Error bars indicate standard deviations. (B) EMSA of the recombinant His6::Fur and the ryhB promoter regions, as indicated in the margin. DNA was incubated with an increasing amount of His6::Fur for 30 min, and then loaded onto a 5% non-denaturing polyacrylamide gel. The gel was stained with SYBR Green EMSA stain and photographed. P ryhB * indicates deletion of the fur box in P ryhB . (C) Assessment of the binding of Fur to the ryhB promoter by using the MM-102 manufacturer FURTA. E. coli H1717 strains carrying the vector control, pT7-7, or the P1

region harboured on pT7-7 are indicated. A red colony (Lac+) is considered to have a FURTA-positive phenotype. RyhB activates CPS biosynthesis In K. pneumoniae CG43, we found that the deletion of fur resulted in elevated CPS production [21, 22]. To investigate ARS-1620 order if RyhB participates in Fur-regulated CPS biosynthesis, the CPS amount was assessed using measuring glucuronic acid content, which served as an indicator for Klebsiella K2 CPS [46], in K. pneumoniae strains, including WT, ΔryhB, Δfur, and ΔfurΔryhB, was quantified. As shown in Figure 2A, although the deletion of ryhB alone did not change on the amount of K2 CPS production, the elevated CPS amount in Δfur cells was abolished by the deletion of ryhB when the bacteria were grown in LB medium. The result indicates

that Fur regulates the expression of RyhB to repress CPS biosynthesis. To confirm the RyhB expression could activate the CPS biosynthesis, the effect of

RyhB induction on CPS amount was determined using an IPTG-inducible vector, pETQ. As shown in Figure 2B, the induced expression of ryhB in K. pneumoniae CG43 increased CPS production, which confirms that RyhB positively regulates CPS biosynthesis. Figure 2 RyhB activates CPS biosynthesis. (A) Comparison of CPS levels in WT, ΔryhB, Δfur, and ΔfurΔryhB strains. Bacterial strains were grown in LB medium at 37°C with agitation. After 16 h of growth, the bacterial glucuronic acid content was determined. *, P < 0.001 compared with WT. (B) WT strains carrying the vector control (pETQ) or pETQ-ryhB were grown in LB with 100 μM IPTG to induce ryhB expression. *, P < 0.001 compared with ALOX15 WT strains carrying pETQ. RyhB increased the transcriptional level of the K2 cps gene cluster To investigate whether RyhB affects the expression of the three cps gene clusters, the mRNA levels of orf1 orf3, and orf16 in Δfur and ΔfurΔryhB strains were measured by quantitative real-time PCR (qRT-PCR). As shown in Figure 3A, compared to the mRNA levels in the Δfur strain, the mRNA levels of orf1 and orf16 were apparent decreased in the ΔfurΔryhB strain, and that of orf3 also had a slight reduction in the ΔfurΔryhB strain. The result suggests that overexpression of RyhB activated the cps gene expression. To confirm our hypothesis, the effect of ryhB induction on the mRNA levels of orf1 orf3, and orf16 was tested using an IPTG-inducible vector, pETQ.