Transcriptional and Translational Control of the Salmonella fliC Gene

Abstract

The flagellin gene fliC encodes the major component of the flagellum in Salmonella enterica serovar Typhimurium. This study reports the identification of a signal within the 5 untranslated region (5UTR) of the fliC transcript required for the efficient expression and assembly of FliC into the growing flagellar structure. Primer extension mapping determined the transcription start site of the fliC flagellin gene to be 62 bases upstream of the AUG start codon. Using tetA-fliC operon fusions, we show that the entire 62-base 5UTR region of fliC was required for sufficient fliC mRNA translation to allow normal FliC flagellin assembly, suggesting that translation might be coupled to assembly. To identify sequence that might couple fliC mRNA translation to FliC secretion, the 5 end of the chromosomal fliC gene was mutagenized by PCR-directed mutagenesis. Single base sequences important for fliC-dependent transcription, translation, and motility were identified by using fliC-lacZ transcriptional and translational reporter constructs. Transcription-specific mutants identified the ؊10 and ؊35 regions of the consensus flagellar class 3 gene promoter. Single base changes defective in translation were located in three regions: the AUG start codon, the presumed ribosomal binding site region, and a region near the very 5 end of the fliC mRNA that corresponded to a potential stem-loop structure in the 5UTR. Motility-specific mutants resulted from base substitutions only in the fliC-coding region. The results suggest that fliC mRNA translation is not coupled to FliC secretion by the flagellar type III secretion system.

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).…”

Genetically Programmable Pathogen Sense and Destroy

“…Type IV system is mostly characterized for the transport of DNA [81,82] whereas type VI secretion system is only recently identified and details of its mechanism are still emerging [73,86]. Flagellar machinery is analogous to the type III secretion apparatus except that it is used by the bacteria to assemble flagella and secrete flagellar components in the medium [123,126,127]. Therefore, I employed flagellar system to secrete CoPy.…”

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

“…Initial assays showed fliC expression of the translational reporter fusion to be elevated by the SL2 deletion ( Table 2, compare TH6235 [row 1] to TH8905 [row 3]), although the standard deviation was too high in these assays to be conclusive. In order to get a more accurate measure on the effect of ⌬SL2 on fliC translation, we introduced mutations in the fliC promoter region that had been isolated in our lab to downregulate fliC gene transcription (1). The purpose of using promoter-down mutations was twofold.…”

“…FlbT acts by directly binding the 5Ј untranslated region (5ЈUTR) of flagellin mRNA to inhibit its translation (2). Recently, we found that transcription of the chromosomal fliC gene without the fliC 5ЈUTR region are nonmotile, suggesting an important role for the fliC 5ЈUTR in normal fliC mRNA expression associated with filament assembly (1). The 5Ј end of the fliC gene was analyzed, and regions essential for transcriptional and translational control were defined.…”

“…The 5Ј end of the fliC gene was analyzed, and regions essential for transcriptional and translational control were defined. A stem-loop region, SL2, of the fliC 5ЈUTR was discovered that when mutated was defective in fliC translation (1).…”