Family 6 Glycosyltransferases inVertebrates and Bacteria:Inactivation and HorizontalGene Transfer May EnhanceMutualism between Vertebratesand Bacteria*□S

Published, JBC Papers in Press, September 24, 2010, DOI 10.1074/jbc.R110.176248

Keith Brew‡1, Percy Tumbale‡2, and K. Ravi Acharya§3

From the ‡Department of Basic Science, College of Medicine, FloridaAtlantic University, Boca Raton, Florida 33431 and the §Department ofBiology and Biochemistry, University of Bath, Claverton Down,Bath BA2 7AY, United Kingdom

Glycosyltransferases (GTs) control the synthesis and struc-tures of glycans. Inactivation and intense allelic variation inmembers of the GT6 family generate species-specific and indi-vidual variations in carbohydrate structures, including histo-blood group oligosaccharides, resulting in anti-glycan anti-bodies that target glycan-decorated pathogens. GT6 genes areubiquitous in vertebrates but are otherwise rare, existing in afew bacteria, one protozoan, and cyanophages, suggesting lat-eral gene transfer. Prokaryotic GT6 genes correspond to oneexon of vertebrate genes, yet their translated protein sequencesare strikingly similar. Bacterial and phage GT6 genes influencethe surface chemistry of bacteria, affecting their interactions,including those with vertebrate hosts.

The surfaces of cells are covered with carbohydrates, so gly-cans have key roles in the interactions between cells and of cellswith the extracellular matrix, regulatory molecules, toxins,viruses, and antibodies (1). In the intestines of mammals,glycans of the outer membranes of Gram-negative bacteriacontribute to the ability of the host to distinguish between com-mensal bacteria and pathogens, a key factor in intestinal home-ostasis (2). Glycans can differ in structure between species andbetween individuals within a species; the histo-blood groups(HBGs)4A, B,AB, andO (see Fig. 1A) are awell studied exampleof phenotypic variation between individuals that results in thedisplay of complex glycans with different structures on cellmembranes and extracellular glycoproteins (3, 4). Because gly-cans are antigenic, individuals produce antibodies directed

against non-self-HBGs, withAorO individuals producing anti-bodies against the B antigen and B or O individuals against theA antigen. The presence of such antibodies indicates exposureto exogenous HBG-like antigens (5). HBG polymorphismappears to reflectmultiple selective effects, including resistanceto bacterial pathogens that bind to HBGs on host cells andinteractions of the immune system with enveloped viruses thatcarry HBGs from a previous host (5).Complex glycans are ubiquitous and have a greater potential

for structural variation than polypeptides and nucleic acidsbecause several different bonds can link pairs of adjacentmonosaccharides. Nevertheless, glycans have received lessattention than other macromolecules partly because they areheterogeneous secondary gene products with structures deter-mined by the specificities of glycosyltransferases (GTs). Besidesbeing antigenic, glycans have vital roles in biological systemsencompassing protein folding and stability, cell adhesion,molecular trafficking and clearance, and signal transduction(6). Their biological and biomedical significance is reflected inthe 1–2% of the open reading frames in all domains that encodeenzymes involved in their synthesis and in the many diseaseslinked to aberrations in glycosylation (6, 7). The Carbohydrate-Active enZymes (CAZy) Database classifies GTs into 89 differ-ent families (92 listed but families 36, 46, and 86 are deleted)based on sequence interrelationships (8, 9). In this minireview,we discuss the properties and evolution of the GT6 family,which includes those responsible for the synthesis of HBGs.Genomedatabases indicate that theGT6 family has an irregulardistribution, diagnostic of a nonlinear evolutionary history;GT6 enzymes are more remarkable for their non-functionalitythan activity in humans and some other primates. The productsof the GT6 family are of particular biomedical interest becauseof their role in interactions of the immune systems of verte-brates with beneficial and pathogenic prokaryotes.

