Tight Regulation of Extracellular Superoxide Points to Its Vital Role in the Physiology of the Globally Relevant Roseobacter Clade

There is a growing appreciation within animal and plant physiology that the reactive oxygen species (ROS) superoxide is not only detrimental but also essential for life. Yet, despite widespread production of extracellular superoxide by healthy bacteria and phytoplankton, this molecule remains associated with stress and death. Here, we quantify extracellular superoxide production by seven ecologically diverse bacteria within the Roseobacter clade and specifically target the link between extracellular superoxide and physiology for two species. We reveal for all species a strong inverse relationship between cell-normalized superoxide production rates and cell number. For exponentially growing cells of Ruegeria pomeroyi DSS-3 and Roseobacter sp. strain AzwK-3b, we show that superoxide levels are regulated in response to cell density through rapid modulation of gross production and not decay. Over a life cycle of batch cultures, extracellular superoxide levels are tightly regulated through a balance of both production and decay processes allowing for nearly constant levels of superoxide during active growth and minimal levels upon entering stationary phase. Further, removal of superoxide through the addition of exogenous superoxide dismutase during growth leads to significant growth inhibition. Overall, these results point to tight regulation of extracellular superoxide in representative members of the Roseobacter clade, consistent with a role for superoxide in growth regulation as widely acknowledged in fungal, animal, and plant physiology.