title: 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.
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ding
authors.affiliation: Department of Life Sciences, Prefectural University of Hiroshima, 562 Nanatsuka, Shobara, Hiroshima, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Uchida
authors.affiliation: Department of Life Sciences, Prefectural University of Hiroshima, 562 Nanatsuka, Shobara, Hiroshima, Japan.
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Aizawa
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Murphy
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Alison
authors.initials: A
authors.lastname: Berezuk
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Cezar M
authors.initials: CM
authors.lastname: Khursigara
authors.affiliation: Department of Biology, University of York, Heslington, York, United Kingdom.
authors.forename: James P J
authors.initials: JP
authors.lastname: Chong
authors.affiliation: Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario, Canada.
authors.forename: Ken F
authors.initials: KF
authors.lastname: Jarrell
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence; D005408:Flagellin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D017017:Methanococcus / Q000378:metabolism*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational
chemical_list: D019843:Archaeal Proteins; D005408:Flagellin