Molecular Phylogenetics and Evolution 68 (2013) 327–339

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Molecular Phylogenetics and Evolution

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Phylogenetic- and genome-derived insight into the evolution of N-glycosylationin Archaea

Lina Kaminski a, Mor N. Lurie-Weinberger b, Thorsten Allers c, Uri Gophna b, Jerry Eichler a,⇑a Department of Life Sciences, Ben Gurion University, Beersheva 84105, Israelb Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv 69978, Israelc School of Biology, University of Nottingham, Nottingham NG7 2UH, UK

a r t i c l e i n f o

Article history:Received 25 January 2013Revised 23 March 2013Accepted 26 March 2013Available online 6 April 2013

Keywords:ArchaeaN-glycosylationOligosaccharyltransferase

1055-7903/$ - see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.ympev.2013.03.024

⇑ Corresponding author. Address: Department oUniversity, P.O. Box 653, Beersheva 84105, Israel. Fax

E-mail address: jeichler@bgu.ac.il (J. Eichler).

a b s t r a c t

N-glycosylation, the covalent attachment of oligosaccharides to target protein Asn residues, is a post-translational modification that occurs in all three domains of life. In Archaea, the N-linked glycans thatdecorate experimentally characterized glycoproteins reveal a diversity in composition and contentunequaled by their bacterial or eukaryal counterparts. At the same time, relatively little is known ofarchaeal N-glycosylation pathways outside of a handful of model strains. To gain insight into the distri-bution and evolutionary history of the archaeal version of this universal protein-processing event, 168archaeal genome sequences were scanned for the presence of aglB, encoding the known archaeal oli-gosaccharyltransferase, an enzyme key to N-glycosylation. Such analysis predicts the presence of AglBin 166 species, with some species seemingly containing multiple versions of the protein. Phylogeneticanalysis reveals that the events leading to aglB duplication occurred at various points during archaealevolution. In many cases, aglB is found as part of a cluster of putative N-glycosylation genes. The pres-ence, arrangement and nucleotide composition of genes in aglB-based clusters in five species of the hal-ophilic archaeon Haloferax points to lateral gene transfer as contributing to the evolution of archaeal N-glycosylation.

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1. Introduction

Originally thought to be a process restricted to Eukarya, it isnow clear that Bacteria and Archaea can also modify proteins viathe addition of oligosaccharides to selected Asn residues, i.e. per-form N-glycosylation (Calo et al., 2010; Nothaft and Szymanski,2010; Larkin and Imperiali, 2011; Eichler, 2013). At present, under-standing of archaeal N-glycosylation lags behind that of the paral-lel process in Eukarya and Bacteria. Nonetheless, analysis of even alimited number of archaeal glycoproteins has made it clear thatarchaeal N-linked glycans show a diversity of content and struc-ture that is not seen elsewhere (Schwarz and Aebi, 2011; Eichler,2013). To date, N-linked glycans decorating glycoproteins or repor-ter peptides from Archaeoglobus fulgidus, Halobacterium salinarum,Haloferax volcanii, Methanococcus maripaludis, Methanococcus vol-tae, Methanothermus fervidus, Pyrococcus furiosus, Sulfolobus acido-caldarius and Thermoplasma acidophilum have been characterized(Wieland et al., 1983; Lechner et al., 1985; Kärcher et al., 1993;Zähringer et al., 2000; Voisin et al., 2005; Abu-Qarn et al., 2007;

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Igura et al., 2008; Kelly et al., 2009; Peyfoon et al., 2010; Nget al., 2011; Matsumoto et al., 2012; Vinogradov et al., 2012). Apartfrom the two similar Methanococcus N-linked glycans, distinct pro-tein-bound oligosaccharides are seen in each species. Moreover, inboth Halobacterium salinarum and Haloferax volcanii, the S-layerglycoprotein is simultaneously modified by two distinct N-linkedglycans (Wieland et al., 1983; Lechner et al., 1985; Guan et al.,2012).

Given the species-specific profile of N-linked glycans that deco-rate archaeal glycoproteins, pathways of oligosaccharide assemblyunique to a given archaeon likely exist. Indeed, in the four Agl(archaeal glycosylation) pathways responsible for N-glycosylationstudied to date, namely those of the halophile Hfx. volcanii, themethanogens M. voltae and M. maripaludis, and the thermoacido-phile S. acidocaldarius (Calo et al., 2010; Jarrell et al., 2010; Albersand Meyer, 2011; Eichler, 2013), few common components areseen. The oligosaccharyltransferase (OST) AglB, responsible fordelivery of the assembled glycan and its precursors from a phos-phorylated dolichol lipid carrier to target protein Asn residues, is,however, present in each pathway. With this is mind, Magidovichand Eichler (2009) relied on the presence of aglB, encoding the onlyOST currently identified in Archaea, to predict the existence of a N-glycosylation pathway in 54 of the 56 species for which completegenome sequence information was available at the time.

328 L. Kaminski et al. / Molecular Phylogenetics and Evolution 68 (2013) 327–339

Today, the number of publicly available archaeal genomesequences, including those of several phylogenetically proximalspecies, is approaching 200. This wealth of genomic data lendsitself to a detailed examination of archaeal N-glycosylation froman evolutionary perspective. Accordingly, the examination ofputative N-glycosylation pathway components across genomelines reported here offers novel insight into the evolution ofthe archaeal version of this universal post-translational proteinmodification.

2. Materials and methods

2.1. Databases

The list of AglB proteins, identified as containing a multi-mem-brane-spanning N-terminal domain and a soluble C-terminal do-main that includes the WWDYG consensus motif implicated inOST function across evolution (Yan and Lennarz, 2002; Maitaet al., 2010; Lizak et al., 2011), was obtained by scanning the fol-lowing: GT family 66 at the Carbohydrate-Active Enzymes data-base (http://www.cazy.org), the Integrated Microbial Genomes –Genome Encyclopedia of Bacteria and Archaea Genomes (IMG/GEBA) (http://img.jgi.doe.gov/cgi-bin/w/main.cgi), using the term‘EC 2.4.1.119’ as query, and the NCBI Protein Database (http://www.ncbi.nlm.nih.gov/protein) sites, using the terms ‘Stt3’ or‘AglB’ as query. These searches were complemented by manualsearches of non-annotated proteins for the presence of WWDXG,a relaxed form of the WWDYG motif.

2.2. Phylogenetic analysis

The sequences of Haloferax AglB proteins were retrieved fromthe IMG/GEBA website utilizing the ‘‘Gene Neighborhood’’ func-tion. Homologs were aligned using MUSCLE (Edgar, 2004). TheHalorubrum lacusprofundi AglB sequence served as an out-group.The alignment was manually edited and ambiguously aligned posi-tions were removed. The tree was then constructed utilizing thePhyML server (http://www.atgc-montpellier.fr/phyml/) (Guindonet al., 2010), using the JTT model + 4 gamma categories to approx-imate the different substitution rates among sites, an estimation ofinvariant sites, and 100 bootstrap trials. A neighbor-joining phylo-genetic tree was generated from the list of euryarchaeal speciescontaining more than one copy of AglB utilizing MEGA 5 software(Tamura et al., 2011). Homologs were aligned using ClustalW (Lar-kin et al., 2007). Robustness of the tree was assessed by a bootstraptest based on 500 pseudo-replicates. Bootstrap values are shownon the nodes of the tree where greater than 50%.

2.3. Calculation of the codon adaptation index (CAI) and effectivenumber of codons (ENC)

Calculation of CAI (Sharp and Li, 1987) and ENC (Wright, 1990)for all clustered agl genes in five Haloferax species was performedutilizing Inca 2.0 (Supek and Vlahovicek, 2004). The CAI calcula-tions required manual indication of highly expressed ribosomalprotein-encoding genes, which were located relying on genomicannotations.

