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

Abstract

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.IMPORTANCE Formation of reactive oxygen species (ROS) through partial reduction of molecular oxygen is widely associated with stress within microbial and marine systems. Nevertheless, widespread observations of the production of the ROS superoxide by healthy and actively growing marine bacteria and phytoplankton call into question the role of superoxide in the health and physiology of marine microbes. Here, we show that superoxide is produced by several marine bacteria within the widespread and abundant Roseobacter clade. Superoxide levels outside the cell are controlled via a tightly regulated balance of production and decay processes in response to cell density and life stage in batch culture. Removal of extracellular superoxide leads to substantial growth inhibition. These findings point to an essential role for superoxide in the health and growth of this ubiquitous group of microbes, and likely beyond.

Keywords: Roseobacter; reactive oxygen species; superoxide; superoxide dismutase.

FIG 1

Cell-normalized extracellular superoxide production rates by seven bacterial species (A to G) within the Roseobacter clade. (H and I) All Roseobacter clade bacterial species, illustrating a strong power law relationship between cell-normalized extracellular superoxide production rates and cell number.

FIG 2

Representative chemiluminescence trace for Ruegeria pomeroyi DSS-3 at three cell loadings. The initial baseline is obtained using a carrier solution consisting of DTPA-treated, aged, filtered seawater with a filter placed in-line. Once a stable baseline is obtained, the pump is stopped, cells are added, and the pump is restarted. This process is repeated with cells added sequentially to the in-line filter. After a steady-state signal is obtained for the last loading, SOD is injected into the AFSW carrier solution to confirm that superoxide was responsible for the chemiluminescence signal. The difference between the AFSW baseline and the +SOD baseline is typically ∼200 luminescence units, and likely represents autooxidation of the MCLA probe. Thus, to obtain the most conservative cell-derived signal and avoid this autooxidation artifact, cell-derived signals are obtained by subtracting the initial AFSW signal from the signal obtained once cells are added to the in-line filter.

FIG 3

Steady-state extracellular superoxide concentrations (A and B) and cell-normalized production rates (C and D) for Ruegeria pomeroyi DSS-3 (blue) and Roseobacter sp. strain AzwK-3b (orange) in serially diluted cultures (0, 10, and 100× diluted) after 6 h of incubation. The same volume of culture, either 0.1 ml (A and C) or 1.0 ml (B and D) was added to the in-line filters for each measurement. Means with different letters are significantly different (P < 0.05); note the lack of an error bar for one treatment of DSS-3, which had an n of 1.

FIG 4

Pseudo-first-order rate constants for decay of an exogenous superoxide spike added as KO₂ within an aged, filtered seawater matrix to Ruegeria pomeroyi DSS-3 and Roseobacter AzwK-3b cultures. Decay rates were obtained in undiluted and diluted (10× and 100×) cultures. Decay followed pseudo-first-order kinetics with spikes 1× to 2× the steady-state concentration measured for each culture (<15 nM).

FIG 5

Steady-state extracellular superoxide concentrations (A and B) and cell-normalized production rates (C and D) for Ruegeria pomeroyi DSS-3 (blue) and Roseobacter sp. strain AzwK-3b (orange) in serially diluted cultures (undiluted and 10× and 100× diluted) at three cell loading levels following 6 h of acclimation to illustrate superoxide production as a function of cell number for each culture.

FIG 6

Superoxide production and decay over the life cycle of Ruegeria pomeroyi DSS-3 in batch culture. (A) Steady-state superoxide levels (in nanomolar) (tan bars) over time through a growth curve indicated by optical density (OD) at 600 nm (blue circles). (B) Pseudo-first-order rate constants for superoxide decay (seconds⁻¹) (tan bars) and half-lives (in minutes) (orange circles) through a growth curve indicated by OD₆₀₀ (blue circles). (C) Net cell-normalized superoxide production rates (in attomoles cell⁻¹ hour⁻¹) (tan bars) and cell-normalized superoxide concentrations (in attomoles/cell) (yellow diamonds) over a growth curve indicated by OD₆₀₀ (blue circles).

FIG 7

Impact of exogenous superoxide dismutase (SOD) on growth as depicted by optical density (OD) at 600 nm for Ruegeria pomeroyi DSS-3 (A) and Roseobacter AzwK-3b (B) at four SOD concentrations (0, 27, 50, and 100 U/ml). (C) Impact of heat-inactivated SOD on growth of Ruegeria pomeroyi DSS-3. Asterisks indicate significance levels of 0.01 to 0.05 (*), 0.001 to 0.01 (**), and 0.0001 to 0.001 (***). Means with different letters are significantly different (P < 0.05).