|The major findings from 1996-present are summarized below.
1. We have developed a PCR based method for amplification of archaeal flagellin genes based on their conserved 5'-sequence and the prediction that they will be present in a multiple tandem arrangement.
2. We have identified from the cloned genes and the N-terminal sequences of purified flagellins that the major flagellins comprising the filament in Methanococcus vannielii were FlaB1 and FlaB2 and that both were made with short leader peptides. This is the first time that the major flagellin species comprising the archaeal filament have been unequivocally identified.
3. We have presented more evidence that archaeal flagellins are related to members of the type IV pilin/transport superfamily widespread in bacteria (). We have identified a gene adjacent to and possibly co-transcribed with the flagellins that is homologous to the gene that encodes PilT in the Pseudomonas aeruginosa type IV pilus system. This protein has ATPase activity and may be involved in the motor of the flagella.
4. We have isolated flagella mutants in M. voltae employing an insertional vector and shown that FlaA has little effect on motility and flagella structure. Insertion of the vector into FlaB2 which causes polar effects on the downstream gene transcription resulted in non flagellated cells confirming that this gene cluster was of major importance in flagella production. These were the first structural mutants isolated in any methanogen. These studies were started in the lab of A. Klein in Marburg Germany during a sabbatical leave as a von Humboldt fellow.
5. We have overexpressed the preflagellin of M. voltae in Escherichia coli and P. aeruginosa and demonstrated that it was not processed by the prepilin peptidase of P. aeruginosa even though it has many characteristics of the prepilin.
6. We have developed an assay using the overexpressed preflagellin to detect the preflagellin peptidase activity responsible for cleavage of the unique leader peptidase from the preflagellins.
7. We have examined the complete published genome sequences of flagellated archaea and reported the complete lack of genes encoding structural proteins of bacterial flagella. This is further evidence to support our hypothesis that archaeal flagella are a unique motility apparatus in both composition and assembly.
8. We have isolated and partially characterized flagella from thermoacidophilic archaea (Thermoplasma and Sulfolobus).
9. In a joint venture with a Russian group (Institute of Protein Research, Puschino,Russia), we were successful in obtaining funds (UNESCO and Human Frontier Science Program) to have a Russian scientist (Dr. Alla Kostyukova) visit my lab on two separate occasions. A paper on flagellin-binding proteins (potential flagellin-specific chapaerones) in Natronobacterium magadii was published using some of the data obtained during these stays.
10. We have cloned and sequenced the flagellins and flagella- associated genes and examined their transcription/co-transcription by northern blotting experiments in mesophilic, thermophilic and hyperthermophilic Methanococcus species (Thomas and Jarrell in preparation). Each organism seems unique in the manner in which the cells transcribe their flagellins, in terms of the number of transcripts and how many of the flagella-associated genes are co-transcribed with the flagellins.
11. We are in the process of overexpressing all of the flagella- associated genes in M. voltae (flaC-flaJ) using vectors that employ a histag for ready purification. The purified proteins are then used to raise antibodies. To date, Nik Thomas has successfully raised antibodies this way to FlaC, D, E, F, H and I. This bank of antibodies with be employed as a detection agents in experiments planned to determine the nature, location and interaction of the flagella-associated gene products.
12. We have presented arguments based on the conservation of sequence surrounding the cleavage site in preflagellins from all archaea examined that the archaeal preflagellin peptidase likely has a limited substrate range similar in many aspects to the prepilin peptidase of type IV pilus systems (Thomas, Chow and Jarrell in preparation).
13. We have isolated mutants in flaH and shown them to be nonflagellated, implicating these genes for the first time in archaeal flagellation (Thomas, Pawson and Jarrell in preparation).
14. We have a developed a technique to enrich for the hook protein of M. voltae and hope to isolate the gene in the near future (Bardy and Jarrell, unpublished).