3. Results and discussion

3.1. In Archaea, the gene encoding AglB is almost universally detected

Given the central role played by AglB in archaeal N-glycosyla-tion, 168 publicly available archaeal genomes were scanned forthe presence of the encoding gene. In this manner, better insight

into the prevalence, and by extension, the importance of N-glycosylation in Archaea, may be obtained. Of the 168 genomesconsidered, 166 contained AglB-encoding sequences (Table 1),including A. fulgidus AF0329, Hfx. volcanii HVO_1530, M. voltaeMVO1749, M. maripaludis MMP1424 and P. furiosus PF0156, AglBproteins all experimentally demonstrated to possess OST activity(Chaban et al., 2006; Abu-Qarn et al., 2007; Igura et al., 2008;VanDyke et al., 2009; Matsumoto et al., 2012). In fact, AglB ispredicted to exist in members of all five archaeal phyla, i.e.Crenarchaeota, Euryarchaeota, Korarchaeota, Nanoarchaeota andThaumarchaeota, further pointing to N-glycosylation as being acommon trait in Archaea. It should, however, be noted that inthe vast majority of cases, neither N-glycosylation nor transcrip-tion of the predicted AglB-encoding gene has been confirmed.Finally, and as previously reported (Magidovich and Eichler,2009), no aglB sequence was detected in either Aeropyrum pernixor Methanopyrus kandleri, suggesting that these species do notperform N-glycosylation. Alternatively, given that Aeropyrumpernix and Methanopyrus kandleri are characterized by an atypicalgene content (Brochier et al., 2004), it is possible that a different,currently unrecognized OST mediates N-glycosylation in thesespecies.

3.2. Multiple versions of AglB appeared throughout evolution

In 31 of the 113 euryarchaeal species considered, and in only 2of the 55 non-euryarchaeal species addressed, two or more aglB se-quences were identified. Of the 31 euryarchaeal species, 14 weremethanogens (out of a total of 49 methanogens considered). Inexamining those methanoarchaeal species containing two or morecopies of aglB, no common phenotypic trait, such as an ability togrow under a given condition, is apparent. On the other hand, inaddressing thermo- and hyperthermophilic euryarchaea, two ormore aglB sequences were identified in all nine Thermococcus spe-cies, in six of the seven Pyrococcus species considered, and in two ofthe three Archaeoglobus species examined. Yet, the possibility thatmultiplicity of AglB in a given species is related to an elevated opti-mal growth temperature is unlikely, since of the 45 crenarchealspecies, all of which are thermo- or hyperthermophiles, only two,belonging to different genera, contain a pair of predicted AglB-encoding genes.

To gain insight into the evolutionary relationship of the multi-ple versions of AglB found in euryarchaeal species, phylogeneticanalysis was performed (Fig. 1). The phylogenetic tree obtained as-signed the multiple AglB proteins into two major groups, termedgroup A, including AglB sequences from the methanogens and A.fulgidus, and group B, including Pyrococcus and Thermococcus AglBsequences. Group A was in turn divided into two major clades(clades a and b) and an additional clade (clade c) containing thetwo Methanobacterium sp. AL-21 AglB sequences, while group Bwas divided into two major clades (clades d and e). A more com-prehensive taxonomic representation of AglB phylogeny can befound in Fig. S1.

Closer analysis of the phylogenetic tree revealed a situationwhereby the multiple versions of AglB encoded by certain speciesappeared at different times during evolution, rather than being theresult of a single duplication event. For example, the two Methan-ocella arvoryzae AglB sequences found adjacent to each other in thegenome likely appeared due to a recent gene duplication event. Athird AglB sequence from this species is assigned to cluster b,reflecting an earlier appearance of two versions of the protein(Fig. 1, arrows). This claim is supported by the fact that a similarpattern of AglB distribution is seen in other species from the samegenus, i.e. Methanocella conradii and Methanocella paludicola. Onthe other hand, the pair of Methanocelleus marisnigri AglB proteinsencoded by genes found at distant positions from each other in the

Table 1Archaeal AglB homologs.

Genus (Phyluma/Classb/Orderc/Familyd) Species AglB

Acidilobus (C/Tp/Ac/Ac) A. saccharovorans ASAC_0278,0710Caldisphaera (C/Tp/Ca/Ca) C. lagunensis Calag_0026,1336Desulfurococcus (C/Tp/De/De) D. amylolyticus SphmelDRAFT_0373

D. fermentans Desfe_0211D. kamchatkensis DKAM_0136D. mucosus Desmu_0294

Ignicoccus (C/Tp/De/De) I. hospitalis Igni_0016Ignisphaera (C/Tp/De/De) I. aggregans Igag_0094Staphylothermus (C/Tp/De/De) S. hellenicus Shell_0596

S. marinus Smar_0223Thermogladius (C/Tp/De/De) T. cellulolyticus TCELL_1363Thermosphaera (C/Tp/De/De) T. aggregans Tagg_0313Hyperthermus (C/Tp/De/Py) H. butylicus Hbut_1205Pyrolobus (C/Tp/De/Py) P. fumarii Pyrfu_1528Fervidicoccus (C/Tp/Fe/Fe) F. fontis FFONT_0123Acidianus (C/Tp/Su/Su) A. hospitalis Ahos_1254Metallosphaera (C/Tp/Su/Su) M. cuprina Mcup_0430

M. sedula Msed_1805M. yellowstonensis MetMK1DRAFT_00024050

Sulfolobus (C/Tp/Su/Su) S. acidocaldarius Saci_1274S. islandicus HVE10/4 SiH_1127S. islandicus L.D.8.5 LD85_1283S. islandicus L.S.2.15 LS215_1264S. islandicus M.14.25 M1425_1167S. islandicus M.16.27 M1627_1231S. islandicus M.16.4 M164_1156S. islandicus REY15A SiRe_1041S. islandicus Y.G.57.14 YG5714_1163S. islandicus Y.N.15.51 YN1551_1688S. solfataricus 98/2 Ssol_2025S. solfataricus P2 SSO1052S. tokodaii ST0940

Thermofilum (C/Tp/Th/Thf) T. pendens Tpen_0640Caldivirga (C/Tp/Th/Thp) C. maquilingensis Cmaq_0438Pyrobaculum (C/Tp/Th/Thp) P. aerophilum PAE3030

P. arsenaticum Pars_1781P. calidifontis Pcal_0997P. islandicum Pisl_0431P. oguniense Pogu_0350Pyrobaculum sp. 1860 P186_1486

Thermoproteus (C/Tp/Th/Thp) T. neutrophilus Tneu_1689T. tenax TTX_0519T. uzoniensis TUZN_0151

Vulcanisaeta (C/Tp/Th/Thp) V. distributa Vdis_2064V. moutnovskia VMUT_0472

Archaeoglobus (E/Ar/Ar/Ar) A. fulgidus AF0040,0329e,0380A. profundus Arcpr_0726,1194A. veneficus Arcve_0568

Ferroglobus (E/Ar/Ar/Ar) F. placidus Ferp_2437Haladaptatus (E/H/H/H) Hap. paucihalophilus ZOD2009_20113Halalkalicoccus (E/H/H/H) Hac. jeotgali HacjB3_10630Haloarcula (E/H/H/H) Har. californiae HAH_00005860

Har. hispanica HAH_1202Har. marismortui rrnAC0431Har. sinaiiensis HAI_00022250Har. vallismortis HAJ_00008880Haloarcula sp. AS7094 pSCM201p1

Halobacterium (E/H/H/H) Hbt. salinarum R1 OE2548FHalobacterium sp. NRC-1 VNG1068GHalobacterium sp. DL1 HalDL1_1649

Halobiforma (E/H/H/H) Hbf. lacisalsi HlacAJ_010100009178Haloferax (E/H/H/H) Hfx. denitrificans HAK_00032060

Hfx. mediterranei HFX_1592Hfx. mucosum HAM_16650Hfx. sulfurifontas HAN_00007740Hfx. volcanii HVO_1530

Halogeometricum (E/H/H/H) Hgm. borinquense Hbor_17000Halomicrobium (E/H/H/H) Hmc. mukohataei Hmuk_2752Halopiger (E/H/H/H) Hpg. xanaduensis Halxa_2340Haloquadratum (E/H/H/H) Hqr. walsbyi C23 Hqrw_3013

