Transcriptional analysis of the flgK and fliD operons of Salmonella typhimurium which encode flagellar hook-associated proteins

In Salmonella typhimurium, three hook-associated proteins, HAP1, HAP2 and HAP3, are known to be essential for formation of flagellar filament. HAP1 and HAP2 are encoded by the flgK and flgL genes, respectively, which together constitute an operon, called the flgK operon. HAP3 is encoded by the fliD gene which forms part of the fliD operon together with the fliS and fliT genes. In the flagellar regulon, the operons are divided into three classes, 1, 2 and 3, based on their positions within a transcriptional hierarchy. Transcriptional analysis suggested that the flgK and fliD operons should belong to class 3, whose expression is dependent on the flagellum-specific sigma factor FliA. However, biochemical data indicated that these HAP proteins are detectable even in the hook-basal body structures produced by the fliA mutant. This work was carried out to resolve this discrepancy. More careful examination of transcription revealed that the fliA mutation reduces but does not eliminate the expression of these operons, whereas a mutation in the flhD operon, which encodes activator proteins for the class 2 operons, eliminates their expression. This suggests that the flgK and fliD operons may be transcribed from both class 2 and class 3 promoters. Primer extension analysis indicated that the promoter region of fliD contains both class 2 and class 3 promoters, while that of flgK contains only a class 3 promoter. Transposon insertion into the flgB operon, which belongs to class 2 and lies upstream of the flgK operon, was found to decrease the expression of the flgK operon to the basal level. This indicates that the flgK operon is transcribed both from its own class 3 promoter and from the class 2 promoter that controls the upstream flgB operon.