Permalink
Cannot retrieve contributors at this time
Fetching contributors…
Cannot retrieve contributors at this time
| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">04266</article-id><article-id pub-id-type="doi">10.7554/eLife.04266</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Genomics and evolutionary biology</subject></subj-group></article-categories><title-group><article-title>Antibacterial gene transfer across the tree of life</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-17601"><name><surname>Metcalf</surname><given-names>Jason A</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17602"><name><surname>Funkhouser-Jones</surname><given-names>Lisa J</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17603"><name><surname>Brileya</surname><given-names>Kristen</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17604"><name><surname>Reysenbach</surname><given-names>Anna-Louise</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-5519"><name><surname>Bordenstein</surname><given-names>Seth R</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="aff" rid="aff3"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Biological Sciences</institution>, <institution>Vanderbilt University</institution>, <addr-line><named-content content-type="city">Nashville</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><institution content-type="dept">Department of Biology</institution>, <institution>Portland State University</institution>, <addr-line><named-content content-type="city">Portland</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><institution content-type="dept">Department of Pathology, Microbiology, and Immunology</institution>, <institution>Vanderbilt University</institution>, <addr-line><named-content content-type="city">Nashville</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>van der Donk</surname><given-names>Wilfred</given-names></name><role>Reviewing editor</role><aff><institution>University of Illinois-Urbana Champaign</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>s.bordenstein@vanderbilt.edu</email></corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>25</day><month>11</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e04266</elocation-id><history><date date-type="received"><day>06</day><month>08</month><year>2014</year></date><date date-type="accepted"><day>16</day><month>10</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Metcalf et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Metcalf et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife04266.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.04266.001</object-id><p>Though horizontal gene transfer (HGT) is widespread, genes and taxa experience biased rates of transferability. Curiously, independent transmission of homologous DNA to archaea, bacteria, eukaryotes, and viruses is extremely rare and often defies ecological and functional explanations. Here, we demonstrate that a bacterial lysozyme family integrated independently in all domains of life across diverse environments, generating the only glycosyl hydrolase 25 muramidases in plants and archaea. During coculture of a hydrothermal vent archaeon with a bacterial competitor, muramidase transcription is upregulated. Moreover, recombinant lysozyme exhibits broad-spectrum antibacterial action in a dose-dependent manner. Similar to bacterial transfer of antibiotic resistance genes, transfer of a potent antibacterial gene across the universal tree seemingly bestows a niche-transcending adaptation that trumps the barriers against parallel HGT to all domains. The discoveries also comprise the first characterization of an antibacterial gene in archaea and support the pursuit of antibiotics in this underexplored group.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.001">http://dx.doi.org/10.7554/eLife.04266.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.04266.002</object-id><title>eLife digest</title><p>Living things inherit most of their genetic material from their parents, so genes tend to be passed on from one generation to the next—from ancestors to descendants. Sometimes, however, DNA is transferred from one organism to another by other means. These events, collectively called horizontal gene transfer, are fairly common in nature; genes have been passed between different species as well as between different groups of organisms. For example, genes that confer resistance to antibacterial drugs have transferred from one species of bacteria to another, and other genes have also ‘jumped’ from bacteria to plants or animals.</p><p>Now Metcalf et al. have studied a gene that first arose in bacteria and that encodes an enzyme called a lysozyme. This enzyme breaks down the outer casing of a bacterial cell: a step that is required for a bacterium to reproduce and divide in two. When Metcalf et al. searched for relatives of the lysozyme gene, they found copies in many other species of bacteria and revealed that this gene has been repeatedly transferred between different bacteria. Members of the lysozyme gene family have also ‘jumped out’ of bacteria and into other organisms at least four times. Metcalf et al. found related lysozyme genes in a plant, an insect, many species of fungi, and a single-celled microbe (called an archaeon) that lives at hot, deep-sea vents.</p><p>A gene family being spread this widely across the tree of life has not been seen before. Nevertheless, as DNA is a common biological language to all living things, it is likely that all the different species that have received a lysozyme gene might use it for similar purposes.</p><p>Metcalf et al. reveal that the lysozyme could be being used as an antibacterial molecule. The archaeon lysozyme can kill a broad range of bacteria; and when the gene was transferred into <italic>Escherichia coli</italic> bacteria, only the bacteria that mutated the lysozyme gene to render it useless were able to survive. Metcalf et al. also revealed that the archaeon microbe produces more of the enzyme if bacteria are present, which allows it to outcompete these bacteria.</p><p>These findings suggest that there may be a number of horizontally transferred genes that have antibacterial activity against a wide range of bacteria. Searching for these genes—particularly in the largely underexplored group of archaea—might reveal new sources for antibiotic drugs to treat bacterial infections.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.002">http://dx.doi.org/10.7554/eLife.04266.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>horizontal gene transfer</kwd><kwd>antibiotic</kwd><kwd>lysin</kwd><kwd>archaea</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>other</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000001</institution-id><institution>National Science Foundation</institution></institution-wrap></funding-source><award-id>DEB1046149</award-id><principal-award-recipient><name><surname>Bordenstein</surname><given-names>Seth R</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>R01GM085163</award-id><principal-award-recipient><name><surname>Bordenstein</surname><given-names>Seth R</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>T32GM07347</award-id><principal-award-recipient><name><surname>Metcalf</surname><given-names>Jason A</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000001</institution-id><institution>National Science Foundation</institution></institution-wrap></funding-source><award-id>DEB1134877</award-id><principal-award-recipient><name><surname>Reysenbach</surname><given-names>Anna-Louise</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Parallel horizontal gene transfer has spread a bacteriolytic gene family to all domains of life, and has bestowed a niche-transcending adaptation in recipients that must deploy antibacterial molecules to survive in a bacterial world.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>HGT is rampant among prokaryotes and phages and is an important mechanism for acquisition of new genes and functions (<xref ref-type="bibr" rid="bib59">Popa and Dagan, 2011</xref>), including the shuttling of antibiotics and antibiotic resistance between bacteria (<xref ref-type="bibr" rid="bib20">Clardy et al., 2009</xref>). Instances of interdomain horizontal transfer of diverse genes between two domains of life or between viruses and their hosts are also increasingly documented (<xref ref-type="bibr" rid="bib52">Nelson et al., 1999</xref>; <xref ref-type="bibr" rid="bib35">Husnik et al., 2013</xref>; <xref ref-type="bibr" rid="bib25">Dunning Hotopp et al., 2007</xref>; <xref ref-type="bibr" rid="bib76">Wu et al., 2013</xref>; <xref ref-type="bibr" rid="bib31">Gladyshev et al., 2008</xref>; <xref ref-type="bibr" rid="bib14">Bratke and McLysaght, 2008</xref>; <xref ref-type="bibr" rid="bib21">Danchin et al., 2010</xref>). While a minority of these transfers have been functionally investigated, the biological activity, selective advantages, and ecological contexts of many interdomain HGT events remain poorly characterized (<xref ref-type="bibr" rid="bib24">Dunning Hotopp 2011</xref>, <xref ref-type="bibr" rid="bib38">Keeling and Palmer, 2008</xref>). In comparison to these intradomain or interdomain highways of HGT (<xref ref-type="bibr" rid="bib12">Beiko et al., 2005</xref>), independent transmission of the same gene family to archaea, bacteria, eukaryotes, and viruses is extremely uncommon and subject to apparently rare events throughout the history of life (<xref ref-type="bibr" rid="bib51">Moran et al., 2012</xref>; <xref ref-type="bibr" rid="bib45">Lundin et al., 2010</xref>; <xref ref-type="bibr" rid="bib41">Koonin et al., 2003</xref>; <xref ref-type="bibr" rid="bib48">McClure, 2001</xref>; <xref ref-type="bibr" rid="bib49">McDonald et al., 2012</xref>). When taken together, genome-enabled studies suggest that horizontal gene transfers (HGTs) are biased and experience a frequency gradient that decreases from within domain > between two domains > between all domains of life (<xref ref-type="bibr" rid="bib17">Bruto et al., 2013</xref>; <xref ref-type="bibr" rid="bib77">Zhaxybayeva and Doolittle, 2011</xref>; <xref ref-type="bibr" rid="bib60">Puigbo et al., 2009</xref>; <xref ref-type="bibr" rid="bib4">Andam and Gogarten, 2013</xref>, <xref ref-type="bibr" rid="bib3">2011</xref>). However, the fact that a large number of organisms from all domains of life have been artificially transformed with genes evolved in other taxa indicates that there is no fundamental obstruction to interdomain HGT when barriers to transfer are deliberately removed.</p><p>One significant question then is why do highways of intra- or interdomain transfers occur more frequently than transfers to all domains in the universal tree of life? There are at least two explanations. First, recurrent transfer of the same gene family may be limited by incompatible mechanics of gene transfer (e.g., transduction, transfection, plasmid exchange, isolation of eukaryotic genome in nucleus, separation of somatic and germline tissues in multicellular eukaryotes) between domains compared to within domains. However, the individual success of gene transfers between any two domains of life, for example archaea and bacteria (<xref ref-type="bibr" rid="bib52">Nelson et al., 1999</xref>; <xref ref-type="bibr" rid="bib71">van Wolferen et al., 2013</xref>), bacteria and eukaryote (<xref ref-type="bibr" rid="bib5">Andersson, 2005</xref>; <xref ref-type="bibr" rid="bib13">Bordenstein, 2007</xref>; <xref ref-type="bibr" rid="bib31">Gladyshev et al., 2008</xref>; <xref ref-type="bibr" rid="bib21">Danchin et al., 2010</xref>), and archaea and eukaryote (<xref ref-type="bibr" rid="bib6">Andersson et al., 2003</xref>; <xref ref-type="bibr" rid="bib66">Schonknecht et al., 2013</xref>), suggests that these barriers may be minimal. Second, the selective barriers against HGT of the same gene to multiple taxa and preservation of the gene through evolutionary time are multifaceted given the potential costs associated with HGT (<xref ref-type="bibr" rid="bib9">Baltrus, 2013</xref>), and that each recipient may not benefit from the trait conferred. In the case of selfish genetic elements, HGT provides a strong benefit to the gene itself, yet these genes can be detrimental to the host organism and select for the evolution of countermeasures to gene propagation (<xref ref-type="bibr" rid="bib74">Werren, 2011</xref>). Thus, it may be necessary for a transferred gene to confer a benefit to its new host in order to be stably maintained in the host genome over the long term. Given these evolutionary dynamics, there may be very few niche-transcending genes (<xref ref-type="bibr" rid="bib75">Wiedenbeck and Cohan, 2011</xref>), defined as genes that are useful in different physiological capabilities, cellular structures, and ecological niches, which repeatedly increase fitness of each recipient across the whole diversity of life and can be stably and repeatedly transferred between very divergent organisms.</p><p>Among the few putative cases, there is a pore-forming toxin domain that appears to have been anciently transferred between diverse lineages (<xref ref-type="bibr" rid="bib51">Moran et al., 2012</xref>). However, the distribution of the transfer across the tree of life is unclear because archaeal sequences were not included in phylogenetic analyses due to low support values. Other candidate genes encode proteins involved in nucleotide metabolism, intramembrane proteolysis, or membrane transport, but the transfer events defy clear interpretations due to their deep antiquity in evolutionary time and the confounding issues of ancient paralogy (<xref ref-type="bibr" rid="bib45">Lundin et al., 2010</xref>; <xref ref-type="bibr" rid="bib41">Koonin et al., 2003</xref>; <xref ref-type="bibr" rid="bib48">McClure, 2001</xref>; <xref ref-type="bibr" rid="bib49">McDonald et al., 2012</xref>). Moreover, these transfers are often not functionally validated in the recipient taxa.</p><p>Here we demonstrate for the first time, to our knowledge, that a functional antibacterial gene family scattered across the tree of life in diverse ecological contexts. This bacterial gene encodes a glycosyl hydrolase 25 (GH25) muramidase, a peptidoglycan-degrading lysozyme that hydrolyzes the 1,4-β-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine in the bacterial cell wall. Typically found in bacteria (<xref ref-type="bibr" rid="bib19">Cantarel et al., 2009</xref>), the lytic enzyme classically functions in cell division and cell wall remodeling (<xref ref-type="bibr" rid="bib72">Vollmer et al., 2008</xref>), while in bacteriophages they lyse bacterial peptidoglycan at the end of the phage life cycle (<xref ref-type="bibr" rid="bib28">Fastrez, 1996</xref>). Although members of the GH25 muramidase family have been noted in other taxa (<xref ref-type="bibr" rid="bib42">Korczynska et al., 2010</xref>; <xref ref-type="bibr" rid="bib53">Nikoh et al., 2010</xref>), extensive analysis of their evolutionary history and functions have not been undertaken. We hypothesized that the transfer of antibacterial genes from bacteria to archaea and to eukaryotes bestows a niche-transcending adaptation that overcomes the barriers against repeated and evolutionarily stable HGT of the same type of gene across the tree of life.</p></sec><sec id="s2" sec-type="results|discussion"><title>Results and discussion</title><sec id="s2-1"><title>A bacterial GH25 muramidase is present in all domains of life</title><p>During a homology search, we uncovered 75 nonredundant sequences (E-values ≤ 10<sup>−12</sup>) of a bacterial GH25 muramidase in disparate taxa across the tree of life, indicating possible HGT of a bacterial gene to both eukaryotic and archaeal species as well as to phages. Putative HGT events were identified in the genomes of the plant <italic>Selaginella moellendorffii</italic> (<xref ref-type="bibr" rid="bib10">Banks et al., 2011</xref>), the deep-sea hydrothermal vent archaeon <italic>Aciduliprofundum boonei</italic> (<xref ref-type="bibr" rid="bib63">Reysenbach et al., 2006</xref>), the pea aphid <italic>Acyrthosiphon pisum</italic> (<xref ref-type="bibr" rid="bib36">International Aphid Genomics Consortium 2010</xref>, <xref ref-type="bibr" rid="bib53">Nikoh et al., 2010</xref>), and several species of fungi such as <italic>Aspergillus oryzae</italic> (<xref ref-type="bibr" rid="bib46">Machida et al., 2005</xref>). We verified the presence of the lysozyme gene in natural populations of selected HGT recipients by PCR and sequencing of the GH25 muramidase domain (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>), including <italic>Aciduliprofundum</italic> field samples harvested from hydrothermal vents worldwide. We detected lysozyme genes in 9 out of 12 field isolates of <italic>Aciduliprofundum</italic> from deep-sea vents in the Atlantic and Pacific oceans, 5 out of 6 species in the plant genus <italic>Selaginella</italic>, and 8 out of 9 aphid species in the subfamily Aphidinae (<xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>). However, it is possible that PCR-negative strains actually do possess lysozyme genes and were simply more divergent than could be detected with our primers. At the protein level, sequenced field isolate lysozymes were relatively similar to each other in each clade, with 74% pairwise identity (385aa alignment) amongst <italic>Aciduliprofundum</italic> sequences, 85.1% (203aa alignment) amongst three intact <italic>Selaginella</italic> sequences, and 87.3% identity (93aa alignment) amongst Aphidinae sequences, with more variable divergence between clades (full identity matrix available in <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2</xref>). We also found lysozymes in two additional WO phages as part of an ongoing next generation sequencing project of <italic>Wolbachia</italic> viruses (unpublished data).