ORIGINAL PAPER Identification of the flagellar chaperone FlgN in the phytopathogen Xanthomonas axonopodis pathovar citri by its interaction with hook-associated FlgK Letı ´cia Khater Æ Marcos C. Alegria Æ Paula F. L. Borin Æ Tu ´lio M. Santos Æ Ca ´ssia Docena Æ Ljubica Tasic Æ Chuck S. Farah Æ Carlos H. I. Ramos Received: 9 January 2007 / Revised: 6 March 2007 / Accepted: 24 March 2007 / Published online: 10 May 2007 Ó Springer-Verlag 2007 Abstract Genome annotation of the plant pathogen Xanthomonas axonopodis pv. citri (Xac), identified fla- gellar genes in a 15.7 kb gene cluster. However, FlgN, a secretion chaperone for hook-associated proteins FlgK and FlgL, was not identified. We performed extensive screen- ing of the X. axonopodis pv. citri genome with the yeast two-hybrid system to identify a protein with the charac- teristics of the flagellar chaperone FlgN. We found a can- didate (XAC1990) encoded by an operon for components of the flagellum apparatus that interacted with FlgK. In order to further support this finding, Xac FlgK and XAC1990 were cloned, expressed, and purified. The re- combinant proteins were characterized by spectroscopic methods and their interaction in vitro confirmed by pull- down assays. We, therefore, conclude that XAC1990 and its homologs in other Xanthomonas species are, in fact, FlgN proteins. These observations extend the sequence diversity covered by this family of proteins. Keywords Flagella  FlgN  Secretion chaperone  Protein–protein interaction  Xanthomonas Abbreviations Xac Xanthomonas axonopodis pathovar citri 3AT 3-Aminotriazole MM Molecular mass A Absorbance CD Circular dichroism SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis Introduction The bacterial flagellum is responsible for motility and early interactions with hosts and is an example of a motor functioning at the molecular level (Vande Broek and Vanderleyden 1995; Moens and Vanderleyden 1996; Finley and Falkow 1997; Minamino and Namba 2004; Honko and Mizel 2005; Journet et al. 2005). It consists of three basic parts: a basal body, a hook, and a filament. The flagellum exports its own extracellular component proteins and assembles them at a distant tip and, together with its cognate sensory transduction system, receives sensory information from the cytoplasm which controls the direction of its high-speed rotation, a process critical for chemotaxis (Macnab 1999). The long helical filament of the flagellum is formed by about 30,000 subunits of flagellin (FliC) and is capped at its extremity by a hook- associated protein, FliD, which acts as a nucleation point Communicated by Jan Tommassen. L. Khater  P. F. L. Borin  T. M. Santos  L. Tasic  C. H. I. Ramos Laborato ´rio Nacional de Luz Sı ´ncrotron, P.O. Box 6192, CEP 13084-971 Campinas, SP, Brazil L. Khater  P. F. L. Borin  L. Tasic  C. H. I. Ramos Universidade Estadual de Campinas, CEP 13083-970 Campinas, SP, Brazil M. C. Alegria  C. Docena  C. S. Farah Departamento de Bioquı ´mica, Instituto de Quı ´mica, Universidade de Sa ˜o Paulo, 05508-000 Sa ˜o Paulo, SP, Brazil C. H. I. Ramos (&) UNICAMP Instituto de Quimica, PO Box 6154, 13084-862 Campinas, SP, Brazil e-mail: carlos.ramos@pq.cnpq.br 123 Arch Microbiol (2007) 188:243–250 DOI 10.1007/s00203-007-0240-y