Haloferax volcanii N-Glycosylation: Delineating the Pathway of dTDP-rhamnose Biosynthesis

In the halophilic archaea Haloferax volcanii, the surface (S)-layer glycoprotein can be modified by two distinct N-linked glycans. The tetrasaccharide attached to S-layer glycoprotein Asn-498 comprises a sulfated hexose, two hexoses and a rhamnose. While Agl11-14 have been implicated in the appearance of the terminal rhamnose subunit, the precise roles of these proteins have yet to be defined. Accordingly, a series of in vitro assays conducted with purified Agl11-Agl14 showed these proteins to catalyze the stepwise conversion of glucose-1-phosphate to dTDP-rhamnose, the final sugar of the tetrasaccharide glycan. Specifically, Agl11 is a glucose-1-phosphate thymidylyltransferase, Agl12 is a dTDP-glucose-4,6-dehydratase and Agl13 is a dTDP-4-dehydro-6-deoxy-glucose-3,5-epimerase, while Agl14 is a dTDP-4-dehydrorhamnose reductase. Archaea thus synthesize nucleotide-activated rhamnose by a pathway similar to that employed by Bacteria and distinct from that used by Eukarya and viruses. Moreover, a bioinformatics screen identified homologues of agl11-14 clustered in other archaeal genomes, often as part of an extended gene cluster also containing aglB, encoding the archaeal oligosaccharyltransferase. This points to rhamnose as being a component of N-linked glycans in Archaea other than Hfx. volcanii.