</p><p>To rule out spurious bacterial contamination in these genomes and to confirm genomic integration of the lysozyme gene, we employed direct sequencing of PCR products amplified using primers inside the lysozyme gene paired with primers outside the gene for <italic>Aciduliprofundum</italic> field samples and <italic>S. moellendorffii</italic>. Incorporation of the lysozyme gene was verified in all cases tested (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>). Additionally, <italic>Aciduliprofundum</italic> samples were grown in strict monocultures as determined by 16S amplicon monitoring. Integration of the <italic>A. pisum</italic> lysozyme has been previously established (<xref ref-type="bibr" rid="bib53">Nikoh et al., 2010</xref>). Flanking genes in the recipient genomes were non-bacterial on either side of the transferred lysozyme in each case (<xref ref-type="fig" rid="fig1">Figure 1</xref>, <xref ref-type="supplementary-material" rid="SD3-data">Supplementary file 3</xref>), with two exceptions. A bordering gene in <italic>A. boonei</italic>, ADP-ribose-1”-monophosphatase (App-1), possesses both bacterial and archaeal homologs and a phylogenetic analysis suggests HGT unrelated to the lysozyme transfer (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3A</xref>). This transfer was likely between archaea and Thermotogae bacteria. The second exception is a GH2 hydrolase gene adjacent to the lysozyme in <italic>A. oryzae</italic>. This hydrolase has bacterial homologs in the phylum Actinobacteria, and recapitulates the same phylogenetic pattern seen in the GH25 muramidase (see below, <xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3B</xref>). Thus, it is likely that the lysozyme and GH2 hydrolase were transferred to fungi in a single event. GH2 hydrolases have a number of carbohydrate-degrading enzymatic abilities (<xref ref-type="bibr" rid="bib19">Cantarel et al., 2009</xref>), including β-galactosidase, β-mannosidase, and β-glucuronidase activities, suggesting that this gene may benefit the fungi by adding additional digestive/nutritive capabilities in Dikarya.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.04266.003</object-id><label>Figure 1.</label><caption><title>Architecture of HGT candidates and surrounding genes.</title><p>Each arrow represents an open reading frame transcribed from either the plus strand (arrow pointing right) or the minus strand (arrow pointing left). The color of the arrow indicates the taxa the gene is found in based on its closest homologs. Black = Eubacteria, purple = virus, red = Archaea, green = Plantae, Orange = Fungi, Blue = Insecta, white = no known homologs, dashed line = present in multiple domains. The length of the arrows and intergenic regions are drawn to scale except where indicated with broken lines. The four paralogs of the lysozyme in <italic>S. moellendorffii</italic> occur on two genomic scaffolds with light green bands connecting homologous genes. Vertical arrows indicate the location of introns in the <italic>A. oryzae</italic> and <italic>A. pisum</italic> lysozymes. Abbreviations: Lys: lysozyme, gpW = phage baseplate assembly protein W, SH3: Src homology domain 3, App-1 = ADP-ribose-1”-monophosphatase, PRT = phosphoribosyltransferase, LD = leucoanthocyanidin dioxygenase; IMP = integral membrane protein. A protein diagram for each lysozyme is drawn to scale with the light gray regions highlighting a conserved protein domain. *<italic>A. pisum</italic> diagram is based on Acyr_1.0 assembly and transcription data (<xref ref-type="bibr" rid="bib53">Nikoh et al., 2010</xref>); the annotation in Acyr_2.0 is different.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.003">http://dx.doi.org/10.7554/eLife.04266.003</ext-link></p></caption><graphic xlink:href="elife04266f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04266.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Presence of HGT lysozyme genes in field samples.</title><p>(<bold>A</bold>) PCR amplifications of portions of the GH25 muramidase domain in the indicated taxa. All amplifications were Sanger sequenced to confirm integration. Primers used are listed in Table S3. Abbreviations: <italic>Sb</italic>: <italic>S. braunii</italic>, <italic>Sm</italic>: <italic>S. moellendorffii</italic>, <italic>Su</italic>: <italic>S. uncinata</italic>, <italic>Ssa</italic>: <italic>S. sanguinolenta</italic>, <italic>Sst</italic>: <italic>S. stauntoniana</italic>, <italic>Sl</italic>: <italic>S. lepidophylla</italic>, E: East Pacific Rise, L: Lao Spreading Center, M: Mid-Atlantic Ridge, <italic>Pu</italic>: <italic>Pleotrichophorus utensis</italic>, <italic>Aa</italic>: <italic>Artemisaphis artemisicola</italic>, <italic>Ue</italic>: <italic>Uroleucon erigeronensis</italic>, <italic>Av</italic>: <italic>Aphis varians</italic>, <italic>Ap</italic>: <italic>Acyrthosiphon pisum</italic>, <italic>Al</italic>: <italic>Aphis lupini</italic>, <italic>Cs</italic>: <italic>Cedoaphis sp.</italic>, <italic>As</italic>: <italic>Aphthargelia symphoricarpi</italic>, <italic>Bs</italic>: <italic>Braggia sp</italic>., - denotes water only control. (<bold>B</bold>) World map with approximate locations of <italic>A. boonei</italic> field samples. Those that tested positive for the GH25 muramidase domain are indicated by green stars and those that tested negative are indicated by red stars. Map is a public domain image from Wikimedia Commons.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.004">http://dx.doi.org/10.7554/eLife.04266.004</ext-link></p></caption><graphic xlink:href="elife04266fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04266.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>PCR amplifications testing genomic integration with primers within and outside of lysozyme genes.</title><p>Primers used are listed in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref> and binding sites are indicated in gene diagrams with small black arrows. All integrations were confirmed with Sanger sequencing. Abbreviations: <italic>Sm</italic>: <italic>S. moellendorffii</italic>, L: Lao Spreading Center, - denotes water only control, CHP = conserved hypothetical protein, App-1 = ADP-ribose-1”monophosphatase, PRT = phosphoribosyltransferase.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.005">http://dx.doi.org/10.7554/eLife.04266.005</ext-link></p></caption><graphic xlink:href="elife04266fs002"/></fig><fig id="fig1s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04266.006</object-id><label>Figure 1—figure supplement 3.</label><caption><title>Protein phylogeny of neighboring genes to transferred lysozymes.</title><p>(<bold>A</bold>) App-1 phylogeny based on alignment of 141aa without indels consisting of top E-value hits to blastp using <italic>A. boonei</italic> App-1 as the query. Taxon of origin for each amino acid sequence is indicated by color. Posterior probability is indicated at all nodes with values above 50. Branch lengths represent number of substitutions per site as indicated by scale bar. Tree is arbitrarily rooted. (<bold>B</bold>) GH2 hydrolase phylogeny based on an alignment of 188aa without indels consisting of top E-value hits to blastp using <italic>A. oryzae</italic> hydrolase as the query.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.006">http://dx.doi.org/10.7554/eLife.04266.006</ext-link></p></caption><graphic xlink:href="elife04266fs003"/></fig></fig-group></p></sec><sec id="s2-2"><title>Non-bacterial GH25 muramidases arose from HGT</title><p>To establish parallel HGT, i.e., the independent transfer of the same gene family to multiple lineages, we conducted a phylogenetic analysis on 86 GH25 muramidase sequences using Bayesian and maximum likelihood inference methods (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). We combined non-redundant <italic>Aciduliprofundum</italic>, <italic>Selaginella</italic>, and WO sequences obtained from PCR and Sanger sequencing with blastp results to reconstruct the phylogeny. Putative instances of HGT are diagrammed in <xref ref-type="fig" rid="fig3">Figure 3</xref>. Additionally, transferred lysozymes in nonbacterial taxa were used as queries to identify homologs and make a second set of phylogenetic trees to confirm the HGT (<xref ref-type="fig" rid="fig2">Figure 2B–D</xref>, <xref ref-type="fig" rid="fig2s1 fig2s2">Figure 2—figure supplements 1 and 2</xref>, <xref ref-type="supplementary-material" rid="SD4-data">Supplementary file 4</xref>). Three key results emerge from these phylogenetic analyses: (i) at least three independent instances of interdomain HGT of the bacterial GH25 muramidase occurred in nonbacterial taxa (<italic>Aciduliprofundum</italic>, <italic>Selaginella</italic>, and <italic>Insecta</italic>) as well as a number of transfers to bacteriophages, (ii) vertical transmission of the transferred gene ensues in some descendant taxa (i.e., <italic>Aciduliprofundum</italic> and <italic>Selaginella</italic>), and (iii) frequent HGT of the muramidase between bacterial clades accompanies the interdomain transfer, indicating unusually frequent and broad-ranging HGT of this niche-transcending gene family.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.04266.007</object-id><label>Figure 2.</label><caption><title>Phylogeny of GH25 muramidase.</title><p>(<bold>A</bold>) Phylogeny based on alignment of 113aa without indels consisting of top E-value hits to blastp using WORiA phage lysozyme as a query. Taxon of origin for each amino acid sequence is indicated by color. Posterior probability (Bayesian phylogeny) and bootstrap values (maximum likelihood phylogeny) are indicated at all nodes with values above 50. Branch lengths represent number of substitutions per site as indicated by scale bar. Tree is arbitrarily rooted. Iterative phylogenies based on top E-value blastp hits to <italic>A. boonei</italic> lysozyme (<bold>B</bold>), <italic>A. pisum</italic> lysozyme (<bold>C</bold>), and <italic>S. moellendorffii</italic> lysozyme (<bold>D</bold>) are also shown.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.007">http://dx.doi.org/10.7554/eLife.04266.007</ext-link></p></caption><graphic xlink:href="elife04266f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04266.008</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Iterative HGT analysis alignments.</title><p>Consensus alignment of GH25 muramidases without indels used in iterative phylogenies in <xref ref-type="fig" rid="fig2">Figure 2</xref> is shown for (<bold>A</bold>) <italic>A. boonei</italic>, (<bold>B</bold>) <italic>A. pisum</italic>, and (<bold>C</bold>) <italic>S. moellendorffii</italic>. Conservation is indicated by amino acid symbol size and bar graphs below the consensus sequence.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.008">http://dx.doi.org/10.7554/eLife.04266.008</ext-link></p></caption><graphic xlink:href="elife04266fs004"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04266.009</object-id><label>Figure 2—figure supplement 2.</label><caption><title>Protein phylogeny of <italic>A. oryzae</italic> GH25 muramidase and relatives.</title><p>Phylogeny based on alignment of 186aa without indels consisting of top E-value hits to blastp using <italic>A. oryzae</italic> lysozyme as a query. Taxon of origin for each amino acid sequence is indicated by color. Posterior probability (Bayesian phylogeny) and bootstrap values (maximum likelihood phylogeny) are indicated at all nodes with values above 50. Branch lengths represent number of substitutions per site as indicated by scale bar. Tree is arbitrarily rooted.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.009">http://dx.doi.org/10.7554/eLife.04266.009</ext-link></p></caption><graphic xlink:href="elife04266fs005"/></fig><fig id="fig2s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04266.010</object-id><label>Figure 2—figure supplement 3.</label><caption><title>DNA phylogeny of A. oryzae GH25 muramidase and relatives.</title><p>Phylogeny based on alignment of 282 bp without indels consisting of top E-value hits to blastn using <italic>A. oryzae</italic> lysozyme exon 2 as a query. Taxon of origin for each nucleic acid sequence is indicated by color. Posterior probability (Bayesian phylogeny) and bootstrap values (maximum likelihood phylogeny) are indicated at all nodes with values above 50. Branch lengths represent number of substitutions per site as indicated by scale bar. Tree is arbitrarily rooted.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.010">http://dx.doi.org/10.7554/eLife.04266.010</ext-link></p></caption><graphic xlink:href="elife04266fs006"/></fig></fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.04266.011</object-id><label>Figure 3.</label><caption><title>Schematic of HGT events.</title><p>Bayesian phylogeny based on the 16S rRNA gene from selected taxa is shown. Colored lines indicate putative horizontal gene transfer events, although other possible HGT patterns cannot be definitively excluded. Posterior probabilities are noted at each node.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.011">http://dx.doi.org/10.7554/eLife.04266.011</ext-link></p></caption><graphic xlink:href="elife04266f003"/></fig></p><p>To statistically validate parallel HGT across the tree of life, we performed a Shimodaira–Hasegawa test (SH-test) (<xref ref-type="bibr" rid="bib68">Shimodaira and Hasegawa, 1999</xref>) by comparing our consensus tree (<xref ref-type="fig" rid="fig2">Figure 2A</xref>) against a hypothetical tree with a binary constraint in which bacterial sequences are monophyletic and separate from monophyletic nonbacterial sequences. In this hypothetical tree consistent with the tree of life, lineage relationships within the bacterial and nonbacterial groups were permissively set as unconstrained. Results indicate that the hypothetical tree is significantly worse than the HGT tree, as expected (p<0.01, D(LH) = −133.9, SD = 31.5). We repeated this analysis with the hypothetical tree compared to 100 randomly sampled HGT trees from maximum likelihood bootstrapping and found the hypothetical tree was also worse than each of these trees (p<0.01). Finally, we performed SH tests between the HGT tree and either 1) a three-domain constraint tree or 2) a monophyletic eukaryote branch constraint tree, and again found that the constraint trees were significantly worse than the HGT tree (p<0.01). Thus, the null hypothesis of vertical descent is rejected, even under the most permissive conditions.</p><p>We observed that each interdomain HGT event (<xref ref-type="fig" rid="fig2">Figure 2</xref>) occurred between taxa that coexist in the same ecological niche, a likely prerequisite for HGT. For instance, the <italic>A. boonei</italic> lysozyme is in a clade dominated by Firmicutes whose members can be common in deep ocean sediments (<xref ref-type="bibr" rid="bib56">Orcutt et al., 2011a</xref>). Indeed, <italic>Bacillus</italic> species have even been found in hydrothermal vents of the same fields in which <italic>Aciduliprofundum</italic> strains were isolated (<xref ref-type="bibr" rid="bib62">Reysenbach et al., 2000</xref>). The <italic>A. pisum</italic> lysozyme clade includes <italic>Wolbachia</italic> prophages and Proteobacteria, which are common endosymbionts of aphids and other insects (<xref ref-type="bibr" rid="bib32">Gomez-Valero et al., 2004</xref>; <xref ref-type="bibr" rid="bib8">Augustinos et al., 2011</xref>; <xref ref-type="bibr" rid="bib73">Wang et al., 2014</xref>). The <italic>S. moellendorffii</italic> plant lysozyme is closely related to Actinobacteria, which are dominant microbes in soil (<xref ref-type="bibr" rid="bib18">Bulgarelli et al., 2013</xref>). These associations, while not proof of HGT, establish interactions that may have facilitated the transfers, although any number of intermediate gene carriers is possible. While the phylogenetic pattern of the GH25 muramidase found in fungi is consistent with HGT (<xref ref-type="fig" rid="fig2">Figure 2A</xref>, <xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2</xref>), the transfer occurred anciently in fungal evolution prior to the divergence of Dikarya, as the domain is present in both Basidiomycota and Ascomycota, but not other fungal phyla. As is the case with most putative ancient transfers, the deep branches of the tree are poorly supported and a definitive donor taxon cannot be established. Additionally, the possibility of multiple ancient transfers between bacteria and fungi or among fungi and plants cannot be excluded. However, a nucleotide-level phylogeny also supports HGT from an ancestral Actinobacterium (<xref ref-type="fig" rid="fig2s3">Figure 2—figure supplement 3</xref>).</p><p>Interestingly, the lysozyme gene in the aphid <italic>A. pisum</italic> consists of a fusion of a bacterial GH25 muramidase domain and a eukaryotic carboxypeptidase domain. The gene includes five introns (<xref ref-type="bibr" rid="bib53">Nikoh et al., 2010</xref>), none of which interrupt the GH25 domain, consistent with a relatively recent HGT event and the absence of the gene from most sequenced insects (<xref ref-type="fig" rid="fig1">Figure 1</xref>). The lysozyme in the fungus <italic>A. oryzae</italic>, meanwhile, contains only a single intron, but it does interrupt the GH25 domain, consistent with the domain’s long association with fungi from the subkingdom Dikarya (<xref ref-type="fig" rid="fig1">Figure 1</xref>). We found no evidence of a GH25 muramidase in 323 sequenced archaeal genomes spanning all the major phyla and sister taxa to <italic>A. boonei</italic> (<xref ref-type="bibr" rid="bib63">Reysenbach et al., 2006</xref>; <xref ref-type="bibr" rid="bib30">Flores et al., 2012</xref>). This lack of homology does not appear to be due to insufficient representation of archaeal diversity, as the 323 members span all of the major phyla: Crenarchaeota (56), Euryarchaeota (205), Nanoarchaeota (10), and Thaumarchaeota (39). Indeed, if vertical descent were assumed for a recent Bayesian phylogeny of archaea with sequenced genomes (<xref ref-type="bibr" rid="bib15">Brochier-Armanet et al., 2011</xref>), this would require at least 10 independent losses of the lysozyme gene, an assumption that is certainly less parsimonious than a single HGT event. Moreover, the relative divergence of the small subunit rRNA gene in <italic>A. boonei</italic> compared to the putative bacterial HGT donors is greater than the relative divergence of the lysozyme gene (<xref ref-type="fig" rid="fig4">Figure 4</xref>), a finding that is inconsistent with both genes being transmitted by vertical descent. Also, there are no other homologs beyond those presented in this study in 132 plant genomes, and only one insect species with additional homologs out of 109 insect genomes. Thus, if the lysozyme were present in the last common ancestor of all domains, it would require the unlikely loss of the gene in dozens of lineages while maintaining it in an exceedingly small number of species. In summary, the presence of a GH25 muramidase in nonbacterial species represents a series of recurrent, independent horizontal gene transfer events derived from diverse, ecologically associated bacteria.<fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.04266.012</object-id><label>Figure 4.</label><caption><title>Comparison of GH25 muramidase and rRNA divergence.</title><p>(<bold>A</bold>) Unrooted Bayesian phylogeny of the GH25 muramidase from <italic>A. boonei</italic> and selected relatives, based on an alignment of 185aa without indels. Taxon of origin for each nucleic acid sequence is indicated by color. Posterior probability is indicated at all nodes with values above 50. Branch lengths represent number of substitutions per site as indicated by scale bar. (<bold>B</bold>) Unrooted Bayesian phylogeny of the 16S rRNA gene for the same taxa as in (<bold>A</bold>), based on an alignment of 1,156 bp without indels.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.012">http://dx.doi.org/10.7554/eLife.04266.012</ext-link></p></caption><graphic xlink:href="elife04266f004"/></fig></p></sec><sec id="s2-3"><title><italic>A. boonei</italic> GH25 muramidase is antibacterial</title><p>We next undertook a series of experiments to test the hypothesis that the transferred muramidase functions as an antibacterial molecule. Since HGT frequently results in pseudogenized and nonfunctional genes (<xref ref-type="bibr" rid="bib40">Kondrashov et al., 2006</xref>; <xref ref-type="bibr" rid="bib53">Nikoh et al., 2010</xref>, <xref ref-type="bibr" rid="bib54">2008</xref>; <xref ref-type="bibr" rid="bib25">Dunning Hotopp et al., 2007</xref>), we first investigated the amino acid sequences for preserved antibacterial action of the transferred lysozymes in nonbacterial genomes. We aligned all 86 GH25 muramidase sequences to identify conserved sites (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). We then mapped the conserved amino acids to a three-dimensional structure prediction of the <italic>A. boonei</italic> GH25 muramidase domain (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). Highly conserved residues (>85% identity between all taxa) invariably mapped to the previously identified active site pocket (<xref ref-type="bibr" rid="bib47">Martinez-Fleites et al., 2009</xref>). Conservation was also evident for structure predictions of other GH25 muramidases in the phylogeny such as <italic>S. moellendorffii</italic> (<xref ref-type="fig" rid="fig5">Figure 5C</xref>).<fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.04266.013</object-id><label>Figure 5.</label><caption><title>Conservation of <italic>A. boonei</italic> GH25 muramidase domain.</title><p>(<bold>A</bold>) Consensus alignment of 86 GH25 muramidases with insertions and deletions removed. Conservation is indicated by amino acid symbol size and bar graphs below the consensus sequence. Active site residues and highly conserved amino acids modeled below are indicated with red and orange asterisks, respectively. (<bold>B</bold>) Space-filling model of the active site face of the predicted structure of <italic>A. boonei</italic> GH25 muramidase domain and (<bold>C</bold>) <italic>S. moellendorffii</italic> GH25 muramidase domain. Active site residues are indicated in red and the eight additional residues most highly conserved across all 86 proteins are orange.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.013">http://dx.doi.org/10.7554/eLife.04266.013</ext-link></p></caption><graphic xlink:href="elife04266f005"/></fig></p><p>Second, we cloned, expressed, and purified the GH25 muramidase domain from the archaeon <italic>A. boonei</italic> as well as from closely related homologs in <italic>Paenibacillus polymyxa</italic> and PhiBP. We obtained each muramidase in a pure elution (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>) and tested for antibacterial action against a range of bacterial species. As predicted, the <italic>A. boonei</italic> GH25 muramidase efficiently killed several species of bacteria in the phylum Firmicutes - the putative donor group of the gene (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). The bacterial inhibition by <italic>A. boonei</italic> GH25 muramidase was more potent than the positive control, chicken egg white lysozyme, and was dose-dependent (<xref ref-type="fig" rid="fig6">Figure 6B</xref>). Bacterial and phage muramidases did not elicit antibacterial killing, similar to cyan fluorescent protein and buffer-only negative controls. Bacteria typically use a large protein complex to limit their lysozymes’ activity to the septum during cell division (<xref ref-type="bibr" rid="bib70">Uehara and Bernhardt, 2011</xref>), and PhiBP phage has a documented spectrum of activity limited only to a <italic>P. polymyxa</italic> strain unavailable for our analyses (<xref ref-type="bibr" rid="bib34">Halgasova et al., 2010</xref>). As expected, the <italic>A. boonei</italic> GH25 muramidase did not exhibit antibacterial activity against Gram-negative species or Gram-positive species outside of the families Bacillaceae and Paenibacillaceae, which was equivalent to the killing range of chicken egg white lysozyme with the exception of the Actinobacterium <italic>Micrococcus luteus</italic> (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2</xref>).<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.04266.014</object-id><label>Figure 6.</label><caption><title>Antibacterial action of <italic>A. boonei</italic> GH25 muramidase domain against Firmicutes.</title><p>(<bold>A</bold>) Bacteria of the specified strain/species incubated overnight on tryptic soy agar after a 20-min liquid preincubation with the proteins indicated. Genera: <italic>B</italic> = <italic>Bacillus</italic>, <italic>P</italic> = <italic>Paenibacillus</italic>. Proteins: CEWL = chicken egg white lysozyme, <italic>P. poly</italic> = <italic>P. polymyxa</italic> lysozyme, PhiBP = bacteriophage PhiBP lysozyme, <italic>A. boo</italic> = GH25 domain of <italic>A. boonei</italic> lysozyme, CFP = cyan fluorescent protein. Images are representative of at least three independent experiments. (<bold>B</bold>) Dose-dependence of <italic>A. boonei</italic> GH25 muramidase antibacterial action. <italic>B. subtilis</italic> colony survival is shown after incubation with <italic>A. boonei</italic> GH25 muramidase at the indicated concentrations for 20 min at 37°C. N = 10 for each concentration. p < 0.001 for linear model fit. Error bars are ± SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.014">http://dx.doi.org/10.7554/eLife.04266.014</ext-link></p></caption><graphic xlink:href="elife04266f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04266.015</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Lysozyme purifications.</title><p>PAGE gel stained with GelCode blue before and after purification of 6x-histidine tagged enzymes using nickel affinity chromatography. L = crude <italic>E.</italic> coli lysate expressing the indicated lysozyme, E = elution after lysozyme purification. <italic>P. poly</italic> = <italic>P. polymyxa</italic> lysozyme, PhiBP = bacteriophage PhiBP lysozyme, <italic>A. boo</italic> = <italic>A. boonei</italic> GH25 domain.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.015">http://dx.doi.org/10.7554/eLife.04266.015</ext-link></p></caption><graphic xlink:href="elife04266fs007"/></fig><fig id="fig6s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04266.016</object-id><label>Figure 6—figure supplement 2.</label><caption><title>Antibacterial test of <italic>A. boonei</italic> GH25 muramidase on non-Firmicutes bacteria.</title><p>Bacteria of the specified strain/species incubated overnight on tryptic soy agar after a 20-min liquid preincubation with the proteins indicated. Genera: L = <italic>Listeria</italic>, <italic>S</italic> = <italic>Staphylococcus</italic>, <italic>E. saccharolyticus</italic> = <italic>Enterococcus, M</italic> = <italic>Micrococcus</italic>, <italic>E. cloacae</italic> = <italic>Enterobacter</italic>, <italic>E. coli</italic> = <italic>Escherichia</italic>, <italic>S</italic> = <italic>Serratia</italic>, <italic>D</italic> = <italic>Deinococcus</italic>. Proteins: CEWL = chicken egg white lysozyme, <italic>P. poly</italic> = <italic>P. polymyxa</italic> lysozyme, PhiBP = bacteriophage PhiBP lysozyme, <italic>A. boo</italic> = GH25 domain of <italic>A. boonei</italic> lysozyme, CFP = cyan fluorescent protein. Images are representative of at least three independent experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.016">http://dx.doi.org/10.7554/eLife.04266.016</ext-link></p></caption><graphic xlink:href="elife04266fs008"/></fig></fig-group></p><p>Third, the <italic>A. boonei</italic> muramidase domain is part of a larger gene (1725 bp) composed of other domains that may broaden or constrain the range of antibacterial activity. To test the full-length gene's function in the absence of genetic tools in this system, we cloned the entire gene into an expression plasmid in <italic>E. coli</italic> and discovered that bacterial colonies grew poorly, with tiny, slow-growing colonies on solid media, and substantial cell death coinciding with a small amount of leaky expression in liquid culture (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). However, two colonies grew to normal size and upon sequencing, we determined that their expression plasmids were disrupted by insertions of 774 bp (mutant 1) and 768 bp (mutant 2) of a native IS1 family transposase from <italic>E. coli</italic> at 21 bp or 266 bp from the start of the lysozyme gene, respectively. These insertions also resulted in a number of premature stop codons in the lysozyme reading frame, disrupting production of the full-length gene (<xref ref-type="fig" rid="fig7">Figure 7B</xref>). Thus, <italic>E. coli</italic> death requires intact and full-length lysozyme, and toxicity is not due to the expression construct itself. In sum, expression of the complete lysozyme resulted in <italic>E. coli</italic> death, while cloned genes with insertion sequences and premature stop codons abolished the lytic capacity of these proteins from within <italic>E. coli</italic> cells, providing evidence for an expanded host range to the antibacterial action.<fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.04266.017</object-id><label>Figure 7.</label><caption><title><italic>E. coli</italic> death following full-length <italic>A. boonei</italic> lysozyme expression.</title><p>(<bold>A</bold>) Live/dead stain of BL21 (DE3) <italic>E. coli</italic> transformed with expression constructs for the full-length lysozyme from <italic>A. boonei</italic> or a control lysozyme WORiA, a bacteriophage infecting <italic>Wolbachia pipientis</italic> strain <italic>w</italic>Ri, after overnight growth without induction. PAGE gels of crude <italic>E. coli</italic> lysates from <italic>E. coli</italic> expressing the indicated lysozyme after 6 hr of induction are also shown with the expected sizes of lysozymes indicated with arrows. (<bold>B</bold>) Structure of original full-length <italic>A</italic><italic>. boonei</italic> lysozyme expression plasmid and two spontaneous knockout mutants caused by insertion of 774 bp (mutant 1) and 768 bp (mutant 2) of IS1 transposase sequences. These insertions also resulted in a number of stop codons in the reading frame of the lysozyme. Knockout mutants grew to normal colony size, while all wild type colonies had intact expression plasmids, grew poorly, and died over time in liquid culture.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.017">http://dx.doi.org/10.7554/eLife.04266.017</ext-link></p></caption><graphic xlink:href="elife04266f007"/></fig></p><p>Fourth, if horizontally transferred lysozymes serve as antibacterials to fend off bacterial niche competitors, two predictions follow: the lysozyme will be upregulated in response to bacterial competition and upregulation may correlate with a relative growth advantage in coculture. We thus cultured <italic>A. boonei</italic> cells in anaerobic marine media (<xref ref-type="bibr" rid="bib63">Reysenbach et al., 2006</xref>) with and without cohabiting <italic>Mesoaciditoga lauensis</italic> (phylum Thermotogae) that was isolated from the same hydrothermal vent field as <italic>Aciduliprofundum</italic> in the Eastern Lau Spreading Center (<xref ref-type="bibr" rid="bib64">Reysenbach et al., 2013</xref>). As expected, we observed a significant increase in <italic>A. boonei</italic> lysozyme expression at four (up to 127% increase) and 12 hr (up to 43% increase) of coculture with <italic>M. lauensis</italic> in comparison to negative control cultures of the singular <italic>A. boonei</italic> (<xref ref-type="fig" rid="fig8">Figure 8A</xref>). Ideally, <italic>A. boonei</italic> wild type and lysozyme knockouts would be employed to test relative fitness and bacterial inhibition. However, genetic manipulation of <italic>A. boonei</italic> is not currently feasible.<fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.04266.018</object-id><label>Figure 8.</label><caption><title>Lysozyme expression and relative fitness during <italic>A. boonei</italic> and <italic>M. lauensis</italic> coculture.</title><p>(<bold>A</bold>) Expression of <italic>A. boonei</italic> GH25 muramidase relative to the control gene elongation factor 1α, after the indicated time of coculture with <italic>M. lauensis (M.l)</italic> at the specified ratio relative to <italic>A. boonei</italic>. *p < 0.05, **p < 0.01, by Mann–Whitney U pairwise comparisons. N = 6 for all samples. Primers are listed in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>. (<bold>B</bold>) Relative fitness of <italic>A. boonei</italic> vs <italic>M. lauensis</italic> in monoculture (N = 5) and coculture (N = 4). (<bold>C</bold>) Growth of <italic>A. boonei</italic> (red) and <italic>M. lauensis</italic> (blue) monocultures over time. Significant differences in cell abundance occur at 24, 52, and 64 hr (p < 0.05), and 56 and 60 hr (p < 0.01) based on pairwise Wilcoxon tests. (<bold>D</bold>) Growth of <italic>A. boonei</italic> and <italic>M. lauensis</italic> in coculture over time. Significant differences in cell abundance occur at 48, 52, and 64 hr (p < 0.05) based on pairwise Wilcoxon tests. Error bars are ±SEM for all panels.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.018">http://dx.doi.org/10.7554/eLife.04266.018</ext-link></p></caption><graphic xlink:href="elife04266f008"/></fig></p><p>Growth experiments of <italic>A. boonei</italic> and <italic>M. lauensis</italic> were continued for 72 hr, during which there was a relative Malthusian fitness (<xref ref-type="bibr" rid="bib43">Lenski et al., 1991</xref>) increase for <italic>A. boonei</italic> in coculture vs. monoculture (<xref ref-type="fig" rid="fig8">Figure 8B</xref>) across the exponential growth phase. This difference is marginally non-significant, perhaps due to low sample sizes (p = 0.11, N = 5, MWU two-tailed test). When the species are cultured separately for 72 hr, <italic>M. lauensis</italic> cell abundance is greater than that of <italic>A. boonei</italic> during 14 out of the 19 sampling points (<xref ref-type="fig" rid="fig8">Figure 8C</xref>, blue circles), indicating that bacteria outperform archaea in monoculture conditions. However, when the two species are cocultured, the cell abundances reverse and <italic>A. boonei</italic> outperforms <italic>M. lauensis</italic> for 14 out of the 19 time points (<xref ref-type="fig" rid="fig8">Figure 8D</xref>, red circles). This competitive frequency difference is significant (Chi-square test, p = 0.0035), complementing the Malthusian fitness increase. Additionally, for each monoculture time point, there are 4.43% fewer <italic>A. boonei</italic> cells on average than <italic>M. lauensis</italic>, while in coculture there are 6.22% more <italic>A. boonei</italic> cells per time point (Mann Whitney U. p = 0.023). Thus, <italic>A. boonei</italic> outcompetes its bacterial competitor in coculture despite a higher monoculture growth rate for the bacteria, although whether lysozyme upregulation is directly responsible for this effect cannot be definitively proven. Other possible explanations for this finding include <italic>A. boonei</italic> scavenging of bacterial waste products, possessing a superior ability to obtain a rate-limiting nutrient, or deploying alternative antibacterial defense mechanisms.</p></sec><sec id="s2-4"><title>Conclusions</title><p>The universal tree represents the evolutionary relationships between cellular domains and establishes the modern foundation for benchmarking the magnitude of HGT across life. Indeed, HGTs have been described between each domain including archaea and bacteria (<xref ref-type="bibr" rid="bib52">Nelson et al., 1999</xref>; <xref ref-type="bibr" rid="bib71">van Wolferen et al., 2013</xref>), bacteria and eukaryote (<xref ref-type="bibr" rid="bib5">Andersson, 2005</xref>; <xref ref-type="bibr" rid="bib13">Bordenstein, 2007</xref>; <xref ref-type="bibr" rid="bib31">Gladyshev et al., 2008</xref>; <xref ref-type="bibr" rid="bib21">Danchin et al., 2010</xref>), and archaea and eukaryote (<xref ref-type="bibr" rid="bib6">Andersson et al., 2003</xref>; <xref ref-type="bibr" rid="bib66">Schonknecht et al., 2013</xref>). Despite these cases and others (<xref ref-type="bibr" rid="bib16">Brown, 2003</xref>; <xref ref-type="bibr" rid="bib77">Zhaxybayeva and Doolittle, 2011</xref>), HGTs are not without limits and often succumb to the selective costs of genomic rearrangements, cytotoxic effects, disruptive insertions, and functional inefficiencies upon integration (<xref ref-type="bibr" rid="bib9">Baltrus, 2013</xref>).