Methanococcus voltae is a mesophilic archaeon with flagella composed of flagellins that are initially made with 11-or 12-amino-acid leader peptides that are cleaved prior to incorporation of the flagellin into the growing filament. Preflagellin peptidase activity was demonstrated in immunoblotting experiments with flagellin antibody to detect unprocessed and processed flagellin subunits. Escherichia coli membranes containing the expressed M. voltae preflagellin (as the substrate) were combined in vitro with methanogen membranes (as the enzyme source). Correct processing of the preflagellin to the mature flagellin was also shown directly by comparison of the N-terminal sequences of the two flagellin species. M. voltae preflagellin peptidase activity was optimal at 37°C and pH 8.5 and in the presence of 0.4 M KCl with 0.25% (vol/vol) Triton X-100.All of the major subgroupings of archaea, including methanogens, extreme halophiles, and sulfur-dependent thermophiles and hyperthermophiles, have members that possess flagella that look superficially like bacterial flagella (10). However, recent evidence has indicated that the archaeal flagellum is a unique motility structure, distinct from that of bacteria in composition and likely assembly (4, 10). One major distinction is that the assembly of archaeal flagella requires the posttranslational cleavage of a short (11-or 12-amino-acid) leader peptide (2, 13) from the precursor form of the flagellin monomer (preflagellin) before its incorporation into the growing filament. Bacterial flagellins are not made with leader peptides (12).Previously, in Methanococcus voltae, four flagellin genes (flaA, flaB1, flaB2, and flaB3) carried by two transcriptional units were identified (13). One transcriptional unit contains only flaA; the other, polycistronic transcriptional unit (at least 5.4 kb in length), containing flaB1, flaB2, flaB3, and a number of presumed flagellum accessory genes, initiates at flaB1 and extends to at least the end of flaG (13; D. P. Bayley, J. D. Correia, and K. F. Jarrell, unpublished data). N-terminal (14), transcriptional (13), and mutational (9) analyses have provided evidence that flaB1 and flaB2 encode the major flagellins in M. voltae. Comparison of the N-terminal sequence of one purified flagellin with the amino acid sequence predicted from the gene sequence (13) confirmed the presence of a 12-amino-acid leader peptide. Similarly, in the related methanogen Methanococcus vannielii, comparison of the N-terminal sequences obtained from the two major flagellins of purified flagellar filaments with the deduced amino acid sequence of the cloned genes indicated the presence of 12-amino-acid leader peptides on the FlaB1 and FlaB2 flagellins (2). The presence of leader peptides on archaeal flagellins indicated that enzymatic activity must be present in archaeal cells to process the preflagellins.Two different substrates were used for the determination of preflagellin peptidase activity. One preflagellin substrate was prepared by the expression of M. voltae Fla...
Microbial life has inhabitedthis planet for approximately 3.7 billion years--3 billion years longer than plants and animals. The development of higher life forms on Earth was dependent on microorganisms, and the continued existence of plant and animal life absolutely requires the continued activity of microbial life. Higher organisms are even known to harbor microbial derivatives in the form of mitochondria and chloroplasts. From the initial production of free 02 by cyanobacteria in the early evolution of the earth to the continuing role that microorganisms play in geochemical cycles, the entire biosphere is dependent on the activities of microorganisms. Certainly, as Carl Woese has said, "to study microorganisms is to study the biosphere" (Woese 1998).Ever since Woese and his colleagues first proposed that there are three basic lines of evolutionary descent (Woese and Fox 1977), an odd collection of microorganisms called The days of the Archaea being considered as just "odd bacteria"adapted to living in extreme environments are long past Ken F. Jarrell (e-mail: jarrellk@post. queensu.ca) is a professor and Jason D. Correia and Nikhil A. Thomas are graduate students in the Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6. Douglas P. Bayley is a postdoctoral fellow in the Department of Microbiology, East Carolina University, Greenville, NC 27858. Jarrell has worked on methanogenic archaea for the past two decades. For the past 10 years, his lab has been studying the unique flagella structure of the archaea. ? 1999 American Institute of Biological Sciences. 530
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