Identification of specific in vivo-induced (ivi) genes in Yersinia ruckeri and analysis of ruckerbactin, a catecholate siderophore iron acquisition system

Abstract

This work reports the utilization of an in vivo expression technology system to identify in vivo-induced (ivi) genes in Yersinia ruckeri after determination of the conditions needed for its selection in fish. Fourteen clones were selected, and the cloned DNA fragments were analyzed after partial sequencing. In addition to sequences with no significant similarity, homology with genes encoding proteins putatively involved in two-component and type IV secretion systems, adherence, specific metabolic functions, and others were found. Among these sequences, four were involved in iron acquisition through a catechol siderophore (ruckerbactin). Thus, unlike other pathogenic yersiniae producing yersiniabactin, Y. ruckeri might be able to produce and utilize only this phenolate. The genetic organization of the ruckerbactin biosynthetic and uptake loci was similar to that of the Escherichia coli enterobactin gene cluster. Genes rucC and rupG, putative counterparts of E. coli entC and fepG, respectively, involved in the biosynthesis and transport of the iron siderophore complex, respectively, were analyzed further. Thus, regulation of expression by iron and temperature and their presence in other Y. ruckeri siderophore-producing strains were confirmed for these two loci. Moreover, 50% lethal dose values 100-fold higher than those of the wild-type strain were obtained with the rucC isogenic mutant, showing the importance of ruckerbactin in the pathogenesis caused by this microorganism.

FIG. 1.

Schematic representation of in vitro expression selection in fish. Approximately 10⁴ cells of Y. ruckeri transcriptional fusions were injected intraperitoneally in rainbow trout (O. mykiss). At 24 and 48 h postinfection, chloramphenicol (6 mg/ml) was injected intramuscularly, and the fish were sacrificed 24 h later. Bacterial cells were recovered from fish tissue, and a new round of infection was carried out. Then, cells were plated on EMB, and Lac⁻ colonies were identified. Triparental mating was used for plasmid rescue from the ivi clones, and sequencing of the cloned fragments was carried out with primers blaseq and catseq-2 from the contiguous bla and cat genes, respectively.

FIG. 2.

Genetic organization of iviI, iviII, and iviIII clones from Y. ruckeri containing the ruckerbactin biosynthetic uptake cluster, and comparison with the E. coli enterobactin gene cluster. Thick arrows represent genes identified in both microorganisms.

FIG. 3.

Anaysis of rucC and rupDGC loci from the ruckerbactin cluster of Y. ruckeri. (A) β-Galactosidase activity of rupDGC (a) and rucC (b) lacZ fusions was measured in cells grown at 18°C (solid bars) and 28°C (open bars) in minimal medium (M9) containing: FeCl₃ (100 μM) (column 1), M9 (column 2), or 2,2′dipyridyl (100 μM) (column 3). The results are the averages of three independent experiments. Note that the data are plotted in different scales. (B) Siderophore production in CAS-agar plates by strains 150RrucC (rucC insertion mutant) (lane 1), 150R (wild type) (lane 2), and 150RrupG (rupG insertional mutant) (lane 3).