Hqr. walsbyi DSM 16790 HQ2681AHalorhabdus (E/H/H/H) Hrd. tiamatea HLRTI_06344

Hrd. utahensis Huta_2808Halorubrum (E/H/H/H) Hrr. lacusprofundi Hlac_1062

(continued on next page)

L. Kaminski et al. / Molecular Phylogenetics and Evolution 68 (2013) 327–339 329

Table 1 (continued)

Genus (Phyluma/Classb/Orderc/Familyd) Species AglB

Haloterrigena (E/H/H/H) Htg. turkmenica Htur_2957Natrialba (E/H/H/H) Nab. magadii Nmag_0927Natrinema (E/H/H/H) Nnm. pellirubrum Natpe_0008Natronobacterium (E/H/H/H) Nbt. gregoryi Natgr_1685Natronomonas (E/H/H/H) Nmn. pharaonis NP3720AMethanobacterium (E/Mtb/Mtb/Mtb) Methanobacterium sp. AL-21 Metbo_0534,0719

Methanobacterium sp. SWAN-1 MSWAN_1855Methanobrevibacter (E/Mtb/Mtb/Mtb) M. ruminantium mru_0391

M. smithii ATCC 35061 Msm_0716M. smithii DSM 2374 METSMIF1_02364M. smithii DSM 2375 METSMIALI_01371

Methanosphaera (E/Mtb/Mtb/Mtb) M. stadtmanae Msp_0368Methanothermobacter (E/Mtb/Mtb/Mtb) M. marburgensis MTBMA_c02090

M. thermautotrophicus MTH1623Methanothermus (E/Mtb/Mtb/Mtm) M. fervidus Mfer_0177Methanocaldococcus (E/Mtc/Mtc/Mtc) M. fervens Mefer_0590

M. infernus Metin_1222M. jannaschii MJ1525Methanocaldococcus sp. FS406-22 MFS40622_0538M. vulcanius Metvu_0243

Methanotorris (E/Mtc/Mtc/Mtc) M. formicicus MetfoDRAFT_0029M. igneus Metig_1797

Methanococcus (E/Mtc/Mtc/Mcc) M. aeolicus Maeo_1409M. maripaludis C5 MmarC5_0154M. maripaludis C6 MmarC6_1249M. maripaludis C7 MmarC7_0669M. maripaludis S2 MMP1424e

M. maripaludis X1 GYY_07955M. vannielii Mevan_0735M. voltae A3 MVO_1038M. voltae PS MVO1749e

Methanothermococcus (E/Mtc/Mtc/Mcc) M. okinawensis Metok_0791Methanocella (E, Mtm, Mtl, Mtl) M. arvoryzae LRC539,541,558

M. conradii Mtc_0182,0183,0205M. paludicola SANAE MCP_2705,2723

Methanocorpusculum (E, Mtm, Mmb, Mcp) M. labreanum Mlab_0662Methanoculleus (E, Mtm, Mmb, Mmc) M. marisnigri Memar_0175,2235Methanofollis (E, Mtm, Mmb, Mmc) M. liminatans Metli_2406Methanoplanus (E, Mtm, Mmb, Mmc) M. limicola Metlim_1216

M. petrolearius Mpet_0084,2443Methanolinea (E, Mtm, Mmb, Mrg) M. tarda MettaDRAFT_0779Methanoregula (E, Mtm, Mmb, Mrg) M. boonei Mboo_0249,1209Methanosphaerula (E, Mtm, Mmb, Mrg) M. palustris Mpal_0785Methanospirillum (E, Mtm, Mmb, Msp) M. hungatei Mhun_2859,3066,3149Methanosaeta (E, Mtm, Msc, MSa) M. concilii MCON_1133,1444

M. harundinacea Mhar_0540,1091,1439,1730M. thermophila Mthe_1164,1498

Methanococcoides (E, Mtm, Msc, MSr) M. burtonii Mbur_1579Methanohalobium (E, Mtm, Msc, MSr) M. evestigatum Metev_1257Methanohalophilus (E, Mtm, Msc, MSr) M. mahii Mmah_0123Methanosalsum (E, Mtm, Msc, MSr) M. zhilinae Mzhil_1653Methanosarcina (E, Mtm, Msc, MSr) M. acetivorans MA_1172,3752,3753,3754

M. barkeri Mbar_A0242,A0243,A0368M. mazei MM_0646,0647,2210

Candidatus Haloredivivus (E/Nnh//) Candidatus Haloredivivus sp. G17 HRED_02810Candidatus Nanosalina (E/Nnh//) Candidatus Nanosalina sp. J07AB43 J07AB43_03340Candidatus Nanosalinarum (E/Nnh//) Candidatus Nanosalinarum J07AB56 J07AB56_11160Pyrococcus (E/Tc/Tc/Tc) P. abyssi PAB0974,1586,2202

P. furiosus COM1 PFC_07420P. furiosus DSM 3638 PF0156e,0411P. horikoshii PH0242,1271P. yayanosii PYCH_17920,19200Pyrococcus sp. NA2 PNA2_0761,1113Pyrococcus sp. ST04 Py04_0309,0456

Thermococcus (E/Tc/Tc/Tc) T. barophilus TERMP_00665,02078,02121T. gammatolerans TGAM_0406,0937T. kodakarensis TK0810,1718T. litoralis OCC_09883, 01289, 05039T. onnurineus TON_0775,1820T. sibiricus TSIB_0007,0418Thermococcus sp. 4557 GQS_05995,06090,01010Thermococcus sp. AM4 TAM4_672,1026Thermococcus sp. CL1 CL1_0839,0859,1904

Picrophilus (E/Tl/Tl/Pi) P. torridus PTO0786Thermoplasma (E/Tl/Tl/Ts) T. acidophilum Ta1136

T. volcanium TVN1212

330 L. Kaminski et al. / Molecular Phylogenetics and Evolution 68 (2013) 327–339

Table 1 (continued)

Genus (Phyluma/Classb/Orderc/Familyd) Species AglB

Aciduliprofundum (E///) A. boonei Aboo_0310Candidatus Micrarchaeum (E///) Candidatus M. acidiphilum UNLARM2_0813Candidatus Parvarchaeum (E///) Candidatus P. acidiphilum BJBARM4_0616

Candidatus P. acidophilus BJBARM5_0254uncultured marine group II euryarchaeote MG2_1283

Candidatus Korarchaeum (K///) Candidatus K. cryptofilum Kcr_1056Nanoarchaeum (N///) N. equitans NEQ155Cenarchaeum (T//Ce/Ce) C. symbiosum CENSYa_1939Candidatus Nitrosoarchaeum (T//Ni/Ni) Candidatus N. koreensis MY1_0015

Candidatus N. limnia BG20 CNitlB_010100007878Candidatus N. limnia SFB1 Nlim_2107

Nitrosopumilus (T//Ni/Ni) Candidatus N. salaria BD31_I1640N. maritimus Nmar_0075Nitrosopumilus sp. MY1 MY1_0015

Candidatus Caldiarchaeum (T///) Candidatus C. subterraneum CSUB_C0660unclassified Archaea halophilic archaeon DL31 Halar_1620

Sequences obtained from the Carbohydrate-Active enZYmes Database (http://www.cazy.org/Home.html) (August, 2012), the Integrated Microbial Genomes – GenomeEncyclopedia of Bacteria and Archaea Genomes (IMG/GEBA) (http://img.jgi.doe.gov/cgi-bin/geba/main.cgi) (August, 2012), UCSC Archaeal Genome Browser (http://archaea.ucsc.edu/) (August, 2012) and the NCBI Protein Database (http://www.ncbi.nlm.nih.gov/protein) (August, 2012) sites.Listed as AglB at CAZy glycosyltransferase group 66 (oligosaccharyltransfases) but lacking the WWDXG motif involved in oligosaccharyltransferase activity: HAH_0492,MSWAN_1515, MSWAN_1516, MTBMA_ c4670, MTBMA_c04680, MTH420, MTH1898, MTH1906, Mfer_0275, Mfer_0623, Mhun_2859, Mhun_3066, Mhun_3149, Mthe_1548.

