Agl16, a thermophilic glycosyltransferase mediating the last step of N-Glycan biosynthesis in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

Abstract

Recently, the S-layer protein of Sulfolobus acidocaldarius was shown to be N-linked with a tribranched hexasaccharide, composed of Man2Glc1GlcNAc2 and a sulfated sugar called sulfoquinovose. To identify genes involved in the biosynthesis and attachment of this glycan, markerless in-frame deletions of genes coding for predicted glycosyltransferases were created. The successful deletion of agl16, coding for a glycosyltransferase, resulted in the S-layer protein and archaellins having reduced molecular weights, as visualized by Coomassie staining or immunoblotting. This analysis indicated a change in the N-glycan composition. Nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses confirmed that the glycan of the S-layer protein from the agl16 deletion mutant was a pentasaccharide, which was missing a terminal hexose residue. High-performance liquid chromatography (HPLC) analyses of the hydrolyzed N-glycan indicated that the missing hexose is a glucose residue. A physiological characterization of the agl16 deletion mutant revealed a significant effect on the growth at elevated salt concentrations. At 300 mM NaCl, the doubling time of the Δagl16 mutant was increased 2-fold compared to that of the background strain. Furthermore, the incomplete glycan structure of the Δagl16 deletion strain affected the assembly and function of the archaellum, as exemplified by semisolid Gelrite plate analysis, in which the motility is decreased according to the N-glycan size.

Fig 1

Proposed activation and transfer pathway of the last hexose residue of the N-glycan in S. acidocaldarius. (A) Proposed biosynthesis and transferase pathway of the last hexose. The structure of the unidentified hexose is shown as glucose. saci0802 encodes a proposed 3-hexulose-6-phosphate synthase; saci0806 is annotated as a phosphoglucomutase, and agl16 codes for a soluble glycosyltransferase. Glycan symbols: blue sphere, glucose; green sphere, mannose; orange sphere, sulfoquinovose; blue square, GlcNAc. (B) Physical map of the gene region of S. acidocaldarius, where the gene coding for the glycosyltransferase Agl16 is located. The illustrated region encompasses saci0802 to saci0808. The outlined gene encodes the glycosyltransferase Agl16. The genes saci0802 and saci0806 are most likely also involved in the activation of the hexose.

Fig 2

Confirmation of the in-frame Δagl16 mutant. (A) Gene deletion was confirmed by PCR using the outside primers against the flanking regions of agl16 and template DNA isolated from the background strain MW001, the mutant lacking agl16, or the plasmid pSVA1233, used for the homologous recombination incorporating the up- and downstream region of deleted agl16. (B) RT-PCR confirmed the deletion of the gene agl16, encoding a glycosyltransferase. The cDNA (C) served as a template in PCR amplification using primers against the internal region of either aglB or agl16. In each case, PCR amplifications were also performed using genomic DNA (G) as a positive control and total RNA (R) as a negative control.

Fig 3

Effects of the agl16 deletion on S. acidocaldarius S-layer glycoprotein. Equivalent amounts of the S-layer protein SlaA from S. acidocaldarius MW001, the Δagl16 strain, the Δagl16 strain with control plasmid pSVA1450, and the Δagl16 strain complemented with pSVA1274 containing agl16 were separated by 11% SDS-PAGE and Coomassie blue stained.

Fig 4

Glycosylation defects on the archaellin FlaB from S. acidocaldarius. Equivalent amounts of cells from S. acidocaldarius MW001 (lane 1), the Δagl16 strain (lane 2), the Δagl16 strain complemented with pSVA1274 (lane 3), the Δagl3 strain (lane 4), and the Δagl3 strain complemented with pSVA1266 (lane 5) were separated by 11% SDS-PAGE and immunoblotted with antibodies raised against FlaB.

Fig 5

Detailed analysis of the S. acidocaldarius Δagl16 mutant background S-layer glycopeptide. The total ion chromatograms of the glycan profiles for the tryptic peptides T24 (A) and T26 (B) are shown. The annotated peaks show that the truncated Hex₂QuiS₁HexNAc₂ glycoform is present on the observed glycopeptides, whereas the full glycan is missing. For reference, a cartoon depicting the full glycan is provided for each spectrum. Glycan symbols: blue sphere, glucose; green sphere, mannose; orange sphere, sulfoquinovose; blue square, GlcNAc.

Fig 6

Effects on the motility from S. acidocaldarius by interfering with the N-glycan assembly. Equivalent amounts of cells (5 to 6 μl) from S. acidocaldarius ΔaapF ΔflaH (lane 1), S. acidocaldarius ΔaapF (lane 2), the background strain S. acidocaldarius MW001(lane 3), S. acidocaldarius Δagl3 (lane 4), S. acidocaldarius Δagl16, and S. acidocaldarius Δagl16 complemented with pSVA1274 (lane 6), were dripped onto a 0.15% Gelrite plate and incubated for 4 and 9 days at 75°C. The panels on the right show the enlarged colony border and the motility zone (taken from the left panel: 0 mM NaCl, 9-day incubation), as well as the N-glycan composition of MW001 and the Δagl16 and Δagl3 mutants. The ability to swim corresponds to the N-glycan size.

Fig 7

TEM analysis of negatively stained cells of MW001 (A), the Δagl3 mutant (B), and the Δagl16 mutant (C) showed the lack of archaella on each of the agl deletion mutants. The MW001 background strain exhibits Aap pili (8 to 10 nm; white arrow) as well as the archaella (10 to 14 nm, black arrow); in the agl deletion mutants, only the Aap pili could be detected. Bars, 200 nm (A) and 100 nm (B and C).

Fig 8

Response to salt stress in the Δagl16 strain. S. acidocaldarius MW001 (black line) or Δagl16 (gray line) were grown in Brock medium at different salt concentrations (0 to 400 mM NaCl). Growth was measured as the optical density at 600 nm (OD₆₀₀). Shown are values obtained from an experiment done in triplicate; repeats generated a similar result.