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

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

Methanococcoides

Me.tha.no.coc.co'i.des. Gr. adj. suff. ‐oides similar to; N.L. neut. n. Methanococcoides organism similar to Methanococcus. Euryarchaeota / Methanomicrobia / Methanosarcinales / Methanosarcinaceae / Methanococcoides The genus Methanococcoides comprises four species, Methanococcoides methylutens, Methanococcoides burtonii, Methanococcoides alaskense, and Methanococcoides vulcani. Cells are irregular cocci, 0.5–3 μm in diameter, occurring singly or in pairs, and may be motile. Clumps of cells can also observed. Cells exhibit a blue‐green autofluorescence under UV illumination. The cell wall consists of a very thin protein S‐layer, approximately 10‐nm thick. Susceptible to lysis by hypotonic or detergent shock. Eurypsychrophilic to mesophilic. Strict anaerobe. Neutrophilic. Halophilic, optimal salinity near seawater. Cells can dismutate methylamines, methanol, glycine betaine, choline, tetramethylammonium, dimethyl sulfide, methyliodide, and N,N‐dimethylethanolamine for growth, but cannot catabolize acetate, dimethylsulfide, H2/CO2, or formate. Methanococcoides spp. are members of the phylum Euryarchaeota, class Methanomicrobia, order Methanosarcinales, and family Methanosarcinaceae. Known habitats are deep‐sea mud volcano, marine anoxic sediment, hypolimnion Ace Lake, mangrove swamp, deep hypersaline anoxic basin, and hydrothermal vents. DNA G + C content (mol%): 40.8–44. Type species: Methanococcoides methylutens Sowers and Ferry 1983, VL17

Archaeal genetics – the third way

For decades, archaea were misclassified as bacteria on account of their prokaryotic morphology. Molecular phylogeny eventually revealed that archaea, like bacteria and eukaryotes, are a fundamentally distinct domain of life. Genome analyses have confirmed that archaea share many features with eukaryotes, particularly in information processing, and therefore can serve as streamlined models for understanding eukaryotic biology. Biochemists and structural biologists have embraced the study of archaea but geneticists have been more wary, despite the fact that genetic techniques for archaea are quite sophisticated. It is high time for geneticists to start asking fundamental questions about our distant relatives