Mammalian GT6 Enzymes

The HBG locus on chromosome 9 has three alleles, A, B, andO. The A and B alleles encode active GTs (HBGTA andHBGTB) that catalyze the transfer of N-acetylgalactosamineand galactose, respectively, from their UDP derivatives into an�-linkage with the 3-OH group of a galactosyl residue on aglycan containing an H antigen (Fig. 1A). In the O allele, aframeshift mutation generates an inactive GT (10). HBGTAand HBGTB are closely similar in structure, differing in only 4of 354 residues (10). Although the A, B, and O alleles predom-inate, giving rise to A, B, AB, and O phenotypes, �100 othervariants have been described with mutations in the coding andnoncoding regions, generating additional phenotypes (www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.cgi). The expressionof HBGs is also modulated by fucosyl-�1,2-transferases (FTs)that catalyze the synthesis of the H antigen. Although the FT inerythrocytes (H-type FT) is present in most individuals, secre-tor FT, which catalyzes H antigen production in saliva, gastro-intestinal secretions,milk, and epithelial cells, is inactive in 20%or more of the population (11). Non-secretors (secretor FT-

* This work was supported in part by a Royal Society (UK) industry fellowship(to K. R. A.). This minireview will be reprinted in the 2010 Minireview Com-pendium, which will be available in January, 2011.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table 1.

1 To whom correspondence may be addressed. E-mail: kbrew@ad.fau.edu.2 Present address: NIEHS, NIH, Research Triangle Park, NC 27709.3 To whom correspondence may be addressed. E-mail: bsskra@bath.ac.uk.4 The abbreviations used are: HBG, histo-blood group; GT, glycosyltrans-

ferase; FT, fucosyl-�1,2-transferase; �3GT, �-galactosyl-�1,3-galactosyl-transferase; FS, Forssman glycolipid synthase; iGb3S, isogloboside 3 syn-thase; BHB, basic-hydrophobic-basic; HGT, horizontal gene transfer.

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negative individuals) have HBGs only on erythrocytes and theirprecursors. The Bombay phenotype, found in a minority ofindividuals, results from inactivity in both H-type and secretorFTs. Although all HBGs are lacking, in these individuals, theyare generally healthy (12). The protective or other role of theABO system is therefore subtle, but correlations have beenfound between various diseases, including pancreatic cancer,and blood groups (e.g. Refs. 3, 5, and 13).

Mammals have genes for three additional GT6 family mem-bers that catalyze the transfer of �-galactose or GalNAc to the3-OH group of a �-linked galactose or GalNAc in an acceptorsubstrate (14). These genes encode a �-galactosyl-�1,3-galac-tosyltransferase (�3GT) that catalyzes the synthesis of thexenoantigen or �-Gal epitope, a �-Gal-�1,3-galactosyl entity(15), Forssman glycolipid synthase (FS) (16), and isogloboside 3synthase (iGb3S) (17) (Fig. 1A). �3GT and iGb3S both catalyzethe formation of a similar structure but in glycoproteins andglycolipids, respectively. In humans, the enzymes expressedfrom the�3GT, FS, and iGb3S genes are inactive as the result offrameshift and missense mutations (14–17). The lack of active�3GT and iGb3S results in the absence of �-Gal epitopes inhuman tissues and the presence of antibodies (�1–3% of theIgG) against the�-Gal epitope in the circulations of all humans.Our closest relatives among the primates, including chimps,gorillas, and orangutans, also produce inactive forms of these

three GTs, but other mammals have active enzymes (14, 18).The inactivation of �3GT and iGb3S is thought to have been anevolutionary adaptation that confers resistance to envelopedviruses derived from mammalian hosts that carry �-Galepitopes on their cell membranes. FS is also inactivated inhumans and our closer relatives among the primates (19); thismay be linked to the effects of Forssman antigen on vulnerabil-ity to toxins (16, 20). The inactivation of FS preceded the diver-gence of the chimpanzees, orangutans, macaques, and rhesusmonkeys, and it has been suggested that the catalytically inac-tive protein may have acquired a new function (19).

Structural Basis of Specificity and Catalysis inMammalian GT6 Enzymes

Like most GTs from vertebrates, GT6 family members aretype 2 membrane proteins with N-terminal cytosolic domains,a transmembrane helix, a stem, and a C-terminal catalyticdomain (14). Their catalytic domains have a GT-A fold, one ofthe two predominant fold types (GT-A and GT-B) of the cata-lytic domains of structurally characterized GTs. Both foldsencompass two Rossmann-like�/�-domains; in theGT-A fold,these are closely associated through a large interaction inter-face, but in theGT-B fold, they aremore distinct and are flexiblylinked. Lairson et al. (21) have recently published a comprehen-sive review of the structures and mechanisms of GTs.