a Phylum: Crenarchaeota, C; Euryarchaeota, E; Korarchaeota, K; Nanoarchaeota, N; Thaumarchaeota, T.b Class: Thermoprotei, Tp; Archaeoglobi, Ar; Halobacteria, H; Methanobacteria, Mtb; Methanococci, Mtc; Methanomicrobia, Mtm; Methanopyri, Mtp; Nanohaloarchaea,

Nnh; Thermococci, Tc; Thermoplasmata, Tl.c Order: Acidilobales, Ac; Caldisphaeraceae, Ca; Desulfurococcales, De; Fervidicoccales, Fe; Sulfolobales, Su; Thermoproteales, Th; Archaeoglobales, Ar; Halobacteriales, H;

Methanobacteriales, Mtb; Methanococcales; Mtc; Methanocellales, Mtl; Methanomicrobiales, Mmb; Methanosarcinales, Msc; Thermococcales, Tc; Thermoplasmatales, Tl;Cenarchaeales, Ce; Nitrosopumilales, Ni; Nitrososphaerales, Nt.

d Family: Acidilobaceae, Ac; Caldisphaera, Ca; Desulfurococcaceae, De; Pyrodictiaceae, Py; Fervidicoccaceae, Fe; Sulfolobaceae, Su; Thermofilaceae, Thf; Thermoproteaceae,Thp; Archaeoglobaceae, Ar; Halobacteriaceae, H; Methanobacteriaceae, Mtb; Methanothermaceae, Mtm; Methanocaldococcaceae, Mtc; Methanococcaceae, Mcc; Methano-cellaceae, Mtl; Methanocorpusculaceae, Mcp; Methanomicrobiaceae, Mmc; Methanoregulaceae, Mrg; Methanospirillaceae, Msp; Methanosaetaceae, Msa; Methanosarcin-aceae, Msr; Thermococcaceae, Tc; Picrophilaceae, Pi; Thermoplasmataceae, Ts; Cenarchaeaceae, Ce; Nitrosopumilaceae, Ni; Nitrososphaeraceae, Nt.

e Experimentally verified to be an oligosaccharyltransferase.

L. Kaminski et al. / Molecular Phylogenetics and Evolution 68 (2013) 327–339 331

genome appeared at yet another point during evolution and clus-tered with homologs from Methanoplanus petroleanus (Fig. 1,arrowheads). Species-specific evolutionary patterns were also seenfor AglB sequences from non-methanogens. For instance, the threeThermococcus litoralis AglB sequences also appeared at distinctpoints during evolution (Fig. 1, diamonds). Indeed, two of these se-quences cluster with their homologs from Thermococcus sibiticusand Thermococcus barophilus, while the third sequence clusterswith AglB from Thermococcus kodakarenesis.

3.3. AglB multiplicity in a given species may carry physiologicalsignificance

The presence of the multiple AglB sequences in a single speciescould be a reflection of differences in OST substrate or target pref-erence, prevalence or availability, possibly as a function of localgrowth conditions. Accordingly, closer examination of the multipleversions of AglB in a given species reveals differences in the con-sensus WWDYG motif. It is conceivable that these modificationsreflect different activities of the various versions of the protein.Accordingly, examination of the phylogenetic distribution of thedistinct versions of AglB found in a single species often suggeststhat these proteins appeared early in evolution. AglB proteins frommembers of the Family Thermococcaceae (Group B) that can be dis-tinguished on the basis of variability at the fourth position of theconsensus WWDYG catalytic motif offer such examples. Withinthe Thermococcaceae, those AglB proteins in which Tyr is replacedwith either His or Gln were all assigned to clade e (i.e., P. furiosusPF0411, Pyrococcus horikoshii PH1271, Thermococcus sp. 4557GQS_05995 and GQS_06090, Thermococcus gammatoleransTGAM_0406, Thermococcus sp. AM4 TAM4_1026, Thermococcussp. CL-1 CL1_0839 and CL1_0859 and Thermococcus onnurineusTON_1820). By contrast, AglB proteins from the same species con-taining Tyr at position four of this catalytic motif were all assigned

to clade d. Similarly, the two Methanocella arvoryzae AglB proteinscontaining the WWDYG motif were assigned to clade a, while thethird AglB, in which this motif was modified to WWDDG, was as-signed to clade b. On the other hand, both AglB proteins from Meth-anoplanus petrolearius and from Methanobacterium sp. AL-21 wereassigned to the same clade, despite presenting differences in thismotif. Likewise, the Methanosaeta harundinacea AglB sequencewhere a modified WWDRG motif is found (Mhar_1439) is assignedto clade b, along with two of the three additional AglB proteins pre-dicted in this organism, each of which contains a WWDYG motif atthis position.

The existence of OSTs possessing unique specificities in a singleorganism offers a strategy for the addition of different N-linkedglycans in a single species. This could be tested in future in genet-ically tractable species containing multiple AglB proteins, such asMethanosarcina or Thermococcales species (Leigh et al., 2011), onceproof of N-glycosylation in these species has been provided. How-ever, while such differential N-glycosylation has been observed inHbt. salinarum and Hfx. volcanii, where S-layer glycoproteinsare simultaneously modified by two distinct N-linked glycans(Wieland et al., 1983; Lechner et al., 1985; Guan et al., 2012), eachspecies only encode a single AglB protein. Nonetheless, it remainspossible that these species contain a second OST that can no longerbe recognized as an AglB ortholog, or alternatively, that relies on adistinct catalytic mechanism. The fact that the two N-linked gly-cans in Hbt. salinarum contain different linking sugars implies theexistence of two OSTs employing different mechanisms ofcatalysis.

3.4. Identification of putative aglB-based N-glycosylation loci

In Hfx. volcanii, one of the few archaeal species for which de-tailed information on N-glycosylation is available, all but one ofthe genes known to participate in the assembly and attachment

Fig. 1. Phylogenetic tree of euryarchaeal AglB sequences. An alignment of 77 AglB sequences from 31 euryarchaeal species containing more than one copy of AglB was used toconstruct a Neighbor-Joining tree. Robustness of the tree was assessed by a bootstrap test based on 500 pseudo-replicates. Bootstrap values are shown on the nodes of thetree where greater than 50%. Each entry lists the species followed by the genome-derived name of AglB, as indicated in Table 1. The limits of the different groups and cladesare marked. The arrows indicate Methanocella arvoryzae AglB sequences, while the meanings of the arrowhead and diamond symbols are provided in the text. Those AglBsequences in which the Tyr of the consensus WWDYG motif is modified are indicated by the full circles.

332 L. Kaminski et al. / Molecular Phylogenetics and Evolution 68 (2013) 327–339

of a pentasaccharide to selected Asn residues of N-glycosylatedproteins are sequestered within an aglB-containing gene clusterbeginning at HVO_1517, encoding AglJ, and extending toHVO_1531, encoding AglM (Yurist-Doutsch and Eichler, 2009; Yur-ist-Doutsch et al., 2010). This gene cluster also includes aglP, aglQ,aglE, aglR, aglS, aglF, aglI and aglG. Only aglD, encoding the GTresponsible for charging the dolichol phosphate carrier with the fi-nal sugar of the pentasaccharide (Abu-Qarn et al., 2007; Guan et al.,

2010), mannose, is found outside this cluster. On the other hand,no N-glycosylation gene clusters (defined as containing aglB andat least three other putative N-glycosylation pathway compo-nent-encoding genes) are seen in M. voltae, M. maripaludis or S. aci-docaldarius, other species where genes involved in N-glycosylationhave also been identified (Chaban et al., 2006; Magidovichand Eichler, 2009; VanDyke et al., 2009; Meyer et al., 2011). Hence,to assess the prevalence of aglB-based N-glycosylation gene

Table 2Clustering of putative N-glycosylation genes around archaeal aglB homologs.

