Characterization of Flagellum Gene Families of Methanogenic Archaea and Localization of Novel Flagellum Accessory Proteins

Thomas, Nikhil A.; Jarrell, Ken F.

doi:10.1128/jb.183.24.7154-7164.2001

Cited by 60 publications

(89 citation statements)

References 54 publications

(58 reference statements)

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“…Initially, we attempted to identify the protein(s) in the purified pilus preparation by in-gel tryptic digestion and analysis of peptides by nLC-MS/MS. These efforts were generally unsuccessful, although a single tryptic peptide corresponding to T [54][55][56][57][58][59][60][61][62][63][64][65][66][67][68] , SNIEILSVSGEGSYK, from MMP0236 was identified in one analysis (data not shown). Reports in the literature of the hydrophobic nature of some Gram-positive pilin proteins led us to explore alternative MS methods to characterize this recalcitrant protein.…”

Section: Resultsmentioning

confidence: 99%

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Genetic and Mass Spectrometry Analyses of the Unusual Type IV-Like Pili of the ArchaeonMethanococcus maripaludis

Nair

et al. 2011

J Bacteriol

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The structure of pili from the archaeon Methanococcus maripaludis is unlike that of any bacterial pili. However, genetic analysis of the genes involved in the formation of these pili has been lacking until this study. Pili were isolated from a nonflagellated (⌬flaK) mutant and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist primarily of subunits with an apparent molecular mass of 17 kDa. In-frame deletions were created in three genes, MMP0233, MMP0236, and MMP0237, which encode proteins with bacterial type IV pilin-like signal peptides previously identified by in silico methodology as likely candidates for pilus structural proteins. Deletion of MMP0236 or MMP0237 resulted in mutant cells completely devoid of pili on the cell surface, while deletion of the third pilin-like gene, MMP0233, resulted in cells greatly reduced in the number of pili on the surface. Complementation with the deleted gene in each case returned the cells to a piliated state. Surprisingly, mass spectrometry analysis of purified pili identified the major structural pilin as another type IV pilin-like protein, MMP1685, whose gene is located outside the first pilus locus. This protein was found to be glycosylated with an N-linked branched pentasaccharide glycan. Deletion and complementation analysis confirmed that MMP1685 is required for piliation.

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Section: Resultsmentioning

confidence: 99%

“…Southern blotting was carried out as previously described (61). For all three deletion screens, the same hybridization temperature of 50°C was used.…”

Section: Vol 193 2011 Pili Of Methanococcus Maripaludis 805mentioning

confidence: 99%

Genetic and Mass Spectrometry Analyses of the Unusual Type IV-Like Pili of the ArchaeonMethanococcus maripaludis

Nair

et al. 2011

J Bacteriol

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“…Two flagellin genes (flgA1 and flgA2) are arranged in tandem at one locus, with the remaining three genes (flgB1, flgB2, and flgB3) clustered in another locus, and all five corresponding gene products have been identified within isolated flagella (9). The estimated lengths of the mRNAs indicate that the flagellin genes within each locus are cotranscribed, but the transcripts did not include the accessory genes as seen in M. voltae and other methanogens (10,38). It was recently determined that the majority of the accessory genes observed in M. voltae are present in H. salinarum next to the flgB locus but are transcribed from a distinct promoter and in the opposite direction (29).…”

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confidence: 94%

Identification and Localization of Flagellins FlaA and FlaB3 within Flagella ofMethanococcus voltae

