Insertional inactivation of the flaH gene in the archaeon Methanococcus voltae results in non-flagellated cells
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O R I GI N A L P A P E R
N. A. Thomas á C. T. Pawson á K. F. Jarrell
Insertional inactivation of the ¯aH gene in the archaeon Methanococcus voltae results in non-¯agellated cells
Received: 10 October 2000 / Accepted: 8 January 2001 / Published online: 8 March 2001 Ó Springer-Verlag 2001
Abstract The marine methanogen Methanococcus voltae possesses two transcriptional units that encode a total of four ¯agellins. Immediately downstream of the ¯agellin genes are a number of ORFs, some of which are cotranscribed with the ¯agellin genes. These putative genes have been named ¯aCDEFGHIJ, although no biochemical data has implicated them in ¯agellar morphogenesis. None of the ¯aC-J genes has homology to any bacterial gene, with the exception of ¯aI, which shows homology to pilT, a gene that encodes a nucleotide binding protein of the type IV pilus family. In this study, insertional mutations in ¯aH of M. voltae were identi®ed. The mutants were non-motile and non-¯agellated as determined by electron microscopy. Southern hybridization experiments con®rmed the insertion of a mutagenic vector into ¯aH and indicated that two, tandem, copies of the vector were present. It is believed that insertion of the vector into ¯aH should disrupt the transcription of ¯aIJ due to polar eects. The ¯aH mutant displayed the same pattern of multiple mRNA transcripts, all originating upstream of ¯aB1, as the wild-type cells. Northern hybridization experiments failed to detect a ¯aHIJ transcript in either wild-type or mutant cells. Immunoblotting experiments indicated, however, that the mutants produced similar amounts of ¯agellin, FlaD and FlaE to wild-type cells. Flagellin localization experiments suggest that the ¯aH mutant is de®cient in ¯agellin secretion and/or assembly. The mutant also displayed similar pre¯agellin peptidase activity to the wild-type cells, indicating that none of the Communicated by W. Goebel N. A. Thomas á C. T. Pawson á K. F. Jarrell (&) Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6 E-mail: [email protected] Tel.: +1-613-5332456 Fax: +1-613-5336796 Part of this work was presented at the 100th Annual General Meeting of the American Society of Microbiology (2000) in Los Angeles, Calif.
genes ¯aHIJ is likely to be the gene that encodes this enzyme, which is required for cleaving the leader peptide from the pre¯agellins prior to their incorporation into the ¯agellar ®lament. This is the ®rst data indicating that the ¯aHIJ gene cluster is essential for ¯agellation in methanogens. Keywords Methanococcus voltae á Flagellum biosynthesis á Flagellum assembly á Insertional inactivation á ¯aHIJ
Introduction The archaeal ¯agellum has been shown to be a unique motility structure (Jarrell et. al. 1996b) that diers from the bacterial ¯agellum in both ultrastructure and composition. Archaeal ¯agella are thin ®lamentous structures (10±13 nm in diameter) that give cells the ability to swim (Alam and Oesterhelt 1984; Jarrell et al. 1996a). The ®laments have been shown to rota
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