</p><p>It follows then that HGTs do not occur at equal rates across the universal tree, but rather experience preferential routes in which the costs of HGT are easier to overcome. The resulting pattern of HGT can be understood as a gradient of decreasing frequency from within domain > between two domains > between all domains of life (<xref ref-type="bibr" rid="bib17">Bruto et al., 2013</xref>; <xref ref-type="bibr" rid="bib77">Zhaxybayeva and Doolittle, 2011</xref>; <xref ref-type="bibr" rid="bib60">Puigbo et al., 2009</xref>; <xref ref-type="bibr" rid="bib3">Andam and Gogarten, 2011</xref>, <xref ref-type="bibr" rid="bib4">2013</xref>). In support of this pattern, the overwhelming evidence of gene transfers between bacteria is counterbalanced by the extreme lack of parallel gene transfers across all extant groups of life. As these parallel transfers are usually ancient and occur in non-model organisms (<xref ref-type="bibr" rid="bib45">Lundin et al., 2010</xref>; <xref ref-type="bibr" rid="bib41">Koonin et al., 2003</xref>; <xref ref-type="bibr" rid="bib48">McClure, 2001</xref>; <xref ref-type="bibr" rid="bib49">McDonald et al., 2012</xref>; <xref ref-type="bibr" rid="bib51">Moran et al., 2012</xref>), they can defy clear interpretations due to their deep antiquity and lack of functional validation.</p><p>One feature that parallel HGTs have in common is that the gene's phenotype must transcend different physiological capabilities, cellular structures, and ecological niches to repeatedly increase the fitness of each recipient across the whole diversity of life. While not traditionally used in the context of parallel HGT across all cellular domains, the term niche-transcending gene appropriately captures these conditions (<xref ref-type="bibr" rid="bib75">Wiedenbeck and Cohan, 2011</xref>). The lysozyme gene family we describe in archaea, bacteria, eukaryotes, and viruses provides one such example because the adaptive benefit of an antibacterial muramidase has repeatedly surmounted the obstacles against recurrent HGT. Indeed, horizontally transferred homologs of the GH25 muramidase exhibit differential tissue expression in <italic>A. pisum</italic> (<xref ref-type="bibr" rid="bib53">Nikoh et al., 2010</xref>) and bacteriolytic activity in the fungus <italic>Aspergillus nidulans</italic> (AN6470.2) (<xref ref-type="bibr" rid="bib11">Bauer et al., 2006</xref>). Thus, the horizontally transferred homologs in eukaryotes confer the same transcriptional and enzymatic activity as in the archaea.</p><p>The muramidase in a thermophilic archaeon is of special note as archaea do not possess murein cell walls (<xref ref-type="bibr" rid="bib2">Albers and Meyer, 2011</xref>), and genes encoding an antibacterial peptide have never before been identified (<xref ref-type="bibr" rid="bib19">Cantarel et al., 2009</xref>). Members of the genus <italic>Aciduliprofundum</italic> are widespread thermoacidophiles in deep-sea hydrothermal vent chimney biofilms (<xref ref-type="bibr" rid="bib30">Flores et al., 2012</xref>) in which bacteria are frequent inhabitants (<xref ref-type="bibr" rid="bib56">Orcutt et al., 2011a</xref>; <xref ref-type="bibr" rid="bib50">Miroshnichenko and Bonch-Osmolovskaya 2006</xref>), including the <italic>M. lauensis</italic> species tested above. Archaea have been largely ignored in the context of antibiotic discovery, likely because of the conjecture that archaea do not compete with bacteria in nature. However, given that they coexist with diverse bacterial species in the environment (<xref ref-type="bibr" rid="bib58">Oren, 2002</xref>; <xref ref-type="bibr" rid="bib37">Kato and Watanabe, 2010</xref>; <xref ref-type="bibr" rid="bib57">Orcutt et al., 2011b</xref>) and can compete for similar resources, there may be significant, unexploited potential for antibiotics in this domain. Only a handful of antimicrobial peptides produced by archaea have been characterized, and those are active only against other archaea (<xref ref-type="bibr" rid="bib55">O'Connor and Shand 2002</xref>) despite the fact that archaea are known to inhibit bacteria in diverse environments (<xref ref-type="bibr" rid="bib7">Atanasova et al., 2013</xref>; <xref ref-type="bibr" rid="bib67">Shand and Levya, 2008</xref>). It is also possible that since <italic>Aciduliprofundum</italic> strains metabolize peptides, the lysozyme enables a nutritive strategy in which lysed bacteria provide nutrients for the archaeon to scavenge.</p><p>Based on this work, we suspect that systematic surveys of archaeal gene products will likely uncover a broad range of antibacterial activities, and may eventually offer novel peptide or small molecule therapeutics. Such antibacterial products may have naturally evolved thermostability that would increase their attractiveness as therapeutics. GH25 muramidases have been demonstrated as effective antibacterials against biofilms of <italic>Streptococcus pneumoniae</italic> (<xref ref-type="bibr" rid="bib23">Domenech et al., 2011</xref>) and related enzymes have proven efficacious in mouse models of bacterial mucosal colonization (<xref ref-type="bibr" rid="bib29">Fenton et al., 2010</xref>), sepsis (<xref ref-type="bibr" rid="bib44">Loeffler et al., 2003</xref>), and endocarditis (<xref ref-type="bibr" rid="bib27">Entenza et al., 2005</xref>).</p><p>In summary, we infer that the evolutionary path to this parallel HGT was paved by the universal drive for nonbacterial taxa to compete in a bacterial world. We predict that similar to the cascade of antibiotic gene transfer discoveries that followed their initial reporting, parallel transfers of genes to all cellular domains and viruses might regularly have antimicrobial functions.</p></sec></sec><sec id="s3" sec-type="materials|methods"><title>Materials and methods</title><p>Unless otherwise stated, reagents were obtained from Fisher Scientific (Waltham, WI).</p><sec id="s3-1"><title>PCR and sequencing</title><p>PCR was performed using GoTaq DNA Polymerase (Promega, Madison, WI) with primers listed in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>. PCR products were electrophoresed using 1% agarose gels in sodium boric acid buffer. Following electrophoresis, gels were dyed with GelRed (Phenix Research, Candler, NC) and imaged on an Alpha Innotech GelRed Imager (Alpha Innotech, San Leandro, CA). Amplified bands were excised from the gels and purified with an SV Wizard Gel Cleanup kit (Promega). Following purification, DNA concentration was measured using the Qubit DNA high sensitivity kit (Life Technologies, Grand Island, NY) and sequencing reactions were performed by Genewiz (South Plainfield, NJ).</p></sec><sec id="s3-2"><title>Bioinformatics</title><p>The lysozyme protein from <italic>Wolbachia</italic> prophage WORiA (ZP_00372884) was used as a query in a blastp search of the NCBI nonredundant protein database using Geneious Pro v5.5.6. All hits with E-values below 10<sup>−12</sup> were collected and duplicate entries were removed. Sequences from field and laboratory samples were added to this collection and aligned with MUSCLE (<xref ref-type="bibr" rid="bib26">Edgar, 2004</xref>), insertions and deletions were removed, and the eight most highly conserved residues from the MUSCLE alignment were mapped to a structure prediction of <italic>A. boonei</italic> lysozyme using PyMOL. Structure prediction was performed using the homology-based modeling tool Phyre2 (<xref ref-type="bibr" rid="bib39">Kelley and Sternberg, 2009</xref>). For phylogenetic analyses, ProtTest (<xref ref-type="bibr" rid="bib1">Abascal et al., 2005</xref>) was used to determine the best model of protein evolution based on the corrected Akaike information criterion (AICc). MrBayes (<xref ref-type="bibr" rid="bib65">Ronquist et al., 2012</xref>) and PhyML (<xref ref-type="bibr" rid="bib33">Guindon et al., 2010</xref>) were used to build a phylogenetic tree with Bayesian and maximum likelihood methods, respectively. For the global lysozyme phylogeny, the best model chosen by ProtTest (LG + I + G) was used to generate the maximum likelihood tree, while the third best model (WAG + I + G; ΔAICc: 74.82) was used to generate the Bayesian tree due to a lack of LG model availability in MrBayes. <italic>S. sanguinolenta</italic> and <italic>S. stauntoniana</italic> lysozymes were excluded from this analysis because frameshift mutations suggest the genes may be evolving in the absence of selection, while Aphidinae lysozymes were not included because of shorter sequences of the GH25 muramidase domain obtained through the use of degenerate primers that would have limited resolution of the tree.</p><p>In an iterative approach, each candidate example of HGT was used as a blastp query against the nr database and the top 15 (<italic>A. boonei</italic>, <italic>A. pisum</italic>, <italic>S. moellendorffii</italic>) or top 75 (<italic>A. oryzae</italic>) E-value hits were subjected to the same phylogenetic analysis as above. Evolutionary models used were <italic>A. boonei</italic>: WAG + I + G on a 148aa indel-free alignment, <italic>A. pisum</italic>: CpREV + I + G on a 190aa indel-free alignment, <italic>S. moellendorffii</italic>: WAG + I + G (ΔAICc: 44.28) on a 200aa indel-free alignment, <italic>A. oryzae</italic>: WAG + G (Bayesian, ΔAICc: 4.49) or LG + G (maximum likelihood) on a 186aa indel-free alignment. The fungal lysozyme was also phylogenetically analyzed on the DNA level using the top 25 E-value blastn hits to exon 2 of the <italic>A. oryzae</italic> lysozyme gene. jModelTest 2 (<xref ref-type="bibr" rid="bib22">Darriba et al., 2012</xref>) was used to determine the best model of nucleic acid evolution (GTR + I + G, ΔAICc: 7.33) of a 282 bp indel-free alignment. The archaea HGT clade was also analyzed phylogenetically with a Bayesian tree of selected taxa using lysozyme protein sequences (WAG + I + G, 185aa) and compared to 16S rRNA (GTR + G, 1,156 bp) for the same strains obtained from SILVA (<xref ref-type="bibr" rid="bib61">Quast et al., 2013</xref>). A schematic representation of putative HGT events was plotted on a Bayesian phylogeny based on the 16 s rRNA gene (GTR + G, 1,226 bp). Representative species used for this phylogeny were <italic>Magnetospirillum magneticum</italic> (Proteobacteria), <italic>Paenibacillus polymyxa</italic> (Firmicutes), <italic>Arthrospira maxima</italic> (Cyanobacteria), Crenarchaeota archaeon SCGC AAA471-B05, <italic>Chthoniobacter flavus</italic> (Verrucomicrobia), <italic>Thermotoga maritima</italic> (Thermotogae), <italic>Pedobacter saltans</italic> (Bacteroidetes), <italic>Streptomyces violaceusniger</italic> (Actinobacteria).</p><p>Statistical support for the HGT hypothesis was assessed with the Shimodaira–Hasegawa test (SH-test) (<xref ref-type="bibr" rid="bib68">Shimodaira and Hasegawa, 1999</xref>) as implemented in RAxML v.8.0.20 (<xref ref-type="bibr" rid="bib69">Stamatakis, 2014</xref>). An unresolved binary constraint tree was generated in MacClade v4.08, in which bacterial sequences are monophyletic, as are nonbacterial sequences, with all other topology unconstrained. This constraint tree was used to generate a maximum likelihood best tree with RAxML, using the same evolutionary models as above. The SH-test was then run comparing the maximum likelihood constrained tree to the unconstrained consensus Bayesian tree or to 100 bootstrap trees from the maximum likelihood analysis from PhyML. This procedure was repeated for a three-domain constraint tree consistent with the tree of life and with a constraint tree in which eukaryotic sequences were monophyletic and other sequences were unconstrained.</p></sec><sec id="s3-3"><title>Lysozyme cloning and purification</title><p><italic>A. boonei</italic> GH25 muramidase domain (ZP_04874596), <italic>P. polymyxa</italic> lysozyme (YP_003869492), and PhiBP lysozyme (CBA18122) were cloned and expressed with a 6x C-terminal histidine tag using an Expresso T7 Cloning and Expression System (Lucigen, Middleton, WI) according to the manufacturer's instructions. We also cloned the <italic>S. moellendorffii</italic> and <italic>A. oryzae</italic> GH25 muramidases, however recombinant 6× histidine-tagged proteins were insoluble when expressed in either <italic>E. coli</italic> or sf9 insect cells and attempts to solubilize them were unsuccessful. Sequence-confirmed expression plasmids and a control plasmid expressing cyan fluorescent protein (CFP) were transformed into HI-Control BL21 (DE3) <italic>E. coli</italic> cells. Cultures at an OD600 of ∼0.5 were induced with 1 mM IPTG for 6 hr, centrifuged, and frozen at −80°C until purification. Frozen pellets were resuspended in lysis buffer containing 10 mM Tris–HCl, pH 7.5, 300 mM NaCl, 0.5% Triton X-100, 0.3% sodium dodecyl sulfate, and 1 mM phenylmethylsulfonylfluoride and sonicated 5 times for 30 s with at least 1 min on ice between sonications. Samples were centrifuged and recombinant proteins were purified from supernatant using HisPur Ni-NTA chromatography cartridges (Thermo Scientific, Waltham, MA) according to manufacturer's instructions. Glycerol at a final concentration of 40% was added to enzymes in elution buffer for storage at −20°C for a maximum of 3 weeks before use in antibacterial assays. Purifications were analyzed with denaturing polyacrylamide gel electrophoresis and stained with GelCode Blue (Thermo Scientific).</p><p>Full-length <italic>A. boonei</italic> lysozyme and WORiA lysozyme were cloned into a pET-20b vector (EMD Millipore, Darmstadt, Germany) with a C-terminal 6× histidine tag and sequence-confirmed plasmids were transformed into BL21 (DE3) <italic>E. coli</italic> (EMD Millipore). Three colonies from each transformation were inoculated into <italic>LB</italic> media and grown to an OD600 of ∼0.5, induced for 4 hr with 1 mM IPTG and harvested for analysis on PAGE gels. Overnight cultures without induction were examined for bacterial death with a BacLight Live/Dead Stain (Life Technologies).</p></sec><sec id="s3-4"><title>Antibacterial assays</title><p>Purified <italic>A. boonei</italic> GH25 muramidase, <italic>P. polymyxa</italic> lysozyme, PhiBP lysozyme, CFP, and commercially purchased CEWL (Sigma-Aldrich, St. Louis, MO) were diluted to 100 μg/ml in buffer EG (60% nickel column elution buffer, 40% glycerol) and filter sterilized. Bacteria to be tested were grown overnight in tryptic soy broth, split 1:10, and incubated to exponential growth before being diluted into each enzyme solution. Samples were incubated with shaking for 20 min at 37°C and then 5 μl was spotted onto tryptic soy agar and incubated overnight at 37°C. To evaluate whether antibacterial activity is dose-dependent, <italic>B. subtilis</italic> was incubated with <italic>A. boonei</italic> GH25 muramidase at 100 μg/ml, 75 μg/ml, 50 μg/ml, 25 μg/ml and 0 μg/ml and 100 μl was spread on tryptic soy agar plates. Replicates of 10 were performed for each concentration, plates were incubated overnight at 37°C, and colonies were counted the following morning. Bacterial strains used in these experiments are listed in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>.</p></sec><sec id="s3-5"><title>A. boonei cultures</title><p><italic>A. boonei</italic> and <italic>M. lauensis</italic> cultures were performed as previously described (<xref ref-type="bibr" rid="bib63">Reysenbach et al. 2006</xref>) with the following modifications: yeast extract was added at 2.0 g/l, pH was adjusted to 4.8, and cultures were incubated at 65°C. For gene expression studies, 8.2 × 10<sup>5</sup> cells were inoculated into 5 ml cultures in 6 replicates each of monocultures and cocultures at 0.1:1, 1:1, and 1:0.1 ratios and 500 μl samples were collected after 4 and 12 hr of co-incubation and frozen for expression analysis. RNA was isolated from frozen samples using an RNeasy Mini Kit (Qiagen) and QIAshredder (Qiagen), DNA contamination was removed with a Turbo DNAfree Kit (Life Technologies), and reverse transcription was performed using a Superscript III first Strand Synthesis System (Life Technologies) along with no-reverse transcriptase controls. Quantitative PCR was performed with GoTaq qPCR Master Mix (Promega) using a CFX96 Real-Time System (Bio-Rad, Hercules, CA). Primers are listed in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>. For competition studies, 5 replicates of 5 ml cultures were inoculated as monocultures or 1:1 cocultures and 175 μl was collected every 4 hr for counting of relative species abundance with a hemocytometer. Relative fitness was calculated based on Malthusian parameters over the period of exponential growth as previously described (<xref ref-type="bibr" rid="bib43">Lenski et al., 1991</xref>).</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We are grateful to members of the Bordenstein and Reysenbach labs for helpful discussions, and particularly to Christine Sislak for <italic>Mesoaciditoga lauensis</italic> cultures, Sarah Bordenstein for WO sequences, and Jessica Hardwicke for assistance with cell counting. We also thank Antonis Rokas, Jennifer Wisecleaver, Kristin Jernigan, and Elise Pfaltzgraff for technical assistance, and Julie Dunning Hotopp and Barton Slatko for helpful feedback on an earlier version of this manuscript, as well as Patrick Abbot for aphid samples and manuscript feedback, and Robert Brucker for support with figures.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>JAM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>LJF-J, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>KB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>A-LR, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con5"><p>SRB, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.04266.019</object-id><label>Supplementary file 1.</label><caption><p>Primers, field samples, and bacterial strains used in this study.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.019">http://dx.doi.org/10.7554/eLife.04266.019</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife04266s001.xlsx"/></supplementary-material><supplementary-material id="SD2-data"><object-id pub-id-type="doi">10.7554/eLife.04266.020</object-id><label>Supplementary file 2.</label><caption><p>Identity matrix of GH25 muramidases.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.020">http://dx.doi.org/10.7554/eLife.04266.020</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife04266s002.xlsx"/></supplementary-material><supplementary-material id="SD3-data"><object-id pub-id-type="doi">10.7554/eLife.04266.021</object-id><label>Supplementary file 3.</label><caption><p>Bordering gene BLAST results.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.021">http://dx.doi.org/10.7554/eLife.04266.021</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife04266s003.xlsx"/></supplementary-material><supplementary-material id="SD4-data"><object-id pub-id-type="doi">10.7554/eLife.04266.022</object-id><label>Supplementary file 4.