Bardy

Mori

Komoriya³

et al. 2002

J Bacteriol

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While they are functionally similar, archaeal flagella have characteristics not typically seen in bacterial flagella (37). Archaeal flagellins are synthesized with a leader peptide (3, 18), which is cleaved prior to the incorporation of the flagellin into the filament, similar to the processing of type IV pilins before incorporation into the pilus (34). A Methanococcus maripaludis protein (FlaK) possessing preflagellin peptidase activity has recently been reported (2). Archaeal flagellins show sequence similarity to type IV pilins at the N termini of the mature proteins (8) and do not demonstrate homology to bacterial flagellins. Archaeal flagella are thinner in diameter (10 to 13 nm [4,16,33]) than bacterial flagella (20 nm [17]) and are always composed of multiple flagellins, which are often glycosylated (6,20,24). Additionally, a search of completely sequenced archaeal genomes failed to identify genes homologous to any genes coding for structural proteins involved in bacterial flagellation (7). This includes, but is not limited to, genes encoding the hook, rod, or ring proteins. All of these characteristics suggest that the structural components of the archaeal flagella are composed of unique, archaeon-specific proteins, possibly fulfilling the same function as those present in bacterial flagella, and that the mode of assembly is likely distinct as well.Methanococcus voltae is a marine organism possessing more than 70 flagella on the cell surface. As is typical of archaeal flagella, M. voltae flagella are composed of multiple flagellins (18). There are four flagellin genes found within two transcriptional units in the M. voltae chromosome, with the first transcriptional unit containing a single flagellin gene, flaA. The second transcriptional unit includes the three remaining flagellin genes (flaB1, flaB2, and flaB3) and the downstream cotranscribed flagellar accessory genes flaCDEFGHIJ (18; N. A. Thomas and K. F. Jarrell, unpublished data). Purified flagella were shown to be composed of two major proteins, flagellins FlaB1 and FlaB2, with molecular masses corresponding to 33 and 31 kDa, respectively (18). Prior to the work presented in this study, the remaining two flagellins (FlaA and FlaB3) remained undetected.Within the flagellated archaea, little work has been done to address the universal presence of multiple flagellins. In Halobacterium salinarum, five flagellin genes are arranged in two different loci. Two flagellin genes (flgA1 and flgA2) are arranged in tandem at one locus, with the remaining three genes (flgB1, flgB2, and flgB3) clustered in another locus, and all five corresponding gene products have been identified within isolated flagella (9). The estimated lengths of the mRNAs indicate that the flagellin genes within each locus are cotranscribed, but the transcripts did not include the accessory genes as seen in M. voltae and other methanogens (10, 38). It was recently determined that the majority of the accessory genes observed in M. voltae are present in H. salinarum next to the flgB locus bu...

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“…The archaella of M. voltae consist of four related archaellins, FlaA, FlaB1, FlaB2, and FlaB3 (114,115). The flaA gene is transcribed separately immediately upstream of a large operon containing flaB1, flaB2, and flaB3, followed by flaCDEFGHIJ (114,116). The major filament proteins are FlaB1 and FlaB2, FlaA is a minor structural component, and FlaB3 is responsible for the curved, hook region of the archaella (115).…”

Section: Pathway Of N-linked Glycosylation In Methanogensmentioning

confidence: 99%

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

Jarrell

Ding

Meyer

et al. 2014

Microbiol Mol Biol Rev

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SUMMARY N-glycosylation of proteins is one of the most prevalent posttranslational modifications in nature. Accordingly, a pathway with shared commonalities is found in all three domains of life. While excellent model systems have been developed for studying N-glycosylation in both Eukarya and Bacteria , an understanding of this process in Archaea was hampered until recently by a lack of effective molecular tools. However, within the last decade, impressive advances in the study of the archaeal version of this important pathway have been made for halophiles, methanogens, and thermoacidophiles, combining glycan structural information obtained by mass spectrometry with bioinformatic, genetic, biochemical, and enzymatic data. These studies reveal both features shared with the eukaryal and bacterial domains and novel archaeon-specific aspects. Unique features of N-glycosylation in Archaea include the presence of unusual dolichol lipid carriers, the use of a variety of linking sugars that connect the glycan to proteins, the presence of novel sugars as glycan constituents, the presence of two very different N-linked glycans attached to the same protein, and the ability to vary the N-glycan composition under different growth conditions. These advances are the focus of this review, with an emphasis on N-glycosylation pathways in Haloferax , Methanococcus , and Sulfolobus .

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Characterization of Flagellum Gene Families of Methanogenic Archaea and Localization of Novel Flagellum Accessory Proteins

Cited by 60 publications

References 54 publications

Genetic and Mass Spectrometry Analyses of the Unusual Type IV-Like Pili of the ArchaeonMethanococcus maripaludis

Genetic and Mass Spectrometry Analyses of the Unusual Type IV-Like Pili of the ArchaeonMethanococcus maripaludis

Identification and Localization of Flagellins FlaA and FlaB3 within Flagella ofMethanococcus voltae

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

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