RcsAB and Fur Coregulate the Iron-Acquisition System via entC in Klebsiella pneumoniae NTUH-K2044 in Response to Iron Availability

Abstract

The iron acquisition system is an essential virulence factor for human infection and is under tight regulatory control in a variety of pathogens. Ferric-uptake regulator (Fur) is one of Fe²⁺-responsive transcription factor that maintains iron homeostasis, and the regulator of capsule synthesis (Rcs) is known to regulate exopolysaccharide biosynthesis. We speculate the Rcs may involve in iron-acquisition given the identified regulator box in the upstream of entC that participated in the biosynthesis of enterobactin. To study the coregulation by RcsAB and Fur of entC, we measured the β-galactosidase activity and relative mRNA expression of entC in WT and mutant strains. The RcsAB- and Fur-protected regions were identified by an electrophoretic mobility shift assay (EMSA) and a DNase I footprinting assay. A regulatory cascade was identified with which Fur repressed rcsA expression and reduced RcsAB and entC expression. Our study demonstrated that entC was coregulated by two different transcriptional regulators, namely, RcsAB and Fur, in response to iron availability in Klebsiella pneumoniae.

Keywords: Fur; Klebsiella pneumoniae; RcsAB; entC; iron-acquisition system.

Figure 1

The K. pneumoniae iron acquisition system is modulated by RcsAB and Fur. CAS assay for K. pneumoniae NTUH-K2044 wild type, wild type with empty plasmid, mutants, and complement strains were assessed as described in Material and Methods. Siderophores secreted from strains removed iron from dye complex giving rise to a reduction in blue color of the solution. Measure the absorbance (A₆₃₀) for loss of blue color. Results were the means of biological triplicates plus standard deviations. Bar graph showed percent siderophore units, calculated as [(Ar – As) / Ar] × 100, where Ar is the absorbance of MM9/CAS solution and As is the sample absorbance. And MM9 can be used as a blank. The percent siderophore units of WT was compared with mutants and the percent siderophore units of mutants were compared with its complement strains. P values were calculated by one-way ANOVA. *P < 0.05. Shown are the averages ± standard deviation (SD) from three independent experiments.

Figure 2

Iron starvation enhanced the activity of the entC promoter. The putative promoter region of entC was cloned into the placZ15 plasmid and then introduced into CCW01 to measure β-galactosidase activity (Miller units) in increasing amounts of Dip. *P < 0.05, compared with the 0 μM Dip group.

Figure 3

RcsAB and Fur coregulate entC expression under different iron levels. (A) The promoter-lacZ fusion for entC was transformed into the CCW01, CCW01:ΔrcsA, CCW01:ΔrcsB, and CCW01:ΔrcsAB that were then grown in LB, LB with 100 μM FeSO₄ and LB with 250 μM Dip. (B) When 250 μM Dip added to LB. entC mRNA levels in WT, Kp:ΔrcsA, Kp:ΔrcsB, and Kp:ΔrcsAB were measured in iron-restricted conditions via RT-qPCR analysis. One-way ANOVA was performed to determine statistically significant differences between each strain and the WT. (C) When 100 μM FeSO₄ added to LB, WT, and Kp:Δfur were examined for entC expression under iron-replete conditions by RT-qPCR analysis. *P < 0.05, compared with the WT. Error bars are standard deviation.

Figure 4

RcsAB and Fur bind the entC promoter. The DNA binding capacities were evaluated by EMSAs. The promoter DNA fragments of entC were incubated with increasing amounts of purified MBP-RcsA (A), His₆-RcsB (B), His₆-RcsB mixed with MBP-RcsA (C), and His₆-Fur (D) and then subjected to polyacrylamide gel electrophoresis.

Figure 5

DNase I footprints of RcsAB and Fur at the entC promoter. The promoter DNA regions of entC were labeled with FAM and incubated with increasing amounts of purified His₆-RcsB (0, 1, 3 μg) (A) and His₆-Fur (B) (0, 0.5, 1 μg). The footprint regions are boxed within lines and marked.

Figure 6

Organization of entC promoter-proximal DNA regions. The DNA sequences were derived from NTUH-K2044. Shown are the translation start sites,−10 and−35 elements, Shine-Dalgamo sequence, RcsAB-binding site, RcsAB box-like sequence, Fur-binding site and Fur box-like sequence.

Figure 7

Fur directly represses rcsA. (A) The DNA binding capacity of Fur was evaluated by EMSA with rcsA. (B) Relative mRNA expression of rcsA in WT and Kp:Δfur was assayed by RT-qPCR in an iron-rich environment. *P < 0.05, compared with the WT.

Figure 8

A model for the regulation by RcsAB and Fur of rcsA and entC in K. pneumoniae. Under iron-replete conditions, apo-Fur combines with Fe²⁺ to form holo-Fur, which strongly represses rcsA and entC. Under iron depletion, upon derepression by apo-Fur of rcsA and entC and activation of RcsAB, the transcription of entC increased.