Abstract
FlhF is a key protein required for complete flagellar synthesis, and its deletion results in the complete absence of a flagella and thus motility in Campylobacter jejuni. However, the specific mechanism still remains unknown. In this study, RNA-Seq, EMSAs, ChIP-qPCR and β-Galactosidase assays were performed to elucidate the novel interactions between FlhF and genes involved in flagellar synthesis. Results showed that FlhF has an overall influence on the transcription of flagellar genes with an flhF mutant displaying down-regulation of most flagellar related genes. FlhF can directly bind to the flgI promoter to regulate its expression, which has significant expression change in an flhF mutant. The possible binding site of FlhF to the flgI promoter was explored by continuously narrowing the flgI promoter region and performing further point mutations. Meanwhile, FlhF can directly bind to the promoters of rpoD, flgS, and fliA encoding early flagellin regulators, thereby directly or indirectly regulating the synthesis of class I, II, and III flagellar genes, respectively. Collectively, this study demonstrates that FlhF may directly regulate the transcription of flagellar genes by binding to their promoters as a transcriptional regulator, which will be helpful in understanding the mechanism of FlhF in flagellar biosynthetic and bacterial flagellation in general.
Highlights
Campylobacter jejuni flagella are considered the main virulence factor playing a key role in many important biological activities, such as motility, chemotaxis, adhesion, secreting virulence and colonization factors (Beeby, 2015; Burnham and Hendrixson, 2018; Subramanian and Kearns, 2019)
To investigate which genes are regulated by FlhF on the transcriptional level, we performed high-throughput RNA sequencing based on the genetic background
A one-way ANOVA analysis showed that class I, III, and II genes were significantly modified when comparing the flhF mutant to the respective wild-type strain (Figure 1B). qRT-polymerase chain reaction (PCR) was performed to verify the results of the RNA-Seq data
Summary
Campylobacter jejuni flagella are considered the main virulence factor playing a key role in many important biological activities, such as motility, chemotaxis, adhesion, secreting virulence and colonization factors (Beeby, 2015; Burnham and Hendrixson, 2018; Subramanian and Kearns, 2019). C. jejuni is a microaerophilic, Gram-negative bacterium, and is the leading cause of foodborne related gastroenteritis worldwide (Flint et al, 2016; Burnham and Hendrixson, 2018). It generates a single unsheathed flagellum at one or both poles of the cell (Hendrixson and Dirita, 2003; Matsunami et al, 2016; Liang and Connerton, 2018). Role of FlhF in Campylobacter more than 50 genes highly regulated by a complex regulatory network that ensures the coordinate expression to construct an intact flagella organelle (Balaban et al, 2009; Grinnage-Pulley et al, 2016; Liang and Connerton, 2018). Flagella have long been extensively studied, there remains a gap in our knowledge as to the regulation mechanisms of flagellar proteins synthesis (Gao et al, 2014)
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