Characterization of novel mechanisms of regulation and resistance to iron in Chromobacterium violaceum

Author(s)

Bianca Bontempi Batista

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2023-07-13

Examining board members

José Freire da Silva Neto; Rogerio Ferreira Lourenço; Célia Maria de Almeida Soares

• Advisor: José Freire da Silva Neto

Abstract

Iron is an essential micronutrient, necessary as a cofactor in several biological reactions, but its intracellular excess can cause damage. In bacteria, iron homeostasis involves regulatory systems that control the uptake, in limitation, and the efflux and storage, in excess, of this metal. In this work, we identified and characterized new regulatory and iron resistance systems in Chromobacterium violaceum, an environmental bacterium that occasionally causes severe infections in humans. Unbiased identification of novel transcription factors and iron detoxification systems was performed by screening a library of 10,000 transposon mutants. The screen on PSA-CAS plates revealed 25 genes with transposon insertion with altered siderophore halos. The mutated genes grouped into different functional categories, and six of them were studied for encoding regulatory systems. Null mutation of csrA (CV_2600) and CV_0635 did not have the same phenotype found in the screen. Insertions and null mutation in the AirSR two-component system (CV_0536 and CV_0537) led to increased siderophore halos. The insertion and null mutation of the VitR regulator led to an increase in siderophore halos, and a decrease in the production of biofilm, violacein, and proteases. We determined that these effects occurred exclusively due to derepression of vioS in the vitR mutant. VioS is an inhibitor of the CviR regulator of the quorum sensing (QS) system CviIR. Indeed, insertion into cviR and null mutation of cviI and cviR led to an increase in siderophore halos. RNA-seq of the ΔcviI and ΔcviR mutants revealed that CviR regulates a set of CviI-dependent and independent genes. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by CviI and CviR. However, genes related to iron homeostasis and other processes were regulated by CviR but not by CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving the VitR regulator acting upstream of the QS system to control siderophore-mediated iron homeostasis in C. violaceum. Screen of the transposon library in iron excess revealed iron-susceptible mutants with transposon insertion in eighteen genes, and two regulatory systems were selected for further characterization. The first system consists of the CV_3659-58-57 operon that encodes the MarR family transcription factor CV_3659 and the cytochrome bd oxidase CV_3658-57. We demonstrated that the regulator CV_3659 represses the expression of its own operon and that the cytochrome bd oxidase is necessary for the resistance of C. violaceum to different metals (zinc and iron), to sulfide and to oxidative and nitrosative stresses. Finally, we characterized two C. violaceum H-NS proteins. RNAseq revealed that the H-NS CV_1363 and CV_1243 silence the expression of several genes in C. violaceum, such as those found in pathogenicity islands. As CV_1363 silences the expression of CV_1243, the effects of CV_1243 on phenotypes and gene regulation were only evidenced with mutation of both H-NS. Phenotypic characterization revealed that the C. violaceum H-NS are involved in violacein production, swimming motility, iron homeostasis, and resistance to acidic pH and high osmolarity. In conclusion, C. violaceum uses several regulatory systems to control iron homeostasis, both siderophore-mediated iron uptake and tolerance to excess of iron. (AU)

• FAPESP's process: 18/19058-2 - Characterization of novel mechanisms of regulation and resistance to iron in Chromobacterium violaceum
• Grantee: Bianca Bontempi Batista
• Support Opportunities: Scholarships in Brazil - Doctorate