Expression of BfrH, a putative siderophore receptor of Bordetella bronchiseptica, is regulated by iron, Fur1, and the extracellular function sigma factor EcfI

Abstract

Iron (Fe) in soluble elemental form is found in the tissues and fluids of animals at concentrations insufficient for sustaining growth of bacteria. Consequently, to promote colonization and persistence, pathogenic bacteria evolved a myriad of scavenging mechanisms to acquire Fe from the host. Bordetella bronchiseptica, the etiologic agent of upper respiratory infections in a wide range of mammalian hosts, expresses a number of proteins for acquisition of Fe. Using proteomic and genomic approaches, three Fe-regulated genes were identified in the bordetellae: bfrH, a gene encoding a putative siderophore receptor; ecfI, a gene encoding a putative extracellular function (ECF) sigma factor; and ecfR, a gene encoding a putative EcfI modulator. All three genes are highly conserved in B. pertussis, B. parapertussis, and B. avium. Genetic analysis revealed that transcription of bfrH was coregulated by ecfI, ecfR, and fur1, one of two fur homologues carried by B. bronchiseptica. Overexpression of ecfI decoupled bfrH from Fe-dependent regulation. In contrast, expression of bfrH was significantly reduced in an ecfI deletion mutant. Deletion of ecfR, however, was correlated with a significant increase in expression of bfrH, due in part to a cis-acting nucleotide sequence within ecfR which likely reduces the frequency of readthrough transcription of bfrH from the Fe-dependent ecfIR promoter. Using a murine competition infection model, bfrH was shown to be required for optimal virulence of B. bronchiseptica. These experiments revealed ecfIR-bfrH as a locus encoding a new member of the growing family of Fe and ECF sigma factor-modulated regulons in the bordetellae.

FIG. 1.

Genetic organization of the ecfIR-bfrH locus. (A) Schematic arrangement of the ecfIR-bfrH locus in B. bronchiseptica. ecfI and ecfR encode a putative ECF sigma factor and a putative sigma factor regulator, respectively. bfrH exhibits homology to genes for OM proteins involved in siderophore uptake. The length of each ORF is denoted above the gene; the molecular mass of each of the predicted polypeptides is denoted below the gene. Positions of the two putative promoters, P_ecfIR and P_bfrH, and the direction of transcription are denoted by arrows. (B) Sequences at P_ecfIR. This promoter contains regions homologous to σ⁷⁰-type promoters. Putative −10 and −35 regions of P_ecfIR are boxed. Sequences homologous to the consensus Fur box of E. coli are underlined; nucleotides in bold are perfectly conserved in RB50 and in the consensus Fur box of E. coli. The translational GTG start codon of ecfI is denoted in bold. (C) Sequences at P_bfrH. This region contains homology to other ECF sigma factor-regulated promoters. Putative −10 and −35 regions of P_bfrH are boxed. Sequences homologous to the consensus Fur box of E. coli are underlined; nucleotides in bold are perfectly conserved in RB50 and in the consensus Fur box of E. coli. The translational stop codon of ecfR and the translational start codon of bfrH are denoted in bold.

FIG. 2.

Fe-dependent transcription of the ecfIR-bfrH locus. Total RNAs were isolated from Fe-replete and Fe-stressed RB50 cultures. Oligonucleotide primer sets used in the reaction mixtures targeted a 218-bp internal region of ecfI, a 288-bp overlap region containing the 3′ end of ecfI and the 5′ end of ecfR (ecfIR), a 488-bp internal region ecfR, a 237-bp region overlapping ecfR, bfrH, and a 93-bp region between ecfR and bfrH (ecfR-bfrH intergenic), a 598-bp internal region of bfrH, a 513-bp internal region of bhuR, and a 402-bp internal region of recA. Amplified DNA from each RT-PCR was resolved in a 2% agarose gel and visualized by ethidium bromide staining. Fe+, Fe-replete conditions; Fe−, Fe-stressed conditions.

FIG. 3.

Regulation of the ecfIR-bfrH locus by fur1. (A) Alignment of Fur1 sequence with sequence of Fur of E. coli. Amino acids within metal binding site 1 are indicated in bold and underlined, and amino acids within metal binding site 2 are denoted only in bold. (B) Fur1-dependent transcription of the ecfIR-bfrH locus. RT-PCR was performed using total RNAs obtained from Fe-replete and Fe-stressed cells, utilizing oligonucleotide primers which targeted the overlap region encompassing the 3′ end of ecfI and the 5′ end of ecfR (ecfIR), bfrH, bhuR, and recA. Amplified DNA from each RT-PCR was resolved in a 2% agarose gel and visualized by ethidium bromide staining. +, Fe-replete conditions; −, Fe-stressed conditions.

FIG. 4.

