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

Abstract

RpoE1, an extracytoplasmic function sigma factor, is a repressor of the flagellar system in Brucella melitensis Unité de Recherche en Biologie Molé culaire (URBM), Faculté s Universitaires Notre-Dame de la Paix Namur (FUNDP), 61 rue de Bruxelles, B-5000 Namur, BelgiumThe genome of Brucella melitensis contains genes coding for the sigma factors RpoD, RpoN, RpoH1, RpoH2, RpoE1 and RpoE2. Previously published data show that B. melitensis is flagellated and that an rpoE1 mutant overexpresses the flagellar protein FlgE. In this study, we demonstrate that mutation of rpoE1 causes an overexpression of the flagellar genes fliF, flgE, fliC, flaF and flbT, correlating with the production of a longer filament and thereby demonstrating that RpoE1 acts as a flagellar repressor. Moreover, mutation of rpoE1 increases the promoter activity of the flagellar master regulator ftcR, suggesting that RpoE1 acts upstream of ftcR. Together, these data show that RpoE1 represses the flagellar synthesis and filament length in B. melitensis. INTRODUCTIONBrucellae are Gram-negative, intracellular pathogenic bacteria that cause brucellosis in a variety of mammals, including humans. For a long time, they were considered as being unflagellated. However, a sheathed flagellum has recently been discovered in Brucella melitensis and flagellar mutants are found to be impaired for infection in vivo (Ferooz & Letesson, 2010;Fretin et al., 2005;Zygmunt et al., 2006).The bacterial flagellum is a complex organelle used for motility and consists of at least three structural elements, the basal body, the hook and the filament (Macnab, 1999). The basal body, working as a motor, is embedded within the cell envelope and is anchored in the cytoplasmic membrane via the MS-ring structure, while the hook and filament, which function as a universal joint and a propeller, respectively, extend outwards from the cells (Minamino et al., 2008).Sigma and anti-sigma factors play important roles in the regulation of the flagellar genes (Smith & Hoover, 2009). The genome of B. melitensis has revealed the presence of genes encoding six sigma factors (rpoD, rpoH1, rpoH2, rpoE1, rpoE2 and rpoN) (Delory et al., 2006). The rpoD gene encodes the housekeeping sigma factor, the rpoH1 and rpoH2 genes encode two s 32 homologues, rpoE1 and rpoE2 encode two extracytoplasmic function (ECF) sigma factors and the rpoN gene encodes a s 54 homologue. The phenotypic characterizations were done on the five nonessential sigma factor mutants DrpoH1, DrpoH2, DrpoE1, DrpoE2 and DrpoN (Delory et al., 2006). Interestingly, at early growth phase in rich liquid medium, DrpoE1 overproduces the flagellar hook protein FlgE, suggesting that the flagellar system synthesis could be downregulated by the ECF sigma factor RpoE1 in B. melitensis (Delory et al., 2006).In this study, we demonstrate that the ECF sigma factor RpoE1 is a flagellar repressor in B. melitensis and that RpoE1 controls the length of the flagellar filament. METHODSBacterial strains and culture conditions. All strains and plasmids used ...

Brucella melitensis MucR, an Orthologue of Sinorhizobium meliloti MucR, Is Involved in Resistance to Oxidative, Detergent, and Saline Stresses and Cell Envelope Modifications

“…Our results clearly indicate that MucR of B. melitensis is also a repressor of flagellar-gene expression and likely acts upstream of FtcR, the flagellar master regulator orthologous to Rem (68) through which MucR of S. meliloti acts (21,67). In B. melitensis 16M, flagellargene expression is very tightly regulated, given that the QS regulator VjbR (27), the sigma factors rpoEI (76,77) and rpoH2 (76), and the cyclic-di-GMP phosphodiesterase BpdA (78) have already been shown to be involved in flagellar regulation. Here, we show that MucR is an additional actor within this complex regulatory network.…”

The Brucella abortus General Stress Response System Regulates Chronic Mammalian Infection and Is Controlled by Phosphorylation and Proteolysis

“…The function of these putative GSR genes in Brucella spp. was largely uncharacterized prior to this study, although it has been reported that a B. melitensis rpoE1 mutant negatively regulates flagellar biosynthesis (18) and is attenuated at 4 weeks postinfection in a mouse model (17). In this study, we have defined a functional role for the B. abortus GSR system in response to acute acid and oxidative stress in vitro and in control of chronic infection in mice (i.e.…”

“…It has been previously reported that a Brucella melitensis 16M ⌬rpoE1 mutant is atten-uated in BALB/c mice at 4 weeks postinfection (17) and exhibits derepressed flagellar biogenesis (17,18). However, this rpoE1 mutant did not have a survival defect in a hydrogen peroxide disk diffusion assay (17).…”

“…; and 3) cell envelope genes, including the flagellar gene cluster, which is repressed by E1 (18), and effectors of LPS O-chain ba14K (28) and pgm (29,30), which are activated by E1 . We note that both ⌬phyR and ⌬rpoE1 mutants appear smooth by both crystal violet and acriflavine staining, suggesting that loss of the GSR system does not result in gross changes in LPS O-chain structure (data not shown).…”

Peculiarities of the regulation of the Brucella flagellum

“…Interestingly, FlbT from S. meliloti could also restore the production of flagellin while the C. cresentus FlbT could not, confirming an opposite role for these two FlbT proteins. In their companion paper (Ferooz et al, 2011b), these authors demonstrate that the alternative sigma factor RpoE1 represses the production of FlgE (hook protein) and FliC (flagellin) via activation of an unknown repressor, which in turn negatively regulates the expression of the master flagellar regulator FtcR, since expression of both flaF and flbT regulator genes was also found to be increased in the rpoE1 mutant. A model combining and summarizing the two findings is presented in Fig.…”