Translation Inhibition of the Salmonella fliC Gene by the fliC 5′ Untranslated Region, fliC Coding Sequences, and FlgM

Abstract

The 5-untranslated region (5UTR) of the fliC flagellin gene of Salmonella contains sequences critical for efficient fliC mRNA translation coupled to assembly. In a previous study we used targeted mutagenesis of the 5 end of the fliC gene to isolate single base changes defective in fliC gene translation. This identified a predicted stem-loop structure, SL2, as an effector of normal fliC mRNA translation. A single base change (؊38C:U) in the fliC 5UTR resulted in a mutant that is defective in fliC mRNA translation and was chosen for this study. Motile (Mot ؉ ) revertants of the ؊38C:T mutant were isolated and characterized, yielding several unexpected results. Second-site suppressors that restored fliC translation and motility included mutations that disrupt a RNA duplex stem formed between RNA sequences in the fliC 5UTR SL2 region (including a precise deletion of SL2) and bases early within the fliC-coding region. A stop codon mutation at position 80 of flgM also suppressed the ؊38C:T motility defect, while flgM mutants defective in anti-28 activity had no effect on fliC translation. One remarkable mutation in the fliC 5UTR (؊15G:A) results in a translation defect by itself but, in combination with the ؊38C:U mutation, restores normal translation. These results suggests signals intrinsic to the fliC mRNA that have both positive and negative effects on fliC translation involving both RNA structure and interacting proteins.Salmonella enterica serovar Typhimurium possess 5 to 10 peritrichous flagella that are utilized for motility (19). The bacterial flagellum is composed of three main substructures: (i) a basal body that transverses the cell membranes and acts a proton-driven rotary motor enabling the flagellum to rotate and propel the bacterium through a liquid environment and across surfaces; (ii) a hook that acts as flexible coupling between the external filament and the basal body; and (iii) a rigid, helical filament that typically extends up to 10 m from the cell surface and acts as a propeller. Flagellum assembly begins with the basal body, proceeds through the hook, and is completed by the filament (20). The assembly of extracellular structures such as the filament and the hook involves the transport of the component proteins through a central channel within the growing flagellum. The cost of the maintenance of a flagellar motility system is high due to the large number of genes and protein subunits and the energy required for flagellum synthesis and functioning, which is ca. 2% of biosynthetic energy expenditure of the cell (19). As a result, the flagellar system of Salmonella is highly regulated at multiple levels, including responses to environmental cues and the developmental stages of the organelle (4). The flagellar biosynthetic and chemotaxis systems require more than 60 genes that are arranged into a transcriptional hierarchy of three promoter classes (class 1, class 2, and class 3) whose expression corresponds to when their products are needed for assembly (16). The class 1 flhDC operon is the maste...

Transcriptional and Translational Control of the Salmonella fliC Gene

“…A novel result that came from this study was the discovery of a potential stem-loop region in the 5ЈUTR that appeared to be required for fliC translation. However, in a separate study, we show that this stem-loop structure in the fliC 5ЈUTR functions to inhibit fliC mRNA translation and that the mutants isolated in the present study enhance this translation-inhibitory activity (31).…”

Control of gene expression by type III secretory activity

“…It has been suggested that the FlgN-dependent enhancement of FlgM translation may allow cells to rapidly down-regulate σ 28 -dependent genes following completion of the flagellar filament [16]. In addition to its role as an anti-sigma factor, FlgM may also regulate FliC (flagellin) expression at the posttranscriptional level [17].…”

Role of FlbT in flagellin production in Brucella melitensis

“…In C. crescentus, FlbT destabilized the flagellin fljK transcript by binding to the 59UTR (Anderson & Gober, 2000). In Salmonella enterica serovar Typhimurium, the 59UTR of the fliC transcript is needed for an efficient translation of fliC mRNA (Aldridge et al, 2006;Rosu et al, 2006) and the very end of the C terminus of FlgM appears to be involved in the regulation of fliC mRNA translation (Rosu et al, 2006). In Helicobacter pylori, the post-transcriptional regulator HP0958 controls the flagellin FlaA synthesis by targeting flaA mRNA to the export apparatus and promoting flagellin translation and secretion (Douillard et al, 2008(Douillard et al, , 2009 Interestingly, the flbT and flaF genes are conserved across the alphaproteobacteria, especially in the Rhizobiales, suggesting that the regulatory mechanism highlighted in this work is conserved (see Supplementary Figs S1 and S3).…”