Introduction
N-glycosylation, the covalent attachment of oligosaccharides to select asparagine residues, is performed by members of all three domains of life [1][2][3][4][5]. Still, understanding of the archaeal version of this protein-processing event remains relatively limited. In the last decade, however, substantial progress has been realized in deciphering pathways of N-glycosylation in several archaeal species, including the halophile Haloferax volcanii [5].
In Hfx. volcanii, the surface (S)-layer glycoprotein, a well-studied glycoprotein and the sole component of the protein-based shell surrounding the cell, is modified by a pentasaccharide comprising a hexose, two hexuronic acids, a methyl ester of hexuronic acid and mannose. Through a series of genetic and biochemical studies, a series of Agl (archaeal glycosylation) proteins involved in the assembly and the attachment of this glycan to the S-layer glycoprotein Asn-13 and Asn-83 positions was described [6][7][8][9][10][11][12]. Most recently, a second glycan composed of a sulfated hexose, two hexoses and a rhamnose was shown to be N-linked to position Asn-498 of the S-layer glycoprotein [13]. Moreover, whereas the Asn-13-and Asn-83-linked pentasaccharide was identified when cells were grown across a range of NaCl concentrations, the novel Asn-498-bound tetrasaccharide was observed when cells were grown in 1.75 M but not 3.4 M NaCl-containing medium.
Relying on bioinformatics, gene deletions and mass spectrometry, Agl5-Agl15 have been identified as components of the pathway responsible for the assembly of the so-called 'low salt' tetrasaccharide N-linked to S-layer glycoprotein Asn-498 [14]. Based on these studies, Agl11-Agl14 were deemed to be involved in the appearance of the final sugar of the 'low salt' tetrasaccharide, rhamnose, on the dolichol-phosphate carrier upon which the glycan is initially assembled.
Rhamnose, a naturally occurring deoxy-hexose, is found in the L-rather than the D-configuration assumed by most other sugars. In Bacteria, plants and fungi, rhamnose is a common component of the cell wall [15][16][17], and was also recently found in viruses [18]. At present, two pathways for synthesizing nucleotideactivated rhamnose are known. In Bacteria, RmlA, RmlB, RmlC and RmlD act sequentially to convert glucose-1-phosphate and deoxy-thymidine triphosphate (dTTP) into thymidine diphosphate (dTDP)-rhamnose [19,20]. Specifically, RmlA, the first enzyme of the pathway, is a glucose-1-phosphate thymidylyltransferase that combines thymidine monophosphate with glucose-1-phosphate to create dTDP-glucose. RmlB, a dTDP-glucose-4,6-dehydratase, then catalyzes the oxidation and dehydration of dTDP-glucose to form dTDP-4-keto 6-deoxy-glucose. RmlC, a dTDP-4-dehydro-6deoxy-glucose-3,5-epimerase, next performs a double epimerization at the C3 and C5 positions of the sugar. Finally, RmlD, a dTDP-4-dehydrorhamnose reductase, catalyzes the last step of the pathway, namely reduction of the C4 keto group of the sugar to yield dTDP-rhamnose. In plants, uridine diphosphate (UDP)rhamnose rather than dTDP-rhamnose is generated by RHM (UDP-L-rhamnose synthase), a single polypeptide that contains all of the enzymatic activities required [21]. Here, UDP-glucose is converted to UDP-4-keto-6-deoxy-glucose by an enzymatic activity similar to bacterial RmlB. Next, and in contrast to the bacterial process, whereby RmlC and RmlD operate sequentially to generate dTDP-rhamnose, plants instead rely on nucleotiderhamnose synthase/epimerase-reductase, a bifunctional enzyme mediating both the epimerization and reduction reactions that lead to the biosynthesis of UDP-rhamnose [21][22][23]. More recently, the same pathway was shown to catalyze UDP-rhamnose biogenesis in large DNA viruses [18]. The two pathways for nucleotide-activated rhamnose biosynthesis are depicted in Fig 1. Although rhamnose has been identified in several archaeal species [13,24,25], studies addressing rhamnose biosynthesis in Archaea are few. In Sulfolobus tokodaii, one of three RmlA homologues was shown to possess sugar-1-phosphate nucleotydyltransferase activity using either glucose-1-phosphate or Nacetylglucosamine-1-phosphate and all four deoxyribonucleoside triphosphates or UTP as substrates [26], while S. tokodaii RmlB and RmlD were reported to be functionally identical to their bacterial counterparts [27]. At the same time, the crystal structure of Methanobacter thermautotrophicus RmlC has been reported [28], as have those of S. tokodaii RmlC and RmlD (PDB 2B9U and 2GGS, respectively). Still, it remains to be determined whether rhamnose is used for glycosylation by these species. Thus, to better understand the biosynthesis of this deoxy-hexose in Archaea, the present study addressed the involvement of Hfx. volcanii Agl11-Agl14 in the biogenesis of nucleotide-activated rhamnose. In addition, the presence and genomic distribution of homologues of genes involved in such activity across the Archaea were considered.

Plasmid construction
To generate a plasmid encoding CBD-Agl11, the agl11 gene was PCR-amplified using primers designed to introduce NdeI and KpnI restriction sites at the 59 and 39 ends of the gene, respectively (primers listed in Table 1). The amplified fragment was digested with NdeI and KpnI and ligated into plasmid pWL-CBD, previously digested with the same restriction enzymes, to produce plasmid pWL-CBD-Agl11. Plasmid pWL-CBD-Agl11 was then introduced into Hfx. volcanii cells. Plasmids encoding CBD-Agl12, CBD-Agl13 and CBD-Agl14 were similarly generated, using the primers listed in Table 1, and also introduced into Hfx. volcanii parent strain cells.

