The Regulatory Protein ChuP Connects Heme and Siderophore-Mediated Iron Acquisition Systems Required for Chromobacterium violaceum Virulence

Abstract

<i>Chromobacterium violaceum</i> is an environmental Gram-negative beta-proteobacterium that causes systemic infections in humans. <i>C. violaceum</i> uses siderophore-based iron acquisition systems to overcome the host-imposed iron limitation, but its capacity to use other iron sources is unknown. In this work, we characterized ChuPRSTUV as a heme utilization system employed by <i>C. violaceum</i> to explore an important iron reservoir in mammalian hosts, free heme and hemoproteins. We demonstrate that the <i>chuPRSTUV</i> genes comprise a Fur-repressed operon that is expressed under iron limitation. The <i>chu</i> operon potentially encodes a small regulatory protein (ChuP), an outer membrane TonB-dependent receptor (ChuR), a heme degradation enzyme (ChuS), and an inner membrane ABC transporter (ChuTUV). Our nutrition growth experiments using <i>C. violaceum chu</i> deletion mutants revealed that, with the exception of <i>chuS</i>, all genes of the <i>chu</i> operon are required for heme and hemoglobin utilization in <i>C. violaceum</i>. The mutant strains without <i>chuP</i> displayed increased siderophore halos on CAS plate assays. Significantly, we demonstrate that ChuP connects heme and siderophore utilization by acting as a positive regulator of <i>chuR</i> and <i>vbuA</i>, which encode the TonB-dependent receptors for the uptake of heme (ChuR) and the siderophore viobactin (VbuA). Our data favor a model of ChuP as a heme-binding post-transcriptional regulator. Moreover, our virulence data in a mice model of acute infection demonstrate that <i>C. violaceum</i> uses both heme and siderophore for iron acquisition during infection, with a preference for siderophores over the Chu heme utilization system.