A cytochrome bd repressed by a MarR family regulator confers resistance to metals, nitric oxide, sulfide, and cyanide in Chromobacterium violaceum

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Abstract
Chromobacterium violaceum is a ubiquitous environmental pathogen. Despite its remarkable adaptability, little is known about the mechanisms of stress resistance in this bacterium. Here, in a screen for iron-susceptible transposon mutants, we identified a cytochrome bd that protects C. violaceum against multiple stresses. The two subunits of this cytochrome bd (CioAB) are encoded by the cioRAB operon, which also encodes a GbsR-type MarR family transcription factor (CioR). A ∆ cioAB mutant strain was sensitive to iron and the iron-requiring antibiotic streptonigrin and showed a decrease in siderophore production. Growth curves and survival assays revealed that the ∆ cioAB strain was also sensitive to zinc, hydrogen peroxide, nitric oxide, sulfide, and cyanide. Expression analysis showed that the promoter activity of the cioRAB operon and the transcript levels of the cioAB genes were increased in a ∆ cioR mutant. CioR bound the promoter region of the cio operon in vitro , indicating that CioR is a direct repressor of its own operon. Expression of the cio operon increased at high cell density and was dependent on the quorum-sensing regulator CviR. As cyanide is also a signal for cio expression, and production of endogenous cyanide is known to be a quorum sensing-regulated trait in C. violaceum , we suggest that CioAB is a cyanide-insensitive terminal oxidase that allows respiration under cyanogenic growth conditions. Our findings indicate that the cytochrome bd CioAB protects C. violaceum against multiple stress agents that are potentially produced endogenously or during interactions with a host. IMPORTANCE: The terminal oxidases of bacterial respiratory chains rely on heme-copper (heme-copper oxidases) or heme (cytochrome bd ) to catalyze the reduction of molecular oxygen to water. Chromobacterium violaceum is a facultative anaerobic bacterium that uses oxygen and other electron acceptors for respiration under conditions of varying oxygen availability. The C. violaceum genome encodes multiple respiratory terminal oxidases, but their role and regulation remain unexplored. Here, we demonstrate that CioAB, the single cytochrome bd from C. violaceum , protects this bacterium against multiple stressors that are inhibitors of heme-copper oxidases, including nitric oxide, sulfide, and cyanide. CioAB also confers C. violaceum resistance to iron, zinc, and hydrogen peroxide. This cytochrome bd is encoded by the cioRAB operon, which is under direct repression by the MarR-type regulator CioR. In addition, the cioRAB operon responds to quorum sensing and to cyanide, suggesting a protective mechanism of increasing CioAB in the setting of high endogenous cyanide production.
Funding Information
  • Fundação de Amparo à Pesquisa do Estado de São Paulo (2018/01388-6, 2021/06894-0, 2021/10577-0)
  • Fundação de Apoio ao Ensino, Pesquisa e Assistência
  • National Institute of Allergy and Infectious Diseases (AI 150041)

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