</label><caption><p>Iterative HGT analysis BLAST results.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04266.022">http://dx.doi.org/10.7554/eLife.04266.022</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife04266s004.xlsx"/></supplementary-material></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Abascal</surname><given-names>F</given-names></name><name><surname>Zardoya</surname><given-names>R</given-names></name><name><surname>Posada</surname><given-names>D</given-names></name></person-group><year>2005</year><article-title>ProtTest: selection of best-fit models of protein evolution</article-title><source>Bioinformatics</source><volume>21</volume><fpage>2104</fpage><lpage>2105</lpage><pub-id pub-id-type="doi">10.1093/bioinformatics/bti263</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Albers</surname><given-names>SV</given-names></name><name><surname>Meyer</surname><given-names>BH</given-names></name></person-group><year>2011</year><article-title>The archaeal cell envelope</article-title><source>Nature Reviews Microbiology</source><volume>9</volume><fpage>414</fpage><lpage>426</lpage><pub-id pub-id-type="doi">10.1038/nrmicro2576</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Andam</surname><given-names>CP</given-names></name><name><surname>Gogarten</surname><given-names>JP</given-names></name></person-group><year>2011</year><article-title>Biased gene transfer in microbial evolution</article-title><source>Nature Reviews Microbiology</source><volume>9</volume><fpage>543</fpage><lpage>555</lpage><pub-id pub-id-type="doi">10.1038/nrmicro2593</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Andam</surname><given-names>CP</given-names></name><name><surname>Gogarten</surname><given-names>JP</given-names></name></person-group><year>2013</year><article-title>Biased gene transfer contributes to maintaining the tree of life</article-title><source>Lateral gene transfer in evolution</source><person-group person-group-type="editor"><name><surname>Gophna</surname><given-names>U</given-names></name></person-group><fpage>263</fpage><lpage>274</lpage></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Andersson</surname><given-names>JO</given-names></name></person-group><year>2005</year><article-title>Lateral gene transfer in eukaryotes</article-title><source>Cellular and Molecular Life Sciences</source><volume>62</volume><fpage>1182</fpage><lpage>1197</lpage><pub-id pub-id-type="doi">10.1007/s00018-005-4539-z</pub-id></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Andersson</surname><given-names>JO</given-names></name><name><surname>Sjogren</surname><given-names>AM</given-names></name><name><surname>Davis</surname><given-names>LA</given-names></name><name><surname>Embley</surname><given-names>TM</given-names></name><name><surname>Roger</surname><given-names>AJ</given-names></name></person-group><year>2003</year><article-title>Phylogenetic analyses of diplomonad genes reveal frequent lateral gene transfers affecting eukaryotes</article-title><source>Current Biology</source><volume>13</volume><fpage>94</fpage><lpage>104</lpage><pub-id pub-id-type="doi">10.1016/S0960-9822(03)00003-4</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Atanasova</surname><given-names>NS</given-names></name><name><surname>Pietila</surname><given-names>MK</given-names></name><name><surname>Oksanen</surname><given-names>HM</given-names></name></person-group><year>2013</year><article-title>Diverse antimicrobial interactions of halophilic archaea and bacteria extend over geographical distances and cross the domain barrier</article-title><source>MicrobiologyOpen</source><volume>2</volume><fpage>811</fpage><lpage>825</lpage><pub-id pub-id-type="doi">10.1002/mbo3.115</pub-id></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Augustinos</surname><given-names>AA</given-names></name><name><surname>Santos-Garcia</surname><given-names>D</given-names></name><name><surname>Dionyssopoulou</surname><given-names>E</given-names></name><name><surname>Moreira</surname><given-names>M</given-names></name><name><surname>Papapanagiotou</surname><given-names>A</given-names></name><name><surname>Scarvelakis</surname><given-names>M</given-names></name><name><surname>Doudoumis</surname><given-names>V</given-names></name><name><surname>Ramos</surname><given-names>S</given-names></name><name><surname>Aguiar</surname><given-names>AF</given-names></name><name><surname>Borges</surname><given-names>PA</given-names></name><name><surname>Khadem</surname><given-names>M</given-names></name><name><surname>Latorre</surname><given-names>A</given-names></name><name><surname>Tsiamis</surname><given-names>G</given-names></name><name><surname>Bourtzis</surname><given-names>K</given-names></name></person-group><year>2011</year><article-title>Detection and characterization of Wolbachia infections in natural populations of aphids: is the hidden diversity fully unraveled?</article-title><source>PLOS ONE</source><volume>6</volume><fpage>e28695</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0028695</pub-id></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Baltrus</surname><given-names>DA</given-names></name></person-group><year>2013</year><article-title>Exploring the costs of horizontal gene transfer</article-title><source>Trends in Ecology & Evolution</source><volume>28</volume><fpage>489</fpage><lpage>495</lpage><pub-id pub-id-type="doi">10.1016/j.tree.2013.04.002</pub-id></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Banks</surname><given-names>JA</given-names></name><name><surname>Nishiyama</surname><given-names>T</given-names></name><name><surname>Hasebe</surname><given-names>M</given-names></name><name><surname>Bowman</surname><given-names>JL</given-names></name><name><surname>Gribskov</surname><given-names>M</given-names></name><name><surname>dePamphilis</surname><given-names>C</given-names></name><name><surname>Albert</surname><given-names>VA</given-names></name><name><surname>Aono</surname><given-names>N</given-names></name><name><surname>Aoyama</surname><given-names>T</given-names></name><name><surname>Ambrose</surname><given-names>BA</given-names></name><name><surname>Ashton</surname><given-names>NW</given-names></name><name><surname>Axtell</surname><given-names>MJ</given-names></name><name><surname>Barker</surname><given-names>E</given-names></name><name><surname>Barker</surname><given-names>MS</given-names></name><name><surname>Bennetzen</surname><given-names>JL</given-names></name><name><surname>Bonawitz</surname><given-names>ND</given-names></name><name><surname>Chapple</surname><given-names>C</given-names></name><name><surname>Cheng</surname><given-names>C</given-names></name><name><surname>Correa</surname><given-names>LG</given-names></name><name><surname>Dacre</surname><given-names>M</given-names></name><name><surname>DeBarry</surname><given-names>J</given-names></name><name><surname>Dreyer</surname><given-names>I</given-names></name><name><surname>Elias</surname><given-names>M</given-names></name><name><surname>Engstrom</surname><given-names>EM</given-names></name><name><surname>Estelle</surname><given-names>M</given-names></name><name><surname>Feng</surname><given-names>L</given-names></name><name><surname>Finet</surname><given-names>C</given-names></name><name><surname>Floyd</surname><given-names>SK</given-names></name><name><surname>Frommer</surname><given-names>WB</given-names></name><name><surname>Fujita</surname><given-names>T</given-names></name><name><surname>Gramzow</surname><given-names>L</given-names></name><name><surname>Gutensohn</surname><given-names>M</given-names></name><name><surname>Harholt</surname><given-names>J</given-names></name><name><surname>Hattori</surname><given-names>M</given-names></name><name><surname>Heyl</surname><given-names>A</given-names></name><name><surname>Hirai</surname><given-names>T</given-names></name><name><surname>Hiwatashi</surname><given-names>Y</given-names></name><name><surname>Ishikawa</surname><given-names>M</given-names></name><name><surname>Iwata</surname><given-names>M</given-names></name><name><surname>Karol</surname><given-names>KG</given-names></name><name><surname>Koehler</surname><given-names>B</given-names></name><name><surname>Kolukisaoglu</surname><given-names>U</given-names></name><name><surname>Kubo</surname><given-names>M</given-names></name><name><surname>Kurata</surname><given-names>T</given-names></name><name><surname>Lalonde</surname><given-names>S</given-names></name><name><surname>Li</surname><given-names>K</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Litt</surname><given-names>A</given-names></name><name><surname>Lyons</surname><given-names>E</given-names></name><name><surname>Manning</surname><given-names>G</given-names></name><name><surname>Maruyama</surname><given-names>T</given-names></name><name><surname>Michael</surname><given-names>TP</given-names></name><name><surname>Mikami</surname><given-names>K</given-names></name><name><surname>Miyazaki</surname><given-names>S</given-names></name><name><surname>Morinaga</surname><given-names>S</given-names></name><name><surname>Murata</surname><given-names>T</given-names></name><name><surname>Mueller-Roeber</surname><given-names>B</given-names></name><name><surname>Nelson</surname><given-names>DR</given-names></name><name><surname>Obara</surname><given-names>M</given-names></name><name><surname>Oguri</surname><given-names>Y</given-names></name><name><surname>Olmstead</surname><given-names>RG</given-names></name><name><surname>Onodera</surname><given-names>N</given-names></name><name><surname>Petersen</surname><given-names>BL</given-names></name><name><surname>Pils</surname><given-names>B</given-names></name><name><surname>Prigge</surname><given-names>M</given-names></name><name><surname>Rensing</surname><given-names>SA</given-names></name><name><surname>Riano-Pachon</surname><given-names>DM</given-names></name><name><surname>Roberts</surname><given-names>AW</given-names></name><name><surname>Sato</surname><given-names>Y</given-names></name><name><surname>Scheller</surname><given-names>HV</given-names></name><name><surname>Schulz</surname><given-names>B</given-names></name><name><surname>Schulz</surname><given-names>C</given-names></name><name><surname>Shakirov</surname><given-names>EV</given-names></name><name><surname>Shibagaki</surname><given-names>N</given-names></name><name><surname>Shinohara</surname><given-names>N</given-names></name><name><surname>Shippen</surname><given-names>DE</given-names></name><name><surname>Sorensen</surname><given-names>I</given-names></name><name><surname>Sotooka</surname><given-names>R</given-names></name><name><surname>Sugimoto</surname><given-names>N</given-names></name><name><surname>Sugita</surname><given-names>M</given-names></name><name><surname>Sumikawa</surname><given-names>N</given-names></name><name><surname>Tanurdzic</surname><given-names>M</given-names></name><name><surname>Theissen</surname><given-names>G</given-names></name><name><surname>Ulvskov</surname><given-names>P</given-names></name><name><surname>Wakazuki</surname><given-names>S</given-names></name><name><surname>Weng</surname><given-names>JK</given-names></name><name><surname>Willats</surname><given-names>WW</given-names></name><name><surname>Wipf</surname><given-names>D</given-names></name><name><surname>Wolf</surname><given-names>PG</given-names></name><name><surname>Yang</surname><given-names>L</given-names></name><name><surname>Zimmer</surname><given-names>AD</given-names></name><name><surname>Zhu</surname><given-names>Q</given-names></name><name><surname>Mitros</surname><given-names>T</given-names></name><name><surname>Hellsten</surname><given-names>U</given-names></name><name><surname>Loque</surname><given-names>D</given-names></name><name><surname>Otillar</surname><given-names>R</given-names></name><name><surname>Salamov</surname><given-names>A</given-names></name><name><surname>Schmutz</surname><given-names>J</given-names></name><name><surname>Shapiro</surname><given-names>H</given-names></name><name><surname>Lindquist</surname><given-names>E</given-names></name><name><surname>Lucas</surname><given-names>S</given-names></name><name><surname>Rokhsar</surname><given-names>D</given-names></name><name><surname>Grigoriev</surname><given-names>IV</given-names></name></person-group><year>2011</year><article-title>The Selaginella genome identifies genetic changes associated with the evolution of vascular plants</article-title><source>Science</source><volume>332</volume><fpage>960</fpage><lpage>963</lpage><pub-id pub-id-type="doi">10.1126/science.1203810</pub-id></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bauer</surname><given-names>S</given-names></name><name><surname>Vasu</surname><given-names>P</given-names></name><name><surname>Persson</surname><given-names>S</given-names></name><name><surname>Mort</surname><given-names>AJ</given-names></name><name><surname>Somerville</surname><given-names>CR</given-names></name></person-group><year>2006</year><article-title>Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>103</volume><fpage>11417</fpage><lpage>11422</lpage><pub-id pub-id-type="doi">10.1073/pnas.0604632103</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Beiko</surname><given-names>RG</given-names></name><name><surname>Harlow</surname><given-names>TJ</given-names></name><name><surname>Ragan</surname><given-names>MA</given-names></name></person-group><year>2005</year><article-title>Highways of gene sharing in prokaryotes</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>102</volume><fpage>14332</fpage><lpage>14337</lpage><pub-id pub-id-type="doi">10.1073/pnas.0504068102</pub-id></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bordenstein</surname><given-names>SR</given-names></name></person-group><year>2007</year><article-title>Evolutionary genomics: transdomain gene transfers</article-title><source>Current Biology</source><volume>17</volume><fpage>R935</fpage><lpage>R936</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2007.09.022</pub-id></element-citation></ref><ref id="bib14"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bratke</surname><given-names>KA</given-names></name><name><surname>McLysaght</surname><given-names>A</given-names></name></person-group><year>2008</year><article-title>Identification of multiple independent horizontal gene transfers into poxviruses using a comparative genomics approach</article-title><source>BMC Evolutionary Biology</source><volume>8</volume><fpage>67</fpage><pub-id pub-id-type="doi">10.1186/1471-2148-8-67</pub-id></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brochier-Armanet</surname><given-names>C</given-names></name><name><surname>Forterre</surname><given-names>P</given-names></name><name><surname>Gribaldo</surname><given-names>S</given-names></name></person-group><year>2011</year><article-title>Phylogeny and evolution of the Archaea: one hundred genomes later</article-title><source>Current Opinion in Microbiology</source><volume>14</volume><fpage>274</fpage><lpage>281</lpage><pub-id pub-id-type="doi">10.1016/j.mib.2011.04.015</pub-id></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brown</surname><given-names>JR</given-names></name></person-group><year>2003</year><article-title>Ancient horizontal gene transfer</article-title><source>Nature Reviews Genetics</source><volume>4</volume><fpage>121</fpage><lpage>132</lpage><pub-id pub-id-type="doi">10.1038/nrg1000</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Bruto</surname><given-names>B</given-names></name><name><surname>Prigent-Combaret</surname><given-names>C</given-names></name><name><surname>Luis</surname><given-names>P</given-names></name><name><surname>Hoff</surname><given-names>G</given-names></name><name><surname>Moënne-Loccoz</surname><given-names>Y</given-names></name><name><surname>Muller</surname><given-names>D</given-names></name></person-group><year>2013</year><article-title>Horizontal acquisition of Prokaryotic genes for eukaryote functioning and niche adaptation</article-title><source>Evolutionary Biology: Exobiology and evolutionary mechanisms</source><person-group person-group-type="editor"><name><surname>Pontarotti</surname><given-names>P</given-names></name></person-group><publisher-name>Springer Berlin Heidelberg</publisher-name><fpage>165</fpage><lpage>179</lpage></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bulgarelli</surname><given-names>D</given-names></name><name><surname>Schlaeppi</surname><given-names>K</given-names></name><name><surname>Spaepen</surname><given-names>S</given-names></name><name><surname>Ver Loren van Themaat</surname><given-names>E</given-names></name><name><surname>Schulze-Lefert</surname><given-names>P</given-names></name></person-group><year>2013</year><article-title>Structure and functions of the bacterial microbiota of plants</article-title><source>Annual Review of Plant Biology</source><volume>64</volume><fpage>807</fpage><lpage>838</lpage><pub-id pub-id-type="doi">10.1146/annurev-arplant-050312-120106</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cantarel</surname><given-names>BL</given-names></name><name><surname>Coutinho</surname><given-names>PM</given-names></name><name><surname>Rancurel</surname><given-names>C</given-names></name><name><surname>Bernard</surname><given-names>T</given-names></name><name><surname>Lombard</surname><given-names>V</given-names></name><name><surname>Henrissat</surname><given-names>B</given-names></name></person-group><year>2009</year><article-title>The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics</article-title><source>Nucleic Acids Research</source><volume>37</volume><fpage>D233</fpage><lpage>D238</lpage><pub-id pub-id-type="doi">10.1093/nar/gkn663</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Clardy</surname><given-names>J</given-names></name><name><surname>Fischbach</surname><given-names>MA</given-names></name><name><surname>Currie</surname><given-names>CR</given-names></name></person-group><year>2009</year><article-title>The natural history of antibiotics</article-title><source>Current