Role of FlbT in flagellin production in Brucella melitensis

Abstract

It was recently demonstrated that the pathogen Brucella melitensis produces a polar sheathed flagellum under the control of the master regulator FtcR. However, the regulatory mechanism controlling the flagellar assembly remains unknown. In this work, we investigate the flagellar hierarchy of B. melitensis as well as the flagellin FliC regulation. We show that a mutation in fliF or flgE (coding for the basal body structure and the hook, respectively) does not affect FliC synthesis, suggesting that production of FliC does not depend on the flagellar assembly. We demonstrate that FlbT is a FliC activator since inactivation of flbT causes a decrease in fliC expression by using a fliC-lacZ translational reporter construct. Moreover, the quantitative realtime PCR and Western blot analysis show a marked decrease in fliC mRNA and FliC protein level, respectively. Conversely, the B. melitensis wild-type strain overexpressing flaF fails to produce FliC, suggesting an opposite function. Interestingly, the expression of the flbT gene in an ftcR or an flbT mutant restores FliC production, demonstrating that FlbT plays a regulatory checkpoint role in FliC synthesis. This mechanism could be conserved in the Rhizobiales since complementation of an flbT or an ftcR mutant with flbT from Sinorhizobium meliloti restores FliC synthesis. INTRODUCTIONThe intracellular pathogen Brucella melitensis is the causative agent of brucellosis in mammals, also known as Malta fever in humans. This intracellular pathogen belongs to the a-2 subgroup of proteobacteria, including the genera Agrobacterium, Rhizobium and Rickettsia, which also live in close association with a eukaryotic host (Batut et al., 2004;Ugalde, 1999). For a long time, brucella was considered to be non-flagellated, but recent studies have demonstrated that B. melitensis produces a polar sheathed flagellum during the beginning of the exponential growth phase in rich medium and that the flagellar genes are required for the establishment of in vivo infection in mice and goats (Ferooz & Letesson, 2010;Fretin et al., 2005;Letesson et al., 2002;Zygmunt et al., 2006).The structure of the bacterial flagellum is classically divided into three main components: the MS-ring in the basal body, the hook and the filament encoded by the fliF, flgE and fliC genes, respectively. The molecular processes involved in assembly of the bacterial flagellum have been extensively studied in the enterobacteria Escherichia coli and Salmonella enterica serovar Typhimurium (Macnab, 1996). In these organisms, the flagellar genes are divided into three different classes hierarchically controlled by a finely tuned mechanism (Chevance & Hughes, 2008; Frye et al., 2006;Kalir et al., 2001). At the first step of flagellar assembly, the class I transcriptional regulator FlhDC activates the transcription of the class II genes which include the genes encoding the structural components of the hook-basal body (HBB) structure, the sigma factor 28 (s 28 also named FliA), which activates the class III genes, and its anti-...

Transcriptional Control of the Lateral-Flagellar Genes of Bradyrhizobium diazoefficiens

“…1C). These results indicated that, as in B. melitensis (38), FlbT L is required for lateral flagellin synthesis.…”

“…We suspected that this role might be fulfilled by FlbT, a protein known as a translational regulator of flagellin synthesis in other bacteria (37,38).…”

“…Moreover, operon I encodes FlbT L , a regulator whose homologs in C. crescentus and B. melitensis regulate the expression of class III genes (37,38). Despite that homology, however, FlbT plays opposite roles in those systems: whereas in C. crescentus FlbT is an inhibitor of the translation of flagellin transcripts, in B. melitensis that protein is required for translation.…”

RpoE1, an extracytoplasmic function sigma factor, is a repressor of the flagellar system in Brucella melitensis

“…2). FliF is the monomer of the MS-ring that is the first part assembled in the flagellum structure, while FlbT and FlaF are two flagellin regulators (Ferooz et al, 2011). Expression of all the flagellar genes tested was found to be higher in DrpoE1 compared with the wild-type strain (Fig.…”

Peculiarities of the regulation of the Brucella flagellum

“…complementation with the regulator in trans restores the production of the flagellin, even in a mutant where the master regulator FtcR has been inactivated (Ferooz et al, 2011a). FlaF, on the other hand, acts in the opposite direction, since FlaF downregulates the production of FliC.…”