Protein purification
To purify the CBD-tagged proteins, 1 ml aliquots of Hfx. volcanii cells transformed to express CBD-Agl11, CBD-Agl12, CBD-Agl13 or CBD-Agl14 were grown to mid-logarithmic phase, harvested and resuspended in 1 ml solubilization buffer (1% Triton X-100, 1.75 M NaCl, 50 mM Tris-HCl, pH 7.2) containing 3 mg/ml DNaseI and 0.5 mg/ml PMSF. The solubilized mixture was nutated for 20 min at 4uC, after which time 50 ml of a 10% (w/v) solution of cellulose was added. After a 120 min nutation at 4uC, the suspension was centrifuged (5,000 rpm for 5 min), the supernatant was discarded and the cellulose pellet was washed four times with wash buffer containing 1.75 M NaCl, 50 mM Tris-HCl, pH 7.2. After the final wash, the cellulose beads were centrifuged (5,000 rpm for 5 min), the supernatant was removed and the pellet, containing cellulose beads linked to CBD-tagged Agl11, Agl12, Agl13 or Agl14, was either subjected to further in vitro assays or resuspended in SDS-PAGE sample buffer, boiled for 5 min, centrifuged (5,000 rpm for 5 min) and subjected to SDS-PAGE and Coomassie Brilliant Blue staining.

Agl11 activity assay
Cellulose-bound CBD-Agl11 were resuspended in reaction buffer containing 1.75 M NaCl, 5 mM MgCl 2 , 50 mM Tris-HCl, pH 7.2 and incubated with 5 mM glucose-1-phosphate and 5 mM dTTP (or UTP) at 42uC. As controls, glucose-1-phosphate, dTTP (or UTP) or both were omitted from the reaction. Aliquots were removed immediately following substrate addition and at several time points up to 40 min and incubated for 10 min at room temperature (RT) with 1 U/ml of pyrophosphatase. The extent of phosphate release was determined using a malachite green-based assay [29]. Briefly, 10 ml aliquots were incubated for 5 min at RT with 850 ml of a Malachite green solution followed by addition of 100 ml of 34% citric acid and incubation for an additional 40 min at RT. Phosphate concentration was calculated using a standard curve based on the 660 nm absorbance of a 0-1000 mM phosphate solution.

Thin layer chromatography
To perform TLC, 10 ml of the products generated in the Agl11 assay described above were spotted onto a Partisil K6 silica gel plate (Whatman, Maidstone, UK). In addition, 10 ml of 2 mM glucose-1-phosphate and dTDP-D-glucose solutions were applied to the same plate as standards. The plates were developed in 95% ethanol/1 M acetic acid (5:2, pH 7.5). The separated spots were detected by spraying the plate with orcinol monohydrate solution (0.1% in 5% H 2 SO 4 in ethanol) and then heating the plate for 10 min at 120uC.

Agl12 activity assay
The dTDP-D-glucose 4,6-dehydratase of Agl12 activity was assayed as described previously [30]. Briefly, cellulose-bound CBD-Agl12 was resuspended in reaction buffer containing 1.75 M NaCl, 5 mM MgCl 2 , 50 mM Tris-HCl, pH 7.2 and incubated at 42uC with 4 mM dTDP-D-glucose or UDP-D-glucose. Aliquots were removed immediately following substrate addition and at several time points up to 40 min and mixed with 750 ml of 100 mM NaOH. Each mixture was incubated for 20 min at 42uC and absorbance at 320 nm was measured.

Combined Agl13 and Agl14 activity assay
Cellulose-bound CBD-Agl13 and CBD-Agl14 were resuspended in reaction buffer containing 1.75 M NaCl, 50 mM Tris-HCl, pH 7.2 and incubated with 4 mM dTDP-4-keto-6- Table 1. Primers used in this study. deoxy-glucose and 10 mM NADPH at 42uC for 20 h. As controls, CBD-Agl13, CBD-Agl14 or NADPH was omitted. After incubation, the mixtures were centrifuged (5,000 rpm for 5 min), and the supernatant was examined by nano-ESI/MS analysis. For nano-ESI/MS analysis, a 10 ml aliquot was dried using a SpeedVac apparatus, resuspended in 10 ml methanol:water (1:1; v/v) containing 10 mM ammonium acetate and injected into a LTQ Orbitrap XL mass spectrometer using static medium NanoES Spray capillaries (Thermo Fisher Scientific, Bremen, Germany). Mass spectra were obtained in the negative mode.

