Ferric enterochelin transport in Yersinia enterocolitica: molecular and evolutionary aspects

Abstract

Yersinia enterocolitica is well equipped for siderophore piracy, encompassing the utilization of siderophores such as ferrioxamine, ferrichrome, and ferrienterochelin. In this study, we report on the molecular and functional characterization of the Yersinia fep-fes gene cluster orthologous to the Escherichia coli ferrienterochelin transport genes (fepA, fepDGC, and fepB) and the esterase gene fes. In vitro transcription-translation analysis identified polypeptides of 30 and 35 kDa encoded by fepC and fes, respectively. A frameshift mutation within the fepA gene led to expression of a truncated polypeptide of 40 kDa. The fepD, fepG, and fes genes of Y. enterocolitica were shown to complement corresponding E. coli mutants. Insertional mutagenesis of fepD or fes genes abrogates enterochelin-supported growth of Y. enterocolitica on iron-chelated media. In contrast to E. coli, the fep-fes gene cluster in Y. enterocolitica consists solely of genes required for uptake and utilization of enterochelin (fep) and not of enterochelin synthesis genes such as entF. By Southern hybridization, fepDGC and fes sequences could be detected in Y. enterocolitica biotypes IB, IA, and II but not in biotype IV strains, Yersinia pestis, and Yersinia pseudotuberculosis strains. According to sequence alignment data and the coherent structure of the Yersinia fep-fes gene cluster, we suggest early genetic divergence of ferrienterochelin uptake determinants among species of the family Enterobacteriaceae.

FIG. 1

Restriction map of pSI10 and various subclones used for protein expression and testing for complementation of growth of E. coli AN272. (+), plasmids which promote AN272 growth; (−), no growth stimulation. Arrows indicate the direction of transcription of genes included in the enterochelin uptake locus. Open inverted triangles represent sites of Tn552kan insertion. Open arrows represent the location of the T7 promoter. Grey shaded boxes upstream of fepDCG, fes, and fepA* (frameshifted fepA) represent potential Fur boxes.

FIG. 2

Comparison of the enterochelin gene cluster of Y. enterocolitica O8 strain WA-C to the E. coli gene cluster encoding proteins involved in biosynthesis and transport of enterochelin. Open arrows indicate the locations of primers used for RT-PCR and LA-PCR cloning procedures. The shaded box between fes and fepC represents the location of the ERIC sequence in Y. enterocolitica.

FIG. 3

Detection of the transcription of fepD-, fes-, and fepA-homologous genes of Y. enterocolitica by RT-PCR. Lane 1, DNA molecular size (MW) markers; lanes 2 and 3, fepD; lanes 4 and 5, fes; lanes 6 and 7, fepA-homologous gene. PCR mixtures in lanes 2, 4, and 6 contained RT, whereas mixtures in lanes 3, 5, and 7 lacked RT and served as negative controls.

FIG. 4

Comparison of the ERIC sequence from the Y. enterocolitica fes-fepDGC DNA region with the consensus (27). The core inverted repeats found in all ERIC sequences are indicated by the arrows.

FIG. 5

T7 protein expression assay with E. coli WM1576 harboring pGP1-2 and other plasmids. Shown are autoradiograms of plasmid-encoded proteins labelled with ³⁵S-amino acids. Lane 1, pACYC184; lane 2, pH-1/C; lane 3, pH-1; lane 4, pB-2. Arrows indicate the presumed expression products: Fes, truncated FepA*, and FepC.

FIG. 6

Southern blotting of ClaI-digested chromosomal DNA of different Y. enterocolitica serovars and E. coli DH5α. The preparation was probed with a labelled PCR product generated from the Yersinia fes gene. Lane 1, E. coli DH5α; lane 2, Y. enterocolitica O3; lane 3, O5; lane 4, O5.27; lane 5, O8; lane 6, O9; lane 7, O13; lane 8, O20; lane 9, Y. pseudotuberculosis I; lane 10, Y. pseudotuberculosis II; lane 11, Y. pseudotuberculosis III; lane 12, Y. pestis pgm⁺.

FIG. 7

Homology tree determined by pairwise alignment of amino acid sequences by the method developed by Wilbur and Lipman (1, 60). (A) Homology tree of E. coli Fes, E. chrysanthemi Fes, Y. enterocolitica Fes, and IroD of S. enterica (∗, Salmonella enterica subsp. enterica serotype Typhi). (B) Homology tree for iron transport proteins belonging to the superfamily of ABC transporters: Y. enterocolitica YfuC, S. marcescens SfuC (2), Y. enterocolitica FepC, E. coli FepC, Y. pestis FepC, and Y. pestis YfeB (8).