Identification and characterization of N-glycosylation and structural genes involved in flagellation of methanogenic archaea from the genus Methanococcus

Abstract

The archaeal flagellum is a unique motility structure. Although functionally similar to the bacterial flagellum, the archaeal flagellum shares more similarities to the bacterial type IV pilus. Using the methanogenic archaea Methanococcus voltae and Methanococcus maripaludis as model organisms, the structural and post-translational requirements for flagellation have been investigated. Known to contain glycosylated flagellin proteins, the N-glycosylation pathway was studied in M. voltae. A number of possible glycosylation component genes, including glycosyl transferases, flippases and an oligosaccharyl transferase were inactivated or deleted in M. voltae and their resulting phenotypes were characterized. Four glycosyl transferases were identified as involved in the assembly of the M. voltae glycan structure, with three of these enzymes, AglA, AglC1 and AglC2, experimentally verified. As well, the oligosaccharyl transferase, AglB, has also been experimentally confirmed and was found to be the homolog of the bacterial and eukaryotic equivalents, PglB and Stt3p, respectively. Disruption of glycan synthesis or attachment resulted in very poorly or non-flagellated cells, implicating for the first time that the glycan structure on archaeal flagellins is necessary for proper flagella assembly and/or stability. These findings also represent the first proven N-glycosylation genes within the domain Archaea. New markerless, in-frame deletion methodology has allowed for advanced studies of a demonstrated and putative set of flagella-related genes in M. maripaludis. This collection of 11 co-transcribed genes, consisting of three flagellin genes (flaB1-flaB3), six genes of unknown function (flaC-flaH) and two genes implicated in flagellin subunit export (flaI and flaJ), make up the fla operon in this organism. Each gene from flaB1-flaI was systematically targeted for deletion and complementation to determine its necessity for flagellation. The analysis showed that both major flagellins, FlaB1 and FlaB2 are required for flagellation, while the minor flagellin, FlaB3, was required for the hook-like region of the flagella filament. FlaC, FlaF, FlaG and FlaH were shown for the first time to be essential for flagellation, while a naturally-occurring, truncated version of FlaD was found not to be required. These results continue to develop our understanding of the archaeal flagellum and the components necessary for its assembly and/or structure.