FIGURE 1. Structure of the catalytic domain of mammalian GT6 and acceptor substrates and products. A, chemical structures of the type 1 H antigen (i),type 1 HBG A (ii), type 1 HBG B (iii), �-Gal epitope (xenoantigen) (iv), and Forssman glycolipid (v). Monosaccharides are color-coded as follows: Gal, red; GalNAc,purple; Fuc, green; GlcNAc, blue; and Glc, black. B, schematic representation of superposition of the �3GT apo-form in cyan (Protein Data Bank code 2JCO) and�3GT in complex with UDP-Gal in brick red (code 2VS5) showing the C-terminal (color-coded) flexible loop. The two invariant Arg and Glu residues are shownin the inset.

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Crystallographic studies have provided extensive informa-tion about the structure and interactions of HBGTA andHBGTB (22–25) and �3GT (26–32) (Fig. 1B), revealing strongconservation of structure and structure-function relationshipswithin the GT6 family. Like most GT-A fold GTs, both requirea divalentmetal ion (Mn2�) for catalytic activity (33). The inter-actions of HBGTA/B and �3GT with donor substrates andmetal cofactor are closely similar. Their N-terminal domainsinteract with the uracil, ribose, and diphosphatemoieties of theUDP-sugar (donor) substrate. An essentialAsp-Val-Asp (DVD)sequencemotif (present as aDXDmotif inmostGT-A foldGTs(21)) at the junction of the two domains (Fig. 2A, region C)interacts directly with the ribose moiety and indirectly, via theMn2�, with theUDP phosphates. The binding of UDP or donorsubstrate increases order in two sections of the polypeptidechain, a region close to the active site and the C-terminal 10residues of both enzymes (Fig. 1B) (25, 27, 28, 31). Thesechanges organize the binding site for acceptor substrates; con-formational changes in disordered loops adjacent to the activesite are a shared feature of many GTs (see Ref. 25). Most of the

interactions with the Gal or GalNAc of the donor substrateinvolve residues of the C-terminal domain, except for an invari-ant Arg of the N-terminal domain (Figs. 1B and 2A, region B)that interacts with the 3-OH group of the donor monosaccha-ride and may be important for galacto versus gluco specificity.Residues that function in acceptor substrate binding and catal-ysis are located in the C-terminal domain, including an invari-ant Glu within a highly conserved region of sequence (Figs. 1Band 2A, region F) and two basic residues close to the C terminus(Fig. 2B, region G). GT6 enzymes catalyze a sequential bi-biordered mechanism in which the UDP-sugar binds, prior toacceptor, to an enzyme-Mn2� complex (28). Inverting GTs arethought to utilize a single displacement Sn2-like mechanism,but themechanisms of retainingGTs are controversial (see Ref.21). The expected mechanism is a double displacement reac-tion involving two inversions in which the monosaccharide isinitially transferred to a nucleophile, such as a protein carboxylgroup, and subsequently to the acceptor (21). Much evidenceindicates that GT6 familymembers do not use this mechanism,but one that is analogous to that of the well characterized gly-

FIGURE 2. Relationship between eukaryotic, bacterial, and phage GT6 enzymes. A, amino acid sequence alignment of selected proteins: HBG A (HuA),bovine �3GT (Bova), C. owczarzaki (Cap), cyanophage P-SSM2 (PSSM2), P. acanthamoebae (Parach), GT6a and GT6b from B. ovatus (BoA and BoB), Bacteroidesstercoris (Bs), and Bacteroides caccae (Bc). B, alignment of C-terminal regions from the putative metal-dependent GTs listed above together with dog FS. Theboxed sequences labeled A–G correspond to regions that interact with substrates in mammalian GT6 enzymes.

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cogen phosphorylases (34, 35), involving cleavage of the UDP–galactose bond, with all substrates present in the active site,generating a planar cationic oxycarbenium ion that is stabilizedby anionic groups. A conformational change in the active site(21) may shift the location of this reactive species in the activesite, possibly via Mn2� or basic residues in the C-terminalregion that interact with UDP. The 3-OH group of the terminalgalactose of the acceptor is deprotonated byUDP, and a bond isformed between this oxygen and the oxycarbenium moiety togenerate the product.A key region in donor substrate specificity is region E in Fig.

