Identification of Novel Genes Involved in N-Glycosylation and Regulation of Archaella Assembly and Exploration of the Effect of N-Glycosylation on Archaella Assembly in Methanococcus Maripaludis
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In this thesis, using Methanococcus maripaludis as a model organism, significant, novel advances have been made to the understanding of the archaeal N-glycosylation pathway as well as archaella regulation and assembly. The archaellum is the swimming organelle in the third domain of life, the Archaea. Genes involved in archaellation are mainly clustered in the fla operon. The structural proteins of archaella, termed archaellins, are usually glycoproteins modified with N-glycans. In M. maripaludis, a euryarchaeon, all three archaellins FlaB1, FlaB2 and FlaB3 are modified at multiple sites with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is a unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-L-erythro-hexos-5-ulo-1,5-pyranose exclusively found in this species. Using FlaB2 as a reporter protein, I genetically identified three novel genes, aglU, aglV and aglW, which are involved in the synthesis of the archaellin N-glycan. AglU, a putative threonine transferase, is thought to be responsible for the transfer of the threonine onto the third sugar residue in the N-glycan. AlgV and AglW, annotated as a methyltransferase and a UDP-glucose 4-epimerase, respectively, are involved in the biosynthesis of the unique terminal sugar. Since it has been shown that disruption of the N-glycosylation pathway caused defects in archaella assembly or function, I explored the specific requirement of N-glycosylation of archaellins on archaellation by eliminating the four N-glycosylation sites in FlaB2 in all possible combinations. As many as three N-glycosylation sites could be removed from FlaB2 without interfering with archaella assembly or function. However, archaella could not be assembled when all four N-glycosylation sites in FlaB2 are eliminated. While there is evidence that archaella synthesis in M. maripaludis is not constitutive, no transcriptional regulators had been identified for the fla operon in any euryarchaeon. I genetically identified and characterized ArnE as the first euryarchaeotic transcriptional activator of the fla operon. Deletion of arnE resulted in the cessation of the transcription and translation of the reporter gene flaB2 and translation of FlaB2 was restored when the mutant was complemented with arnE in trans. Electrophoretic mobility shift assays confirmed the specific binding of purified ArnE to DNA fragments from the fla promoter.