004, 214: 81-88. 10.1007/s00427-003-0379-8.View ArticleGoogle Scholar
- Keeling PJ, Palmer JD: Horizontal gene transfer in eukaryotic evolution. Nat Rev Gen. 2008, 9: 605-618. 10.1038/nrg2386.View ArticleGoogle Scholar
- Sharp PM, Cowe E, Higgins DG, Shields DC, Wolfe KH, WRIGHT F: Codon usage patterns in Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens; a review of the considerable within-species diversity. Nuc Acid Res. 1988, 16: 8207-8211. 10.1093/nar/16.17.8207.View ArticleGoogle Scholar
- Groisman EA, Saier MH, Ochman H: Horizontal transfer of a phosphatase gene as evidence for mosaic structure of the Salmonella genome. EMBO J. 1992, 11: 1309-1316.PubMedPubMed CentralGoogle Scholar
- Médigue C, Rouxel T, Vigier P, Hénaut A, Danchin A: Evidence for horizontal gene transfer in Escherichia coli speciation. J Mol Biol. 1991, 222: 851-856. 10.1016/0022-2836(91)90575-Q.View ArticlePubMedGoogle Scholar
- Delorme C, Godon JJ, Ehrlich SD, Renault P: Mosaic structure of large regions of the Lactococcus lactis subsp. cremoris chromosome. Microbiology. 1994, 140: 3053-3060. 10.1099/13500872-140-11-3053.View ArticlePubMedGoogle Scholar
- Ikemura T: Codon usage and tRNA content in unicellular and multicellular organisms. Mol Biol Evol. 1985, 2: 13-34.PubMedGoogle Scholar
- Koonin EV, Makarova KS, Aravind L: Horizontal gene transfer in prokaryotes: quantification and classification. Annu Rev Microbiol. 2001, 55: 709-742. 10.1146/annurev.micro.55.1.709.View ArticlePubMedPubMed CentralGoogle Scholar
- Kondrashov FA, Koonin EV, Morgunov IG, Finogenova TV, Kondrashova MN: Evolution of glyoxylate cycle enzymes in Metazoa: evidence of multiple horizontal transfer events and pseudogene formation. Biol Dir. 2006, 1: 31-10.1186/1745-6150-1-31.View ArticleGoogle Scholar
- Syvanen M: Horizontal gene transfer: evidence and possible consequences. Annu Rev Genet. 1994, 28: 237-261. 10.1146/annurev.ge.28.120194.001321.View ArticlePubMedGoogle Scholar
- Grunau C, Boissier J: No evidence for lateral gene transfer between salmonids and schistosomes. Nat Genet. 2010, 42: 918-919. 10.1038/ng1110-918.View ArticlePubMedGoogle Scholar
- Cohen O, Rubinstein ND, Stern A, Gophna U, Pupko T: A likelihood framework to analyse phyletic patterns. Philos Trans R Soc Lond B. 2008, 363: 3903-3911. 10.1098/rstb.2008.0177.View ArticleGoogle Scholar
- Cohen O, Pupko T: Inference and characterization of horizontally transferred gene families using stochastic mapping. Mol Biol Evol. 2010, 27: 703-713. 10.1093/molbev/msp240.View ArticlePubMedGoogle Scholar
- Ochman H, Lawrence JG, Groisman EA: Lateral gene transfer and the nature of bacterial innovation. Nature. 2000, 405: 299-304. 10.1038/35012500.View ArticlePubMedGoogle Scholar
- Cohen O, Gophna U, Pupko T: The complexity hypothesis revisited: connectivity rather than function constitutes a barrier to horizontal gene transfer. Mol Biol Evol. 2011, 28: 1481-1489. 10.1093/molbev/msq333.View ArticlePubMedGoogle Scholar
- Thomas T, Rusch D, Demaere MZ, Yung PY, Lewis M, Halpern A, Heidelberg KB, Egan S, Steinberg PD, Kjelleberg S: Functional genomic signatures of sponge bacteria reveal unique and shared features of symbiosis. ISME J. 2010, 4: 1557-1567. 10.1038/ismej.2010.74.View ArticlePubMedGoogle Scholar
- Pearson WR: Searching protein sequence libraries: Comparison of the sensitivity and selectivity of the Smith-Waterman and FASTA algorithms. Genomics. 1991, 11: 635-650. 10.1016/0888-7543(91)90071-L.View ArticlePubMedGoogle Scholar
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