Genus (Phylum/Class/Order/Familya) Species Members of putative N-glycosylation cluster

Acidilobus (C/Tp/Ac/Ac) A. saccharovorans n.d.Desulfurococcus (C/Tp/De/De) D. fermentans n.d.

D. kamchatkensis n.d.D. mucosus n.d.

Ignicoccus (C/Tp/De/De) I. hospitalis n.d.Ignisphaera (C/Tp/De/De) I. aggregans n.d.Staphylothermus (C/Tp/De/De) S. hellenicus n.d.

S. marinus n.d.Thermogladius (C/Tp/De/De) T. cellulolyticus n.d.Thermosphaera (C/Tp/De/De) T. aggregans n.d.Hyperthermus (C/Tp/De/Py) H. butylicus n.d.Pyrolobus (C/Tp/De/Py) P. fumarii n.d.Fervidicoccus (C/Tp/Fe/Fe) F. fontis n.d.Acidianus (C/Tp/Su/Su) A. hospitalis n.d.Metallosphaera (C/Tp/Su/Su) M. cuprina Mcup_0425,0426,0427,0430

M. sedula Msed_1805,1808,1809,1810,1811,1814,1816M. yellowstonensis MetMK1DRAFT_00024050,00024120,00024130,00024150,00024220

Sulfolobus (C/Tp/Su/Su) S. acidocaldarius n.d.S. islandicus HVE10/4 n.d.S. islandicus L.D.8.5 n.d.S. islandicus L.S.2.15 n.d.S. islandicus M.14.25 n.d.S. islandicus M.16.27 n.d.S. islandicus M.16.4 n.d.S. islandicus REY15A n.d.S. islandicus Y.G.57.14 n.d.S. islandicus Y.N.15.51 n.d.S. solfataricus 98/2 n.d.S. solfataricus P2 n.d.S. tokodaii n.d.

Thermofilum (C/Tp/Th/Thf) T. pendens n.d.Caldivirga (C/Tp/Th/Thp) C. maquilingensis n.d.Pyrobaculum (C/Tp/Th/Thp) P. aerophilum n.d.

P. arsenaticum n.d.P. calidifontis n.d.P. islandicum n.d.P. oguniense n.d.Pyrobaculum sp. 1860 n.d.

Thermoproteus (C/Tp/Th/Thp) T. neutrophilus n.d.T. tenax n.d.T. uzoniensis n.d.

Vulcanisaeta (C/Tp/Th/Thp) V. distributa n.d.V. moutnovskia n.d.

Archaeoglobus (E/Ar/Ar/Ar) A. fulgidus AF0035,0038,0039,0040,0043,0044,0045/0321,0322,0323a,0323b,0324,032,0326,0327,0328,0329

A. profundus Arcpr_1194,1195,1196,1201,1202,1203,1204,1207,1214A. veneficus Arcve_0544,0545,0546,0552,0556,055,0562,0566,0567,0568

Ferroglobus (E/Ar/Ar/Ar) F. placidus n.d.Haladaptatus (E/H/H/H) Hap. paucihalophilus ZOD2009_20058,20063,20073,20083,20098,20113Halalkalicoccus (E/H/H/H) Hac. jeotgali HacjB3_10595,10600,10620,10625,10630Haloarcula (E/H/H/H) Har. californiae HAH_00005730,00005780,00005820,00005840,00005850,00005860

Har. hispanica HAH_1202,1203,1206,1208,1210,1214Har. marismortui rrnAC0419,0421,0427,0429,0430,0431Har. sinaiiensis HAI_00022150,00022180,0002210,0002230,0002240,00022250Har. vallismortis HAJ_00008880,00008890,00008900,00008920

Halobacterium (E/H/H/H) Hbt. salinarum R1 OE2524R,2528R,2529F,2530F,2535R,2537F,2546F,254,2548FHalobacterium sp. NRC-1 VNG1048G,1053G,1054G,1055G,1059C,1062G,1066C,1067G,1068GHalobacterium sp. DL1 HalDL1DRAFT_1630,1631,1632,1633,1634,1639,1640,1641,1642,1643,1644,1645,

1646,1647,1649Halobiforma (E/H/H/H) Hbf. lacisalsi HlacAJ_010100009153,010100009163,010100009168,010100009173,010100009178Haloferax (E/H/H/H) Hfx. denitrificans HAK_000032050,00032060,000032070,000032080,000032090,

000032110,000032120,000032130,000032150Hfx. mediterranei HFX_1580,1581,1582,1587,1591,1592Hfx. mucosum HAM_16650,16660,16700,16750,16760,16770Hfx. sulfurifontas HAN_00007730,00007740,00007750,00007760,00007780,00007790,00007800,

00007810,00007850,00007860,00007870Hfx. volcanii HVO_1517b,1522b,1523b,1523.1b,1524,1525b,1526b,1527b,1528b,1529b,1530,1531b

Halogeometricum (E/H/H/H) Hgm. borinquense Hbor_16990,17000,17010,17020,17030,17040,17050,17060,17070,17100,17110,17120,17130,17140,17180,17190,17200,17210

Halomicrobium (E/H/H/H) Hmc. mukohataei Hmuk_2752,2753,2754,2756,2757,2758,Halopiger (E/H/H/H) Hpg. xanaduensis Halxa_2340,2341,2342,2344,2348,2349,2351,2352,2355,2357,2538,2361,2368,

2369,2371,2372,2379,2380,2381Haloquadratum (E/H/H/H) Hqr. walsbyi C23 Hqrw_3012,3013,3016,3017,3021,3023,3029,3036,3040,3043,3044,3045

Hqr. walsbyi DSM 16790 HQ2680A,2681A,2682A,2683A,2686A,2687A,2691A,2692A,2694AHalorhabdus (E/H/H/H) Hrd. tiamatea n.d.

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L. Kaminski et al. / Molecular Phylogenetics and Evolution 68 (2013) 327–339 333

Table 2 (continued)

Genus (Phylum/Class/Order/Familya) Species Members of putative N-glycosylation cluster

Hrd. utahensis n.d.Halorubrum (E/H/H/H) Hrr. lacusprofundi Hlac_1062,1063,1065,1067,1069,1071,1073,1074,1075Haloterrigena (E/H/H/H) Htg. turkmenica Htur_2947,2949,2954,2955,2956,2957Natrialba (E/H/H/H) Nab. magadii Nmag_0916,0917,0922,0924,0925,0926,0927Natrinema (E/H/H/H) Nnm. pellirubrum NatpeDRAFT_0005,0006,0007,0008Natronobacterium (E/H/H/H) Nbt. gregoryi NatgrDRAFT_1666,1669,1670,1675,1682,1683,1684,1685Natronomonas (E/H/H/H) Nmn. pharaonis n.d.Methanobacterium (E/Mtb/Mtb/Mtb) Methanobacterium sp. AL-21 Metbo_0719,0720,0721,0722,0723,0725,0726,0727,0729,0734

Methanobacterium sp.SWAN-1

n.d.

Methanobrevibacter (E/Mtb/Mtb/Mtb) M. ruminantium n.d.M. smithii ATCC 35061 n.d.M. smithii DSM 2374 n.d.M. smithii DSM 2375 n.d.

Methanosphaera (E/Mtb/Mtb/Mtb) M. stadtmanae n.d.Methanothermobacter (E/Mtb/Mtb/Mtb) M. marburgensis n.d.

M. thermautotrophicus n.d.Methanothermus (E/Mtb/Mtb/Mtm) M. fervidus n.d.Methanocaldococcus (E/Mtc/Mtc/Mtc) M. fervens n.d.

M. infernus n.d.M. jannaschii n.d.Methanocaldococcus sp.FS406-22

n.d.

M. vulcanius n.d.Methanotorris (E/Mtc/Mtc/Mtc) M. formicicus n.d.

M. igneus n.d.Methanococcus (E/Mtc/Mtc/Mcc) M. aeolicus n.d.

M. maripaludis C5 n.d.M. maripaludis C6 n.d.M. maripaludis C7 n.d.M. maripaludis S2 n.d.M. maripaludis X1 n.d.M. vannielii n.d.M. voltae n.d.M. voltae n.d.

