Effects of N-Glycosylation Site Removal in Archaellins on the Assembly and Function of Archaella in Methanococcus maripaludis

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.

Effects of growth conditions on archaellation and N-glycosylation in Methanococcus maripaludis

“…Transmission electron microscopy of Methanococcus maripaludis cells was performed as described previously (Ding et al, 2015). Briefly, cells were collected from an overnight culture by centrifugation, resuspended in 2 % (w/v) NaCl and placed on 200-mesh carbon-coated copper grids.…”

“…Methanococcus maripaludis is considered a model archaeal organism (Leigh et al, 2011) and one of the best-studied archaea with regard to its archaella (Chaban et al, 2007;Ding et al, 2015;Jarrell et al, 2011;VanDyke et al, 2009). This study reports on a variety of growth conditions and the effect they have on archaellation.…”

“…Mutations in agl genes that result in non-glycosylated archaellins or archaellins modified with only a single sugar glycan are non-archaellated. Whilst multiple sites of N-glycosylation are found in each archaellin, archaella can be formed when three of the four glycosylation sites of FlaB2 are removed, but not if all four sites are eliminated (Ding et al, 2015).…”

Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar

“…Though N-linked glycosylation is widespread in Archaea, the study of N-linked glycosylation pathways by both genetic and structural methodologies is concentrated on a few model species (4), including the thermoacidophile Sulfolobus acidocaldarius (5-9), the halophile Haloferax volcanii (10)(11)(12)(13)(14), and the methanogens Methanococcus voltae (15,16) and Methanococcus maripaludis (17)(18)(19)(20)(21)(22)(23). In general, the archaeal N-linked glycosylation pathway contains aspects of both the eukaryotic and bacterial systems, with additional domain-specific traits that include unique glycan structures, a variety of linking sugars, and unusual dolichol carri- Citation Siu S, Robotham A, Logan SM, Kelly JF, Uchida K, Aizawa S-I, Jarrell KF.…”

“…In S. acidocaldarius, mutants producing a truncated N-glycan were shown to have severely reduced motility (7), although recent studies indicate that strains engineered to lack all six glycosylation sites in the archaellin FlaB were still motile (9). Recent studies have also shown that for FlaB2 of M. maripaludis, at least one of the four sequons used for N-glycan attachment must be occupied for archaellum assembly to occur (22), and for H. volcanii all sequons are necessary (14).…”