Reverse transcriptase polymerase chain reaction (RT-PCR)
RT-PCR performed as previously described [31]. Briefly, RNA from Hfx. volcanii cells was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA). cDNA was prepared for each sequence from the corresponding RNA (2 mg) using random hexamers (150 ng) in a SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). The single-stranded cDNA was then used as PCR template in a reaction containing forward and reverse primers to sequences within agl13 and agl11, respectively (Table 1). In control reactions, genomic DNA or RNA served as template, or no nucleic acid was added to the reaction. The generation of PCR products was assessed by electrophoresis in 1% agarose followed by detection using ethidium bromide.

Bioinformatics analysis
Predicted archaeal RmlABCD proteins were identified using Hfx. volcanii Agl11, Agl12, Agl13 and Agl14 as query in a BLAST Cellulose-bound CBD-Agl11 or cellulose beads alone (blank) were resuspended in reaction buffer and incubated in the presence of dTTP and glucose-1-phosphate, with each substrate separately or without both substrates. Aliquots removed immediately after substrate addition and up to 40 min later were incubated with pyrophosphatase and the extent of phosphate release was measured 29]. The results represent average of triplicates 6 standard deviation for one of three repeats of the experiment. C. The assay products obtained after a 5 h incubation at 42uC were separated by TLC, along with glucose-1-phosphate and dTDP-glucose standards, as described in Experimental Procedures. doi: 10 RmlC and RmlD homologues, respectively. Archaeal RmlA-, RmlB-, RmlC-and RmlD-encoding genes were deemed as being clustered with the oligosaccharyltransferase-encoding aglB gene based upon the presence of these genes within previously identified aglB-based glycosylation gene clusters [32], or when rmlA, rmlB, rmlC and rmlD were clustered and found 10 genes or less away from clusters containing aglB and other glycosylation-or sugar processing-related genes.

