AglB, catalyzing the oligosaccharyl transferase step of the archaeal N-glycosylation process, is essential in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

Abstract

Sulfolobus acidocaldarius, a thermo-acidophilic crenarchaeon which grows optimally at 76 °C and pH 3, exhibits an astonishing high number of N-glycans linked to the surface (S-) layer proteins. The S-layer proteins as well as other surface-exposed proteins are modified via N-glycosylation, in which the oligosaccharyl transferase AglB catalyzes the final step of the transfer of the glycan tree to the nascent protein. In this study, we demonstrated that AglB is essential for the viability of S. acidocaldarius. Different deletion approaches, that is, markerless in-frame deletion as well as a marker insertion were unsuccessful to create an aglB deletion mutant. Only the integration of a second aglB gene copy allowed the successful deletion of the original aglB.

Keywords: AglB; Archaea; Crenarchaeota; N-glycosylation; Sulfolobus.

Figure 1

Topology model of AglB from Sulfolobus acidocaldarius. (A) The model is based on the TMHMM prediction server 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Transmembrane segments are indicated in yellow boxes. Asparagine residues within a predicted glycosylation site are indicated by red circle, the N-glycan attachment sites in S. acidocaldarius are not confirmed. External loops are shown in bold numbers. The conserved WWDYG and DxxK motif within the external loops 7 are circled in red. (B–D) Structural alignment of full AglB from S. acidocaldarius with the crystal structure of the soluble domain of AglB from Pyrococcus furiosus (pdb: 2zagD). (B) Alignment was performed by the SWISS-MODEL program (http://swissmodel.expasy.org/); Modeled are the residues 473–749 of the template (pdb: 2zagD)(Igura et al. 2007). Estimated per-residues inaccuracy is visualized using a color gradient from blue (more reliable regions) to red (potentially regions), indicating the low sequence identity of 17.67%. (C) Modeled structure of AglB form S. acidocaldarius compared with (D) the crystal structure of P. furiosus (AA 473–749); the conserved WWDYG and DxxK motifs are shown in blue and yellow, respectively. (E) Alignment of the WWDYG and DxxK/MxxI motifs and their flanking regions of selected OTase from the three domains of life. The amino acid sequences of the STT3, PglB, and AglB proteins were retrieved from the InterPro database, and aligned with the ClustalW program. In 2014, over 1434 sequences are grouped to the family IPR003674, including 844 Stt3, 225 PglB, and 358 AglB; in 2012, 827 sequences were grouped to this family; (530 Stt3, 96 PglB, and 201 AglB). Representative sequences from eukaryal Stt3 proteins and the bacterial PglB from Campylobacter jejuni were selected. The AglB sequences from the crenarchaeota S. acidocaldarius DSM639 and S. solfataricus P2; the euryarchaeota Methanococcus voltae strain A3, M. maripaludis strain A2 and P. furiosus DSM 3638 Hfx. volcanii DS2, Archaeoglobus fulgidus DSM4304; from the nanoarchaeota N. equitans strain Kin4-M; from the korachaeota K. cryptofilum strain OPF8; and from the thaumarchaeota Nitrosopumilus maritimus SCM1 were selected for the alignment. Highlighted amino acids belong to the WWDYG or DxxK/MxxI motif.

Figure 2

A Genetic neighborhood of the archaeal and bacterial gene coding for OTases. Physical map of the gene region of Sulfolobus acidocaldarius MW001, S. islandicus Rey15 A, Hfx. volcanii DS2, and Campylobacter jejuni in which the gene coding for oligosaccharyl transferase is located. Illustrated are the genes saci1257 until saci1280, sire_1024 till sire_1048, hvol1523 till hvol1548, and the Campylobacter jejuni subsp. jejuni NCTC 11168 pgl gene cluster. Dark gray displayed genes encode the archaeal or bacterial OTase aglB or pglB, respectively. Framed genes code for GTase or other proteins involved in the N-glycosylation process. Upstream of aglB from S. acidocaldarius MW001 the genes sulA and sulB encoding the sulfolobicins are found. Downstream of the aglB from S. acidocaldarius MW001 and S. islandicus Rey15A genes coding for the translational machinery are located.