2A (HAAI in �3GT); the corresponding sequences in HBGTAand HBGTB, LGGF and MGAF, respectively, determine theirrespective specificities for UDP-GalNAc and UDP-Gal (22).The dominant role of this region in donor substrate specificityin �3GT was demonstrated by the identification of a mutantthat is specific for UDP-GalNAc rather than UDP-Gal byscreening a library of mutants with alternative sequences forresidues 280–288. This mutant had the sequence AGGLreplacing HAAI; structural studies suggested that the smallerside chains generate a pocket on the protein surface that canaccommodate the 2-acetamido group of GalNAc (36).Turcot-Dubois et al. (14) have investigated genes encoding dif-

ferent GT6 family members in non-mammalian vertebrates.These species have genes that may be orthologs of blood groupenzymes andothers that encodeFS-like or iGb3S/�3GT-likeGTs.Atypical members of the GT6 family in mammals were identifiedthat have substitutions for highly conserved active-site residues,including theDXDmotif (14).Twoof thesewereexpressedbutdidnot display GT activity, so their functions are presently unknown(14). It is possible that they are pseudogenes or encode proteinsthat have lost catalytic activity, becoming binding proteins (lec-tins) like somemembers of glycoside hydrolase family 18 in verte-brates and invertebrates (37).

GT6 Family Members from Other Eukaryotes andProkaryotes

With new information from genome sequences, genes wereidentified that encode members of the GT6 family in two non-vertebrate eukaryotes, Branchiostoma floridae (an amphioxus)and Capsaspora owczarzaki (a unicellular opisthokont that is asymbiont of a snail) (38), plus 16 species of bacteria and thecyanophage P-SSM2 (supplemental Table 1); many otherhomologs are also in the human intestinal and marine metage-nomes (39). The distribution pattern in eukaryotes is puzzlingbecause the amphioxus is a chordate like the vertebrates, butC. owczarzaki represents an early branch from the line leadingto metazoa and fungi. If the genes from eukaryotes, bacteria,and phages were related to vertebrate GT6 genes by the normalevolutionary process (vertical gene transfer), the GT6 familywould have a broad distribution in eukaryotes and prokaryotes.However, GT6 genes are absent in currently sequencedgenomes of a wide array of other invertebrates, plants, fungi,protozoa, archaea, andmost bacteria. Thus, outside of the chor-dates, the GT6 family has a sporadic distribution, suggestingthe horizontal transfer of GT6 genes between eukaryotes andprokaryotes aswell as between bacteria. It is interesting to com-pare the distribution of GT6 family members with that of

another retaining GT family, GT8. These enzymes catalyze theformation of Gal-�1,4, Gal-�1,3 and Glc-�1,2 linkages andinclude GTs from plants, fungi, vertebrates, invertebrates, pro-tozoa, bacteria, and viruses. The GT8 family encompasses gly-cogenin and bacterial �1,4-galactosyltransferases related toLgtC (40). The GT8 family has a continuous distribution, con-sistent with evolution through vertical gene transfer.Comparisons of the catalytic domain sequences of eukary-

otic GT6 family members with the complete sequences of theirprokaryotic counterparts show a high level of identity, up to35% in some pairwise comparisons. Many, but not all, highlyconserved sites are components of the active sites in the struc-turally characterized mammalian enzymes (Fig. 2A). The bac-terial and phage GT6 enzymes lack the non-catalytic domainsof the vertebrate proteins and are also truncated relative to theminimal functional mammalian catalytic domains by �47 res-idues. In fact, with a single exception, the coding sequences ofthe prokaryotic GT6 enzymes correspond closely to the finalexon in the genes for the vertebrate enzymes, consistent withhorizontal transfer of this exon of the vertebrate gene (38). Phy-logenetic trees constructed using GT6 protein sequences indi-cate that they form two clades, one containing the phage,eukaryotic, and Parachlamydia acanthamoebae GT6 enzymesand the second containing the other bacterial GT6 enzymes(Fig. 3).The significance of the 47 extra residues in the vertebrate

catalytic domains is unclear; an extension of similar length ispresent in GT6 from C. owczarzaki, but it is not significantlysimilar in sequence to the extension in the vertebrate catalyticdomains. Therefore, although this part of the mammalian GT6catalytic domains is well structured and tightly associated withthe rest of the domain, it does not appear to be functionallyimportant.

