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. 2020 Jan 17;10:3063. doi: 10.3389/fmicb.2019.03063

FIGURE 3.

FIGURE 3

BrpT and BrpS constitute a negative feedback loop. (A,B) Total RNA was isolated from biofilms of the V. vulnificus strains grown in microtiter plates. The levels of brpR, brpT, brpA, and cabA transcripts (A) or brpS transcript (B) were determined by qRT-PCR, and the parent strain was set to 1. Error bars represent the SD from three independent experiments. ∗∗, p < 0.005 relative to the parent strain; ns, not significant. Parent and Parent (pJK1113), parent strain; ΔbrpS, brpS mutant; ΔbrpT (pJK1113), brpT mutant; ΔbrpT (pJK1113::brpT), complemented strain. (C) A 358-bp DNA fragment of the brpS upstream region (2.5 nM) was radioactively labeled and incubated with increasing amounts of BrpT as indicated in the presence of c-di-GMP (50 μM). For competition analysis, the same but unlabeled 358-bp DNA fragment was used as self-competitor DNA. Various amounts of self-competitor DNA were added as indicated to a reaction mixture containing the probe DNA before the addition of BrpT. (D) A 388-bp DNA fragment of the brpT upstream region (2.5 nM) was radioactively labeled and incubated with increasing amounts of BrpS as indicated in the presence of c-di-GMP (50 μM). For competition analysis, the same but unlabeled 388-bp DNA fragment was used in the same manner as described above, except that BrpS was added. B, bound DNA; F, free DNA.