Regulation of bfrH by EcfI. (A) Effects of overexpression of EcfI on expression of bfrH. RT-PCR was performed using total RNAs isolated from Fe-replete and Fe-stressed cells. Oligonucleotide primer sets used in the reaction mixtures targeted a 218-bp region of ecfI, a 288-bp overlap region encompassing the 3′ end of ecfI and the 5′ end of ecfR (ecfIR), a 237-bp region overlapping ecfR, bfrH, and a 93-bp region between ecfR and bfrH (ecfR-bfrH intergenic), a 598-bp internal region of bfrH, a 513-bp internal region of bhuR, and a 402-bp internal region of recA. Amplified DNA from each RT-PCR was resolved in a 2% agarose gel and visualized by ethidium bromide staining. +, Fe-replete conditions; −, Fe-stressed conditions. (B) Expression of bfrH in RB50ΔecfI. qRT-PCR was performed on total RNAs isolated from Fe-replete and Fe-stressed cells, using oligonucleotides targeting sequences within bfrH. Data are expressed as means and standard errors and were obtained by calculating the relative SQ of the respective mRNA after normalizing to the amount of recA mRNA expressed by the cell. *, statistically significantly different from Fe-stressed RB50 (P < 0.05). (C) β-Galactosidase activities of RB50(pJB3.1) and RB50(pDJM41Δ) cultured under Fe-repelete (Fe+) and Fe-stressed (Fe−) conditions. *, statistically significantly different from Fe-replete RB50(pJB3.1) (P < 0.05). (D) Western immunoblot of BfrH. OMs were prepared from Fe-replete and Fe-stressed cells. OMs were resolved in a 7.5% SDS-PAGE gel and immunoblotted with anti-BfrH-peptide rabbit polyclonal antibodies. +, Fe-replete conditions; −, Fe-stressed conditions; rBfrH, whole-cell extract of IPTG-induced BL21(DE3)(pLysS)(pJB7.1); Vec, whole-cell extract of the IPTG-induced vector control [BL21(DE3)(pLysS)(pET21a)].

FIG. 5.

Regulation of bfrH by ecfR. qRT-PCR analysis was performed using total RNAs isolated from Fe-replete and Fe-stressed cells and oligonucleotides targeting an internal sequence of bfrH (A and C) or targeting the intergenic region between ecfR and bfrH (B). Data are expressed as means and standard errors and were obtained by calculating the relative SQ of the respective mRNA after normalizing to the amount of recA mRNA expressed by the cell. Fe+, Fe-replete conditions; Fe−, Fe-stressed conditions. *, statistically significantly different from Fe-stressed RB50 (P < 0.05); #, statistical significance between Fe-stressed RB50ΔecfR(pecfI) and Fe-stressed RB50(pecfI) (P < 0.05).

FIG. 6.

BfrH is required for optimal virulence of RB50. (A) Growth of RB50 (solid symbols, dotted lines) and RB50ΔbfrH::Kan (open symbols, solid lines) cultured in SS liquid medium supplemented with 36 μM FeSO₄ (+Fe; squares) or left unsupplemented (−Fe; circles). Samples of each culture were taken at time points between 0 and 50 h, and the OD₆₀₀ was used as a measure of growth. (B) Two groups (n = 9) of female BALB/c mice (4 to 6 weeks old) were inoculated intranasally with 20 μl of PBS or with PBS containing a 1:1 mixture of 2.5 × 10⁵ CFU of RB50 and 2.5 × 10⁵ CFU of RB50ΔbfrH::Kan. The amount of colonization by each strain in the lungs, trachea, and nasal cavity of each mouse was determined on days 5 and 10 postinfection, and the CFU of RB50 and RB50ΔbfrH::Kan were used to determine the mean CI ± SEM. A paired two-tailed t test was used to determine statistical significance. *, P < 0.05; **, P < 0.001.

FIG. 7.

Genetic and expression model of the ecfIR-bfrH locus. Under conditions of Fe sufficiency, Fur1 represses transcription of the ecfIR-bfrH locus. Upon encountering Fe-limiting conditions, Fur1 dissociates from P_ecfIR and possibly also from P_bfrH, thus derepressing expression of ecfI and ecfR and enabling readthrough transcription from P_ecfIR into bfrH. At the genetic level, a cis-acting element (solid black line) in ecfR limits readthrough transcription of bfrH from P_ecfIR. A transduction signal initiated by BfrH after binding of the extracellular inducing ligand (an unknown siderophore) is transmitted through EcfR and EcfI to highly upregulate expression of bfrH from P_bfrH. The extracellular inducing ligand for the ecfIR-bfrH locus is believed to be an unknown xenosiderophore. OM, outer membrane; IM, cytoplasmic membrane; RNAP, RNA core polymerase.