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. 2021 Jan 21;11:601921. doi: 10.3389/fmicb.2020.601921

FIGURE 1.

FIGURE 1

The kpf gene cluster encoding type 1-like fimbriae is regulated by Fur. (A) kpf cluster comprises kpfR, kpfA, kpfB, kpfC, and kpfD genes and is transcribed as a polycistronic mRNA. cDNA synthesized from total RNA of K. pneumoniae was used in PCR reactions using the primer pairs represented in the scheme. The resulting amplicons were analyzed by agarose gel electrophoresis and confirmed the predicted sizes of 847 and 3,773 base pairs (bp). The red spot on the scheme indicates the putative Fur box sequence identified on kpf cluster. (B) Partial sequence of kpfR showing the initial codon (ATG, double underlined) and the Fur-binding sequence located inside the coding region of kpfR (highlighted in blue). Also indicated on the promoter region of the cluster are the –35 (in green) and –10 (in yellow) domains of the housekeeping Sigma factor 70, the predicted transcription initiation site at position + 1 (cytosine in red), and a putative IHF-binding consensus sequence (in gray; lowercase nucleotides are not identical to the consensus sequence). (C) Fur Titration Assay (FURTA) validated the Fur box on kpfR of K. pneumoniae, as indicated by the E. coli H1717 red colonies on MacConkey plates, similar to the FURTA-positive control (Lac+). (D) DNA Electrophoretic Mobility Shift Assay (EMSA) confirms the direct interaction of the K. pneumoniae Fur protein on the putative Fur box found on kpfR. Fur-DNA probe complexes are formed (mobility shifts, indicated by closed arrowheads) as increased concentrations of purified His-Fur protein from K. pneumoniae were incubated with the probes (DNA fragment containing the Fur box of kpfR). Fur interaction depends on divalent cations, since the addition of EDTA chelator abolished the mobility shift of the probe (open arrowheads). No mobility shift is observed in the EMSA with the negative control (DNA probe without Fur box), indicated by open arrowheads). (E) RT-qPCR analyses showed that Fur represses the expression of the kpfR gene on K. pneumoniae cells cultured under iron-depleted condition when compared to the control condition (bacteria cultured in LB medium only). Since kpfR belongs to the kpf gene cluster, Fur modulates the expression of the entire cluster according to the availability of iron in the culture medium. *p ≤ 0.05.