Abstract
In Methanococcus maripaludis S2, the swimming organelle, the archaellum, is composed of three archaellins, FlaB1S2, FlaB2S2 and FlaB3S2. All three are modified with an N-linked tetrasaccharide at multiple sites. Disruption of the N-linked glycosylation pathway is known to cause defects in archaella assembly or function. Here, we explored the potential requirement of N-glycosylation of archaellins on archaellation by investigating the effects of eliminating the 4 N-glycosylation sites in the wildtype FlaB2S2 protein in all possible combinations either by Asn to Glu (N to Q) substitution or Asn to Asp (N to D) substitutions of the N-glycosylation sequon asparagine. The ability of these mutant derivatives to complement a non-archaellated ΔflaB2S2 strain was examined by electron microscopy (for archaella assembly) and swarm plates (for analysis of swimming). Western blot results showed that all mutated FlaB2S2 proteins were expressed and of smaller apparent molecular mass compared to wildtype FlaB2S2, consistent with the loss of glycosylation sites. In the 8 single-site mutant complements, archaella were observed on the surface of Q2, D2 and D4 (numbers after N or Q refer to the 1st to 4th glycosylation site). Of the 6 double-site mutation complementations all were archaellated except D1,3. Of the 4 triple-site mutation complements, only D2,3,4 was archaellated. Elimination of all 4 N-glycosylation sites resulted in non-archaellated cells, indicating some minimum amount of archaellin glycosylation was necessary for their incorporation into stable archaella. All complementations that led to a return of archaella also resulted in motile cells with the exception of the D4 version. In addition, a series of FlaB2S2 scanning deletions each missing 10 amino acids was also generated and tested for their ability to complement the ΔflaB2S2 strain. While most variants were expressed, none of them restored archaellation, although FlaB2S2 harbouring a smaller 3-amino acid deletion was able to partially restore archaellation.
Highlights
N-glycosylation is a prevalent protein modification found in all three domains of life in which the attachment of the glycan is via the nitrogen atom in asparagine residues located in the acceptor glycoprotein [1,2,3,4,5,6]
In Hfx. volcanii H53, changing the sequence of the major archaellin flgA at any of the 3 examined sequons so that the encoded amino acid changed from Asn to Gln led to mutant forms of the protein that could not rescue the swimming defect of an flgA deletion strain, suggesting that each glycosylation site was necessary for archaellation [18]
This is not the case for M. maripaludis S2. Previous work in this methanogen showed that a strain that had a spontaneous mutation in flaB2S2 which led to the loss of the 2nd N-glycosylation site of the archaellin that is normally decorated with the N-linked tetrasaccharide, was, still archaellated and motile [43]
Summary
N-glycosylation is a prevalent protein modification found in all three domains of life in which the attachment of the glycan is via the nitrogen atom in asparagine residues located in the acceptor glycoprotein [1,2,3,4,5,6]. Signal peptide removal is critical for incorporation of archaellins into the filament [37, 39] It appears that archaellins are commonly modified with N-linked glycans and interruption of the normal N-glycosylation pathway leads to defects in archaella assembly or function [1,40]. The first sequon 26NTS28 located in the N-terminal conserved region was previously reported to be unoccupied with glycan while the remaining four, located in the hypervariable region, were modified with tetrasaccharide (Fig. 1, [11]) For these experiments, we eliminated the 4 occupied sequons (66NIT68, 110NLT112, 119NTT121 and 124NWS126, designated as the 1st, 2nd, 3rd and 4th N-glycosylation site, respectively) in all possible combinations (creating single-, double-, triple- and quadruplesite mutations in FlaB2S2). We generated a series of FlaB2S2 scanning deletions in an attempt to determine regions of the molecule that were essential for assembly of archaella
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