The Iron-Regulated iupABC Operon Is Required for Saprophytic Growth of the Intracellular Pathogen Rhodococcus equi at Low Iron Concentrations

Abstract

Rhodococcus equi is a facultative intracellular pathogen which proliferates rapidly in both manure-enriched soil and alveolar macrophages. Although both environments are characterized by extremely low concentrations of free iron, very little is known regarding the strategies employed by R. equi to thrive under these conditions. This paper reports the characterization of an R. equi transposome mutant that fails to grow at low iron concentrations. The transposome was shown to be inserted into iupA, the first gene of the iupABC operon encoding an ABC transport system highly similar to siderophore uptake systems. Disruption of the iupA gene also resulted in a failure of R. equi to utilize heme and hemoglobin as a source of iron. Introduction of the iupABC operon in trans restored the wild-type phenotype of the mutant strain. iupABC transcripts were 180-fold more abundant in R. equi grown in iron-depleted medium than in organisms grown in iron-replete medium. Proliferation of the iupABC mutant strain in macrophages was comparable to that of the wild-type strain. Furthermore, the iupABC mutant was not attenuated in mice, showing that the iupABC operon is not required for virulence.

The Hydroxamate Siderophore Rhequichelin Is Required for Virulence of the Pathogenic Actinomycete Rhodococcus equi

“…3). As was observed previously, the transcription levels of iupS, iupT, and iupA increased by two to three orders of magnitude following growth of R. equi in LMMϪFe compared to growth in LMM, whereas transcription levels of iupU and 16S rRNA were not affected (29,30) (Fig. 3).…”

“…Transcription of rhbC is regulated by iron. Transcription of the rhequibactin biosynthetic genes iupS and iupT, as well as that of the iupABC operon, which encodes a siderophore uptake system, is upregulated when R. equi is grown in medium containing low iron concentrations (29,30), highlighting their role in iron acquisition. R. equi was grown in LMM (iron replete) and LMMϪFe (iron depleted) to determine whether rhbC transcription is also controlled by the concentration of iron.…”

“…We previously showed that R. equi produces a soluble catecholatecontaining siderophore, rhequibactin, and a nondiffusible siderophore (29,30). Disruption of iupS and iupU, required for biosynthesis of the former and latter compounds, respectively, prevented growth under iron-limiting conditions but did not attenuate virulence in macrophages or mice, strongly suggesting that R. equi produces a third siderophore that provides the pathogen with iron following infection (29,30).…”

“…Pelleted bacteria were lysed using TRIzol (Sigma) and physically disrupted with zirconia beads in a MagNALyser instrument (Roche). Total RNA was isolated by chloroform extraction followed by DNA digestion with Turbo DNase (Ambion) and application to a Qiagen RNeasy column, with a second, in-column, DNA digestion with the RNase-free DNase as previously described (29).…”

“…Transcriptional regulation of genes encoding siderophore-producing NRPS (rhbC, iupS, iupT, and iupU) (30) and a siderophore ABC transporter (iupA) (29) in response to the iron concentration in the medium. R. equi was grown in iron-replete LMM medium (white bars) or in iron-depleted LMMϪFe medium (black bars).…”