Results
Agl11 is a glucose-1-phosphate thymidylyltransferase/ uridylyltransferase As a first step towards defining the precise function of Agl11, a BLAST homology-based search was conducted using Hfx. volcanii Agl11 as query. This revealed the homology of Agl11 to RmlA, the bacterial glucose-1-phosphate thymidylyltransferase (EC 2.7.7.24) that catalyzes the formation of dTDP-glucose from dTTP and glucose 1-phosphate [33]. For instance, Agl11 shared 53% identity, with 100% coverage and an E-value of 8e-120, to RmlA from the bacterium Sulfobacillus acidophilus TPY. To biochemically confirm that Agl11 indeed acts as does RmlA, Hfx. volcanii cells were transformed with a plasmid encoding Agl11 bearing an Nterminally-fused CBD tag [34]. The presence of the CBD tag allows for cellulose-based purification compatible with the hypersaline conditions in which Hfx. volcanii grow. PCR amplification using DNA extracted from the transformed strain as template, together with forward and reverse primers directed against regions within the CBD and agl11 sequences, respectively, confirmed uptake of the plasmid (not shown). Cellulose-based purification of an extract prepared from the transformed cells captured a single 55 kDa protein, corresponding to the predicted molecular mass of the 17 kDa CBD moiety and the 38 kDa Agl11 protein (Fig 2A).
The predicted glucose-1-phosphate thymidylyltransferase activity of purified Agl11 was next considered. Glucose-1-phosphate thymidylyltransferase, like RmlA, transfers the deoxy-thymidine monophosphate (dTMP) group of dTTP to glucose-1-phosphate to yield dTDP-glucose and pyrophosphate. Hence, the actions of Agl11 as a glucose-1-phosphate thymidylyltransferase was tested using a malachite green-based assay to detect the formation of phosphate following the conversion of pyrophosphate into inorganic phosphate upon addition of pyrophosphatase [29]. The assay revealed that Agl11 was able to generate phosphate only when incubated with dTTP and glucose-1-phosphate but not with either substrate alone or without both substates (Fig 2B). Thin layer chromatography (TLC) was also employed to further confirm the glucose-1-phosphate thymidylyltransferase activity of Agl11. In these experiments, the product generated upon incubation of Agl11 with dTTP and glucose-1-phosphate migrated to the same position as a dTDP-glucose standard (Fig 2C). Similar results were obtained when UTP was used in place of dTTP (not shown). As such, Agl11 acts as a glucose-1-phosphate thymidylyltransferase and a glucose-1-phosphate uridylyltransferase, namely the first enzyme in the biosynthesis of nucleotide activatedrhamnose in bacteria and in plants, respectively.
To test whether Agl12 indeed acts as a dTDP-glucose-4,6dehydratase, working downstream to Agl11 in the biosynthesis of dTDP-rhamnose, Hfx. volcanii cells were transformed to express CBD-tagged Agl12. Again, successful transformation was verified by PCR amplification using DNA from the transformed strain as template, together with forward and reverse primers directed against regions within the CBD and agl12 sequences, respectively (not shown). Cellulose-based purification of an extract prepared from Hfx. volcanii cells transformed to express CBD-Agl12 captured a 51 kDa species, corresponding to the predicted molecular mass of the 17 kDa CBD moiety and the 34 kDa Agl12 protein (Fig 3A).
Cellulose-purified CBD-Agl12 was incubated in the absence or presence of dTDP-glucose, the product of the Agl11-catalyzed reaction, and the formation of dTDP-4-keto-6-deoxy-glucose was assessed spectrophotometrically, following the increase in absorption at 320 nm, indicative of the formation of the product keto group. dTDP-4-keto-6-deoxy-glucose was only generated when Agl12 was combined with dTDP-glucose, confirming that Agl12 is indeed a dTDP-glucose-4,6-dehydratase, like RmlB (Fig 3B). When CBD-Agl12 was combined with UDP-glucose, the product generated when UDP-rhamnose serves as substrate, no UDP-4keto-6-deoxy-glucose was formed (Fig 3C).
To determine whether Agl13 and Agl14 indeed participate in the biosynthesis of dTDP-rhamnose by acting as RmlC and RmlD, respectively, Hfx. volcanii cells were transformed to express CBD-tagged versions of Agl13 and Agl14. Here as well, each transformation was verified by PCR amplification using DNA from the transformed strain as template, together with forward and reverse primers directed against regions within the CBD and agl13, or the CBD and agl14 sequences, respectively (not shown). Following transformation, cellulose-based purification of extracts prepared from Hfx. volcanii cells transformed to express either CBD-Agl13 or CBD-Agl14 was conducted. Following SDS-PAGE separation of cellulose-captured proteins, only bands corresponding to CBD-Agl13 or CBD-Agl14 were observed (Fig 4 inset, left  and right panels, respectively).
The ability of Agl13 and Agl14 to act as RmlC and RmlD respectively in the production of dTDP-rhamnose was next considered in a combined assay. Briefly, dTDP-4-keto-6-deoxyglucose was incubated together with CBD-tagged Agl13 and Agl14, along with NADPH, the substrate for the dehydrogenase reaction putatively catalyzed by Agl14. The appearance of dTDPrhamnose was revealed by nano-electrospray ionization mass spectrometry (nano-ESI/MS) [18], since initial attempts to detect dTDP-rhamnose formation spectrophotometrically as previously described [38,39] were unsuccessful. Nano-ESI/MS analysis revealed the formation of a m/z 547.07 peak corresponding to dTDP-rhamnose (m/z 547.07 calculated [M-H] 2 mass) and a peak at m/z 569.05 corresponding to the sodium adduct (m/z 569.05 calculated [M-2H+Na] 2 mass) (Fig 4). In the absence of CBD-Agl13, CBD-Agl14 or NADPH, peaks corresponding to dTDP-4-keto-6-deoxy-glucose were observed (m/z 545.06 calculated [M-H] 2 mass); no peaks corresponding to dTDP-rhamnose were seen (Fig S1A-C, respectively). In the absence of dTDP-4keto-6-deoxy-glucose, no peaks corresponding to either sugar were detected (Fig S1D).