Figure 3

Confirmation of the integration and segregation of the aglB deletion plasmid pSVA1204 or pSVA1204 in Sulfolobus acidocaldarius MW001 and S. islandicus E2331, respectively. (A) First panel: The integration of the deletion plasmid pSVA1203 in the gene aglB of S. acidocaldarius MW001 (aglB::pSVA1203) was monitored by PCR using the outer primers of the upstream and downstream region of aglB and the genomic DNA from two-first selection colonies (aglB::pSVA1203). DNA from the background strain MW001 and the plasmid pSVA1203 were used as control, showing a PCR fragment corresponding to the flanking region including or excluding the aglB gene, respectively. Second panel: The segregation of pSVA1203 (second selection) was confirmed by PCR using the outer primers of the flanking region of aglB and the genomic DNA from second selection colonies. All PCR fragments gained from genomic DNA of second selection colonies correspond to the full-length aglB gene (3500 bp). (B) First panel: Integration of the aglB deletion plasmid pSVA1204 in S. islandicus E2331 (aglB::pSVA1204) was confirmed by PCR using the outside primers against the flanking regions of aglB and DNA isolated from one-first selection colony (aglB::pSVA1204). DNA isolated from the background strain E2331 or plasmid pSVA1204 were used as controls. Second panel: The segregation of the pSVA1204 (second selection) was monitored using the same flanking primer and the genomic DNA from second selection colonies. All PCR products have the calculated size of 3500 bp corresponding to the flanking regions including the full-length aglB gene.

Figure 4

Genomic integration of a second aglB gene copy in the saci1162 locus. (A) Left panel: Physical map of the homologous integration of pSVA1241 into the genome of Sulfolobus acidocaldarius via the saci1162 upstream region. Integrated plasmid is indicated by the black line above the genes. Right panel: PCR using a forward primer binding upstream of the integration site and a reverse primer binding to an internal plasmid site (primer binding sites are indicated in the physical map as arrows) confirmed the integration of the plasmid via the upstream region of the colonies 139 and 167. (B) Left panel: Physical map of the integration of pSVA1241 via the aglB region in to the genome of S. acidocaldarius. Integrated plasmid is indicated by the black line above the genes. PCR, using primer binding to an internal plasmid region and to the downstream aglB region (primer binding sites are indicated in the physical map as arrows), confirmed the integration directly with aglB of the colonies 135-138 and 165, 166, and 168. (C) The segregation of the plasmid pSVA1241 was confirmed by PCR using primers binding outside of the saci1162 flanking regions on genomic DNA derived from second selection colonies. PCR products correspond either to the wild type saci1162 (4337 bp) or the saci1162::aglB mutants (3977 bp). Plasmid pSVA1241 (P) used for the homologous recombination and the genomic DNA of the wild type strain (WT) were used as a control.

Figure 5

Confirmation and growth of the MW099 (ΔaglB, saci1162::aglB) strain. Colony PCR using second selection colony derived from the strains integrated the deletion plasmid pSVA1203 in the aglB site: MW001aglB::pSVA1203 strain (A) or the MW098 aglB::pSVA1203 strain (B) with the forward primer against the upstream region and the reverse primer against the downstream region of aglB revealed the presence of ΔaglB PCR fragments only in the colonies 14 and 16 derived from the MW098 aglB::pSVA1203 strain. None ΔaglB PCR fragment could be detected in the MW001 aglB::pSVA1203 background strain. (C) Growth of the MW001 background strain (rectangle) and the MW099 (ΔaglB, saci1162::aglB) strain (triangles) in Brock medium supplemented with 0.1% xylose (filled symbols) or 0.1% maltose (open symbols) were measured by the optical density at 600 nm.