The yersiniabactin biosynthetic gene cluster of Yersinia enterocolitica: organization and siderophore-dependent regulation - PubMed (original) (raw)

The yersiniabactin biosynthetic gene cluster of Yersinia enterocolitica: organization and siderophore-dependent regulation

C Pelludat et al. J Bacteriol. 1998 Feb.

Abstract

The ability to synthesize and uptake the Yersinia siderophore yersiniabactin is a hallmark of the highly pathogenic, mouse-lethal species Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica 1B. We have identified four genes, irp1, irp3, irp4, and irp5, on a 13-kb chromosomal DNA fragment of Y. enterocolitica 08, WA-314. These genes constitute the yersiniabactin biosynthetic gene cluster together with the previously defined irp2. The irp1 gene consists of 9,486 bp capable of encoding a 3,161-amino-acid high-molecular-weight protein 1 (HMWP1) polypeptide with a predicted mass of 384.6 kDa. The first 3,000 bp of irp1 show similarity to the corresponding regions of the polyketide synthase genes of Bacillus subtilis and Streptomyces antibioticus. The remaining part of irp1 is most similar to irp2, encoding HMWP2, which might be the reason for immunological cross-reactivity of the two polypeptides. Irp4 was found to have 41.7% similarity to thioesterase-like protein of the anguibactin biosynthetic genes of Vibrio anguillarum. Irp5 shows 41% similarity to EntE, the 2,3-dihydroxybenzoic acid-activating enzyme utilized in enterobactin synthesis of Escherichia coli. Irp4 and Irp5 are nearly identical to YbtT and YbtE, recently identified in Y. pestis. irp3 has no similarity to any known gene. Inactivation of either irp1 or irp2 abrogates yersiniabactin synthesis. Mutations in irp1 or fyuA (encoding yersiniabactin/pesticin receptor) result in downregulation of irp2 that can be upregulated by the addition of yersiniabactin. A FyuA-green fluorescent protein translational fusion was downregulated in an irp1 mutant. Upregulation was achieved by addition of yersiniabactin but not desferal, pesticin, or pyochelin, which indicates high specificity of the FyuA receptor and autoregulation of genes involved in synthesis and uptake of yersiniabactin.

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Figures

FIG. 1

FIG. 1

Genetic organization of the Y. enterocolitica O8 WA-314 irp2-fyuA gene cluster. The genes are depicted as boxes. Arrows above indicate the direction of transcription. E, _Eco_RI; S, _Sal_I.

FIG. 2

FIG. 2

Common amino acids motifs found in HMWP1 and HMWP2.

FIG. 3

FIG. 3

Southern hybridization of chromosomal DNAs from Yersinia and E. coli strains with the irp1 probe. The chromosomal DNA was digested with Eco_RI, and the resulting fragments were separated on a 1% agarose gel prior to Southern blotting. Hybridization was performed with a DIG-labeled PCR probe generated with primers i8513 and i8730. Lane 1, Y. pestis KUMA; lane 2, Y. pestis KIM; lane 3, Y. pestis KIM Δ_pgm; lane 4, Y. pseudotuberculosis 346; lane 5, Y. pseudotuberculosis 201; lane 6, Y. pseudotuberculosis PB1; lane 7, Y. enterocolitica 8081; lane 8, Y. enterocolitica WA-CS; lane 9, Y. enterocolitica Y5.27; lane 10, Y. enterocolitica Y-96-C; lane 11, Y. enterocolitica Y-108-C; lane 12, E. coli Phi; lane 13; E. coli DH5α.

FIG. 4

FIG. 4

Comparison of the region from bp 1041 to 1191 of irp1 in Y. enterocolitica 8081, Y. enterocolitica WA-CS (I), Y. pestis KUMA, Y. pestis KIM, Y. pseudotuberculosis 346, Y. pseudotuberculosis PB1, and E. coli Phi (II). Nonmatching bases are boldfaced and underlined.

FIG. 5

FIG. 5

CAS agar plate showing siderophore-producing Y. enterocolitica WA-CS (A; with halo) and mutant WA-CS irp1::Kanr (B).

FIG. 6

FIG. 6

Expression of HMWP1 and -2 in WA-CS irp1::Kanr and WA fyuA mutants. SDS-PAGE (7.5% gel) of total-cell proteins from iron-starved strains WA-CS (lane 1), WA-CS irp1::Kanr (lane 2), and WA fyuA (lane 3).

FIG. 7

FIG. 7

SDS-PAGE (7.5% gel) (A) and corresponding immunoblot (B) with cell lysates of WA-CS (lane 1), WA-CS irp1::Kanr (lane 2), and WA-CS irp1::Kanr with an addition of purified yersiniabactin (lane 3). The strains were grown under iron starvation in NBD medium. Western blotting was performed with HMWP polyclonal antibodies kindly provided from Elisabeth Carniel.

FIG. 8

FIG. 8

Effects of pesticin and yersiniabactin on expression of the HMWPs. SDS-PAGE (7.5% gel) of total-cell proteins of iron-starved strains WA-CS and WA-CS irp1::Kanr with addition of pesticin and culture supernatant containing yersiniabactin. Lane 1, WA-CS in NBD medium; lane 2, WA-CS in NBD medium with pesticin (1:1,024); lane 3, WA-CS in NBD medium with yersiniabactin supernatant (1:50); lane 4, WA-CS irp1::Kanr in NBD medium; lane 5, WA-CS irp1::Kanr in NBD medium with pesticin; lane 6, WA-CS irp1::Kanr in NBD medium with yersiniabactin supernatant.

FIG. 9

FIG. 9

Fluorescence of FyuA-GFP in Y. enterocolitica WA-CS (- -) and WA-CS irp1::Kanr (—) in NB medium (A), NBD medium (B), NBD medium plus purified yersiniabactin (C), and NBD medium plus desferrioxamine B (D). (E) Positive (WA-CS[pGFP mut 3]; -- --) and negative (WA-CS; -- —— --) controls. Au, arbitrary units.

FIG. 10

FIG. 10

Chemical structures of yersiniabactin, pyochelin, and anguibactin.

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