Properties and Structure-Function Relationships ofBacterial GT6 Enzymes

At present, little information is available on the properties ofprokaryotic GT6 family members. Wang and co-workers (41)first expressed GT6 from Escherichia coli strain O86 and foundthat it is a galactosyltransferase that catalyzes the synthesis ofHBG B antigens. This enzyme was shown to catalyze a step inthe synthesis of the bacterial O antigen because gene disruptionmodified its structure (42). The same group cloned GT6 fromHelicobacter mustelae and showed that it is a GalNAc-transfer-ase that can produce HBG A antigen structures (43). Theseenzymes were found to be useful for efficient enzymatic glycansynthesis and were applied for this purpose in the presence ofMn2� ions, as used with mammalian enzymes.

Comparison of the sequences of the bacterial and mamma-lian GT6 enzymes shows some striking differences (Fig. 2A).First, the DXD motif of the eukaryotic GTs is replaced in thebacterial proteins by NXN, with the single exception of GT6from P. acanthamoebae; also cyanophage P-SSM2 GT6 retainsthe DXD motif. In the mammalian enzymes, the aspartates ofthe DXD motif are essential for activity (24, 33). The genomeof Bacteroides ovatus, a Gram-negative commensal bacteriumof the distal mammalian gut, encodes two GT6 family mem-bers; Tumbale and Brew (39) expressed one of these (desig-

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nated BoA) (Fig. 2) and found it to be a GalNAc-transferasewith a specificity similar to human blood group GTA. Thisenzyme does not require a divalent metal ion for activity and isnot inhibited by EDTA. It was suggested that the change of theDXD sequence to NXN in the bacterial GTs may be associatedwith a loss of metal ion dependence. In mammalian GT6, themetal ion functions in substrate binding and catalysis, suggest-ing that the bacterial enzymes may differ in catalytic mecha-nism. However, the effects of mutating selected residues (Fig.2A, regions C and E–G) in the B. ovatus enzyme suggest thatthere are strong similarities in structure-function relationshipswith the mammalian GT6 group (39).At present, BoA is the only bacterial GT6 that has been char-

acterized with regard to structure-function relationships.Based on the replacement of the DXD motif by NXN in thesebacterial enzymes, it appears that, apart from the Parachlamy-dia enzyme, all may be metal-independent, whereas cyanoph-age GT6 and its close relatives from the marine metagenomedatabase are expected to be metal-dependent. Although func-tional studies of more bacterial enzymes are needed, theirapparent metal independence suggests that their catalyticmechanisms differ from those of the well studied metal-depen-dent enzymes frommammals or that the metal ion is function-

ally replaced by a substructure of the protein. The strong con-servation of mammalian active-site residues in the bacterialenzymes suggests that the active-site structures in these groupsare similar, consistent with the results of mutational studieswith B. ovatus GT6 (39).A second unique feature of the bacterial GT6 enzymes (other

than that from Parachlamydia) is that the C-terminal regionsof their sequences align poorly with those of other familymem-bers and also with each other (Fig. 2, compare A and B). Thisregion has a distinctive composition with high proportions ofbasic and nonpolar amino acids; truncation of this region inBoA shows that it is not required for activity (39). These fea-tures are reminiscent of previous observations with the unre-lated bacterial GT8 enzymes. The C-terminal regions of bacte-rial GT8 enzymes show little similarity to each other apart fromhaving a high content of basic and aromatic amino acids. In twoof these enzymes, LgtC andWaaJ, the C-terminal regions werefound to have a role in interactions with the bacterial cell mem-brane, and they appear to form amphipathic�-helices that bindto the cell membrane (40, 44). Although the bacterial GT6enzymes do not have a high content of aromatic residues, pro-tein-membrane interactions can also be mediated by unstruc-tured regions of polypeptide that typically have basic-hydro-phobic-basic (BHB) amino acid sequence motifs (45). Brzeskaet al. (46) have identified BHB lipid-binding regions in variousproteins, including myosin I from Acanthamoebae and Dictyo-stelium; their web-based program (helixweb.nih.gov/bhsearch)uses the Wimley and White hydrophobicity scale (47) to scanprotein sequences for BHBmembrane-binding regions. Analy-sis of bacterial GT6 sequences indicates that their C-terminalregions contain such motifs, suggesting that bacterial GT6enzymes may interact with the cytoplasmic face of the cellmembrane through these regions. This is consistent with therole ofGT6 enzymes fromGram-negative bacteria in catalyzingsteps in the synthesis of theO antigen component of the lipopo-lysaccharide component of the outer membrane; this is synthe-sized on the cytosolic side of the cell membrane prior to assem-bly with the lipid A-core oligosaccharide intermediate (48).