Biology</source><volume>19</volume><fpage>R437</fpage><lpage>R441</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2009.04.001</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Danchin</surname><given-names>EG</given-names></name><name><surname>Rosso</surname><given-names>MN</given-names></name><name><surname>Vieira</surname><given-names>P</given-names></name><name><surname>de Almeida-Engler</surname><given-names>J</given-names></name><name><surname>Coutinho</surname><given-names>PM</given-names></name><name><surname>Henrissat</surname><given-names>B</given-names></name><name><surname>Abad</surname><given-names>P</given-names></name></person-group><year>2010</year><article-title>Multiple lateral gene transfers and duplications have promoted plant parasitism ability in nematodes</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>107</volume><fpage>17651</fpage><lpage>17656</lpage><pub-id pub-id-type="doi">10.1073/pnas.1008486107</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Darriba</surname><given-names>D</given-names></name><name><surname>Taboada</surname><given-names>GL</given-names></name><name><surname>Doallo</surname><given-names>R</given-names></name><name><surname>Posada</surname><given-names>D</given-names></name></person-group><year>2012</year><article-title>jModelTest 2: more models, new heuristics and parallel computing</article-title><source>Nature Methods</source><volume>9</volume><fpage>772</fpage><pub-id pub-id-type="doi">10.1038/nmeth.2109</pub-id></element-citation></ref><ref id="bib23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Domenech</surname><given-names>M</given-names></name><name><surname>Garcia</surname><given-names>E</given-names></name><name><surname>Moscoso</surname><given-names>M</given-names></name></person-group><year>2011</year><article-title>In vitro destruction of Streptococcus pneumoniae biofilms with bacterial and phage peptidoglycan hydrolases</article-title><source>Antimicrobial Agents and Chemotherapy</source><volume>55</volume><fpage>4144</fpage><lpage>4148</lpage><pub-id pub-id-type="doi">10.1128/AAC.00492-11</pub-id></element-citation></ref><ref id="bib24"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dunning Hotopp</surname><given-names>JC</given-names></name></person-group><year>2011</year><article-title>Horizontal gene transfer between bacteria and animals</article-title><source>Trends in Genetics</source><volume>27</volume><fpage>157</fpage><lpage>163</lpage><pub-id pub-id-type="doi">10.1016/j.tig.2011.01.005</pub-id></element-citation></ref><ref id="bib25"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dunning Hotopp</surname><given-names>JC</given-names></name><name><surname>Clark</surname><given-names>ME</given-names></name><name><surname>Oliveira</surname><given-names>DC</given-names></name><name><surname>Foster</surname><given-names>JM</given-names></name><name><surname>Fischer</surname><given-names>P</given-names></name><name><surname>Munoz Torres</surname><given-names>MC</given-names></name><name><surname>Giebel</surname><given-names>JD</given-names></name><name><surname>Kumar</surname><given-names>N</given-names></name><name><surname>Ishmael</surname><given-names>N</given-names></name><name><surname>Wang</surname><given-names>S</given-names></name><name><surname>Ingram</surname><given-names>J</given-names></name><name><surname>Nene</surname><given-names>RV</given-names></name><name><surname>Shepard</surname><given-names>J</given-names></name><name><surname>Tomkins</surname><given-names>J</given-names></name><name><surname>Richards</surname><given-names>S</given-names></name><name><surname>Spiro</surname><given-names>DJ</given-names></name><name><surname>Ghedin</surname><given-names>E</given-names></name><name><surname>Slatko</surname><given-names>BE</given-names></name><name><surname>Tettelin</surname><given-names>H</given-names></name><name><surname>Werren</surname><given-names>JH</given-names></name></person-group><year>2007</year><article-title>Widespread lateral gene transfer from intracellular bacteria to multicellular eukaryotes</article-title><source>Science</source><volume>317</volume><fpage>1753</fpage><lpage>1756</lpage><pub-id pub-id-type="doi">10.1126/science.1142490</pub-id></element-citation></ref><ref id="bib26"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Edgar</surname><given-names>RC</given-names></name></person-group><year>2004</year><article-title>MUSCLE: multiple sequence alignment with high accuracy and high throughput</article-title><source>Nucleic Acids Research</source><volume>32</volume><fpage>1792</fpage><lpage>1797</lpage><pub-id pub-id-type="doi">10.1093/nar/gkh340</pub-id></element-citation></ref><ref id="bib27"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Entenza</surname><given-names>JM</given-names></name><name><surname>Loeffler</surname><given-names>JM</given-names></name><name><surname>Grandgirard</surname><given-names>D</given-names></name><name><surname>Fischetti</surname><given-names>VA</given-names></name><name><surname>Moreillon</surname><given-names>P</given-names></name></person-group><year>2005</year><article-title>Therapeutic effects of bacteriophage Cpl-1 lysin against Streptococcus pneumoniae endocarditis in rats</article-title><source>Antimicrobial Agents and Chemotherapy</source><volume>49</volume><fpage>4789</fpage><lpage>4792</lpage><pub-id pub-id-type="doi">10.1128/AAC.49.11.4789-4792.2005</pub-id></element-citation></ref><ref id="bib28"><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Fastrez</surname><given-names>J</given-names></name></person-group><year>1996</year><article-title>Phage lysozymes</article-title><source>Lysozymes: model enzymes in Biochemistry and Biology</source><person-group person-group-type="editor"><name><surname>Jolles</surname><given-names>P</given-names></name></person-group><publisher-name>Birkhauser Verlag</publisher-name><fpage>35</fpage><lpage>64</lpage></element-citation></ref><ref id="bib29"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fenton</surname><given-names>M</given-names></name><name><surname>Casey</surname><given-names>PG</given-names></name><name><surname>Hill</surname><given-names>C</given-names></name><name><surname>Gahan</surname><given-names>CG</given-names></name><name><surname>Ross</surname><given-names>RP</given-names></name><name><surname>McAuliffe</surname><given-names>O</given-names></name><name><surname>O'Mahony</surname><given-names>J</given-names></name><name><surname>Maher</surname><given-names>F</given-names></name><name><surname>Coffey</surname><given-names>A</given-names></name></person-group><year>2010</year><article-title>The truncated phage lysin CHAP(k) eliminates <italic>Staphylococcus aureus</italic> in the nares of mice</article-title><source>Bioengineered Bugs</source><volume>1</volume><fpage>404</fpage><lpage>407</lpage><pub-id pub-id-type="doi">10.4161/bbug.1.6.13422</pub-id></element-citation></ref><ref id="bib30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Flores</surname><given-names>GE</given-names></name><name><surname>Wagner</surname><given-names>ID</given-names></name><name><surname>Liu</surname><given-names>Y</given-names></name><name><surname>Reysenbach</surname><given-names>AL</given-names></name></person-group><year>2012</year><article-title>Distribution, abundance, and diversity patterns of the thermoacidophilic ‘deep-sea hydrothermal vent euryarchaeota 2’</article-title><source>Frontiers in Microbiology</source><volume>3</volume><fpage>47</fpage><pub-id pub-id-type="doi">10.3389/fmicb.2012.00047</pub-id></element-citation></ref><ref id="bib31"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gladyshev</surname><given-names>EA</given-names></name><name><surname>Meselson</surname><given-names>M</given-names></name><name><surname>Arkhipova</surname><given-names>IR</given-names></name></person-group><year>2008</year><article-title>Massive horizontal gene transfer in bdelloid rotifers</article-title><source>Science</source><volume>320</volume><fpage>1210</fpage><lpage>1213</lpage><pub-id pub-id-type="doi">10.1126/science.1156407</pub-id></element-citation></ref><ref id="bib32"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gomez-Valero</surname><given-names>L</given-names></name><name><surname>Soriano-Navarro</surname><given-names>M</given-names></name><name><surname>Perez-Brocal</surname><given-names>V</given-names></name><name><surname>Heddi</surname><given-names>A</given-names></name><name><surname>Moya</surname><given-names>A</given-names></name><name><surname>Garcia-Verdugo</surname><given-names>JM</given-names></name><name><surname>Latorre</surname><given-names>A</given-names></name></person-group><year>2004</year><article-title>Coexistence of Wolbachia with Buchnera aphidicola and a secondary symbiont in the aphid Cinara cedri</article-title><source>Journal of Bacteriology</source><volume>186</volume><fpage>6626</fpage><lpage>6633</lpage><pub-id pub-id-type="doi">10.1128/JB.186.19.6626-6633.2004</pub-id></element-citation></ref><ref id="bib33"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Guindon</surname><given-names>S</given-names></name><name><surname>Dufayard</surname><given-names>JF</given-names></name><name><surname>Lefort</surname><given-names>V</given-names></name><name><surname>Anisimova</surname><given-names>M</given-names></name><name><surname>Hordijk</surname><given-names>W</given-names></name><name><surname>Gascuel</surname><given-names>O</given-names></name></person-group><year>2010</year><article-title>New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0</article-title><source>Systematic Biology</source><volume>59</volume><fpage>307</fpage><lpage>321</lpage><pub-id pub-id-type="doi">10.1093/sysbio/syq010</pub-id></element-citation></ref><ref id="bib34"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Halgasova</surname><given-names>N</given-names></name><name><surname>Ugorcakova</surname><given-names>J</given-names></name><name><surname>Gerova</surname><given-names>M</given-names></name><name><surname>Timko</surname><given-names>J</given-names></name><name><surname>Bukovska</surname><given-names>G</given-names></name></person-group><year>2010</year><article-title>Isolation and characterization of bacteriophage PhiBP from <italic>Paenibacillus polymyxa</italic> CCM 7400</article-title><source>FEMS Microbiology Letters</source><volume>305</volume><fpage>128</fpage><lpage>135</lpage><pub-id pub-id-type="doi">10.1111/j.1574-6968.2010.01908.x</pub-id></element-citation></ref><ref id="bib35"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Husnik</surname><given-names>F</given-names></name><name><surname>Nikoh</surname><given-names>N</given-names></name><name><surname>Koga</surname><given-names>R</given-names></name><name><surname>Ross</surname><given-names>L</given-names></name><name><surname>Duncan</surname><given-names>RP</given-names></name><name><surname>Fujie</surname><given-names>M</given-names></name><name><surname>Tanaka</surname><given-names>M</given-names></name><name><surname>Satoh</surname><given-names>N</given-names></name><name><surname>Bachtrog</surname><given-names>D</given-names></name><name><surname>Wilson</surname><given-names>AC</given-names></name><name><surname>von Dohlen</surname><given-names>CD</given-names></name><name><surname>Fukatsu</surname><given-names>T</given-names></name><name><surname>McCutcheon</surname><given-names>JP</given-names></name></person-group><year>2013</year><article-title>Horizontal gene transfer from diverse bacteria to an insect genome enables a tripartite nested mealybug symbiosis</article-title><source>Cell</source><volume>153</volume><fpage>1567</fpage><lpage>1578</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2013.05.040</pub-id></element-citation></ref><ref id="bib36"><element-citation publication-type="journal"><person-group person-group-type="author"><collab>International Aphid Genomics Consortium</collab></person-group><year>2010</year><article-title>Genome sequence of the pea aphid Acyrthosiphon pisum</article-title><source>PLOS Biology</source><volume>8</volume><fpage>e1000313</fpage><pub-id pub-id-type="doi">10.1371/journal.pbio.1000313</pub-id></element-citation></ref><ref id="bib37"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kato</surname><given-names>S</given-names></name><name><surname>Watanabe</surname><given-names>K</given-names></name></person-group><year>2010</year><article-title>Ecological and evolutionary interactions in syntrophic methanogenic consortia</article-title><source>Microbes and Environments/JSME</source><volume>25</volume><fpage>145</fpage><lpage>151</lpage><pub-id pub-id-type="doi">10.1264/jsme2.ME10122</pub-id></element-citation></ref><ref id="bib38"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Keeling</surname><given-names>PJ</given-names></name><name><surname>Palmer</surname><given-names>JD</given-names></name></person-group><year>2008</year><article-title>Horizontal gene transfer in eukaryotic evolution</article-title><source>Nature Reviews Genetics</source><volume>9</volume><fpage>605</fpage><lpage>618</lpage><pub-id pub-id-type="doi">10.1038/nrg2386</pub-id></element-citation></ref><ref id="bib39"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kelley</surname><given-names>LA</given-names></name><name><surname>Sternberg</surname><given-names>MJ</given-names></name></person-group><year>2009</year><article-title>Protein structure prediction on the Web: a case study using the Phyre server</article-title><source>Nature Protocols</source><volume>4</volume><fpage>363</fpage><lpage>371</lpage><pub-id pub-id-type="doi">10.1038/nprot.2009.2</pub-id></element-citation></ref><ref id="bib40"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kondrashov</surname><given-names>FA</given-names></name><name><surname>Koonin</surname><given-names>EV</given-names></name><name><surname>Morgunov</surname><given-names>IG</given-names></name><name><surname>Finogenova</surname><given-names>TV</given-names></name><name><surname>Kondrashova</surname><given-names>MN</given-names></name></person-group><year>2006</year><article-title>Evolution of glyoxylate cycle enzymes in Metazoa: evidence of multiple horizontal transfer events and pseudogene formation</article-title><source>Biology Direct</source><volume>1</volume><fpage>31</fpage><pub-id pub-id-type="doi">10.1186/1745-6150-1-31</pub-id></element-citation></ref><ref id="bib41"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Koonin</surname><given-names>EV</given-names></name><name><surname>Makarova</surname><given-names>KS</given-names></name><name><surname>Rogozin</surname><given-names>IB</given-names></name><name><surname>Davidovic</surname><given-names>L</given-names></name><name><surname>Letellier</surname><given-names>MC</given-names></name><name><surname>Pellegrini</surname><given-names>L</given-names></name></person-group><year>2003</year><article-title>The rhomboids: a nearly ubiquitous family of intramembrane serine proteases that probably evolved by multiple ancient horizontal gene transfers</article-title><source>Genome Biology</source><volume>4</volume><fpage>R19</fpage><pub-id pub-id-type="doi">10.1186/gb-2003-4-3-r19</pub-id></element-citation></ref><ref id="bib42"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Korczynska</surname><given-names>JE</given-names></name><name><surname>Danielsen</surname><given-names>S</given-names></name><name><surname>Schagerlof</surname><given-names>U</given-names></name><name><surname>Turkenburg</surname><given-names>JP</given-names></name><name><surname>Davies</surname><given-names>GJ</given-names></name><name><surname>Wilson</surname><given-names>KS</given-names></name><name><surname>Taylor</surname><given-names>EJ</given-names></name></person-group><year>2010</year><article-title>The structure of a family GH25 lysozyme from <italic>Aspergillus fumigatus</italic></article-title><source>Acta Crystallographica Section F, Structural Biology and Crystallization Communications</source><volume>66</volume><fpage>973</fpage><lpage>977</lpage><pub-id pub-id-type="doi">10.1107/S1744309110025601</pub-id></element-citation></ref><ref id="bib43"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lenski</surname><given-names>RE</given-names></name><name><surname>Rose</surname><given-names>MR</given-names></name><name><surname>Simpson</surname><given-names>SC</given-names></name><name><surname>Tadler</surname><given-names>SC</given-names></name></person-group><year>1991</year><article-title>Long-term Experimental evolution in <italic>Escherichia-Coli</italic> .1. Adaptation and divergence during 2,000 generations</article-title><source>American Naturalist</source><volume>138</volume><fpage>1315</fpage><lpage>1341</lpage><pub-id pub-id-type="doi">10.1086/285289</pub-id></element-citation></ref><ref id="bib44"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Loeffler</surname><given-names>JM</given-names></name><name><surname>Djurkovic</surname><given-names>S</given-names></name><name><surname>Fischetti</surname><given-names>VA</given-names></name></person-group><year>2003</year><article-title>Phage lytic enzyme Cpl-1 as a novel antimicrobial for pneumococcal bacteremia</article-title><source>Infection and Immunity</source><volume>71</volume><fpage>6199</fpage><lpage>6204</lpage><pub-id pub-id-type="doi">10.1128/IAI.71.11.6199-6204.2003</pub-id></element-citation></ref><ref id="bib45"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lundin</surname><given-names>D</given-names></name><name><surname>Gribaldo</surname><given-names>S</given-names></name><name><surname>Torrents</surname><given-names>E</given-names></name><name><surname>Sjoberg</surname><given-names>BM</given-names></name><name><surname>Poole</surname><given-names>AM</given-names></name></person-group><year>2010</year><article-title>Ribonucleotide reduction - horizontal transfer of a required function spans all three domains</article-title><source>BMC Evolutionary Biology</source><volume>10</volume><fpage>383</fpage><pub-id pub-id-type="doi">10.1186/1471-2148-10-383</pub-id></element-citation></ref><ref