Methanothermococcus (E/Mtc/Mtc/Mcc) M. okinawensis n.d.Methanocella (E, Mtm, Mtl, Mtl) M. arvoryzae LRC537,539,541,542,543,544,545,547,548,549,550,551,552,553,555,558

M. conradii Mtc_0169,0171,0172,0182,0183,0186,0187,0188,0189,0190,0191,0193,0197,0198,0199,0201,0202,0203,0205,206

M. paludicola SANAE MCP_2704,2705,2706,2707,2708,2709,2710,2711,2714,2715,2716,2717,2718,2719,2720,2723

Methanocorpusculum (E, Mtm, Mmb, Mcp) M. labreanum Mlab_0662,0663,664,665,666Methanoculleus (E, Mtm, Mmb, Mmc) M. marisnigri Memar_0175,0183,0184,0185,0186,0187,0188,0189,0192Methanofollis (E, Mtm, Mmb, Mmc) M. liminatans n.d.Methanoplanus (E, Mtm, Mmb, Mmc) M. limicola n.d.

M. petrolearius n.d.Methanolinea (E, Mtm, Mmb, Mrg) M. tarda MettaDRAFT_0779,0781,0782,0783,0784,0785,0786,0787Methanoregula (E, Mtm, Mmb, Mrg) M. boonei Mboo_0249,0250,0252,0253,0254,0255Methanosphaerula (E, Mtm, Mmb, Mrg) M. palustris n.d.Methanospirillum (E, Mtm, Mmb, Msp) M. hungatei Mhun_2852,2853,2854,2855,2856,2857,2858,2859/

3065,3066,3067,3072,3073,3074,3075,3076,3077,3078,3079,3080,3084,3090/3138,3145,3147,3149,3151,3154,3161

Methanosaeta (E, Mtm, Msc, MSa) M. concilii n.d.M. harundinacea Mhar_1091,1093,1094,1095,1096,1097,1098,1099,1100,1101,1102,1103,1104,1106,1110M. thermophila n.d.

Methanococcoides (E, Mtm, Msc, MSr) M. burtonii Mbur_1579,1581,1582,1583,1584,1585,1586,1587,1590,1593,1594,1597,1603,1604,1605,1607,1608,1612,1613,1615,1617

Methanohalobium (E, Mtm, Msc, MSr) M. evestigatum Metev_1236,1237,1242,1244,1250,1252,1253,1254,1255,1257Methanohalophilus (E, Mtm, Msc, MSr) M. mahii n.d.Methanosalsum (E, Mtm, Msc, MSr) M. zhilinae Mzhil_1638,1639,1640,1641,1642,1643,1645,1648,1649,1651,1652,1653,1655,1656Methanosarcina (E, Mtm, Msc, MSr) M. acetivorans MA_1172,1173,1174,1175,1176,1177,1179,1180,1181,1183,1184,1185,1186,1187/

3752,3753,3754,3755,3756,3757,3758,3764,3766,3767,3769a, 3769b,3777,3778,3779,3780,3781

M. barkeri Mbar_A0229,A0230,A0231,A0232, A0233,A0234,A0235,A0236,A0237,A0238,A0239,A0240,A0241,A0242, A0243/A0366,A0368,A0369,A0373,A0374,A0375

M. mazei MM_0646,0647,0648,0649,0650,0651,0652,0653,0654,656,657,658,659,660/2208,2210,2213,2214,2215,2216,2217,2221,2222,2223

Candidatus Haloredivivus (E/Nnh//) Candidatus Haloredivivus sp.G17

HRED_02640,02670,02680,02720,02810

Candidatus Nanosalina (E/Nnh//) Candidatus Nanosalina sp.J07AB43

J07AB43_03180,03190,03200,03210,03240,03270,03310,03320,03330,03340

Candidatus Nanosalinarum (E/Nnh//) Candidatus NanosalinarumJ07AB56

J07AB56_11160,11200,11210,11240,11250

Pyrococcus (E/Tc/Tc/Tc) P. abyssi PAB1411,1410,1409,0973,0974/0783,0784,0785,0787,0789,0790.1nn,0973,0795,0796,1587,1586

334 L. Kaminski et al. / Molecular Phylogenetics and Evolution 68 (2013) 327–339

Table 2 (continued)

Genus (Phylum/Class/Order/Familya) Species Members of putative N-glycosylation cluster

P. furiosus COM1 n.d.P. furiosus DSM 3638 n.d.P. horikoshii n.d.P. yayanosii PYCH_17860,17870,17880,17900,17910,17920Pyrococcus sp. NA2 PNA2_1113,1114,1115,1120,1121Pyrococcus sp. ST04 Py04_0454,0455,0456,0457,0461,0465

Thermococcus (E/Tc/Tc/Tc) T. barophilus TERMP_02119,02121,02122,02123,02124,02129/2078,2079,2080,2084,2089,2091,2094,2096,2097,2099,2100

T. gammatolerans n.d.T. kodakarensis TK1708,1711,1712,1713,1714,1715,1716,1717,1718,1719,1720,1721,

1722,1723,1725,1731,1732,1733T. litoralis OCC_01289,01319,01324,01329,01334,01354/05039,05049,05054,05059,05064T. onnurineus TON_1818,1819,1820,1821,1822,1823T. sibiricus TSIB_2044,2045,2047,2048,2049,2050,2054,2059,2061,0003,0004,0005,0006,0007Thermococcus sp. 4557 GQS_05950,05955,05960,05975,05995,06000,0600/06075,06080,06085,06090Thermococcus sp. AM4 TAM4_1088,1094,1026,1040Thermococcus sp. CL1 CL1_0827,0828,0830,0834,0838,0839,0840,0841/0850,0856,0857,0859

Picrophilus (E/Tl/Tl/Pi) P. torridus n.d.Thermoplasma (E/Tl/Tl/Ts) T. acidophilum n.d.

T. volcanium n.d.Aciduliprofundum (E///) A. boonei n.d.Candidatus Micrarchaeum (E///) Candidatus M. acidiphilum n.d.Candidatus Parvarchaeum (E///) Candidatus P. acidiphilum n.d.

Candidatus P. acidophilus n.d.uncultured marine group IIeuryarchaeote

n.d.

Candidatus Korarchaeum (K///) Candidatus K. cryptofilum n.d.Nanoarchaeum (N///) N. equitans n.d.Cenarchaeum (T//Ce/Ce) C. symbiosum A n.d.Candidatus Nitrosoarchaeum (T//Ni/Ni) Candidatus N. koreensis n.d.

Candidatus N. limnia BG20 n.d.Candidatus N. limnia SFB1 n.d.

Nitrosopumilus (T//Ni/Ni) Candidatus N. salaria n.d.N. maritimus n.d.Nitrosopumilus sp. MY1 n.d.

Candidatus Caldiarchaeum (T///) Candidatus C. subterraneum n.d.unclassified Archaea halophilic archaeon DL31 Halar_1591,1600,1601,1610,1611,1612,1613,1615,1616,1620

Glycosylation-related annotation at the Integrated Microbial Genomes – Genome Encyclopedia of Bacteria and Archaea Genomes (IMG/GEBA) (http://img.jgi.doe.gov/cgi-bin/geba/main.cgi) (August, 2012), UCSC Archaeal Genome Browser (http://archaea.ucsc.edu/) (August, 2012) and the NCBI Protein Database (http://www.ncbi.nlm.nih.gov/protein) (August, 2012) sites.Cluster is defined as including AglB and at least 3 other putative proteins involved in glycosylation; AglB in bold.n.d., not detected.

a The abbreviations used from the different phyla, classes, orders, and families are provided in the legend to Table 1.b Sequences other than AglB experimentally confirmed as participating in N-glycosylation.

L. Kaminski et al. / Molecular Phylogenetics and Evolution 68 (2013) 327–339 335

clustering across the Archaea, those regions down- and upstreamof genes annotated as encoding AglB were examined (Table 2).