Functions of GT6 Family Members across theEvolutionary Spectrum

Bacterial glycans display greater diversity and have little sim-ilarity in chemical structure compared with eukaryotic glycans.Nevertheless, somebacteria have surface structures that resem-ble those frommammals and other vertebrates and are thoughtto facilitate their survival in eukaryotic hosts. TheO antigens ofGram-negative bacteria are highly variable in structure, gener-ating variations in the chemistry of the bacterial surfacebetween different species and strains and within a single strainunder different conditions (48). Changes in the complement ofGTs and other enzymes are the ultimate source of these differ-ences in structure and can arise from evolutionary changes inGT specificity, gene loss or inactivation, and the acquisition ofnew GT genes by horizontal gene transfer (HGT). The GT6genes in bacteria appear to be an example of a GT gene ofprobable vertebrate origin participating in O antigen synthesisand enhancing molecular mimicry of host glycans. Based oncurrent information on three bacterial GT6 enzymes, it appears

FIGURE 3. Phylogenetic tree constructed using amino acid sequences ofGT6 family members from diverse sources. The tree was generated usingthe Phylogeny.fr Web site using standard settings apart from the use ofMrBayes (100,000 generations) for constructing the phylogenetic tree (60).The tree is displayed in radial style, and the sequences from the bacterialspecies that have NXN rather than DXD motifs are enclosed in the shaded area.Eukaryotes: bovine �1,3 galactosyltransferase (Boa), C. owczarzaki ATCC30864 (Cap), dog FS, and human HBG A synthase (HuA); bacteria: Acineto-bacter calcoaceticus (Ac), Acinetobacter johnsonii (Aj), B. caccae (Bc), B. ovatusGT1 (Bo1), B. ovatus GT2 (Bo2), B. stercoris (Bs), E. coli Wcmb (Ec), Francisellaphilomiragia (Fp), H. mustelae (Hm), Haemophilus somnus 2336 (Hs), P. acan-thamoebae (Parach), Psychrobacter sp. PRwf-1 (Ps), S. deleyianum (Sd), and twoGTs from Subdoligranulum variabile (Sv1 and Sv2); and phage: Prochlorococcusphage P-SSM2 (PSSM2). The sources of the sequences are given in supple-mental Table 1.

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that they catalyze the synthesis of either HBG A or B-like gly-cans; current knowledge of structure-function relationships inthe GT6 family suggests that the other bacterial GT6 enzymeshave similar specificities. Consequently, their mimicry of hostglycans and avoidance of immune responses will be confined tohosts that carry the corresponding HBG phenotype. Therefore,eluding immune responses from the host may not be the pri-mary role of GT6 genes in commensal bacteria (5, 7). As dis-cussed previously, the polymorphism of ABOHBGs in humanshas been suggested to arise from selective pressure to protectthe species against pathogens that utilize a particularHBGanti-gen as a receptor combined with protection, via antibodiesagainst other HBGs, against enveloped viral pathogens thatcarry HBGs from another host (5). HBGs have othermore indi-rect effects; for example, A or B antigens can modulate sialicacid-mediated interactions between pathogens, includingmalarial parasites and eukaryotic cell membranes (49). Theselective advantage to a bacterium of carryingA or B antigens isless clear, but it may be linked to mutualism. For example, theymay represent a major source of non-self-HBG antigens thatpromote the production of anti-HBG antibodies that protecttheir hosts from enveloped viruses and other pathogens. Therecognition of commensal bacteria by their host is mutuallybeneficial, and perturbation of their cell surface chemistry canlead to inflammatory bowel disease (50); glycans such as HBGantigens appear to contribute to this recognition process (2).Also, some enteric pathogens, including noroviruses (51) andE. coli heat-labile and cholera-derived toxins (52–54), initiallybind to HBGs on mammalian intestinal cells, and it may beadvantageous for HBG-decorated intestinal bacteria to protecttheir hosts by acting as decoy receptors.Cyanophage P-SSM2 GT6 is part of an array of P-SSM2

