Characterization of flagellum gene families of methanogenic archaea and localization of novel flagellum accessory proteins.
Archaeal flagella are unique motility structures, and the absence of bacterial structural motility genes in the complete genome sequences of flagellated archaeal species suggests that archaeal flagellar biogenesis is likely mediated by novel components. In this study, a conserved flagellar gene family from each of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii has been characterized. These species possess multiple flagellin genes followed immediately by eight known and supposed flagellar accessory genes, flaCDEFGHIJ. Sequence analyses identified a conserved Walker box A motif in the putative nucleotide binding proteins FlaH and FlaI that may be involved in energy production for flagellin secretion or assembly. Northern blotting studies demonstrated that all the species have abundant polycistronic mRNAs corresponding to some of the structural flagellin genes, and in some cases several flagellar accessory genes were shown to be cotranscribed with the flagellin genes. Cloned flagellar accessory genes of M. voltae were successfully overexpressed as His-tagged proteins in Escherichia coli. These recombinant flagellar accessory proteins were affinity purified and used as antigens to raise polyclonal antibodies for localization studies. Immunoblotting of fractionated M. voltae cells demonstrated that FlaC, FlaD, FlaE, FlaH, and FlaI are all present in the cell as membrane-associated proteins but are not major components of isolated flagellar filaments. Interestingly, flaD was found to encode two proteins, each translated from a separate ribosome binding site. These protein expression data indicate for the first time that the putative flagellar accessory genes of M. voltae, and likely those of other archaeal species, do encode proteins that can be detected in the cell.
Thomas
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NA
Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Jarrell
K F
KF
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000096:biosynthesis / Q000235:genetics*; D002451:Cell Compartmentation; D004926:Escherichia coli / Q000235:genetics; D005407:Flagella / Q000235:genetics*; D005408:Flagellin / Q000235:genetics; D019766:Genes, Archaeal; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D017017:Methanococcus / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D011498:Protein Precursors / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D019843:Archaeal Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C403641:preflagellin; D005408:Flagellin