In the 45 crenarcheal species considered, aglB-based gene clus-tering was only observed in the three species belonging to theGenus Metallosphaera. Given the broad geographic distribution ofMetallosphaera cuprina (sulfuric hot spring in Tengchong, Yunnan,China; Liu et al., 2011), Metallosphaera sedula (Thermal pond, Pisc-iarelli Solfatara, Naples, Italy; Huber et al., 1989) and Metallosphae-ra yellowstonensis (acidic geothermal springs in YellowstoneNational Park; Kozubal et al., 2008), it would appear that N-glyco-sylation gene clustering occurred prior to division of an ancestorinto the three species. In the Euryarchaeota, aglB-based glycosyla-tion gene clustering was detected in 26 of the 29 available haloar-chaeal genomes, with only the two Halorhabdus species andNatronomonas pharaonis not presenting such an arrangement. Thisis not unexpected, since gene clusters appear to be better con-served in haloarchaea than other archaeal groups (Berthon et al.,2008). In the 49 methanoarchaeal species examined, aglB-basedglycosylation gene clustering was observed largely along genuslines, with some genera in a given family displaying aglB-basedglycosylation gene clustering and others in the same familynot. Indeed, even within a given methanoarchaeal genus, onlysome species presented such gene clustering. In the thermo- and

hyperthemophilic euryarchaeota, aglB-based glycosylation geneclustering was seen in the three Archaeoglobus species, in allThermococcus species apart from T. gammatolerans, and in four ofthe seven Pyrococcus species. No aglB-based clustering was seenin the Korarchaeota, Nanoarchaeota or Thaumarchaeota.

In most cases where aglB-based glycosylation gene clusteringwas observed, aglB itself corresponds to one edge of the cluster.In a limited number of cases, an additional glycosylation geneadjacent to aglB serves this role. Where multiple AglB sequencesare found, some species presented each aglB in a glycosylationgene clusters, others organized only some of the multiple AglB se-quences into such clusters, while yet other species containingmultiple AglB sequences did not cluster N-glycosylation genesaround aglB at all. In each of the three Methanocella species, themultiple versions of AglB were all found in a common genecluster.

Finally, the distribution of genes known or believed to mediateN-glycosylation in other Archaea suggests that N-glycosylationgene clusters not anchored by aglB may also exist. For example,N-glycosylation roles have been demonstrated for the products ofM. maripaludis MMP1079-MMP1088, while AglB is encoded byMMP1424 (Chaban et al., 2006, 2009; Shams-Eldin et al., 2008;Jones et al., 2012).

Fig. 3. Phylogenetic tree of Haloferax AglB proteins. The phylogenetic relationshipsof AglB from Hfx. volcanii, Hfx. denitrificans, Hfx. mediterranei, Hfx. mucosum and Hfx.sulfurifontas is presented. The Hrr. lacusprofundi AglB sequence served as an out-group. Numbers represent the percent of bootstrap support for each node.

Fig. 2. Schematic representation of aglB-based gene clusters in five Haloferax species. The positions of agl genes in Hfx. volcanii and their homologs in Hfx. denitrificans, Hfx.mediterranei, Hfx. mucosum and Hfx. sulfurifontas are indicated, as are those of other glycosylation-related genes. The genes are arbitrarily drawn in terms of size. In thosespecies where the orientation of the gene cluster is opposite to that in Hfx. volcanii, a double-headed arrow is found next to the species name. The legend describes themeaning of the coloring scheme employed.

336 L. Kaminski et al. / Molecular Phylogenetics and Evolution 68 (2013) 327–339

3.5. Evolutionary insight into N-glycosylation in Haloferaxspeciesderived from gene cluster comparison and gene content

To demonstrate how the phenomenon of aglB-based glycosyla-tion gene clustering can be used for making predictions related toN-glycosylation in a given species and to gain insight into the evo-lution of this post-translational modification, the five Haloferaxspecies for which genomic information is presently available wereconsidered. In terms of their geography, the five species are founddistally of one another, with Hfx. denitrificans originally havingbeen isolated from a saltern in California, USA (Tomlinson et al.,1986), Hfx. mediterranei from a saltern near Alicante, Spain (Rodri-guez-Valera et al., 1980), Hfx. mucosum from Shark Bay, Australia(Allen et al., 2008), Hfx. sulfurifontis from a sulfur spring in Okla-homa, USA (Elshahed et al., 2004) and Hfx. volcanii from the DeadSea (Mullakhanbhai and Larsen, 1975).

At present, the pathway of N-glycosylation has been delineatedin Hfx. volcanii, based on a series of genetic and biochemical stud-ies. In the Agl pathway in this species, aglJ, aglG, aglI and aglE en-code GTs that sequentially add four nucleotide-activated sugarsto a common dolichol phosphate carrier on the inner face of theplasma membrane (Abu-Qarn et al., 2008; Plavner and Eichler,2008; Yurist-Doutsch et al., 2008; Guan et al., 2010; Kaminskiet al., 2010). Once the tetrasaccharide-bearing dolichol phosphatehas been translocated across the membrane, AglB delivers the gly-can to target protein Asn residues (Abu-Qarn et al., 2007). At thesame time, the only N-glycosylation pathway component encodedby a gene outside the aglB-based glycosylation gene cluster, AglD,adds the final sugar of the N-linked pentasaccharide, mannose, to adistinct dolichol phosphate (Abu-Qarn et al., 2007; Yurist-Doutschand Eichler, 2009; Guan et al., 2010). Mannose-charged dolicholphosphate is ‘flipped’ across the membrane in a process involvingAglR (Kaminski et al., 2012), at which point AglS delivers the man-nose to the Asn-bound tetrasaccharide (Cohen-Rosenzweig et al.,2012). In additon, AglF, AglM and AglP serve various sugar process-ing roles (Magidovich et al., 2010; Yurist-Doutsch et al., 2010).

Examination of the genomes of Hfx. denitrificans, Hfx. mediterra-nei, Hfx. mucosum and Hfx. sulfurifontas reveals aglB-based geneclusters containing homologs to many of the Hfx. volcanii agl genes(Fig. 2). For example, the Hfx. denitrificans aglB-based gene clusteris almost identical to its Hfx. volcanii counterpart, except for thepresence of transposases in the latter. The Hfx. sulfurifontas aglB-based gene cluster also contains several homologs to Hfx. volcaniiagl sequences, albeit differently arranged. In addition, the Hfx. sul-furifontas aglB-based gene cluster contains sequences encoding GTs

and other sugar-processing proteins not found in the comparableHfx. volcanii cluster. Thus, based on the composition of the Hfx. den-itrificans aglB-based gene cluster, it can be predicted that N-linkedglycans in this species will be highly similar if not identical to theN-linked pentasaccharide decorating glycoproteins in Hfx. volcanii.By the same reasoning, one would expect a somewhat different N-glycan in Hfx. sulfurifontas. In considering the identical aglB-basedgene clusters seen in Hfx. mediterranei and Hfx. mucosum, not onlyare far fewer glycosylation-related genes observed, the few homo-logs of Hfx. volcanii Agl protein-encoding genes are distributed dif-ferently than in the Hfx. volcanii cluster. As such, the N-glycanspredicted to decorate Hfx. mediterranei and Hfx. mucosum glycopro-teins are expected to be identical, yet significantly differing fromwhat is found in the other Haloferax strains.

The organization of aglB and other agl genes within each clusteroffers evolutionary insight into the N-glycosylation process. Thesimilarities of the aglB-based gene clusters in Hfx. volcanii, Hfx.

Table 3G + C content of Haloferax agl genes.

