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This study focused on FlhF and FlhG, which control the spatial and numerical regulation of flagella in many exoflagellated bacteria, in the spirochete <jats:italic>Leptospira<\/jats:italic>. In contrast to <jats:italic>flhF<\/jats:italic> which seems to be essential in <jats:italic>Leptospira<\/jats:italic>, we demonstrated that <jats:italic>flhG<jats:sup>\u2212<\/jats:sup><\/jats:italic> mutants in both the saprophyte <jats:italic>L. biflexa<\/jats:italic> and the pathogen <jats:italic>L. interrogans<\/jats:italic> were less motile than the wild\u2010type strains in gel\u2010like environments but not hyperflagellated as reported previously in other bacteria. Cryo\u2010electron tomography revealed that the distance between the flagellar basal body and the tip of the cell decreased significantly in the <jats:italic>flhG<jats:sup>\u2212<\/jats:sup><\/jats:italic> mutant in comparison to wild\u2010type and complemented strains. Additionally, comparative transcriptome analyses of <jats:italic>L. biflexa flhG<jats:sup>\u2212<\/jats:sup><\/jats:italic> and wild\u2010type strains showed that FlhG acts as a negative regulator of transcription of some flagellar genes. We found that the <jats:italic>L. interrogans flhG<jats:sup>\u2212<\/jats:sup><\/jats:italic> mutant was attenuated for virulence in the hamster model. Cross\u2010species complementation also showed that <jats:italic>flhG<\/jats:italic> is not interchangeable between species. Our results indicate that FlhF and FlhG in <jats:italic>Leptospira<\/jats:italic> contribute to governing cell motility but our data support the hypothesis that FlhF and FlhG function differently in each bacterial species, including among spirochetes.<\/jats:p>","DOI":"10.1111\/mmi.14831","type":"journal-article","created":{"date-parts":[[2021,10,19]],"date-time":"2021-10-19T22:00:09Z","timestamp":1634680809000},"page":"1392-1406","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Role of the major determinant of polar flagellation FlhG in the endoflagella\u2010containing spirochete <i>Leptospira<\/i>"],"prefix":"10.1111","volume":"116","author":[{"given":"Lenka","family":"Fule","sequence":"first","affiliation":[{"name":"Institut Pasteur Biology of Spirochetes Unit Paris France"},{"name":"Pasteur International Unit Integrative Microbiology of Zoonotic Agents Institut Pasteur de Montevideo Montevideo, 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