Bacteria use siderophores to scavenge iron from environmental or host sources. The iron acquisition systems of Chromobacterium violaceum, a ubiquitous environmental bacterium that can cause infections in humans, are still unknown. In this work, we demonstrated that C. violaceum produces putative distinct endogenous siderophores, here named chromobactin and viobactin, and showed that they are each required for iron uptake and virulence. An in silico analysis in the genome of C. violaceum revealed that genes related to synthesis and uptake of chromobactin (cba) and viobactin (vba) are located within two secondary-metabolite biosynthetic gene clusters. Using a combination of gene deletions and siderophore detection assays, we revealed that chromobactin and viobactin are catecholate siderophores synthesized from the common precursor 2,3-dihydroxybenzoate (2,3-DHB) on two nonribosomal peptide synthetase (NRPS) enzymes (CbaF and VbaF) and taken up by two TonB-dependent receptors (CbuA and VbuA). Infection assays in mice revealed that both the synthesis and the uptake of chromobactin or viobactin are required for the virulence of C. violaceum, since only the mutant strains that do not produce any siderophores or are unable to take up both of them were attenuated for virulence. In addition, the mutant strain unable to take up both siderophores showed a pronounced attenuation of virulence in vivo and reduced neutrophil extracellular trap (NET) formation in in vitro assays, suggesting that extracellularly accumulated siderophores modulate the host immune response. Overall, our results revealed that C. violaceum uses distinct endogenous siderophores for iron uptake and its establishment in the host.

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.

Here we present the establishment of an inducible system based on the dimerizable Cre recombinase (DiCre) for controlled gene expression in the protozoan parasite Leishmania. Rapamycin-induced DiCre activation promoted efficient flipping and expression of gene products in a time and dose-dependent manner. The DiCre flipping activity induced the expression of target genes from both integrated and episomal contexts broadening the applicability of the system. We validated the system by inducing the expression of both full length and truncated forms of the checkpoint protein Rad9, which revealed that the highly divergent C-terminal domain of Rad9 is necessary for proper subcellular localization. Thus, by establishing the DiCre-based inducible system we have created and validated a robust new tool for assessing gene function in Leishmania.