IroN, a novel outer membrane siderophore receptor characteristic of Salmonella enterica

Abstract

Speciation in enterobacteria involved horizontal gene transfer. Therefore, analysis of genes acquired by horizontal transfer that are present in one species but not its close relatives is expected to give insights into how new bacterial species were formed. In this study we characterize iroN, a gene located downstream of the iroBC operon in the iroA locus of Salmonella enterica serotype Typhi. Like iroBC, the iroN gene is present in all phylogenetic lineages of S. enterica but is absent from closely related species such as Salmonella bongori or Escherichia coli. Comparison of the deduced amino acid sequence of iroN with other proteins suggested that this gene encodes an outer membrane siderophore receptor protein. Mutational analysis in S. enterica and expression in E. coli identified a 78-kDa outer membrane protein as the iroN gene product. When introduced into an E. coli fepA cir fiu aroB mutant on a cosmid, iroN mediated utilization of structurally related catecholate siderophores, including N-(2,3-dihydroxybenzoyl)-L-serine, myxochelin A, benzaldehyde-2,3-dihydroxybenzhydrazone, 2-N,6-N-bis(2,3-dihydroxybenzoyl)-L-lysine, 2-N,6-N-bis(2,3-dihydroxybenzoyl)-L-lysine amide, and enterochelin. These results suggest that the iroA locus functions in iron acquisition in S. enterica.

FIG. 1

Restriction map of a DNA region located at about four centisomes on the S. enterica serotype Typhi chromosome. Positions and sizes of inserts carried in cosmids (pTY908 and pTY2117) or plasmids depicted were determined previously (5). Arrows above the map indicate positions and orientations of open reading frames identified by sequence analysis. E, EcoRI.

FIG. 2

Percentage sequence identity determined by pairwise alignment of amino acid sequences from IroN, PfeA, BfeA, FepA, and Cir with the program CLUSTAL.

FIG. 3

Multiple sequence alignment of IroN, PfeA, FepA, and BfeA with the program CLUSTAL. Dashes represent gaps introduced by the program to improve the alignment; identical amino acids are indicated by asterisks; dots indicate amino acids with similar properties.

FIG. 4

Phylogenetic distribution of genes of the iroA locus. The phylogenetic tree on the left was established by Reeves and coworkers (28). (A) Restriction map of the region from S. enterica serotype Typhi AJB70 (E, EcoRI). Positions of genes (arrows) identified in the iroA locus and of DNA fragments used as probes (black bars) are indicated. (B) Results of hybridization with these DNA probes. +, hybridization signal; −, no hybridization signal.

FIG. 5

Outer membrane profiles of bacterial strains which carry the iroN gene (lanes 3, 5, and 6) or in which iroN is lacking (lanes 7 and 8) or inactivated (lane 2). The position of IroN is indicated by an arrow. Positions and sizes of bands from standard proteins are indicated (lanes 1 and 4). (A) SDS-PAGE of outer membrane preparations of S. enterica serotype Typhimurium AJB64 (lane 2) and IR715 (lane 3) grown under iron limitation. (B) Outer membrane profiles of E. coli H5058 (lanes 7 and 8) and H5058(pTY908) (lanes 5 and 6) grown in LB supplemented with 0.2 mM 2,2′-dipyridyl (lanes 6 and 8) or 0.04 mM FeSO₄ (lanes 5 and 7).

FIG. 6

Structures of catecholate siderophores used in this study.

FIG. 7

Growth curves of S. enterica serotype Typhimurium (A) and E. coli (B) strains in NBD broth culture without supplements or supplemented with myxochelin A (MyxA). Growth was measured as optical density at 620 nm (OD620).