PMC

RpoN-3V5 complements the rpoN deletion mutant. The pBAD-derivative plasmid pRpoN carrying arabinose-inducible RpoN-3V5 complements two known RpoN-dependent traits in the rpoN mutant, Hcp expression (A) and motility (B).

The new sRNA flaX downstream of VC2188. (A) RNA-Seq data show a substantial increase of coverage in the downstream region of flaA. (B) Northern blot analysis confirmed the existence of flaX. (C) Mutation in flaX attenuated motility. (D) Motility of the flaX mutant was complemented by expressing flaX in trans on a sRNA expression vector pNM12. Arabinose (0.02%) was used for inducing the expression of flaX.

VasH, the enhancer-binding protein for RpoN, controls the expression of hcp but not the major cluster genes vipA and vipB. Relative gene expression was compared in wild type V52 and the vasH mutant by qPCR. The 16 S ribosomal RNA gene rrsA was used as a control sample. RNA was extracted in exponential phase cultures OD₆₀₀ = 0.5.

A sample of ChIP-Seq and RNA-Seq data covering the flagellar region and consensus RpoN-binding motif. (A) Genomic structures are shown above the peak panel. The height of each peak corresponds to the number of reads bound to the corresponding ChIP-site. (B) Motif was generated based on all ChIP-peaks using MEME program. The height of each letter represents the occurrence frequency at each location. (C) RNA-Seq data show difference in regulation of flagellar genes by RpoN. The RpoN-dependence (expression ratio) is shown at the bottom with the left axis. The absolute expression for each gene in the rpoN mutant is shown on the top with the right axis. Genes with high-RpoN-dependence and low-expression in the rpoN mutant require RpoN for expression, whereas genes with high-expression in the rpoN mutant likely have other promoters independent of RpoN.

RpoN is required for the expression of Hcp but not VipA or VipB. (A) Protein levels of Hcp, VipA (VCA0107) and VipB (VCA0108) by western blot analysis. The rpoN mutant was transformed with the plasmid pRpoN or the empty vector pBAD18. Cultures were grown aerobically in LB medium at 37°C to exponential phase (OD₆₀₀ = 0.5), and RpoN was induced by the addition of arabinose (0.1%) for 30 minutes. Samples were taken at different time points and protein expression was detected by western blot analysis. (B) Transcriptional levels of hcp and and vipAB and ChIP-binding of their promoter regions. The rrsA gene encoding 16 S RNA was used as an internal control sample to normalize difference in total RNA quantity among samples for qPCR. VgrG2 is located downstream of hcp2 and used as a control sample for ChIP assay. Monoclonal antibody to RpoN-3V5 was used for ChIP assay.