ABSTRACT The metabolism of pathogenic microorganisms has to be flexible as the efficient replication of pathogenic bacteria in the host depends on an active adaptation process. Human infection by the bacterial pathogen Vibrio cholerae, requires multiple adaptation processes that ultimately lead to robust virulence factor production in the small intestine. Although a link between the metabolic status and virulence factor expression has been described in several pathogens, it has not been investigated in detail in V. cholerae. Similarly, the role of membrane bioenergetics in bacterial virulence is not well understood, although most pathogenic organisms extensively utilize membrane respiration for part of their physiology. Our previous results suggest that changes in membrane bioenergetics affect virulence gene expression in V. cholerae. More specifically, loss of the main V. cholerae respiration enzyme (NQR) leads to changes in virulence gene transcription. Here, we propose to perform an investigation into the link between membrane respiration and virulence gene regulation in V. cholerae, with an emphasis on reactive oxygen species (ROS) and the quinone pool. It has been well documented that multiple virulence-associated transcription factors sense oxidative stress in V. cholerae via post-translational modifications such as thiol-switches. We have previously established NQR as a source of both periplasmic as well as cytoplasmic ROS. Moreover, we recently developed a method to produce varying levels of ROS in V. cholerae by manipulating the expression level of NQR and in parallel, the level of cytoplasmic ROS in live cells can be monitored using a novel, fluorophore-based approach. This system enables us to quantitatively correlate ROS levels with the expression of genes encoding for virulence factors in V. cholerae. Overall, we expect to gain important insights into the metabolic capacity of this organism per se and its relation to virulence gene expression as an important aspect of pathogenesis. Our rationale for this project is that the generation of fundamental information concerning V. cholerae metabolism and its link to virulence factor production will provide potential opportunities for novel treatment strategies for infections.