Identification of AlcR, an AraC-type regulator of alcaligin siderophore synthesis in Bordetella bronchiseptica and Bordetella pertussis - PubMed (original) (raw)

Identification of AlcR, an AraC-type regulator of alcaligin siderophore synthesis in Bordetella bronchiseptica and Bordetella pertussis

E Pradel et al. J Bacteriol. 1998 Feb.

Abstract

A Fur titration assay was used to isolate DNA fragments bearing putative Fur binding sites (FBS) from a partial Bordetella bronchiseptica genomic DNA library. A recombinant plasmid bearing a 3.5-kb DNA insert was further studied. Successive deletions in the cloned fragment enabled us to map a putative FBS at about 2 kb from one end. Sequence analysis revealed the presence of an FBS upstream from a new gene encoding an AraC-type transcriptional regulator. The deduced protein displays similarity to PchR, an activator of pyochelin siderophore and ferripyochelin receptor synthesis in Pseudomonas aeruginosa. Homologous genes in Bordetella pertussis and Bordetella parapertussis were PCR amplified, and sequence comparisons indicated a very high conservation in the three species. The B. pertussis and B. bronchiseptica chromosomal genes were inactivated by allelic exchange. Under low-iron growth conditions, the mutants did not secrete the alcaligin siderophore and lacked AlcC, an alcaligin biosynthetic enzyme. Alcaligin production was restored after transformation with a plasmid bearing the wild-type gene. On the basis of its role in regulation of alcaligin biosynthesis, the new gene was designated alcR. Additional sequence determination showed that alcR is located about 2 kb downstream from the alcABC operon and is transcribed in the same orientation. Two tightly linked open reading frames, alcD and alcE, were identified between alcC and alcR. AlcE is a putative iron-sulfur protein; AlcD shows no homology with the proteins in the database. The production of major virulence factors and colonization in the mouse respiratory infection model are AlcR independent.

PubMed Disclaimer

Figures

FIG. 1

FIG. 1

Mapping of the region of pEP279 conferring a Lac+ phenotype in the FURTA. Subclones of the 3.5-kb _Pst_I insert were constructed by deletions in pEP279 using the following restriction enzymes: HII, _Hin_cII; K, _Kpn_I; N, _Nar_I; P, _Pst_I; S, _Sma_I; SI, _Sac_I; and SII, _Sac_II. The recombinant plasmid-associated Lac phenotypes are indicated on the right. The deduced localization of the FBS is also shown (black box).

FIG. 2

FIG. 2

Nucleotide sequence of alcR of B. bronchiseptica and deduced amino acid sequence of AlcR. Selected restriction enzyme sites, the putative FBS, and possible initiation codons are indicated (underlined). Differences detected in the B. pertussis and B. parapertussis sequences are also shown (underlined and labelled and , respectively).

FIG. 3

FIG. 3

Alignment of the deduced C-terminal amino acid sequences of AlcR of B. bronchiseptica and PchR of P. aeruginosa. The final 180 residues of each protein are shown. The putative helix-turn-helix and AraC signature motifs (underlined), exact matches, and conserved changes (+) are indicated.

FIG. 4

FIG. 4

Effect of alcR inactivation on protein and siderophore production in B. pertussis and B. bronchiseptica as determined by SDS-PAGE analysis. (A) WCLs of cultures grown in iron-limited SS medium. Lanes: 1, BPSM (wild type); 2, BPEP184 alcR::Kmr; 3, BPEP184(pEP301); 4, BPEP184(pBBR1MCS); 5, BB1015 (wild type); 6, BBEP205 alcR::Kmr; 7, BBEP205(pEP301); 8, BBEP205(pBBR1MCS). Siderophore (Sid) production in matching culture supernatants tested by the CAS assay is indicated below the gel (+, high level of activity; −, no siderophore activity detected). (B) WCLs of BB1015 (lanes 1 and 3) and BBEP205 alcR::Kmr (lanes 2 and 4) grown in iron-rich SS medium (SS+Fe) (lanes 1 and 2) or iron-limited SS medium (SS-Fe) (lanes 3 and 4). (C) Soluble proteins prepared from BPSM (lane 5) and BPEP184 alcR::Kmr (lane 6) grown in iron-limited SS medium. For both gels, the molecular masses of markers in lane MM are 97.4, 66.2, 45, and 31 kDa, from top to bottom. The 60-kDa iron-repressed protein is indicated (arrowheads).

FIG. 5

FIG. 5

Physical map of the alc locus of B. bronchiseptica. Blocks representing ORFs are drawn to scale from the 4.3-kb alcABC sequence determined by Giardina et al. (20) and from the sequence data for the 3.5-kb _Pst_I DNA fragment presented in this study. _Pst_I restriction sites (P), FBS upstream from alcA and alcR (black bars), and _bcr_′, the 5′ extremity of a putative bicyclomycin resistance gene downstream from alcR, are indicated.

FIG. 6

FIG. 6

(A) Flanking sequences of the alcD gene and deduced amino acid sequences. The tight genetic organization of the alcC, alcD, and alcE genes is shown. The _Pst_I restriction site (underlined), the 3′-terminal sequence of alcC (lowercase), and the deduced C-terminal sequence of AlcC and the alcD and alcE putative coding sequences and predicted translation products (uppercase) are shown. A potential RBS upstream from alcE is also indicated (underlined). (B) Partial amino acid sequence alignments of the putative AlcE protein with P. putida TdnA1. C and H residues forming the predicted iron-sulfur reaction center in TdnA1 (underlined) and conservative changes (+) are indicated.

Similar articles

Cited by

References

    1. Agiato L A, Dyer D W. Siderophore production and membrane alterations by Bordetella pertussis in response to iron starvation. Infect Immun. 1992;60:117–123. - PMC - PubMed
    1. Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. - PubMed
    1. Armstrong S K, Clements M O. Isolation and characterization of Bordetella bronchiseptica mutants deficient in siderophore activity. J Bacteriol. 1993;175:1144–1152. - PMC - PubMed
    1. Beall B, Sanden G N. Cloning and initial characterization of the Bordetella pertussis fur gene. Curr Microbiol. 1995;30:1–4. - PubMed
    1. Beall B, Sanden G N. A Bordetella pertussis fepA homologue required for utilization of exogenous ferric enterobactin. Microbiology. 1995;141:3193–3205. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources