Characterization of the flgG operon of Rhodobacter sphaeroides WS8 and its role in flagellum biosynthesis Bertha Gonza ´lez-Pedrajo a,1 , Javier de la Mora a , Teresa Ballado a , Laura Camarena b , Georges Dreyfus a, * a Departamento de Gene ´tica Molecular, Instituto de Fisiologı ´a Celular, Universidad Nacional Auto ´noma de Me ´xico, Ap. Postal 70-243, 04510 Me ´xico, D.F., Mexico b Departamento de Biologı ´a Molecular, Instituto de Investigaciones Biome ´dicas, Universidad Nacional Auto ´noma de Me ´xico, Ap. Postal 70-228, 04510 Me ´xico, D.F., Mexico Received 16 May 2002; received in revised form 26 July 2002; accepted 2 September 2002 Abstract In this work, we show evidence regarding the functionality of a large cluster of flagellar genes in Rhodobacter sphaeroides. The genes of this cluster, flgGHIJKL and orf-1, are mainly involved in the formation of the basal body, and flgK and flgL encode the hook-associated proteins HAP1 and HAP3. In general, these genes showed a good similarity as compared with those reported for Salmonella enterica. However, flgJ and flgK showed particular features that make them unique among the flagellar sequences already reported. flgJ is only a third of the size reported for flgJ from Salmonella; whereas flgK is about three times larger than any other flgK sequence previously known. Our results indicate that both genes are functional, and their products are essential for flagellar assembly. In contrast, the interruption of orf-1, did not affect motility suggesting that this sequence, if functional, is not indispensable for flagellar assembly. Finally, we present genetic evidence suggesting that the flgGHIJKL genes are expressed as a single transcriptional unit depending on the sigma-54 factor. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Rhodobacter sphaeroides; Bacterial flagellum; Bacterial motility; Hook – basal body complex 1. Introduction Swimming and swarming motility in many species of bacteria is achieved by rotating flagella (for recent reviews, see Refs. [1,2]). The flagellum is a complex propulsive organelle composed of three main substructures: a long helical filament, hook, and basal body. Between the hook and the filament are two junction proteins, FlgK and FlgL, also known as HAPs (for hook-associated proteins), which act as structural adapters for connecting these functionally different hook and filament homopolymers [3,4]. The basal body is a complex structure to which many different functions are associated (e.g. protein export as well as motor rotation and switching), and is composed of many proteins organized into a set of rings and a central rod. The inner MS ring, formed by subunits of the FliF protein [5], is localized in the cytoplasmic membrane, and serves as a mounting plate for components involved in motor rotation and torque generation. The C ring is a bell-like structure that projects into the cytoplasmic surface of the MS ring, and it contains the three motor/switch proteins responsible for determining the direction of flagellar rotation [6]. The rod is formed by four proteins: FlgB, FlgC and FlgF in the proximal portion, while FlgG constitutes the distal rod. The function of the rod is to allow transmission of torque to the external structures [1]. The L ring (FlgH) lies in the plane of the outer lipopolysaccharide membrane, and the P ring (FlgI) is in the plane of the peptidoglycan layer [7,8]. These rings are connected forming a stable cylindrical structure, which is believed to act as a molecular bushing for the central rod [9,10]. FlgH, FlgI, and FlgA are the only flagellar proteins in which a typical N-terminal signal sequence is present; consequently, they are thought to be 0167-4781/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S0167-4781(02)00504-3 * Corresponding author. Tel.: +52-55-5622-5618; fax: +52-55-5622- 5611. E-mail address: gdreyfus@ifisiol.unam.mx (G. Dreyfus). 1 Present address: Department of Molecular Biophysics and Biochem- istry, Yale University, New Haven, CT 06520-8114, USA. www.bba-direct.com Biochimica et Biophysica Acta 1579 (2002) 55 – 63