id="bib46"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Machida</surname><given-names>M</given-names></name><name><surname>Asai</surname><given-names>K</given-names></name><name><surname>Sano</surname><given-names>M</given-names></name><name><surname>Tanaka</surname><given-names>T</given-names></name><name><surname>Kumagai</surname><given-names>T</given-names></name><name><surname>Terai</surname><given-names>G</given-names></name><name><surname>Kusumoto</surname><given-names>K</given-names></name><name><surname>Arima</surname><given-names>T</given-names></name><name><surname>Akita</surname><given-names>O</given-names></name><name><surname>Kashiwagi</surname><given-names>Y</given-names></name><name><surname>Abe</surname><given-names>K</given-names></name><name><surname>Gomi</surname><given-names>K</given-names></name><name><surname>Horiuchi</surname><given-names>H</given-names></name><name><surname>Kitamoto</surname><given-names>K</given-names></name><name><surname>Kobayashi</surname><given-names>T</given-names></name><name><surname>Takeuchi</surname><given-names>M</given-names></name><name><surname>Denning</surname><given-names>DW</given-names></name><name><surname>Galagan</surname><given-names>JE</given-names></name><name><surname>Nierman</surname><given-names>WC</given-names></name><name><surname>Yu</surname><given-names>J</given-names></name><name><surname>Archer</surname><given-names>DB</given-names></name><name><surname>Bennett</surname><given-names>JW</given-names></name><name><surname>Bhatnagar</surname><given-names>D</given-names></name><name><surname>Cleveland</surname><given-names>TE</given-names></name><name><surname>Fedorova</surname><given-names>ND</given-names></name><name><surname>Gotoh</surname><given-names>O</given-names></name><name><surname>Horikawa</surname><given-names>H</given-names></name><name><surname>Hosoyama</surname><given-names>A</given-names></name><name><surname>Ichinomiya</surname><given-names>M</given-names></name><name><surname>Igarashi</surname><given-names>R</given-names></name><name><surname>Iwashita</surname><given-names>K</given-names></name><name><surname>Juvvadi</surname><given-names>PR</given-names></name><name><surname>Kato</surname><given-names>M</given-names></name><name><surname>Kato</surname><given-names>Y</given-names></name><name><surname>Kin</surname><given-names>T</given-names></name><name><surname>Kokubun</surname><given-names>A</given-names></name><name><surname>Maeda</surname><given-names>H</given-names></name><name><surname>Maeyama</surname><given-names>N</given-names></name><name><surname>Maruyama</surname><given-names>J</given-names></name><name><surname>Nagasaki</surname><given-names>H</given-names></name><name><surname>Nakajima</surname><given-names>T</given-names></name><name><surname>Oda</surname><given-names>K</given-names></name><name><surname>Okada</surname><given-names>K</given-names></name><name><surname>Paulsen</surname><given-names>I</given-names></name><name><surname>Sakamoto</surname><given-names>K</given-names></name><name><surname>Sawano</surname><given-names>T</given-names></name><name><surname>Takahashi</surname><given-names>M</given-names></name><name><surname>Takase</surname><given-names>K</given-names></name><name><surname>Terabayashi</surname><given-names>Y</given-names></name><name><surname>Wortman</surname><given-names>JR</given-names></name><name><surname>Yamada</surname><given-names>O</given-names></name><name><surname>Yamagata</surname><given-names>Y</given-names></name><name><surname>Anazawa</surname><given-names>H</given-names></name><name><surname>Hata</surname><given-names>Y</given-names></name><name><surname>Koide</surname><given-names>Y</given-names></name><name><surname>Komori</surname><given-names>T</given-names></name><name><surname>Koyama</surname><given-names>Y</given-names></name><name><surname>Minetoki</surname><given-names>T</given-names></name><name><surname>Suharnan</surname><given-names>S</given-names></name><name><surname>Tanaka</surname><given-names>A</given-names></name><name><surname>Isono</surname><given-names>K</given-names></name><name><surname>Kuhara</surname><given-names>S</given-names></name><name><surname>Ogasawara</surname><given-names>N</given-names></name><name><surname>Kikuchi</surname><given-names>H</given-names></name></person-group><year>2005</year><article-title>Genome sequencing and analysis of <italic>Aspergillus oryzae</italic></article-title><source>Nature</source><volume>438</volume><fpage>1157</fpage><lpage>1161</lpage><pub-id pub-id-type="doi">10.1038/nature04300</pub-id></element-citation></ref><ref id="bib47"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Martinez-Fleites</surname><given-names>C</given-names></name><name><surname>Korczynska</surname><given-names>JE</given-names></name><name><surname>Davies</surname><given-names>GJ</given-names></name><name><surname>Cope</surname><given-names>MJ</given-names></name><name><surname>Turkenburg</surname><given-names>JP</given-names></name><name><surname>Taylor</surname><given-names>EJ</given-names></name></person-group><year>2009</year><article-title>The crystal structure of a family GH25 lysozyme from <italic>Bacillus anthracis</italic> implies a neighboring-group catalytic mechanism with retention of anomeric configuration</article-title><source>Carbohydrate Research</source><volume>344</volume><fpage>1753</fpage><lpage>1757</lpage><pub-id pub-id-type="doi">10.1016/j.carres.2009.06.001</pub-id></element-citation></ref><ref id="bib48"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McClure</surname><given-names>MA</given-names></name></person-group><year>2001</year><article-title>Evolution of the DUT gene: horizontal transfer between host and pathogen in all three domains of life</article-title><source>Current Protein & Peptide Science</source><volume>2</volume><fpage>313</fpage><lpage>324</lpage><pub-id pub-id-type="doi">10.2174/1389203013381062</pub-id></element-citation></ref><ref id="bib49"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McDonald</surname><given-names>TR</given-names></name><name><surname>Dietrich</surname><given-names>FS</given-names></name><name><surname>Lutzoni</surname><given-names>F</given-names></name></person-group><year>2012</year><article-title>Multiple horizontal gene transfers of ammonium transporters/ammonia permeases from prokaryotes to eukaryotes: toward a new functional and evolutionary classification</article-title><source>Molecular Biology and Evolution</source><volume>29</volume><fpage>51</fpage><lpage>60</lpage><pub-id pub-id-type="doi">10.1093/molbev/msr123</pub-id></element-citation></ref><ref id="bib50"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Miroshnichenko</surname><given-names>ML</given-names></name><name><surname>Bonch-Osmolovskaya</surname><given-names>EA</given-names></name></person-group><year>2006</year><article-title>Recent developments in the thermophilic microbiology of deep-sea hydrothermal vents</article-title><source>Extremophiles</source><volume>10</volume><fpage>85</fpage><lpage>96</lpage><pub-id pub-id-type="doi">10.1007/s00792-005-0489-5</pub-id></element-citation></ref><ref id="bib51"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Moran</surname><given-names>Y</given-names></name><name><surname>Fredman</surname><given-names>D</given-names></name><name><surname>Szczesny</surname><given-names>P</given-names></name><name><surname>Grynberg</surname><given-names>M</given-names></name><name><surname>Technau</surname><given-names>U</given-names></name></person-group><year>2012</year><article-title>Recurrent horizontal transfer of bacterial toxin genes to eukaryotes</article-title><source>Molecular Biology and Evolution</source><volume>29</volume><fpage>2223</fpage><lpage>2230</lpage><pub-id pub-id-type="doi">10.1093/molbev/mss089</pub-id></element-citation></ref><ref id="bib52"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nelson</surname><given-names>KE</given-names></name><name><surname>Clayton</surname><given-names>RA</given-names></name><name><surname>Gill</surname><given-names>SR</given-names></name><name><surname>Gwinn</surname><given-names>ML</given-names></name><name><surname>Dodson</surname><given-names>RJ</given-names></name><name><surname>Haft</surname><given-names>DH</given-names></name><name><surname>Hickey</surname><given-names>EK</given-names></name><name><surname>Peterson</surname><given-names>JD</given-names></name><name><surname>Nelson</surname><given-names>WC</given-names></name><name><surname>Ketchum</surname><given-names>KA</given-names></name><name><surname>McDonald</surname><given-names>L</given-names></name><name><surname>Utterback</surname><given-names>TR</given-names></name><name><surname>Malek</surname><given-names>JA</given-names></name><name><surname>Linher</surname><given-names>KD</given-names></name><name><surname>Garrett</surname><given-names>MM</given-names></name><name><surname>Stewart</surname><given-names>AM</given-names></name><name><surname>Cotton</surname><given-names>MD</given-names></name><name><surname>Pratt</surname><given-names>MS</given-names></name><name><surname>Phillips</surname><given-names>CA</given-names></name><name><surname>Richardson</surname><given-names>D</given-names></name><name><surname>Heidelberg</surname><given-names>J</given-names></name><name><surname>Sutton</surname><given-names>GG</given-names></name><name><surname>Fleischmann</surname><given-names>RD</given-names></name><name><surname>Eisen</surname><given-names>JA</given-names></name><name><surname>White</surname><given-names>O</given-names></name><name><surname>Salzberg</surname><given-names>SL</given-names></name><name><surname>Smith</surname><given-names>HO</given-names></name><name><surname>Venter</surname><given-names>JC</given-names></name><name><surname>Fraser</surname><given-names>CM</given-names></name></person-group><year>1999</year><article-title>Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of <italic>Thermotoga maritima</italic></article-title><source>Nature</source><volume>399</volume><fpage>323</fpage><lpage>329</lpage><pub-id pub-id-type="doi">10.1038/20601</pub-id></element-citation></ref><ref id="bib53"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nikoh</surname><given-names>N</given-names></name><name><surname>McCutcheon</surname><given-names>JP</given-names></name><name><surname>Kudo</surname><given-names>T</given-names></name><name><surname>Miyagishima</surname><given-names>SY</given-names></name><name><surname>Moran</surname><given-names>NA</given-names></name><name><surname>Nakabachi</surname><given-names>A</given-names></name></person-group><year>2010</year><article-title>Bacterial genes in the aphid genome: absence of functional gene transfer from Buchnera to its host</article-title><source>PLOS Genetics</source><volume>6</volume><fpage>e1000827</fpage><pub-id pub-id-type="doi">10.1371/journal.pgen.1000827</pub-id></element-citation></ref><ref id="bib54"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nikoh</surname><given-names>N</given-names></name><name><surname>Tanaka</surname><given-names>K</given-names></name><name><surname>Shibata</surname><given-names>F</given-names></name><name><surname>Kondo</surname><given-names>N</given-names></name><name><surname>Hizume</surname><given-names>M</given-names></name><name><surname>Shimada</surname><given-names>M</given-names></name><name><surname>Fukatsu</surname><given-names>T</given-names></name></person-group><year>2008</year><article-title>Wolbachia genome integrated in an insect chromosome: evolution and fate of laterally transferred endosymbiont genes</article-title><source>Genome Research</source><volume>18</volume><fpage>272</fpage><lpage>280</lpage><pub-id pub-id-type="doi">10.1101/gr.7144908</pub-id></element-citation></ref><ref id="bib55"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>O'Connor</surname><given-names>EM</given-names></name><name><surname>Shand</surname><given-names>RF</given-names></name></person-group><year>2002</year><article-title>Halocins and sulfolobicins: the emerging story of archaeal protein and peptide antibiotics</article-title><source>Journal of Industrial Microbiology & Biotechnology</source><volume>28</volume><fpage>23</fpage><lpage>31</lpage><pub-id pub-id-type="doi">10.1038/sj/jim/7000190</pub-id></element-citation></ref><ref id="bib56"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Orcutt</surname><given-names>BN</given-names></name><name><surname>Bach</surname><given-names>W</given-names></name><name><surname>Becker</surname><given-names>K</given-names></name><name><surname>Fisher</surname><given-names>AT</given-names></name><name><surname>Hentscher</surname><given-names>M</given-names></name><name><surname>Toner</surname><given-names>BM</given-names></name><name><surname>Wheat</surname><given-names>CG</given-names></name><name><surname>Edwards</surname><given-names>KJ</given-names></name></person-group><year>2011a</year><article-title>Colonization of subsurface microbial observatories deployed in young ocean crust</article-title><source>The ISME Journal</source><volume>5</volume><fpage>692</fpage><lpage>703</lpage><pub-id pub-id-type="doi">10.1038/ismej.2010.157</pub-id></element-citation></ref><ref id="bib57"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Orcutt</surname><given-names>BN</given-names></name><name><surname>Sylvan</surname><given-names>JB</given-names></name><name><surname>Knab</surname><given-names>NJ</given-names></name><name><surname>Edwards</surname><given-names>KJ</given-names></name></person-group><year>2011b</year><article-title>Microbial ecology of the dark ocean above, at, and below the seafloor</article-title><source>Microbiology and Molecular Biology Reviews</source><volume>75</volume><fpage>361</fpage><lpage>422</lpage><pub-id pub-id-type="doi">10.1128/MMBR.00039-10</pub-id></element-citation></ref><ref id="bib58"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Oren</surname><given-names>A</given-names></name></person-group><year>2002</year><article-title>Molecular ecology of extremely halophilic Archaea and Bacteria</article-title><source>FEMS Microbiology Ecology</source><volume>39</volume><fpage>1</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1111/j.1574-6941.2002.tb00900.x</pub-id></element-citation></ref><ref id="bib59"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Popa</surname><given-names>O</given-names></name><name><surname>Dagan</surname><given-names>T</given-names></name></person-group><year>2011</year><article-title>Trends and barriers to lateral gene transfer in prokaryotes</article-title><source>Current Opinion in Microbiology</source><volume>14</volume><fpage>615</fpage><lpage>623</lpage><pub-id pub-id-type="doi">10.1016/j.mib.2011.07.027</pub-id></element-citation></ref><ref id="bib60"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Puigbo</surname><given-names>P</given-names></name><name><surname>Wolf</surname><given-names>YI</given-names></name><name><surname>Koonin</surname><given-names>EV</given-names></name></person-group><year>2009</year><article-title>Search for a 'Tree of Life' in the thicket of the phylogenetic forest</article-title><source>Journal of Biology</source><volume>8</volume><fpage>59</fpage><pub-id pub-id-type="doi">10.1186/jbiol159</pub-id></element-citation></ref><ref id="bib61"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Quast</surname><given-names>C</given-names></name><name><surname>Pruesse</surname><given-names>E</given-names></name><name><surname>Yilmaz</surname><given-names>P</given-names></name><name><surname>Gerken</surname><given-names>J</given-names></name><name><surname>Schweer</surname><given-names>T</given-names></name><name><surname>Yarza</surname><given-names>P</given-names></name><name><surname>Peplies</surname><given-names>J</given-names></name><name><surname>Glockner</surname><given-names>FO</given-names></name></person-group><year>2013</year><article-title>The SILVA ribosomal RNA gene database project: improved data processing and web-based tools</article-title><source>Nucleic Acids Research</source><volume>41</volume><fpage>D590</fpage><lpage>D596</lpage><pub-id pub-id-type="doi">10.1093/nar/gks1219</pub-id></element-citation></ref><ref id="bib62"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Reysenbach</surname><given-names>AL</given-names></name><name><surname>Banta</surname><given-names>AB</given-names></name><name><surname>Boone</surname><given-names>DR</given-names></name><name><surname>Cary</surname><given-names>SC</given-names></name><name><surname>Luther</surname><given-names>GW</given-names></name></person-group><year>2000</year><article-title>Microbial essentials at hydrothermal vents</article-title><source>Nature</source><volume>404</volume><fpage>835</fpage><pub-id pub-id-type="doi">10.1038/35009029</pub-id></element-citation></ref><ref id="bib63"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Reysenbach</surname><given-names>AL</given-names></name><name><surname>Liu</surname><given-names>Y</given-names></name><name><surname>Banta</surname><given-names>AB</given-names></name><name><surname>Beveridge</surname><given-names>TJ</given-names></name><name><surname>Kirshtein</surname><given-names>JD</given-names></name><name><surname>Schouten</surname><given-names>S</given-names></name><name><surname>Tivey</surname><given-names>MK</given-names></name><name><surname>Von Damm</surname><given-names>KL</given-names></name><name><surname>Voytek</surname><given-names>MA</given-names></name></person-group><year>2006</year><article-title>A ubiquitous thermoacidophilic archaeon from deep-sea hydrothermal vents</article-title><source>Nature</source><volume>442</volume><fpage>444</fpage><lpage>447</lpage><pub-id pub-id-type="doi">10.1038/nature04921</pub-id></element-citation></ref><ref id="bib64"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Reysenbach</surname><given-names>AL</given-names></name><name><surname>Liu</surname><given-names>Y</given-names></name><name><surname>Lindgren</surname><given-names>AR</given-names></name><name><surname>Wagner</surname><given-names>ID</given-names></name><name><surname>Sislak</surname><given-names>CD</given-names></name><name><surname>Mets</surname><given-names>A</given-names></name><name><surname>Schouten</surname><given-names>S</given-names></name></person-group><year>2013</year><article-title>Mesoaciditoga lauensis gen. nov., sp. nov., a moderate thermoacidophilic Thermotogales from a deep-sea hydrothermal vent</article-title><source>International Journal of Systematic and Evolutionary Microbiology</source><volume>63</volume><fpage>4724</fpage><lpage>4729</lpage><pub-id pub-id-type="doi">10.1099/ijs.0.050518-0</pub-id></element-citation></ref><ref