A quorum-sensing regulatory cascade for siderophore-mediated iron homeostasis in Chromobacterium violaceum.

Iron is a transition metal used as a cofactor in many biochemical reactions. In bacteria, iron homeostasis involves Fur-mediated de-repression of iron uptake systems, such as the iron-chelating compounds siderophores. In this work, we identified and characterized novel regulatory systems that control siderophores in the environmental opportunistic pathogen Chromobacterium violaceum. Screening of a 10,000-transposon mutant library for siderophore halos identified seven possible regulatory systems involved in siderophore-mediated iron homeostasis in C. violaceum. Further characterization revealed a regulatory cascade that controls siderophores involving the transcription factor VitR acting upstream of the quorum-sensing (QS) system CviIR. Mutation of the regulator VitR led to an increase in siderophore halos, and a decrease in biofilm, violacein, and protease production. We determined that these effects occurred due to VitR-dependent de-repression of vioS. Increased VioS leads to direct inhibition of the CviR regulator by protein-protein interaction. Indeed, insertion mutations in cviR and null mutations of cviI and cviR led to an increase of siderophore halos. RNA-seq of the cviI and cviR mutants revealed that CviR regulates CviI-dependent and CviI-independent regulons. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by both CviI and CviR. However, genes related to iron homeostasis and many other processes were regulated by CviR but not CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving QS that controls siderophore-mediated iron homeostasis in C. violaceum.IMPORTANCEThe iron-chelating compounds siderophores play a major role in bacterial iron acquisition. Here, we employed a genetic screen to identify novel siderophore regulatory systems in Chromobacterium violaceum, an opportunistic human pathogen. Many mutants with increased siderophore halos had transposon insertions in genes encoding transcription factors, including a novel regulator called VitR, and CviR, the regulator of the quorum-sensing (QS) system CviIR. We found that VitR is upstream in the pathway and acts as a dedicated repressor of vioS, which encodes a direct CviR-inhibitory protein. Indeed, all QS-related phenotypes of a vitR mutant were rescued in a vitRvioS mutant. At high cell density, CviIR activated classical QS-dependent processes (violacein, proteases, and antibiotics production). However, genes related to iron homeostasis and type-III and type-VI secretion systems were regulated by CviR in a CviI- or cell density-independent manner. Our data unveil a complex regulatory cascade integrating QS and siderophores in C. violaceum.

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

Chromobacterium violaceum is an environmental Gram-negative beta-proteobacterium that causes systemic infections in humans. C. violaceum 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 C. violaceum to explore an important iron reservoir in mammalian hosts, free heme and hemoproteins. We demonstrate that the chuPRSTUV genes comprise a Fur-repressed operon that is expressed under iron limitation. The chu 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 C. violaceum chu deletion mutants revealed that, with the exception of chuS, all genes of the chu operon are required for heme and hemoglobin utilization in C. violaceum. The mutant strains without chuP 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 chuR and vbuA, 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 C. violaceum uses both heme and siderophore for iron acquisition during infection, with a preference for siderophores over the Chu heme utilization system.