Identification of a Putative Acetyltransferase Gene, MMP0350, Which Affects Proper Assembly of both Flagella and Pili in the ArchaeonMethanococcus maripaludis

Abstract

Glycosylation is a posttranslational modification utilized in all three domains of life. Compared to eukaryotic and bacterial systems, knowledge of the archaeal processes involved in glycosylation is limited. Recently, Methanococcus voltae flagellin proteins were found to have an N-linked trisaccharide necessary for proper flagellum assembly. Current analysis by mass spectrometry of Methanococcus maripaludis flagellin proteins also indicated the attachment of an N-glycan containing acetylated sugars. To identify genes involved in sugar biosynthesis in M. maripaludis, a putative acetyltransferase was targeted for in-frame deletion. Deletion of this gene (MMP0350) resulted in a flagellin molecular mass shift to a size comparable to that expected for underglycosylated or completely nonglycoslyated flagellins, as determined by immunoblotting. Assembled flagellar filaments were not observed by electron microscopy. Interestingly, the deletion also resulted in defective pilus anchoring. Mutant cells with a deletion of MMP0350 had very few, if any, pili attached to the cell surface compared to a nonflagellated but piliated strain. However, pili were obtained from culture supernatants of this strain, indicating that the defect was not in pilus assembly but in stable attachment to the cell surface. Complementation of MMP0350 on a plasmid restored pilus attachment, but it was unable to restore flagellation, likely because the mutant ceased to make detectable flagellin. These findings represent the first report of a biosynthetic gene involved in flagellin glycosylation in archaea. Also, it is the first gene to be associated with pili, linking flagellum and pilus structure and assembly through posttranslational modifications.

N-Linked Glycosylation in Archaea: a Structural, Functional, and Genetic Analysis

“…This correlation is very well defined for both M. voltae and M. maripaludis (71,119,229). Any deletion in a GT, OST, or biosynthetic gene that resulted in nonglycosylated archaellins or archaellins decorated with a glycan consisting of only the first sugar led to cells that were nonarchaellated.…”

“…There is one exception involving agl genes and piliation, and this is for mmp0350 mutants. MMP0350 is likely an acetyltransferase for the second sugar, as the deletion of mmp0350 results in a one-sugar glycan (229). These cells are nonarchaellated, as expected, but also have difficulty in maintaining pilus attachment to the cell surface.…”

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

“…The majority of these genes lay in a large cluster (mmp1079 to mmp1088) comprised of multiple operons (18,19,21). However, the deletion of one gene not located near this cluster, mmp0350, which encodes a putative acetyltransferase, was also previously shown to affect glycan structure and archaellum formation (27). In the immediate vicinity of mmp0350 are a number of other genes annotated as potentially involved in the biosynthesis of the glycan sugars or assembly of the glycan.…”

“…Previously, mutants containing markerless inframe deletions of mmp0350 and mmp0354 were reported (21,27). Using the same methods established by Moore and Leigh (32), mutants containing markerless in-frame deletions of mmp0351, mmp0352, mmp0353, mmp0355, mmp0357, mmp0358, and mmp0359 were generated in M. maripaludis Mm900.…”

“…These include AglXYZ (MMP1081 to MMP1083), responsible for the acetamidino group modification at C-3 of the third sugar (18), AglU (MMP1084), a threonine transferase that modifies the third sugar at C-6, and AglV (MMP1085), a methyltransferase for the methyl group at C-5 in the fourth sugar (19). MMP0350 was also identified as a putative acetyltransferase involved in biosynthesis of the second sugar (27). Additionally, in vitro assays using heterologously expressed proteins have identified other M. maripaludis gene products that are involved in acetamido sugar biosynthesis (28,29), although genetic analysis of these is still lacking.…”