Ferric Uptake Regulator Fur Coordinates Siderophore Production and Defense against Iron Toxicity and Oxidative Stress and Contributes to Virulence in Chromobacterium violaceum

Abstract

Iron is a highly reactive metal that participates in several processes in prokaryotic and eukaryotic cells. Hosts and pathogens compete for iron in the context of infection. Chromobacterium violaceum, an environmental Gram-negative bacterial pathogen, relies on siderophores to overcome iron limitation in the host. In this work, we studied the role of the Ferric Uptake Regulator Fur in the physiology and virulence of C. violaceum. A Δfur null mutant strain showed decreased growth and fitness under regular in vitro growth conditions and presented high sensitivity to iron and oxidative stresses. Furthermore, the absence of fur caused derepression of siderophore production and reduction in swimming motility and biofilm formation. Consistent with these results, the C. violaceum Δfur null mutant was highly attenuated for virulence and liver colonization in mice. In contrast, a manganese-selected spontaneous fur mutant showed only siderophore overproduction and sensitivity to oxidative stress, indicating that Fur remained partially functional in this strain. We found that mutations in genes related to siderophore biosynthesis and a putative CRISPR/Cas locus rescued the Δfur growth defects, indicating that multiple Fur-regulated processes contribute to maintaining bacterial cell fitness. Overall, our data indicated that Fur is conditionally essential in C. violaceum mainly by protecting cells from iron overload and oxidative damage. The requirement of Fur for virulence highlights the importance of iron in the pathogenesis of C. violaceum. IMPORTANCE Maintenance of iron homeostasis is vital to bacteria and their hosts, avoiding scarcity or toxicity of this metal. Iron sequestration by host proteins is a crucial strategy to combat bacterial infections. In bacteria, the Ferric Uptake Regulator Fur coordinates the expression of several iron-related genes. Sometimes, Fur can also regulate several other processes. In this work, we performed an in-depth phenotypic characterization of fur mutants in the human opportunistic pathogen Chromobacterium violaceum. We determined that fur is a conditional essential gene necessary for proper growth under regular conditions and is fully required for survival under iron and oxidative stresses. Fur also controlled several virulence-associated traits, such as swimming motility, biofilm formation and siderophore production. Consistent with these results, a C. violaceum fur null mutant showed attenuation of virulence. Therefore, our data established Fur as a major player required for C. violaceum to manage iron, including during infection in the host.

Functional characterization of Fur in iron metabolism, oxidative stress resistance and virulence of Riemerella anatipestifer

“…RecA repairs oxidative DNA damage by combining with the RecF-like pathway or RecBCD pathway [ 8 , 9 ]. In most Gram-negative bacteria, iron homeostasis is regulated by the ferric uptake regulator Fur [ 11 – 15 ]. When the concentration of intracellular free iron is high, the iron-associated Fur dimer binds to the promoter region of iron uptake genes to inhibit transcription, thus reducing iron intake.…”

The zinc transporter ZnuABC is critical for the virulence of Chromobacterium violaceum and contributes to diverse zinc-dependent physiological processes

“…pyridylmethyl) ethylenediamine (TPEN). For iron, we used FeSO 4 (iron sufficiency) or 2,2'dipyridyl (2,2'-DP) (iron deficiency), as previously defined for C. violaceum (27,28).…”

“…Swimming motility. The swimming motility assay was performed in M9CH 0.3% agar plates as previously described (27). When indicated, the M9CH plates were supplemented with 1 mM EDTA and/or 250 µM ZnCl 2 .…”

“…All strains were cultured in glass tubes without agitation in LB medium with or without 100 µM EDTA at 37°C for 24 h, before staining with 0.1% (w/v) crystal violet. Stained biofilms were resuspended with 100% ethanol (1 ml), followed by measurement of OD 600 (27). The experiments were performed in six biological replicates.…”

“…Mouse virulence assays. 6-week-old female BALB/c mice were used to inject intraperitoneally 10 6 CFU from the C. violaceum strains, as previously determined (27,28,31,37). Animal survival was followed up for 10 days post infection.…”