Energy starved Candidatus Pelagibacter ubique substitutes light-mediated ATP production for endogenous carbon respiration

Abstract

Previous studies have demonstrated that Candidatus Pelagibacter ubique, a member of the SAR11 clade, constitutively expresses proteorhodopsin (PR) proteins that can function as light-dependent proton pumps. However, exposure to light did not significantly improve the growth rate or final cell densities of SAR11 isolates in a wide range of conditions. Thus, the ecophysiological role of PR in SAR11 remained unresolved. We investigated a range of cellular properties and here show that light causes dramatic changes in physiology and gene expression in Cand. P. ubique cells that are starved for carbon, but provides little or no advantage during active growth on organic carbon substrates. During logarithmic growth there was no difference in oxygen consumption by cells in light versus dark. Energy starved cells respired endogenous carbon in the dark, becoming spheres that approached the minimum predicted size for cells, and produced abundant pili. In the light, energy starved cells maintained size, ATP content, and higher substrate transport rates, and differentially expressed nearly 10% of their genome. These findings show that PR is a vital adaptation that supports Cand. P. ubique metabolism during carbon starvation, a condition that is likely to occur in the extreme conditions of ocean environments.

Figure 1. Respiration decreases in the light compared to the dark as organic carbon becomes limiting.

Cell density and oxygen concentration of HTCC1062 grown under continuous darkness (filled circles, black bars) or light∶dark (12∶12 hr cycles) (open triangles, white bars) during logarithmic and stationary growth phase. The light source was a mixture of cool white and GRO-LUX fluorescent light bulbs, the intensity was of ca. 250–300 µmol photons m⁻² s⁻¹. Cells were grown in glass air-tight sealed bottles (transparent for light treatments or opaque for dark) that were submerged in a 17°C water bath in order to maintain constant temperature.

Figure 2. Under carbon-limiting conditions, cells in the light are able to maintain their morphology, while cells in darkness became smaller.

Scanning electron microscopy of HTCC1062 cells grown either in continuous light, 70 µmol photons m⁻² s⁻¹ (A, B, C, D) or in darkness (E, F, G, H), two, five and eight days after entering stationary phase. Pictures on day five were taken at two different magnifications (∼30000× (B and F) and 105000× (C and G)); higher magnification was used to show the pili formed by dark grown cells. Original high resolution pictures are available on Supporting Information.

Figure 3. PR contributes a higher percentage to the total energetic budget of the cells as the period of starvation lengthens.

Sequential measurements of cellular ATP content performed over 35 min, including 4 dark to light shifts. A) growth curve of HTCC1062 cells showing the four time points of the ATP assays (filled black dots). B) ATP content/cell (mean±range of duplicate samples) five minutes after exposure to either dark (triangles, grey background) or light (squares, white background) at the four time points shown in A. C) Percent increase in ATP/cell as a result of light exposure (mean±s.d. of the last three dark to light shifts in each graph). The light source, utilized during the dark-light shifts, was a white lamp covered by a green filter that transmits light mainly in the 500–580 nm wavelength range at ca. 70 µmol photons m⁻² s⁻¹.

Figure 4. Taurine-uptake transport capacity of starved Candidatus Pelagibacter ubique cells is 65% more efficient in the light than in darkness.

Uptake of radio-labeled ¹⁴C-taurine (1 µM final concentration) by HTCC1062 cells in light, (80 µmol photons m⁻² s⁻¹, open circles) versus dark (closed circles; mean±s.d. of triplicate measurements). Assays were conducted at room temperature (22°C) in artificial seawater media.