agl11 and agl13 are co-transcribed
To obtain further insight into the actions of Agl11-Agl14, the transcription of each gene was addressed. Specifically, given that agl11 is found adjacent to agl13 in the Hfx. volcanii genome and that both are similarly oriented (Fig 5A), the co-transcription of these genes was considered. Accordingly, RT-PCR amplification was performed using primers directed at regions corresponding to the beginning of agl13 and the end of agl11 together with cDNA produced from RNA isolated from Hfx. volcanii cells. A PCR product of approximately 1500 bp, consistent with the genomic sizes of agl13 (471 bp) and agl11 (1074 bp), was observed ( Fig 5B).  Given the identification of an rmlABCD gene cluster in Hfx. volcanii, similar clusters were sought in other available archaeal genomes. Towards this aim, the 166 completed archaeal genomes listed at the Joint Genome Institute Database for Integrated Microbial Genomes (January, 2014) were subjected to a BLAST search seeking homologues of Hfx. volcanii Agl11, Agl12, Agl13 and Agl14. In addition, these genomes were also scanned for genes encoding proteins listed as EC 2.7.7.24 (RmlA), EC 4.2.1.46 (RmlB), EC 5.1.3.13 (RmlC) or EC 1.1.1.133 (RmlD). In this manner, 69 genomes were shown to encode an rmlABCD gene cluster, including Hfx. volcanii. Of these, 16 included rmlABCD as part of a previously defined larger cluster anchored by aglB, encoding the archaeal oligosaccharyltransferase [32] (Table 2). In addition, 19 species were found to encode partial rmlABCD gene clusters, where two or three of these genes are clustered (Table S1). Of these species, four (Methanobrevibacter ruminantium, Methanosarcina acetivorans, Sulfolobus islandicus Y.G.57.14 and Sulfolobus solfataricus P2) also encode a complete rmlABCD gene cluster.

