Fig. 2: Phosphorylation of serine 46 residue in HPr2 is essential for FruR activation.

A In vitro transcription assays were performed to assess the effects of HPr2 and F1P on FruR regulation. A linear DNA template (1 μg) covering −219 to +272 bp relative to the transcription start site (TSS) of the fru promoter was incubated with E. coli RNAP holoenzyme (1 μg) in the absence or presence of FruR (4 μg), F1P (1 mM), HPr2 (4 μg), or HPr2(S46D) (4 μg) as indicated. The resulting RNA products were purified and annealed with a HEX-labelled primer, which binds to the region from +84 to +105 relative to the TSS, followed by extension using a cDNA synthesis kit as described in the “Methods” section. A 142 bp HEX-labelled control DNA, and the 105 bp fruR transcript are indicated by arrowheads. Fragment sizes were determined by comparison to the internal molecular weight standards. B A lacZ reporter assay was performed in E. coli ER2566, harboring a plasmid carrying E. coli lacZ and F. prausnitzii fruR(K73E), transcriptionally fused to the fru promoter and the constitutive cat promoter, respectively. A plasmid expressing phosphomimetic mutant HPr2 (H15D, H15A, or H15AS46D) from the cat promoter was co-transformed to verify the effects of HPr2 phosphorylation on transcriptional activation. Relative lacZ expression was determined based on Miller units in the HPr2(H15D) expressing strain. C A lacZ reporter assay was conducted in an HPrK-expressing strain, ER2566Δpts::hprK, harboring the plasmids used in panel (B) and grown in M9 medium supplemented with 0.2% glycerol. Relative lacZ expression was determined as in panel (B). Statistical significance was determined using the Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001, n.s.: no statistical significance). Data are presented as means and SD (n  =  5, independent measurements).