id="bib65"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ronquist</surname><given-names>F</given-names></name><name><surname>Teslenko</surname><given-names>M</given-names></name><name><surname>van der Mark</surname><given-names>P</given-names></name><name><surname>Ayres</surname><given-names>DL</given-names></name><name><surname>Darling</surname><given-names>A</given-names></name><name><surname>Hohna</surname><given-names>S</given-names></name><name><surname>Larget</surname><given-names>B</given-names></name><name><surname>Liu</surname><given-names>L</given-names></name><name><surname>Suchard</surname><given-names>MA</given-names></name><name><surname>Huelsenbeck</surname><given-names>JP</given-names></name></person-group><year>2012</year><article-title>MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space</article-title><source>Systematic Biology</source><volume>61</volume><fpage>539</fpage><lpage>542</lpage><pub-id pub-id-type="doi">10.1093/sysbio/sys029</pub-id></element-citation></ref><ref id="bib66"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schonknecht</surname><given-names>G</given-names></name><name><surname>Chen</surname><given-names>WH</given-names></name><name><surname>Ternes</surname><given-names>CM</given-names></name><name><surname>Barbier</surname><given-names>GG</given-names></name><name><surname>Shrestha</surname><given-names>RP</given-names></name><name><surname>Stanke</surname><given-names>M</given-names></name><name><surname>Brautigam</surname><given-names>A</given-names></name><name><surname>Baker</surname><given-names>BJ</given-names></name><name><surname>Banfield</surname><given-names>JF</given-names></name><name><surname>Garavito</surname><given-names>RM</given-names></name><name><surname>Carr</surname><given-names>K</given-names></name><name><surname>Wilkerson</surname><given-names>C</given-names></name><name><surname>Rensing</surname><given-names>SA</given-names></name><name><surname>Gagneul</surname><given-names>D</given-names></name><name><surname>Dickenson</surname><given-names>NE</given-names></name><name><surname>Oesterhelt</surname><given-names>C</given-names></name><name><surname>Lercher</surname><given-names>MJ</given-names></name><name><surname>Weber</surname><given-names>AP</given-names></name></person-group><year>2013</year><article-title>Gene transfer from bacteria and archaea facilitated evolution of an extremophilic eukaryote</article-title><source>Science</source><volume>339</volume><fpage>1207</fpage><lpage>1210</lpage><pub-id pub-id-type="doi">10.1126/science.1231707</pub-id></element-citation></ref><ref id="bib67"><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Shand</surname><given-names>RF</given-names></name><name><surname>Leyva</surname><given-names>KJ</given-names></name></person-group><year>2008</year><article-title>Archaeal Animicrobials: an Undiscovered Country</article-title><source>Archaea: new models for Prokaryotic Biology</source><person-group person-group-type="editor"><name><surname>Blum</surname><given-names>P</given-names></name></person-group><publisher-name>Caister Academic Press</publisher-name><fpage>233</fpage><lpage>243</lpage></element-citation></ref><ref id="bib68"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shimodaira</surname><given-names>H</given-names></name><name><surname>Hasegawa</surname><given-names>M</given-names></name></person-group><year>1999</year><article-title>Multiple comparisons of log-likelihoods with applications to phylogenetic inference</article-title><source>Molecular Biology and Evolution</source><volume>16</volume><fpage>1114</fpage><lpage>1116</lpage><pub-id pub-id-type="doi">10.1093/oxfordjournals.molbev.a026201</pub-id></element-citation></ref><ref id="bib69"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Stamatakis</surname><given-names>A</given-names></name></person-group><year>2014</year><article-title>RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies</article-title><source>Bioinformatics</source><volume>30</volume><fpage>1312</fpage><lpage>1313</lpage><pub-id pub-id-type="doi">10.1093/bioinformatics/btu033</pub-id></element-citation></ref><ref id="bib70"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Uehara</surname><given-names>T</given-names></name><name><surname>Bernhardt</surname><given-names>TG</given-names></name></person-group><year>2011</year><article-title>More than just lysins: peptidoglycan hydrolases tailor the cell wall</article-title><source>Current Opinion in Microbiology</source><volume>14</volume><fpage>698</fpage><lpage>703</lpage><pub-id pub-id-type="doi">10.1016/j.mib.2011.10.003</pub-id></element-citation></ref><ref id="bib71"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van Wolferen</surname><given-names>M</given-names></name><name><surname>Ajon</surname><given-names>M</given-names></name><name><surname>Driessen</surname><given-names>AJ</given-names></name><name><surname>Albers</surname><given-names>SV</given-names></name></person-group><year>2013</year><article-title>How hyperthermophiles adapt to change their lives: DNA exchange in extreme conditions</article-title><source>Extremophiles</source><volume>17</volume><fpage>545</fpage><lpage>563</lpage><pub-id pub-id-type="doi">10.1007/s00792-013-0552-6</pub-id></element-citation></ref><ref id="bib72"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vollmer</surname><given-names>W</given-names></name><name><surname>Joris</surname><given-names>B</given-names></name><name><surname>Charlier</surname><given-names>P</given-names></name><name><surname>Foster</surname><given-names>S</given-names></name></person-group><year>2008</year><article-title>Bacterial peptidoglycan (murein) hydrolases</article-title><source>FEMS Microbiology Reviews</source><volume>32</volume><fpage>259</fpage><lpage>286</lpage><pub-id pub-id-type="doi">10.1111/j.1574-6976.2007.00099.x</pub-id></element-citation></ref><ref id="bib73"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wang</surname><given-names>Z</given-names></name><name><surname>Su</surname><given-names>XM</given-names></name><name><surname>Wen</surname><given-names>J</given-names></name><name><surname>Jiang</surname><given-names>LY</given-names></name><name><surname>Qiao</surname><given-names>GX</given-names></name></person-group><year>2014</year><article-title>Widespread infection and diverse infection patterns of Wolbachia in Chinese aphids</article-title><source>Insect Science</source><volume>21</volume><fpage>313</fpage><lpage>325</lpage><pub-id pub-id-type="doi">10.1111/1744-7917.12102</pub-id></element-citation></ref><ref id="bib74"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Werren</surname><given-names>JH</given-names></name></person-group><year>2011</year><article-title>Selfish genetic elements, genetic conflict, and evolutionary innovation</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>108</volume><supplement>Suppl2</supplement><fpage>10863</fpage><lpage>10870</lpage><pub-id pub-id-type="doi">10.1073/pnas.1102343108</pub-id></element-citation></ref><ref id="bib75"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wiedenbeck</surname><given-names>J</given-names></name><name><surname>Cohan</surname><given-names>FM</given-names></name></person-group><year>2011</year><article-title>Origins of bacterial diversity through horizontal genetic transfer and adaptation to new ecological niches</article-title><source>FEMS Microbiology Reviews</source><volume>35</volume><fpage>957</fpage><lpage>976</lpage><pub-id pub-id-type="doi">10.1111/j.1574-6976.2011.00292.x</pub-id></element-citation></ref><ref id="bib76"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wu</surname><given-names>B</given-names></name><name><surname>Novelli</surname><given-names>J</given-names></name><name><surname>Jiang</surname><given-names>D</given-names></name><name><surname>Dailey</surname><given-names>HA</given-names></name><name><surname>Landmann</surname><given-names>F</given-names></name><name><surname>Ford</surname><given-names>L</given-names></name><name><surname>Taylor</surname><given-names>MJ</given-names></name><name><surname>Carlow</surname><given-names>CK</given-names></name><name><surname>Kumar</surname><given-names>S</given-names></name><name><surname>Foster</surname><given-names>JM</given-names></name><name><surname>Slatko</surname><given-names>BE</given-names></name></person-group><year>2013</year><article-title>Interdomain lateral gene transfer of an essential ferrochelatase gene in human parasitic nematodes</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>110</volume><fpage>7748</fpage><lpage>7753</lpage><pub-id pub-id-type="doi">10.1073/pnas.1304049110</pub-id></element-citation></ref><ref id="bib77"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhaxybayeva</surname><given-names>O</given-names></name><name><surname>Doolittle</surname><given-names>WF</given-names></name></person-group><year>2011</year><article-title>Lateral gene transfer</article-title><source>Current Biology</source><volume>21</volume><fpage>R242</fpage><lpage>R246</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2011.01.045</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.04266.023</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>van der Donk</surname><given-names>Wilfred</given-names></name><role>Reviewing editor</role><aff><institution>University of Illinois-Urbana Champaign</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Antibacterial Gene Transfer Across the Tree of Life” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by Diethard Tautz (Senior editor), Wilfred van der Donk (Reviewing editor), and 3 reviewers (Peter Gogarten, Anthony Poole, and David Baltrus). The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>The study provides clear evidence for the transfer of the GH25 muramidase between the three domains of life. Phylogenetic analyses establish that the eukaryotic and archaeal versions of the gene have bacterial neighbors and that the transfers occurred more recently than the splitting of the domains. Furthermore, the use of phenotypic tests as well as expression in <italic>E. coli</italic> establishes that the GH25 muramidase is functional across its range and not a pseudogene or other non-functional relic. The assessment of genomic context also supports that the GH25 muramidase gene has been transferred from bacteria to archaeal and eukaryote recipients.</p><p>The potential antibacterial role of the GH25 muramidase gene in the archaeal recipient Aciduliprofundum boonei is addressed by showing that the product of the transferred gene exhibits antibiotic properties against expected species in purified form. Experimental work in a non-model system that lacks standard genetic tools is challenging, so these experiments along with the data in <italic>E. coli</italic> provide a good means of assessing function.</p><p>Finally, the authors test whether the gene can impact competitors. Again, without the capacity to undertake direct knockouts, the authors do an admirable job of testing this, by examining relative growth in both monocultures and cocultures with Mesoaciditoga lauensis, a bacterium known to cohabit the environments from which A. boonei has been isolated.</p><p>All reviewers agreed that this study is of high interest to the readership of <italic>eLife</italic>, but request you address the following points in a revised manuscript:</p><p>1) One question stems from the observation that not all field isolates appear to have lysozyme genes. If there are isolates that genuinely lack the GH25 muramidase genes, these would make a good control. It would be helpful if the authors could comment on whether they think the isolates definitely lack the GH25 muramidase gene, and whether they have tried the growth and coculture assays with any of these isolates.</p><p>2) The clarity of the manuscript would benefit greatly from the inclusion of a single phylogeny (recA-like genes?) across all domains of life with arrows actually showing putative transfer directions. The included phylogenies are great, but don't necessarily give an intuitive sense of how distant these transfers are. The inclusion of non-lysozyme containing taxa would really help tell the story to a wider audience.</p><p>3) The SH tests of phylogenies are significantly different and demonstrate that the constrained tree is a poorer fit than the unconstrained tree. However, this only demonstrates that at least one transfer has occurred while the study is about numerous independent transfers...it is possible to sequentially constrain the tree and perform independent SH tests for each potential transfer, which would make a stronger story.</p><p>4) In regards to the competition assays, the reviewers realize that the authors are dealing with intractable genetic systems, but were wondering if there might be cleaner ways of comparing growth between microbes, instead of independent vs. together. The current comparison is confounded because a variety of lysozyme independent reasons could be responsible for a shift in competitive outcome in co-culture (i.e. cross-feeding of archaea on bacterial waste products). Would it be possible to isolate lysozyme resistant bacteria and perform paired competition assays with sensitive and resistant versions? The reviewers felt that the story might be good enough as is without such experiments but wanted to share their thoughts with the authors. Regardless, the authors should point out the caveats of their approach in the paper.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.04266.024</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) One question stems from the observation that not all field isolates appear to have lysozyme genes. If there are isolates that genuinely lack the GH25 muramidase genes, these would make a good control. It would be helpful if the authors could comment on whether they think the isolates definitely lack the GH25 muramidase gene, and whether they have tried the growth and coculture assays with any of these isolates</italic>.</p><p>The idea of using an <italic>Aciduliprofundum</italic> strain without GH25 muramidase in coculture experiments is an excellent one. Unfortunately, it is not clear that the strains that lacked GH25 muramidase by PCR truly do not possess the gene. Two species of <italic>Aciduliprofundum</italic> have been fully sequenced, T469 from the Lau Spreading Center in the Western Pacific Ocean and Mar08-339 from the Mid-Atlantic Ridge. The lysozyme sequences from these two strains have only 58% pairwise identity at the nucleotide level, suggesting that there has been substantial divergence between geographically disparate species. Thus, although PCR using degenerate primers designed for both sequenced species was able to detect close relatives in the same vent fields, we suspect that the PCR-negative <italic>Aciduliprofundum</italic> strains likely also have the lysozyme. We have updated the text to reflect this possibility.</p><p><italic>2) The clarity of the manuscript would benefit greatly from the inclusion of a single phylogeny (recA-like genes?) across all domains of life with arrows actually showing putative transfer directions. The included phylogenies are great, but don't necessarily give an intuitive sense of how distant these transfers are. The inclusion of non-lysozyme containing taxa would really help tell the story to a wider audience</italic>.</p><p>We thank the reviewer for the suggestion and agree that this phylogeny would be a good way to illustrate the HGT events described in the manuscript. We have added it as <xref ref-type="fig" rid="fig3">Figure 3</xref>.</p><p><italic>3) The SH tests of phylogenies are significantly different and demonstrate that the constrained tree is a poorer fit than the unconstrained tree. However, this only demonstrates that at least one transfer has occurred while the study is about numerous independent transfers...it is possible to sequentially constrain the tree and perform independent SH tests for each potential transfer, which would make a stronger story</italic>.</p><p>The reviewers are correct that additional SH tests would strengthen the statistical support for multiple HGT events. We have now performed two more SH tests between the lysozyme HGT tree and either 1) a three-domain constraint tree, or 2) a constraint tree in which the eukaryote branch is monophyletic and other branches are unconstrained. In both cases, the constrained trees were statistically inferior to the HGT trees at P < 0.01. SH tests using monophyletic constraints on individual HGT clades (i.e., plant and bacteria) are not suitable in these individual cases because the transferred genes are monophyletic in both the constraint tree and the HGT tree.</p><p>However, the recurrent patchy distribution of these lysozyme genes and the comparison between branch lengths in lysozyme phylogenies compared to 16S phylogenies further support the hypothesis of HGT, as discussed in the manuscript.</p><p><italic>4) In regards to the competition assays, the reviewers realize that the authors are dealing with intractable genetic systems, but were wondering if there might be cleaner ways of comparing growth between microbes, instead of independent vs. together. The current comparison is confounded because a variety of lysozyme independent reasons could be responsible for a shift in competitive outcome in co-culture (i.e. cross-feeding of archaea on bacterial waste products). Would it be possible to isolate lysozyme resistant bacteria and perform paired competition assays with sensitive and resistant versions? The reviewers felt that the story might be good enough as is without such experiments but wanted to share their thoughts with the authors. Regardless, the authors should point out the caveats of their approach in the paper</italic>.</p><p>We thank the reviewers for this perceptive analysis. We agree that there may be reasons other than lysozyme-dependent inhibition of bacterial competitors for the results seen in our coculture assays. We have added these caveats to the manuscript in addition to the original ones already stated. While the experiments suggested are interesting, the poor availability of cultured microbes that can subsist in these extreme culture conditions, as well as the need to test such strains for lysozyme susceptibility, make such studies outside the scope of the current work.</p></body></sub-article></article> |