Hfx. volcanii Hfx. denitrificans Hfx. sulfurifontis Hfx. mediterranei Hfx. mucosum

aglJa 0.61 0.60 0.62 0.62 0.62aglP 0.45 0.44 –b – –aglQ 0.48 0.47 – – –aglE 0.47 0.47 0.62 – –aglR 0.47 0.46 0.49 0.61 0.62aglS 0.41 0.41 – – –aglF 0.55 0.54 0.54 – –aglI 0.58 0.58 0.51 0.64 0.64aglG 0.54 0.56 – – –aglB 0.63 0.62 0.62 0.62 0.63aglM 0.66 0.65 0.67 – –aglD 0.70 0.71 0.71 0.64 0.67Genomec 0.62 0.66 0.66 0.60 0.62

a Genes are listed in the order they appear in the Hfx. volcanii agl gene cluster, except for aglD, which is found elsewhere in the genome.b Gene not detected.c G + C content as listed at http://img.jgi.doe.gov/cgi-bin/w/main.cgi.

Table 4Codon usage bias in Haloferax agl genes.

Hfx. volcanii Hfx. denitrificans Hfx. sulfurifontis Hfx. mediterranei Hfx. mucosum

aglJa 40.89b 41.82 38.74 35.79 32.890.506c 0.4802 0.5435 0.6255 0.7186

aglP 51.25 51.54 –d – –

0.1748 0.1551

aglQ 56.37 55.82 – – –

0.1718 0.1624

aglE 56.37 55.30 40.76 – –

0.1831 0.1628 0.5159

aglR 57.21 55.91 56.22 39.60 41.30

0.1806 0.1658 0.2015 0.5275 0.5337

aglS 56.37 49.76 – – –

0.1718 0.1246

aglF 46.98 48.19 50.83 – –

0.4565 0.4065 0.3378aglI 47.67 46.94 56.74 34.16 33.05

0.358 0.3436 0.2089 0.6864 0.7078

aglG 43.91 42.63 – – –0.4306 0.4613

aglB 38.28 38.84 38.01 37.72 34.970.5495 0.5371 0.5328 0.6469 0.6948

aglM 33.90 34.35 32.51 – –0.6683 0.61 0.675

Genome 33.78 34.17 34.33 42.41 40.090.6585 0.6511 0.6494 0.5533 0.5974

s.d.e ±5.95 ±6.64 ±6.67 ±7.06 ±6.28±0.1199 ±0.1293 ±0.1284 ±0.1121 ±0.0990

a Genes are listed in the order they appear in the Hfx. volcanii agl gene cluster.b ENC value – values between one and two standard deviations higher than the genomic average are in bold, while values more than two standard deviations higher than

the genomic average are in bold and underlined.c CAI value – values between one and two standard deviations lower than the genomic average are in bold, while values more than two standard deviations lower than the

genomic average are in bold and underlined.d Gene not detected.e s.d. = standard deviation.

L. Kaminski et al. / Molecular Phylogenetics and Evolution 68 (2013) 327–339 337

denitrificans and Hfx. sulfurifontas and in Hfx. mediterranei and Hfx.mucosum are in agreement with the grouping of these species intoseparate clades, based on genomic segment loss and gain studies(Lynch et al., 2012). AglB protein phylogeny (Fig. 3) is congruentwith the Haloferax tree generated by this earlier study. At the sametime, homologs of Hfx. volcanii aglD, the only component of the N-glycosylation pathway in this species found outside the aglB-basedgene cluster, were detected in Hfx. denitrificans (HAK_00016980),Hfx. mediterranei (HAL_00010870), Hfx. mucosum(HAM_00012150) and Hfx. sulfurifontas (HAN_00024650). In eachcase, the identical six downstream and three upstream genesbordered this GT-encoding gene. However, in the case of the Hfx.

volcanii aglD and its Hfx. denitrificans and Hfx. sulfurifontas homo-logs, this region was expanded to include the 9 upstream and the28 downstream genes (not shown).

At the same time, variations in the composition of agl genes inthe aglB-based clusters of the different Haloferax strains, togetherwith the concept that differences in N-linked glycosylation couldprovide adaptive advantages, raise the possibility that lateral genetransfer (LGT) played a role in the evolution of AglB-based N-glyco-sylation in this genus. With the exception of Haloquadratum wal-sbyi, haloarchaea are characterized by a high genomic G + Ccontent (typically 65% G + C) (Hartman et al., 2010). The G + C con-tents of aglB and aglM tend to resemble the genomic average. This

338 L. Kaminski et al. / Molecular Phylogenetics and Evolution 68 (2013) 327–339

is not the case for other agl genes, which, in several species, presenta highly unusual base composition (Table 3). For instance, severalof the clustered genes encoding Agl homologs in Hfx. volcanii,Hfx. denitrificans and Hfx. sulfurifontas are A + T-rich, relative tothe rest of the genome (i.e. a difference of P10% from the genomicmean), indicative of fairly recent horizontal acquisition. Further-more, these genes also have extremely high effective number of co-dons (ENC) values and low codon adaptation index (CAI) values,indicative of the use of codons that are rare in the respective gen-omes (Table 4). It is noteworthy that the CAI of aglB is substantiallyhigher in Hfx. mediterranei and Hfx. mucosum than in the other spe-cies, implying higher levels of gene expression. Combined with thefact that the other N-glycosylation genes in these two genomes ap-pear to be ancestral rather than recently acquired, it would appearthat N-glycosylation is a more fundamental trait of Hfx. mediterra-nei and Hfx. mucosum. Additionally, there is high within-clustervariation in nucleotide composition in the agl clusters of Hfx. volca-nii and Hfx. denitrificans, indicating that agl genes were recruitedfrom different sources, in agreement with the protein-based phy-logenies of these genes, which show conflicting evolutionary histo-ries (Fig. S2).

4. Conclusions

In 1976, the Hbt. salinarum S-layer glycoprotein became the firstnon-eukaryal N-glycosylated protein reported (Mescher andStrominger, 1976). Over the next fifteen years, advances in describ-ing both the structures of glycans N-linked to archaeal glycopro-teins and archaeal N-glycosylation pathways were made. Morerecently, the availability of complete genome sequences, togetherwith the development of appropriate molecular tools and tech-niques led to renewed interest in this topic. In the last decade, con-siderable progress has been made in addressing genes and proteinsinvolved in N-glycosylation in several species. Today, alongsidesuch efforts being conducted at the molecular level, insight intothe archaeal version of this universal post-translational modifica-tion can now be gleaned at the genome level.

As discussed here, virtually all Archaea encode for componentsof a N-glycosylation pathway, pointing to such protein processingas being a common event in this life form. Moreover, even thoughrelatively few examples have been experimentally characterized, itis already abundantly clear that archaeal N-glycosylation involvesmore variety in terms of sugars, glycan structures, and by exten-sion, biosynthetic pathways than seen elsewhere. By focusing ona single component of the archaeal N-glycosylation pathway, itwas shown that gene duplication and modification had occurredat numerous different points during evolution. Furthermore, com-parison of the organization and content of N-glycosylation genes infive members of the same genus revealed that substantial LGT hadoccurred over the course of time.

Despite advances made in deciphering pathways of archaealN-glycosylation, numerous unanswered questions remain. For in-stance, one can ask what species-specific changes allow the archa-eal oligosaccharyltransferase, AglB, to accommodate such a widerange of glycan structures. Do Archaea encountering similar envi-ronmental extremes decorate their proteins with similar N-linkedglycans? How common is the ability to modify N-glycosylation inresponse to changing surroundings, a phenomenon recently ob-served in Hfx. volcanii? As new species appeared, did N-glycosyla-tion change at the same rate? Finally, one can ask whether it willbecome possible to describe the composition of the N-linked gly-cans decorating archaeal glycoproteins based on their glycosyla-tion gene content. Examining archaeal N-glycosylation from thegenomic perspective will help address these and elucidate other

facets of the archaeal version of this universal protein-processingevent.

Acknowledgments

The authors thank Sam Haldenby for his early contributions tothis project. JE is supported by grants from the Israel Science Foun-dation (30/07) and the US Army Research Office (W911NF-11-1-520). UG is supported by grants from the Israel Science Foundation(201/12) and the German-Israeli Project Cooperation (DIP). TA hasbeen supported by a Royal Society University Research Fellowship.LK is the recipient of a Negev-Zin Associates Scholarship.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2013.03.024.

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