genes for enzymes involved in LPS synthesis that are absent inthe genomes of twoother cyanophages, P-SSM4andP-SSM7. Ithas been suggested that in P-SSM2, they affect infection andestablishment of the prophage stage by altering the surface of itshost cyanobacterium to prevent other phages from binding(55). Many GT6 genes similar to that of P-SSM2 are found inthe Environmental Samples Database, mostly in the marinemetagenome. Their sequences group into two clades: one thatis 60–84% identical in protein sequence to P-SSM2 GT6,whose members are probably from currently unidentified cya-nophages, and one that consists of GT6 family members thatare �40% identical to those from P-SSM2. Based on sequencesin key substrate interaction regions, themembers of each groupappear to be similar in substrate specificity to each other,whereas the two groups may differ in both donor and acceptorsubstrate specificity; whether the second group is from phageand/or bacterial sources is unclear. Other homologs in thehuman intestinalmetagenome have sequence characteristics ofbacterial GT6 enzymes (39).Members of the GT6 family fall into several evolutionary

subgroups. One consists of the vertebrate enzymes with dis-tinct multidomain structures, and a second consists of the bac-terial enzymes, apart from P. acanthamoebae, which have BHBmembrane-binding and NXN sequence motifs. Phylogenetictrees constructed using sequences from all available sources,including environmental databases, show a clear division into

two principal clades with DXD andNXN sequencemotifs, withthe deepest branches in the DXD group leading to cyanophageP-SSM2, P. acanthamoebae, andC. owczarzaki. These analysesdo not provide a robust answer regarding whether the ancestorof the GT6 family was from the metal-dependent or metal-independent GT6 groups. GT6 from P. acanthamoebae strainHall’s coccus is unique among the bacterial enzymes in having aDXD motif. This bacterium normally infects free-living amoe-bae but can also infect humanmacrophages, pneumocytes, andlung fibroblasts (56) and has been linked to pneumonia andother diseases (57). It has aGT6 that ismost similar to cyanoph-age P-SSM2GT6, and its products maymediate interactions ofthis Parachlamydia strain with mammalian and other hosts.GT6 from Sulfurospirillum deleyianum is, at present, ananomaly because it is from a species that does not inhabit thegastrointestinal tract or skin of vertebrates but is from a sulfur-reducing bacterium that is found in mud, lake material, andsubsurface ground water (58).The features of the GT6 family reviewed here indicate that

HGT involving eukaryotes, bacteria, and phages occurred dur-ing their evolutionary development; although it seems likelythat the direction is eukaryote to prokaryote, the evidence is notconclusive. Examples of HGT from eukaryotes to prokaryoteshave been reported previously, including a fructose-bisphos-phate aldolase gene between red algae andmarine cyanophages(59). It is interesting that the deeper branches in sequence-based phylogenetic trees (Fig. 3) lead to endosymbionts withpotential for mediating HGT: P. acanthamoebae, associatedwith acanthamoebae and humans, and C. owczarzaki, a symbi-ont of the snail Biomphalaria glabrata, which is also an inter-mediate host of Schistosoma mansoni, the causative agent ofschistosomiasis. Capsaspora is a predator of S. mansoni (38).The presence of many GT6 homologs from uncharacterizedspecies in the marine metagenome indicates that there is moreto be discovered about the distribution and evolution of theGT6 family.

Acknowledgments—We thank Professor Louise N. Johnson for con-structive comments and Dr. Nethaji Thiyagarajan for generatingFig. 1B.

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Keith Brew, Percy Tumbale and K. Ravi AcharyaBacteria

Horizontal Gene Transfer May Enhance Mutualism between Vertebrates and Family 6 Glycosyltransferases in Vertebrates and Bacteria: Inactivation and

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