Discussion
In addition to the pentasaccharide linked to select Asn residues of the Hfx. volcanii S-layer glycoprotein, it was recently shown that at least one additional Asn can be modified by a novel tetrasaccharide [14]. While many of the enzymes involved in the assembly of the N-linked pentasaccharide have been characterized biochemically [9,10,12,40], virtually nothing is known of the enzymes responsible for the assembly of the N-linked tetrasaccharide. As such, this study reports the first biochemical analysis of enzymes contributing to this novel N-glycosylation pathway. The results reveal that Agl11 is a glucose-1-phosphate thymidylyltransferase, Agl12 is a dTDP-glucose-4,6-dehydratase, Agl13 is a dTDP-4-dehydro-6-deoxy-glucose-3,5-epimerase and Agl14 is a dTDP-4-dehydrorhamnose reductase.
While rhamnose is a common component of both the bacterial and the plant cell wall, different biosynthetic pathways are employed in each case, leading to the generation of differentially nucleotide-activated species. At the same time, it is not clear which of these strategies Archaea employ for nucleotide-activated rhamnose biogenesis. Indeed, numerous examples of Archaea relying on the same biochemical pathways as used by either their bacterial or eukaryal counterparts have been reported, as have examples of archaeal pathways comprising selected aspects of the parallel bacterial and eukaryal processes or even biosynthetic pathways unique to this form of life [41][42][43][44][45][46][47][48][49][50]. In the case of Hfx. volcanii, the current study revealed that Agl11-Agl14 are homologous to RmlA-D, enzymes that catalyze the conversion of glucose-1-phosphate to dTDP-rhamnose in Bacteria [19,20]. Indeed, examination of available archaeal genomes detected the presence of RmlA-D in numerous species, pointing to Archaea and Bacteria as relying on the same route for nucleotide-activated rhamnose generation. At the same time, no gene encoding a homologue of the bifunctional nucleotide-rhamnose synthase/ epimerase-reductase used in eukaryal UDP-rhamnose biosynthesis was detected in Archaea. Still, the fact that several archaeal species encode only a partial rmlABCD cluster (Table S1) raises the  TERMP_02079  TERMP_02080  TERMP_02084  TERMP_02089  TERMP_02078   Thermococcus onnurineus  TON_1842  TON_1843  TON_1848  TON_1851  TON_1820   Thermococcus sibiricus  TSIB_2044  TSIB_2045  TSIB_2047  TSIB_2048  TSIB_0007   Thermogladius cellulolyticus  TCELL_0180  TCELL_0179  TCELL_0177  TCELL_0178 Thermogladius shockii Des1633_00001920 Des1633_00001910 Des1633_00001890 Des1633_00001900 Thermoproteus tenax TTX_1336 TTX_1335 TTX_1333 TTX_1334 Thermosphaera aggregans Tagg_0563  Tagg_0562  Tagg_0560  Tagg_0561   1 Clustering with aglB is defined as occurring when rmlABCD are part of a gene cluster containing aglB as described in ref. [39] or #10 genes away from such aglB-based clusters. doi:10.1371/journal.pone.0097441.t002 possibility that those enzymes present are recruited for the synthesis of molecules other than dTDP-rhamnose.
In addition to determining the route of nucleotide-activated rhamnose biosynthesis in Hfx. volcanii, the present study also represents the first biochemical characterization of components of a second N-glycosylation pathway recently identified in this species [14]. Based on earlier work revealing the presence of one or more genes encoding AglB, the oligosaccharyltransferase of the archaeal N-glycosylation machinery, in all but two of 168 genomes considered, it would appear that this protein-processing event is common in Archaea [40]. Yet, the diverse composition of the few N-linked archaeal glycans characterized to date points to archaeal N-glycosylation as largely relying on species-specific pathways [5]. The finding that some species contain rmlABCD homologues as part of a larger gene cluster containing aglB and other sugarrelated genes implies that as in Hfx. volcanii, rhamnose is a component of N-linked glycans in these other Archaea as well. Continued investigation into archaeal protein glycosylation will test this prediction.
Finally, the simultaneous modification of the same protein by two completely different N-linked glycans has only been reported to date in two haloarchaeal species, namely Halobacterium salinarum and Hfx. volcanii [13,51]. Of these, it is only in Hfx. volcanii that two N-glycosylation pathways have been identified [14]. Moreover, it was shown that N-glycosylation by both pathways occurs as a function of salt levels in the growth medium [13]. At present, it is not clear why the Hfx. volcanii S-layer glycoprotein is modified by two distinct N-linked glycans in 1.75 M NaCl-containing medium but not when cells are grown at higher salinity, nor what advantages such differential N-glycosylation offer the cell. The results obtained in this study will help answer these and other outstanding questions related to Hfx. volcanii N-glycosylation. Figure S1 Agl13, Agl14 and NADPH are required for the conversion of dTDP-4-keto-6-deoxy-glucose into dTDPrhamnose. Reactions were conducted as described in the legend to Figure 4, albeit in the absence of cellulose-bound CBD-Agl13 (A), CBD-Agl14 (B) or NADPH (C). In each case, nano-ESI/MS analysis detected peaks corresponding to dTDP-4-keto-6-deoxyglucose (m/z 545.06 calculated [M-H] 2 mass) but not peaks corresponding to dTDP rhamnose (m/z 547.07 calculated [M-H] 2 mass). When the reaction was conducted in the absence of dTDP-4-keto-6-deoxy-glucose (D), no peaks corresponding to either sugar were detected. As standards, 10 ml of 1 mM dTDP-4-keto-6deoxy-glucose (E) and dTDP rhamnose (F) solutions were examined by nano-ESI/MS. (TIF)