Identification and characterization of alcR, a gene encoding an AraC-like regulator of alcaligin siderophore biosynthesis and transport in Bordetella pertussis and Bordetella bronchiseptica

Abstract

A Bordetella bronchiseptica iron transport mutant was isolated following an enrichment procedure based on streptonigrin resistance. The mutant displayed a growth defect on iron-restricted medium containing ferric alcaligin as the sole iron source. In addition to the apparent inability to acquire iron from the siderophore, the mutant failed to produce alcaligin as well as two known iron-regulated proteins, one of which is the AlcC alcaligin biosynthesis protein. A 1.6-kb KpnI-PstI Bordetella pertussis DNA fragment mapping downstream of the alcaligin biosynthesis genes alcABC restored both siderophore biosynthesis and expression of the iron-regulated proteins to the mutant. Nucleotide sequencing of this complementing 1.6-kb region identified an open reading frame predicted to encode a protein with strong similarity to members of the AraC family of transcriptional regulators, for which we propose the gene designation alcR. Primer extension analysis localized an iron-regulated transcription initiation site upstream of the alcR open reading frame and adjacent to sequences homologous to the consensus Fur repressor binding site. The AlcR protein was produced by using an Escherichia coli expression system and visualized in electrophoretic gels. In-frame alcR deletion mutants of B. pertussis and B. bronchiseptica were constructed, and the defined mutants exhibited the alcR mutant phenotype, characterized by the inability to produce and transport alcaligin and express the two iron-repressed proteins. The cloned alcR gene provided in trans restored these siderophore system activities to the mutants. Together, these results indicate that AlcR is involved in the regulation of Bordetella alcaligin biosynthesis and transport genes and is required for their full expression.

FIG. 1

Identification of the B. pertussis DNA region restoring alcaligin production to B. bronchiseptica mutant BRM10. Phenotypic complementation by B. pertussis recombinant cosmid pCP1.11 and plasmid subclones was evaluated on CAS siderophore indicator agar. BRM10 carrying the designated plasmids was scored as follows: +, siderophore activity was produced; −, no siderophore activity was detected. The alcABC alcaligin biosynthesis genes and the newly identified alcR gene are indicated. Abbreviations: B, BamHI; K, KpnI; P, PstI; S, SalI; Sm, SmaI; X, XhoI.

FIG. 2

SDS-PAGE analysis of wild-type B. bronchiseptica B013N and mutant derivative BRM10. Strain B013N, streptonigrin-resistant mutant BRM10 harboring the plasmid vector pRK415, and the complementing 1.6-kb B. pertussis KpnI-PstI DNA fragment as pP9KP were grown in parallel under iron-replete (+) and iron-depleted (−) conditions, and cell fractions were prepared as described in Materials and Methods. (A) Soluble cell fractions showing the iron-repressed ca. 59-kDa AlcC protein (arrowhead). (B) Total membrane fractions; the 79-kDa iron-repressed protein, which is absent in mutant BRM10(pRK415), migrates as the middle species of a protein triplet (arrowhead). The migration positions of molecular mass protein standards are shown on the left in kilodaltons.

FIG. 3

Nucleotide sequence of alcR. The nucleotide sequence (upper strand) of the 1,200-bp region of the B. pertussis 1.6-kb KpnI-PstI DNA fragment and the predicted amino acid sequence of AlcR in the one-letter code are shown. The transcription initiation sites (+1) determined by primer extension analysis using the designated antisense primer (arrow) are indicated. Sequences similar to the consensus Fur binding site (Fur Box) and the positions of the NgoAIV restriction endonuclease sites used to construct deletion mutants PM10 and BRM11 are shown.

FIG. 4

Primary amino acid sequence alignments of translated B. pertussis DNA alcR sequences with selected high-scoring AraC family members identified in BLAST database searches. The partial sequences shown represent the highest-scoring carboxy-terminal segments of similarity. The helix-turn-helix DNA binding and AraC signature motifs (lines), amino acid positions in the protein sequences as reported in GenBank (numbers), and residues which match the consensus (boxes) are indicated. The GenBank accession numbers are as follows: B. pertussis AlcR, AF018255; P. aeruginosa PchR, L11657; Y. pestis YbtA, U50452; P. putida XylS, M10143, M15819, and M20635; Y. pestis LcrF, M86690; and E. coli K-12 AraC, V00259.

FIG. 5

Localization of the iron-regulated P_alcR promoter of alcR. RNA was isolated from B. pertussis wild-type strain UT25Sm1 (WT) and B. pertussis mutant PM-4, in which the alcA promoter-operator region is deleted, grown in iron-replete (+Fe) and iron-depleted (−Fe) conditions. Transcriptional initiation sites (+1) corresponding to two nucleotides, C and T (at positions 124 and 125, respectively), were mapped by primer extension analysis using the antisense primer shown in Fig. 3.

FIG. 6

Expression of the alcR protein product in E. coli. A T7 polymerase-promoter system was used to express alcR from the 1.6-kb KpnI-PstI B. pertussis DNA fragment as described in Materials and Methods. (A) Autoradiogram of ³⁵S-labelled translational products. (B) Proteins visualized by Coomassie blue staining. Lanes: pET-3, BL21(DE3) cells carrying the plasmid vector control; pET-3KP, cells carrying the alcR gene. U, protein samples from uninduced cells; I, protein samples after induction of cells by addition of IPTG. The migration position of the 31-kDa molecular mass standard is indicated on the left. The AlcR polypeptide is indicated on the right (arrowheads). The labelled protein in panel A migrating at approximately 32 kDa is presumed to be the vector-encoded β-lactamase.

FIG. 7

Alcaligin utilization bioassays. Promotion of growth of B. bronchiseptica by exogenously supplied alcaligin in iron-restricted medium was performed as detailed in Materials and Methods. Alcaligin biosynthesis mutant BRM3 is wild type with respect to ferric alcaligin transport. The diameters of the zones of growth include the 6 mm contributed by the sample well. Bacteria supplied with diluent alone showed no growth stimulation. Standard deviations are indicated as vertical bars; no standard deviation bars are shown if the values were less than 0.4.